Cell Culture Bioprocess Engineering

Cell Culture Bioprocess Engineering

Wei-Shou HuDepartment of Chemical Engineering and Material ScienceUniversity of MinnesotaMinneapolis, MN

With Contributions From:

Weichang ZhouGargi SethSadettin OzturkChun Zhang

Cell Culture BioproCess engineering

Copyright © 2012 by Wei-Shou Hu

ISBN: 978-0-9856626-0-8

http://www.cellprocessbook.com/

prefaCe

For over two decades, we have assembled innovative guest lecturers to share their research and best-practices at our annual cell culture bioprocessing short course at the University of Minnesota. This course was created for industrial practitioners of the production of biologics. This book is the culmination of two decades of accumulated expertise, practical know-how and insight into future trends. There have been many books and courses on cell culture technology covering topics from a technical or business perspective. The goal of this course and this book is to bring new knowledge from cutting-edge research into the very practical setting of today’s industrial laboratories. A second goal of this course is to prepare industrial practitioners and students from different academic disciplines to collaborate in today’s cross-disciplinary teams. In the course of delivering a molecule from a gene sequence in the laboratory to a product in the manufacturing plant, scientists and engineers must quickly communicate, troubleshoot and innovate. The fundamental knowledge for practicing industrial cell culture spans from cell biology and physiology to process engineering principles in stoichiometry, reactor kinetics and scale up. Thus, we have designed this course for students of diverse backgrounds. The book is used in the classroom of our annual course. The layout of the book is thus designed to facilitate the delivery of information. The left panels are graphs, tables, diagrams, highlights of key points and space for note taking; while the right panels are descriptive text. This course has been given around the world: in Europe, East and South Asia, South America and as an internal course at many corporations. Over three thousand industrial biotechnologists have taken this course. With the technology of biologics production spreading to wider regions of the world, this book will meet a timely need of many who practice the technology but cannot attend the course in Minnesota. The book is published in an electronic form to allow for more frequent future updates, and for easy distribution to the parts of the world where the biologics manufacturing is quickly expanding.

Wei-Shou HuDepartment of Chemical Engineering and Material ScienceUniversity of Minnesota

aCknowledgements

Theauthoringofthisbookhasbeeninfluencedbymanywhohavelecturedinthesummer course at the University of Minnesota over the years. Foremost, thanks go to Anthony J. Sinskey, Michael C. Flickinger, Donald McClure and Fredrick Srienc who started the course with me originally. Konstantin Konstantinov, James Piret, James N. Thomas, Randall Kaufman, Florian Wurm, John Aunins, Michael Betenbaugh, Sadettin Ozturk, Matthew Croughan, Weichang Zhou, Chun Zhang and Gargi Seth all contributed to enrich the course. Many former and current members of my research laboratory at the University of Minnesota contributed to the preparation of course materials. These include Derek Adams, Marlene Castro, Bhanu Chandra Mulukutla, Anushree Chatterjee, Anna Europa, Patrick Fu, Chetan Gadgil, Mugdha Gadgil, Anshu Gambhir, Patrick Hossler, Claire Hypolite, Nitya M. Jacob, Kathryn Johnson, Anne Kantardjieff, Edmund Kao, Anurag Khetan, Rashmi Korke, Huong Le, Jongchan Lee, Marcela de Leon Gatti, Sarika Mehra, Jason D. Owens, Yonsil Park, Gargi Seth, Shikha Sharma, Kartik Subramanian, Siguang Sui, Katie Wlaschin, and Kathy Wong. Gargi Seth, Sadettin Ozturk, Weichang Zhou and Chun Zhang, whose participation in the course led to the development of new chapters, are noted as contributors. This book, which began as a set of lecture notes, has gone through many years of refinementinorganizationbymanyskillfulhands.KimberlyDurandfirsttookthenotestodigital form in a CD ROM. Ruth Patton, Radha Dalal, Katherine Matthews, Heather Wooten, Kirsten Keefe, Jessica Raines-Jones, Kimberly Coffee and Kaitlyn Pladson continued to shape it. At the long last, Erin Fenton and Jenna Novotny took it to current form. Kimberly Durandalsocoordinatedourfinalpublicationefforts. This book is dedicated to the students, fellows and staff formerly and currently in my laboratory at the University of Minnesota. It is through working with them that the materials used in the book were distilled. It was also through their educating me with new knowledge, new concepts, and new tools that this book took its shape. I must also thank my dear friend and close colleague, Miranda Yap of Bioprocess Technology Institute, Singapore, with whom I have had a wonderful and long collaboration. Finally, I wish for my lovely family, Jenny, Kenny and my wife, Sheau-Ping to share the joy of the book’s completion.

Wei-Shou HuDepartment of Chemical Engineering and Material ScienceUniversity of Minnesota

Contents in Brief

Overview of Cell Culture Technology . . . . . . . . . . . . . . . . . . . . . . . 1

Cell Biology for Bioprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Cell Physiology for Process Engineering . . . . . . . . . . . . . . . . . . . . . 57

Medium Design for Cell Culture Processing . . . . . . . . . . . . . . . . . . 97

Cell Line Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Stoichiometry and Kinetics of Cell Cultivation . . . . . . . . . . . . . . . . 147

Cell Culture Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Metabolic Flux Analysis in Cell Culture Systems . . . . . . . . . . . . . . 175

Cell Culture Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Oxygen Transfer in Cell Culture Bioreactors . . . . . . . . . . . . . . . . . 213

Fedbatch Culture and Dynamic Nutrient Feeding . . . . . . . . . . . . . 233

Cell Retention and Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Scaling Up and Scaling Down for Cell Culture Bioreactors . . . . . . 263

Cell Culture Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

ACKNOWLEDGEMENTS | VII

OVERVIEW | 1

In the past decade we have seen continuous growth in mammalian cell culture bioprocessing, driven primarily by the expansion of therapeutic antibody production in the pharmaceutical industry. The range and quantity of products havebothsignificantly increasedoverthepast tenyears. Also fueling this growth are the increasing numbers of therapeutic protein candidates in the drug development pipeline that can potentially render many more untreatable diseases treatable.

Recombinant therapeutic proteins have yielded major advances in healthcare. Their societal impacts may even rival those of antibiotics, whose discovery and clinical applications transformed much of modern medicine. Microbial fermentation technology enabled pharmaceutical industry to make penicillin widely available between 1950 and 1970. Today, we see cell culture processing technology enabling this new class of protein biologics to reach needy patients.

As with any product, manufacturers are under continual pressure to produce more with less. In

Cell Culture Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Cell Culture Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Virus Vaccines and Protein Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Protein Molecule as Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Industrial Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Biosimilars or Follow-on-Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Alternative Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Product Quality and Process Robustness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Critical Feature of rDNA Proteins from Mammalian Cells . . . . . . . . . . . . . . . . . 14Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Cell Culture Engineering

Overview of Cell Culture Technology

OVERVIEW OF CELL CULTURE TECHNOLOGY | 2

the case of cell culture bioprocess technology, we see increasing demand for therapeutic proteins, coupled with the strain of often prohibitively high investment costs for new manufacturing facilities. Thus, we must constantly re-evaluate, streamline and refine, to increase production without theluxury of totally new and improved facilities.

As we look to the future of cell culture processing, it is useful to look back at the history and development ofpenicillin.Thisspecificcasehighlightslessonsthatare almost universally relevant for the manufacturing of other products. Today’s innovations will all travel through some variation of these phases, from the moment of discovery, expansion and distribution, maturation and even demise of the product. Pencillin is also representative of the many strides made in the broader field of microbial naturalproducts that preceded today’s protein biologics.

Sir Alexander Fleming’s discovery of penicillin began a new chapter in biotechnology. In the twenty-five years following the first clinical applications(pioneered by Edward Penley Abraham) both the product titer and the production volume of penicillin increased almost exponentially. This rapid expansion in production quantities and titer was then followed by a period of slower but steady growth over the next fifty years.

The roughly three orders of magnitude increase in production volume and product concentration was the result of relentless effort on the part of process scientists and engineers. These engineers looked for hidden opportunities for strain improvement, media development, and much more. As a result, we have seen steady productivity growth due to improvements in oxygen transfer, heat transfer, and mixing characteristics. Additional advances in on-line sensing, sterility control, equipment reliability and process control all contributed to technological success.

It should be noted that the success of process technology also eventually drove down the price. Penicillin G is no longer produced in the United States; the cost of production is now

Fig. 1.1: Historical trend of penicillin titer and value


dramatically lower in other parts of the world.

Now, two decades after the first introductionof therapeutic biologics, we have seen titers in large manufacturing processes increase from tens of milligrams per liter to more than five grams per liter for many immunoglobulinproducts today. Although little published information is available, the production cost has also decreased by at least an order of magnitude since the beginning of cell culture products.

A graph of historical data for cell culture products plotted one or two decades from now will likely resemble that of penicillin. Cell culture production today is likely around the transition from the exponential growth stage to the steady and slower growth period. However, it is important to note that, in terms of both absolute quantity of product produced and economic value, the slower and steadier phase is as critical as the early rapid growth stage for the product life cycle.

Even for penicillin, there were tremendous process enhancements after the initial rapid growth phase. Due to these improvements, major medicines became affordable for the world’s population. The next question for bioprocess scientists and engineers is: How can cell culture processing accomplish what the antibiotics industry has achieved for our society?

Bioprocess scientists and engineers possess genomics and genome engineering tools that were not previously available to antibiotic researchers or even to the early innovators of cell culture processes. These new genome-wide investigative and engineering tools will greatly facilitate the designing and engineering of cells with desired growth and production characteristics. Process technologists will need to harness the power of genomics and genome engineering to enhance productivity and process robustness. This will also facilitate the expansion of biosimilars (i.e., “Follow-on” biologics) and make many medicines available to needy patients around the world at an affordable cost.

Much of the process technology employed in cell culture biologics was developed for antibiotic


production. In transforming cell culture products from laboratory discovery to clinical reality, many innovations in the design and engineering of gene constructs, cells, products, and processes have been conceived and implemented. These technologies are also likely to help new technologies move forward. The next generation technology that will benefitmost from cell culture innovations is stem cell based therapy. This technology is still in its infancy, but its significant potential impact on our societywillcompel cell culture technologists to push the evelope.

Cell culture processes have been used to produce viral vaccines for over half a century. Virus production in animals or in tissues has been in practice for over two centuries. The most notable example is the pox vaccine from cow. Most of the tissue-based production methods have since been replaced by cell culture processes. A tissue system that is still in use is the chick egg. This process is begun by seeding a virus into 10-day-old embryos in chicken eggs. A few days later, the replicated virus is then isolated from infected embryos.

Early cell culture processes were an extension of tissue culture, using primary cells explanted from various tissues (such as chick embryos and monkey kidneys) for the virus to infect and replicate. The primary cells used in virus production have mostly been replaced by cell strains or even cell lines, which can be cultivated over many generations to build up stocks (or a cell bank) for routine use to ensure consistent quality.

Most viruses used as vaccines have been inactivated by formalin treatment to render the virus incapable of infection. However, the treated virus particles retain a small degree of immunogenicity to elicit the immune response in vaccine applications.

There are cases in which live attenuated viruses are used. These attenuated viruses have been adapted,

Cell Culture Products

Virus Vaccines and Protein Therapeutics


often by the prolonged cultivation in a non-human host species so that the adapted strain is no longer virulent to humans. These viruses are still capable ofreplication,whichsignificantlyreducesthedoserequired for immunization. However, they also carry a very low, but non-zero, risk of reverting to their wild type form and causing an infection in the patient.

Vaccine technology predated modern cell culture for recombinant protein production by over two decades. Although recombinant therapeutic proteins propelled the advances in cell culture technology, proteins derived from tissues, and even cell culture, were used for therapeutic purposes even before the arrival of recombinant DNA technology. These examples include insulin, urokinase, Factor VIII, and interferon.

The generation of recombinant DNA therapeutic proteins, such as human growth hormone and insulin, were first produced in microorganisms.The next wave were human proteins, which naturally circulate in human blood and require post-translational modifications, such as complexdisulfide-bond formation and glycosylation.These proteins can not be replicated in microbial systems. For the production of those proteins, mammalian cells must be, and were, employed.

Initially, hybridoma cells were used. These are fusion products of the non-antibody-secreting, but continuously proliferating, myeloma cell and the antibody-secreting, but non-dividing, lymphocyte. This soon gave way to recombinant DNA technology. After the introduction of tissue plasminogen activation (tPA) by Genentech in 1987, erythropoietin (EPO) and Factor VIII also reached the market in following years.

Antibody products and antibody-based fusion proteins have since blossomed. They make up the bulk of the protein drugs in clinical use.

Table 1. Principal Viral Vaccines Used in Prevention of Human Virus Diseases

Disease Source of vaccineCondition of virus

PoliomyelitisTissue culture (human diploid cell line, monkey kidney)

Live attenuated, inactivated

Measles Tissue culture (chicken embryo) Live attenuated

Mumps Tissue culture (chicken embryo) Live attenuated

RubellaTissue culture (duck embryo, rabbit, or human diploid)

Live attenuated

Smallpox (vaccinia)

Lymph from calf or sheep (glycerolated, lyophilized) Live vaccinia

Smallpox (vaccinia)

Chorioallantois, tissue cultures (lyophilized) Vaccinia

Yellow fever Tissue cultures and eggs (17D strain) Live attenuated

Influenza

Highly purified subunit forms of chicken embryo allantoic fluid (formalinized UV irradiated)

Inactivated

Influenza Cell culture (MDCK, Vero) Attenuated

Rabies Duck embryo or human diploid cells Inactivated

Adenovirus Human diploid cell cultures Live attenuated

Japanese B encephalitis

Mouse brain (formalinized), cell culture

Inactivated

Venezuelan equine cephalomyelitis

Guinea pig heart cell culture Live attenuated

Eastern equine Chicken embryo cell culture Inactivated

Western equine Chicken embryo cell culture Inactivated

Russian spring - summer encephalitis

Mouse brain (formalinized) Inactivated

Cell Culture Products


Table 3. Non-Antibody Products Produced in Mammalian CellsTrade name Type Therapeutic Use Manufacturer U.S.

approval year

Host

Aldurazyme Laronidase Mucopolysaccharid-eosis I Genzyme 2006 CHOCerezyme β-glucocerebrosidase Gaucher’s disease Genzyme 1994 CHOMyozyme -galactosidase Pompe disease Genzyme 2006 CHOFabrazyme -galactosidase Fabry disease Genzyme 2003 CHONaglazyme N-acetylgalactosamie

4-sulfataseMucopolysaccharideosis VI BioMarin Pharmaceutical 2005 CHO

Orencia Ig-CTLA4 fusion Rheumatoid arthritis Bristol-Myers Squibb 2005 CHOLuveris Luteinizing hormone Infertility Serono 2004 CHOActivase Tissue plasminogen

activatorAcute myocardial infraction Genentech 1987 CHO

Epogen/Procrit

EPO Anemia Amgen/Ortho Biotech 1989 CHO

Aranesp EPO (engineered) Anemia Amgen 2001 CHOPulmozyme Deoxyribonuclease I Cystic fibrosis Genentech 1993 CHOAvonex Interferon-β Relapsing multiple sclerosis Biogen Idec 1996 CHORebif Interferon-β Relapsing multiple sclerosis Serono 2002 CHOFollistim/Gonal-F

Follicle stimulating hormone

Infertility Serono/NV Organon 1997 CHO

Benefix Factor IX Hemophillia A Wyeth 2000 CHOEnbrel TNF receptor fusion Rheumatoid arthritis Amgen, Wyeth 1998 CHOTenecteplase Tissue plasminogen

activator (engineered)

Myocardial infraction Genentech 2000 CHO

ReFacto Factor VIII Hemophilia A Wyeth 2000 CHOAdvate Factor VIII

(engineered)Hemophilia A Baxter 2003 CHO

Table 2. Therapeutic Protein Biologics Produced in Non-Mammalian Host

Activity/UseGranulocyte colony-stimulating factor (Neupogen)

White blood cell growth for Neutropenia

Insulin (Humulin) Diabetesα-Interferon (Intron-A) Anticancer, viral infectionsSomatropin [human growth hormone] (Humatrope) Growth deficiencies

Somatropin [human growth hormone] (Protopin/Nutropin)

Growth deficiencies

Interleukin-2 (Proleukin) Kidney Cancer


Table 4 . Therapeutic Antibody ProductsTrade name mAb type Therapeutic Use Manufacturer U.S.

approval year

Host

Orthoclone OKT3

Muromomab CD3 Reversal of acute kidney transplant rejection

Johnson & Johnson 1986 Hybridoma

ReoPro Anti-Abciximab Prevention of blood clots Centocor 1994 SP2/0 Rituxan Anti-CD20 mAb Non-Hodgkin’s lymphoma Genentech, Biogen

IDEC1997 CHO

Zenapax (Daclizumab)

Humanized, anti-α-subunit T cell IL-2 receptor

Prevention of acute kidney transplant rejection

Protein Design Labs

1997 NS0

Simulect (Basiliximab)

Chimeric, anti-α-chain T cell IL-2 receptor

Prophylaxis of acute organ rejection in allogeneic renal transplantation

Novartis 1998

Synagis (Palivizumab)

Humanized, anti-A antigen of RSV

Prophylaxis of lower-respiratory-tract disease

MedImmune 1998 CHO

Remicade Anti-TNF- - mAb Active Crohn’s disease Centocor 1998 SP2/0Herceptin Anti-HER2 mAb Metastatic breast cancer Genentech 1998 CHOMylotarg Anti-CD33 Acute myeloid leukemia Wyeth 2000 CHOCampath Anti-CD52 mAb Chronic lymphocytic

leukemiaMillennium, Berlex, Genzyme

2001 CHO

Zevalin Anti-CD20 murine mAb

Non-Hodgkins lymphoma Biogen IDEC 2002 CHO

Humira Anti-TNF- mAb Rheumatoid arthritis Abbott 2002 CHOXolair Humanized, Anti-

IgE mAbModerate/severe asthema Genentech 2003 CHO

BEXXAR Anti- CD20 mAb Follicular non-Hodgkins lymphoma

GSK 2003 CHO

Raptiva Anti-CD11a mAb Chronic psoriasis Genentech 2003 CHOErbitux Chimeric antibody

raised against human EGF receptor

EGF receptor–expressing metastatic colorectal cancer

Imclone Systems, Bristol-Myers Squibb, Merck

2004 CHO

Avastin Anti-VEGF Metastatic colorectal cancer and lung cancer

Genetech 2004 CHO

Soliris Antibody binding to C5

Paroxysmal nocturnal hemoglobinuria

Alexion 2007 NS0

Vectibix Anti-EGFR mAb Metastatic colorectal cancer Amgen 2006 CHO

Protein Molecule as Therapeutics The early generation of protein therapeutics consisted of all molecules native to humans. Many antibody molecules retained part of the sequence of the immunized species (e.g., mouse or rabbit), although later generations of antibody molecules were all humanized or were human antibodies. Some products are engineered molecules with altered amino acid sequences that enhance their drug characteristics.


Subsequent products entail fusion proteins, in which domains (or fragments) of different human protein molecules are joined. A prominent example is the fusion molecule of the Fc fragment of IgG and the TNFα binding fragment of the TNFα receptor.This molecule was developed by then Immunex (nowAmgen)forinhibitionofTNFαtosuppressitsinflammatoryeffect.Anotherclassofproductentailscompletely foreign proteins, such as recombinant protein or designer proteins, which have enhanced potency for eliciting an immune response.

Table 5 . Industrial Cell LinesMajor Cell Strains and Lines for Human Biologics ProductionHuman Vaccines

Primary CellsGreen monkey kidney cells (no longer used)Chicken embryo cells

Cell strains MRC5 (human lung fibroblast)

Cell line Vero (monkey kidney epithelial cell), MDCK

Recombinant ProteinsSpecies cell line derived fromHuman HEK 293, Per C6Mouse C-127, NSO, hybridoma cells, SP2/0Chinese Hamster CHO

Syrian hamster BHK

Table 6 . Cell Lines Used in the Production of Veterinarian Vaccines*Vaccines Cell lineBovine viral diarrhea virus MDBKBovine parainfluenza virus type 3 MDBKBovine rhinotracheitis virus MDBKBovine respiratory syncytial virus MDBK

Feline leukemia virus FL72Feline panleukopenia virus CRFKFeline chlamydia CRFKCanine parvovirus CRFKCanine distemper VeroCanine adenovirus type 2 VeroEhrlichia canis DH82Rabies BHK-21Eastern equine encephalitis virus VeroWestern equine encephalitis virus VeroEquine rotavirus MA104Equine rhinopneumonitis virus type 1 and 4 Equine Dermal Equine influenza virus MDCKFoot and mouth disease virus BHK-21Swine parvovirus ST, PKSwine influenza virus MDCK*This table was provided by Terry Ng, 2001 . Organisms in italics are intracellular parasitic bacteria.


For the production of traditional viral vaccines, human diploid cell strains are the primary production vehicle. Viral products differ from protein products, in that the viral genome, along with the entire virus particle, is injected into the patient to elicit a response. Even though the virus particle is inactivated by formalin or other treatment, there is still a potential risk of recombination between the virus genome and the host cell genome that may result in the transmission of activated oncogenic or foreign genetic elements to the patient. Therefore, the vast majority of virus vaccines are still produced in normal diploid human cells. Vero and MDCK cells (along with chick embryos) are notable exceptions of non-human continuous cell lines used for human vaccine production.

For veterinary vaccines, the selection of host cells is vastly wider. Both cell lines and tissue-derived cell strains with limited life spans are widely used.

For the production of recombinant therapeutic proteins, the cell lines that are primarily used are of rodent origin and include mouse, chinese hamster, and syrian hamster cells. Human cells are only used for a handful of products. The vast majority is produced using chinese hamster ovary (CHO) cells.

Industrial Cell Lines

Biosimilars or Follow-on-Biologics Two decades after the introduction of mammalian cell-based therapeutic proteins, many of those medicines’ patents have expired. A number of commercially successful therapeutic proteins will go off patent between 2013 and 2017, including the blockbuster drugs Remicade and Humira. These prospects certainly have helped to draw in investments to follow-on biologics. Generic versions of those protein therapeutics have begun to reach patients throughout the world. The terms “biosimilar” or “follow-on biologic” refer to products that are marketed after the expiration of patents. They are expected to have similar properties to existing biologic products. Sandoz was the first companyto launch a biosimilar-human growth hormone,


Table 7 . Approved Biosimilars in the EUGeneric Name

Product Launch

Recombinant human EPO-α

Medice Arzneimittel Putter (Germany)

2007

BinocritRecombinant human EPO-α

Sandoz (Austria) 2007

Epoetin alfa Hexal

Recombinant human EPO-α

Hexal Biotech (Germany)

2007

Retacrit Hospira Enterprises 2007

SilapoSTADA Arzneimittel (Germany)

2007

Somatropin growth hormone

Sandoz(Austria) 2006

ValtropinSomatropin growth hormone

Biopartners (Germany) 2006

Table 8 . Marketed Biosimilars in IndiaCompany Brand Name Biosimilar Launch

Ranbaxy Ceriton Epoetin 2003Dr Reddy’s Grastim G-CSF 2001

Reditux MabThera 2007Wockhardt Wosulin Insulin 2003

Wepox Epoetin 2001Biovac-B Hepatitis B 2000

Biocon Insurgen Insulin 2004BioMab-EGFR MabThera 2006Recosulin Epoetin 2004

Intas Pharmaceuticals

Epofit, Erykine Epoetin 2005

Neukine G-CSF 2004Shantha Biotechnics

Shanpoietin Epoetin 2005

Shanferon IFN α 2b 2002Shankinase Strptokinase 2004Shanvac B Hepatitis B 1997

Omnitrope, in both Europe and the United States.

Follow-on biologics differ from traditional generic drugs,inthattheirbiologicalactivity,ortheefficacyoftheiractiveingredient,isnotaseasilydefinedasthe traditional chemical and natural product drugs. Traditional drugs, like penicillin and statins, have very clearlydefinedchemicalstructuresthatalsoconfertheir biochemical activities. Protein therapeutics, on the other hand, cannot be entirely characterized by their chemical composition, or primary sequences. Therefore, their biological equivalency to their patented and branded counterparts cannot be established simply by structural similarity or identity.

The status of molecular folding, glycan composition, etc. may affect their activity profoundly. The particular host cell line that is used, as well as the production process, may affect subtle aspects of the protein’s properties, thus posing a greater uncertainty about the “quality” of the product produced by manufacturers of those follow-ons. While a biosimilar’s approval pathway has been established in Europe, the U.S. has yet to lay down any guidelines.


Table 9 . Marketed Biosimilars in ChinaCompany BiosimilarsDragon Pharmaceuticals Epoetin, filgrastim

Dongbao Insulin, G-CSFAnhui Anke Biotechnology HGH, interferon alpha

Amyotop G-CSF, IL-11

GeneLeuk Biotech G-CSF, PEG filgrastim, interferon

HangzhouJiuyan Gene G-CSF, IL-11

Viral vaccines are administered to patients in relatively minute quantities because a small amount of antigen proteins is sufficient to elicit immuneresponse. Cytokine, growth factors, or enzyme-types of proteins (such as EPO, human growth hormone or tPA) are also given in small doses, in terms of protein quantity. Depending on the market size, the production facility of these products may be relatively small. The biological effect of antibody products is largely based on their binding to antigen; this event requires antibody and antigen molecules to be in some stoichiometric ratio to elicit downstream target killing or neutralization. Antibodies are large molecules, as are many antibody-based fusion proteins. Thus, many antibody products are administered in relatively high doses. Thus, the product vessels, and the size of the manufacturing plant for antibody products, tend to be larger.

The manufacturing process of protein therapeutics is rather similar to that for traditional biochemical, such as antibiotics and E. coli-based recombinant proteins. A typical process entails a couple of seed expansion reactor cultures before reaching the production reactor. The process cycle tends to be longer. Many cell culture manufacturing processes are operated in fed-batch modes that last ten to fifteen days. Some are operated as continuousperfusion processes and last from two to six months.

The recovery process of cell culture products is simpler than that for bacterial-based recombinant

Table 10. Dose of Some Antibody Product

ProductDisease Indication Company

Approximate Formulation Configuration

Amevive Psoriasis Biogen 7 .5mg / 0 .5ml; 15mg / 0 .5ml

Enbrel RA Amgen 25mg

Heceptin Breast Cancer Genentech 440mg / 30cc

Humira Rheumatoid arthritis Abbott 40mg (1ml

prefilled syringe)

Remicade Crohn’s disease, RA

Johnson & Johnson, Centocor

100 mg / 20cc

Rituxan NHL Genetech/Idec 100mg / 10cc; 500mg / 50cc

SynagisRespiratory syncytial virus

MedImmune 100mg

Xolair Allergic Asthma

Genetech/Tanox/Novatis 150mg / 5cc

Manufacturing


Alternative Technologies Mammalian cells, especially CHO and myeloma cells such as NS0 and SP1/0, have been the workhorse for the production of protein therapeutics that require post-translational modifications (e.g.,glycosylation,γ-carboxylation,etc).Althoughthosepost-translationalmodifications cannot be carriedout in bacterial systems (primarily E. coli), there are a number of host systems that are capable of performing N- and O-glycosylation and other post-translationalmodifications.Theyhavebeenexploredas the production vehicles of therapeutic proteins.

Other host cells used for biopharmaceutical production include E.coli and Sacchromyces cerevisiae. Alternative production systems include:

• Insect cell culture

• Yeast ( Pichia )

• Transgenic animals

• Transgenic plants

proteins. The vast majority of processes now employ a medium with a relatively low concentration of proteins, to ease the purification operation. Withthe high product concentrations in the range of 5 – 10 g/L, the product molecule should be the predominant protein in the medium at the end of cell culture process. The product isolation and purification process is substantially simplerthan separating intracellular protein products.

Manufacturing Plants• Genentech’s Vacaville Facility, California

• Started construction in 2004, started operation in 2009. Currently inoperative due to capacity reasons

• Investment: $800 million• Eight 25,000-liter bioreactor• Production of Herceptin, Avastin and Rituxan

• Bristol Myer Squibb, Devens, Masschusetts• Started construction in 2007, validation in 2011• Investment: $750 million• Six 20,000- liter bioreactors, one purification strain• Production of Orencia and other biologics

• Biogen IDEC LSM Facility• 245,000 ft2 production• Multi-product facility• Six 15,000L production reactor capacity

Fig. 2.2: Flow chart of a typical recombinant antibody production process


Alternative Technologies

Insect cells were explored as a production vehicle for therapeutic proteins. The glycoforms of the proteins produced in insect systems are rather different from those produced in mammals. Overall, such efforts have largely subsided. However, for other applications, such as protein production for toxicity studies and for veterinary vaccine production, the insect cell culture remains attractive because the cultivation is relatively straightforward and the process development time can be relatively short.

Table 11 . Insect Cell CultureApplication CommentsBasic research Hundreds of genes have been expressed

using baculovirus .Bioproduction Using baculovirus expression systems .

Gene therapy BV may be used as the gene-delivery vehicle .

Bioreagent production

A number of bioreagent suppliers use BV to make target proteins, viral components and other compounds for the research market .

Insect Cell Culture

Yeast The yeast in the genus Pichia is capable of synthesizing N-glycans that are not the mannose-rich types produced in Saccharomyces. They have been used in the production of recombinant proteins, including serum albumin. Advances have been made in ‘humanizing’ the glycosylation characteristics in Pichia systems for the production oftherapeuticproteins.Glycofi(Merck)hasworkedtowards a multistep genetic engineering process wherenon-humanglycosylationenzymeswerefirsteliminated and human glycosylation reactions were then introduced. A titer of ~ 1.4 g/L of recombinant proteins has been reported. With further improved secretion capacities and glycosylation patterns, these engineered yeast strains may be capable of producing proteins with consistent glycosylation patterns, or even with uniform glycans.

Table 12 .Product Company Use Status

Medway (recombinant human serum albumin)

Mitsubishi Tanabe Pharma Corporation, Osaka, Japan

Blood expander

On the market in Japan

Hepatitis B vaccine

Shantha Biotechnics Ltd ., India

Hepatitis B On the market in India

Interferon-alpha

Shantha Biotechnics Ltd ., India

Hepatitis C/Cancer

On the market in India

DX-88 Dyax Corporation, Cambridge, Mass .

Hereditary angioedema (HAE), a debilitating condition characterized by acute attacks of inflammation.

BLA submitted

Recombinant Human Insulin

Biocon, India Diabetes, all types

On the market in India

Recombinant collagen

Fibrogen Inc ., South San Francisco

Medical research reagents and dermal filler

On the market

Botulism vaccine

USAMRIID/DynPort

Botulism vaccine product

Phase I (U .S .)

Anti-thrombolytic

ThromboGenics Ltd .

Thrombosis Tx Phase II


Transgenic Animals Transgenic organisms for the production of biotherapeutics have been in development for two decades. These production systems require a low initial capital investment and have a relativelyeasypurificationprocessforglycosylatedproducts. However, so far, the FDA has approved only one product, ATryn, which is produced in transgenic goat’s milk by GTC Biotherapeutics.

An advantage of transgenic animal production is its high titer in milk, on the order of 2 – 10 g/L. However, over the years, the titer in cell culture processes has also increased to 5 – 10 g/L range, thereby diminishing this particular advantage of transgenic animal production.

Table 13 . Transgenic Animal Products Approved or Under DevelopmentSpecies Company Product Status CommentsGoat GTC Biotherapeutics,

MA ATryn- recombinant human antithrombin-alpha

Approved Glycosylation patterns differ slightly ( involves N-glycolylneuramic acid- not seen in humans), but was not a regulatory hurdle; Predicted sales of $6-$10 million in 2009.

Goat PharmAthene, MD Protexia- recombinant human butyrylcholinesterase (BChE)

Development

Rabbit Pharming, Netherlands

Rhucin-Recombinant human C1 esterase inhibitor

Phase 3 trials For the treatment of hereditary angiodema .

Chickens (eggs)

Origen Therapeutics, Medarex Inc ., CA

mAb Pilot Studies Functional Mabs produced at 3 mg/egg; some differences in glycosylation; Half life in mouse serum half that of natural antibodies (reduced from 200-100h)

Product Quality and Process Robustness

Critical Feature of rDNA Proteins from Mammalian Cells

In spite of its dominance as the production vehicle for therapeutic proteins, the mammalian cell system does have some shortcomings in its process characteristics. Compared to microbial systems, mammalian cell systems have a slow growth rate and a relatively low achievable cell concentration. The product titer is also substantially lower than that of extracellular protein produced using fungal systems. Finally, the optimal range of growth environments for mammalian cells is much narrower than the range for either plant or microbial systems.

After years of research effort, the low productivity that used to be associated with the low cell and product concentrations has largely been overcome.

• Folding and disulfide bond

• Glycosylation

• N or O - glycosylation

• Sulfation or phosphorylation of glycans

• Affect solubility, clearance and biological activities

• Other post-translational modifications

• Y-carbonxylation

• Lipidation

• Phosphorylation


Product Quality and Process Robustness

Through cell adaptation and media development, the complex nutritional requirements for mammalian cell growth have been greatly simplified. Now,the relatively low tolerance of mammalian cells to their chemical and physical environment has not prevented highly stressed conditions from beingused inthe finalproductionstage.Whathasbeen lagging is our ability to control the quality, notably the glycosylation profile, of the product.

The mammalian cell system is chosen for protein production, almost invariably for its capability of post-translationalmodificationsontheproduct(suchastheformationofmultipledisulfidebondsoftPAandthe glycosylation of Factor VIII and EPO). Major post-translationalmodificationscommonlyseeninproteintherapeutics, such as disulfide bond formation,N- and O-glycosylation, and phosphorylation, all involve extensive enzymatic reactions in the endoplasmic reticulum or in the Golgi apparatus.

The level of those enzymes, as well as the supply of precursors and co-factors, affects the outcome of those reactions. The enzyme levels vary with cell clone and growth stage, while the supply of precursors and cofactors change with the chemical environment. These variations cause fluctuations in the glycans attached to N-(asparagin) sites or to O- (serine or tyosine) sites.

For a given glycoprotein, regardless of whether it is produced in culture or present in circulation, the glycans attached to different molecules are not identical. Rather, they are a mixture of different, but related, forms. In fact, most glycoproteins in blood circulation also have hetergeneous glycans. The structure of glycan and the extent of glycosylation on the protein molecule affect the blood circulation half-life of the protein. In some cases, the glycan structure even affects the protein’s biological functions. Thus, confiningglycandistributiontoanacceptablerangeis important for the quality control of the product.

The glycosylation pathway is long and complex, and takes place in multiple compartments in the cell. Producing a glycoprotein product with a defined range of glycan structures throughout

Tissue Plasminogen Activator (tPA)1 • Single polypeptide chain (70 kDa) or

proteolytically cleaved at ARG276.

• Multiple N-linked carbohydrates: ASN117 (high mannose), ASN184 (50% complex multiantenary, 50% unoccupied), THR61 (O linked fucose) .

• Contains 35 cysteine residues, 17 pairs of disulfide bonds. CYS83 can form a disulfide with other free thiols depending upon the growth medium and buffer composition.

• May form high molecular weight aggregate (complexes with protease inhibitors) and proteolytically cleaved tPA.

Erythropoietin • Contains 40% carbohydrate, only 2 disulfide

bonds .

• 3 N-linked ASN (24,38,83), 1 O linked (SER126) glycosylation sites.

• O-linked site not essential for in vitro or in vivo activity.

• Sialic acid residues (average 10 moles/mole Epo) responsible for preserving pharmacokinetic behavior . Muteins lacking 2 or 3 N-linked sites are poorly secreted .

• N linked glycosylation and sialylation is critical to optimal secretion, structure, in vivo potency.


a product’s life cycle is still a challenge.

Glycosylation may affect the folding of the protein molecule, but it does not affect its structure. Other post-translationalmodificationsmayaffectproteinstructure.Failuretoformadisulfidebondorthemis-pairingofadisulfidebondbothgiverisetoanalteredprotein structure or the improper cross-linking (multimer formation) among different molecules. A lack of γ-carboxylation or phosphorylationalso drastically changes a protein’s properties.

Errors in protein synthesis caused by amino acid mis-incorporation have been reported. Many production cell lines have multiple copies of the product gene; a non-silent mutation (i.e., a mutation causing a change of an amino acid in the protein) in one of those genes will inevitably result in the presence of some fraction of mutated protein molecules.

An alteration of the amino acid structure may also result from chemical modifications afterbeing secreted into the medium. After being secreted into culture medium, the product protein molecules are also subjected to modification byenzymes released by cells, which are either actively secreted or released from lysed cells. Extracellular proteolytic cleavage can give rise to degradation of Factor VIII, or can alter the ratio of single chain/double chain molecules of tPA and Protein C. Also, the sialic acid moiety in glycans may be cleaved by sialidase released from lysed cells. Table 14 summarizes some more commonly-seen alterations in protein molecules in cell culture processes.

In the past decade we have seen the productivity of recombinant cells reaching or even exceeding the production rate of professional secretors in our body (such as liver cells or antibody- and insulin-secreting cells). We have also seen the product titer in the bioreactor approaching the concentrations ofantibodyinascitesfluid.Astheproductivityandproduct concentration of cell culture processes approaching its “natural” biological counter parts, we must also be cautious and ask ourselves whther we are pushing cell’s protein folding and processing machinery to operate at its limit.

Table 14. In Process Structural Alternations to Mammalian Protein Biologics Glycoform

Site occupancy

Possibly caused by stochasticity of glycosylation process

Altered sialic acid content

Uncapped galactosyl residue, High mannose

Glycan distribution out of range

Amino acid alterations in protein

Mis-incorporation (codon misreading)

Error rate of amino acid incorporation during translation (1/1000)

Deamidation (Asparagine)

Most likely occurred in culture fluid, may be affected by process conditions, or even product titer

Loss of terminal amino acid • lysine in C-terminus

of heavy chain IgG, enzymatic cleavage

• cyclization of N terminus glutamine

Glycation (addition of reducing sugar (glucose) to amino acids)

Protein aggregation

May be caused by folding in ER or agglomeration in culture or in down stream processing


The unprecedented high productivity is achieved by operating the reactor at conditions that are neither optimal for growth nor the natural homeostatic state. Rather, they are often highly stressed to favor producing the product of our design. In today’s production cultures, both the intracellular and the extracellular environments are extremely harsh. While protein synthesis and secretion is boosted to a nearly unprecedented level, the cellular machinery for protein quality control may not be operated at the same level of stringency as it is for optimal growth. Process technologists must bear in mind that quality consistency and process robustness must be the highest priority when pushing productivity higher.

Over half a century, cell culture processes have evolved from tissue and small-scale cell culture for vaccine production to large scale manufacturing process for protein production. Therapeutic proteins, especially antibody and antibody-based proteins, are the dominant products. The continuing pressure to meet increasing demand for products has led to many process innovations and refinements over the past two decades. The celland product concentrations in today’s process are nearly two orders of magnitude higher than they were at the dawn of the recombinant protein era. The success of this technology has also shifted the focus from production quantity to product quality.

Concluding RemarksCell culture processes now aim to provide optimal growth conditions for cell expansion, while often employing highly-stressed conditions for the finalproduction stage. All must be accomplished without compromising the quality of product produced. Achieving those aims through process innovation will be critical in the next phase of the technology, wherein follow-ons or biosimilars will have an increasing presence. Cell culture engineering efforts in the past quarter century have transformed bioprocess technology. The advances made in cell culture technology will greatly facilitate the development of the emerging stem cell and other cell therapy.


CELL ENGINEERING| 19CELL BIOLOGY | 19

The cells commonly used for the production of biologics are derived from different tissues of different species. Thus, they can vary widely at the genomic level. Their differences are even visible microscopically, with various numbers of chromosomes. However, at a physiological and transcriptome level, cells from the same tissue of different species are strikingly similar. Their similarity is much greater than different cell types from the same animal.Forexample,chickenembryofibroblastslookmorphologically very similar to human fibroblastsfrom the lung or foreskin, while the epithelial MDCK cells look ratherdifferentfromdogfibroblastseventhough they are both derived from the same species.

Table 1 . Cells Commonly Used in Bioprocessing

Species Cell Type TissueIsolated

W1 - 38 Human Fibroblast LungMRC - 5 Human Fibroblast LungFS - 4 Human Fibroblast ForeskinHEK 293 Human Epithelial KidneyVero Monkey Epithelial KidneyMDCK Dog Epithelial KidneyNS/SP2/0 Mouse Lymphoid Myeloma

CHO Chinese Hamster Epithelial Ovary

BHK Syrian Hamster Epithelial Kidney

Cells: Source, Composition and Structure

Cell Biology for Bioprocessing

Cells: Source, Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Cell Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Cell Composition and Chemical Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Cell Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Cytoplasm and Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27Transport Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Major Mechanism of Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Extracellular Matrices and Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Growth, Death and Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47Cell Cycle and Growth Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Senescence and Telomeres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

Cell Source

20 | CELL BIOLOGYCELL BIOLOGY | 20

Even though there are about two hundred types of cells in a vertebrate animal, most cells that are used for the production of biologics are either epithelial or fibroblast in nature. These two cell types aremore amenable to isolation from tissues and to in vitro culture, as demonstrated during the early explorations on tissue cell isolation more than half a century ago. NSO and CHO are the two prominent host cell lines used for therapeutic recombinant protein production. They exhibit different behaviors and were derived from two different tissues and two different species. CHO cells were isolated from the ovary of a Chinese hamster; NSO cells were isolated from a mouse myeloma. Cells used for recombinant protein production are primarily epithelial and lymphatic.

Both fibroblasts and epithelial cells are frequentlyused for viral vaccine production. These cells differ in both their functions and tissue locations. Epithelial cellslinethe“boundary”oftissues,whilefibroblastsmake up a larger part of the connective tissue. Epithelial cells form tightly connected sheets, which often get damaged, die, and are replenished by “new” ones. Thus, many of them are constantly growing in vivo. Conversely, fibroblasts are mostly quiescent.They migrate into wounds and begin to grow only when they are stimulated by various cues. Lymphatic cells, especially the terminally-differentiated plasma cells (from which myeloma cells are derived), are needed to secrete antibodies against a particular antigen, but only for a limited period of time after the host’s exposure to the antigen. They undergo apoptosis days after their differentiation into active antibody-secreting cells, so that the host does not continue to have unnecessary or maybe even harmful antibody molecules in circulation. Such native characteristics are often still evident in culture.

• Among the ~200 different types of cells, fibroblasts, epithelial cells and myeloma cells are most frequently used cell types in biologics production

• Cells in culture bare closer physiological and morphological characteristics of the tissue they were derived from than the species

• Cells in vivo may be in a quiescent state or in a proliferative state, but are all adapted to rapid proliferation in culture

CELL ENGINEERING| 21CELL BIOLOGY | 2120 | CELL BIOLOGY

Most cells in culture have a diameter of about 12 – 18 µm. Some types of stem cells are rather small and have only a small amount of cytoplasm. In contrast, liver cells (i.e., hepatocytes) in some species are rather large, with an average cellular diameter of 20 µm. A typical cell has nearly 80% of its mass as water. Proteins make up the next largest portion of cell mass, after water.

Other than water and proteins, the other cellular constituents are present in much smaller amounts and rarely exceed 10% of the total dry mass. Lipids make up various membranes of the cell, including the cytoplasmic membrane and the membrane enclosing all organelles. Lipids, thus, constitute a significantportion (about 5-8% of total dry mass) of cell mass.

Carbohydrates (such as glycogen) are used to store energy in some cells. However, not all cells have a large amount of free carbohydrates. Carbohydrate molecules that serve as energy sources are quickly metabolized to become intermediates in energy metabolism. Most carbohydrate moieties that remain in their carbohydrate forms exist as part of nucleotides or are conjugated to proteins or lipids.

The size of a haploid genome in a typical mammalian cell is about 3 Gbp. That equates to about 5 pg of DNA for a diploid cell. However, DNA is not the most abundant nucleic acid in the cell. RNAs are far more abundant than DNA in a cell and include messenger RNA (mRNA), ribosomal RNA (rRNA), and others. Ribosomal RNA, which is a major constituent of the cell’s protein synthesis machinery, constitutes over 90% of all RNA in the cell.

Since water constitutes the largest fraction of all cell materials, the chemical species that are present at a high concentration in the cytosol are also major cellular constituents. Combined, all minerals contribute a significant (~5%) proportion of the dry mass.

The concentrations of some ions are vastly different inside the cell versus outside the cell. Maintaining these concentration gradients is critical for cell functions. The concentration ratio between intracellular and extracellular K+ and Na+ is in the range of 15 to

Table 2. Typical Composition of a Cell

Mammalian Cell E. coli

pg / Cell Range % %Wet weight 3,000 3,000 - 8,000Dry weight 600 300 - 1,200Protein 250 200 - 300 10-20 15Carbohydrate 150 40 - 200 1 - 5 2Lipid 120 100 - 200 1 - 2 2DNA 10 8 - 17 0 .3 1RNA 25 20 - 40 0 .7 6Water 80 - 85 70

Volume 4 x 10-9 cm3

Diameter 18 μm 0.5-2 μm

Table 3 . Cellular and Extracellular Fluids Ion Concentration

Plasma (mmole / l) Interstitial (mmole / l)

Intracellular osmolality (mmole / l)

Na+ 140 14

K+ 4 4 140

Ca++ 1 1 10-4

Mg++ 0 .8 0 .7 20

Cl- 110 110 50

• >10 fold concentation difference for K+ and Na+ across the plasma membrane

• Opposite direction of concentration gradient for K+ and Na+

• Extremely low concentration of Mg++ in intracellular fluid

• Total osmolality ̴ 280 mOsm

Cell Composition and Chemical Environment


Cell Membrane

30. Conversely, their direction of concentration gradient is opposite: the concentration of K+ and Na+ should be far higher inside and outside the cell, respectively. The solutes in a solution exert osmotic pressure,whichistypicallyquantifiedbyosmolality.Theosmolalityofcellularfluidisabout280mM(ormOsm). A typical medium has its osmolality at the same level as in cells, to avoid incurring osmotic stress.

Fig.2.1: A phospholipid molecule with glycerol as backbone, with an ethanol amine, a saturated and an unsaturatted fatty acid.

Cultured mammalian cells have long been thought as being extremely fragile to mechanical stresses because their cellular materials are surrounded only by cytoplasmic membrane; the only thing preventing the cellular content from dissolving into the aqueous environment is that lipid bilayer. Yet in a modern manufacturing plant, these tiny cells thrive in bioreactors of tens of cubic meters in volume under such highly turbulent conditions. The membrane surrounding a cell is not merely a double-layer of lipids, and the integrity of a cell is not merely dictated by its membrane wrapping.

The lipids which make up the lipid bilayer are amphipathic. They have a hydrophilic head group, and a hydrophobic tail group made of fatty acids. When suspended in an aqueous solution, amphipathic molecules can form micelles. In such micelles, the hydrophilic.

Lipid Bilayer Composition

Phospholipids • Constitute the majority (35-70%)

Glycolipids • Neutral glycolipids (e.g. galactocerebroside)

Gangliosides • Have sialic acids

Four types of phospholipids • Three have glycerol as backbone,Phosphotidyl ethanol amine, Phosphotidyl serine and Phosphotidyl choline

• Serine as backbone


Lipid BilayerAlipidbilayermembranebehaveslikeafluid.Ifthelipidmoleculesinaspecificlocationarelabeledwithafluorescentdye,thefluorescencedispersesshortlythereafter due to molecular diffusion (instead of staying in the same place as in a solid). The lateral diffusioncoefficientofaphospholipidmoleculeinabilayer membrane is about 10-8 cm2/s. A lipid molecule doesnotflip-flop(orchangeitssideofalipidbilayer)without the aid of membrane-bound phospholipid translocator. Gas species diffuse about equally fast in a lipid bilayer as they do in water. Even large protein molecules diffuse in a lipid bilayer membrane.

Fatty acids make up the hydrophobic tail. At very mild temperatures these acids undergo a phase transitionfromafluidtoanorderedstructure.Thus,lipid bilayers also undergo phase transition to form a “liquid crystal” at a relatively moderate temperature. This tightly-packed, ordered structure acts as a very good barrier to keep most molecules from freely passing in or out of the cell. The permeability of most biological molecules across a lipid bilayer membrane is rather low. Even the smallest nutrient, such as glucose and simple amino acids, cannot pass by fast enough to support cell growth.

All major biological macromolecules (e.g., DNA, proteins, and polysaccharides) are biopolymers made of covalently-bonded monomers. A lipid bilayer membrane is a not a polymer, rather, it is an assembly of phospholipids. The non-covalent nature of phospholipids within the cell membrane allows it to be very dynamic: expanding, shrinking, breaking, and fusing rapidly. The lipid bilayer also envelops various organelles to compartmentalize regions in the cell for specialized functions. Many of those organelles are in a constant dynamic process of membrane budding and fusion. For example, in trafficking between organelles and in proteinsecretion, the “cargo” is carried inside membrane vesicles while transiting from one organelle to another. This process occurs without the need to break up and re-form a larger number of covalent bonds.

Three types of lipids make up a lipid bilayer membranes in cells and organelles: phospholipids,

Characteristics of a Lipid Bilayer• The lipid bilayer is a fluid

• As temperature decreases, the bilayer transitions from a fluid state to a gel state

• The degree of fatty acid “unsaturation” affects the transition temperature of membrane from a fluid state to a gel state

• The magnitude of diffusion of various solutes in the cell membrane resembles that of a liquid

Fig. 2.2: Lipid bilayer membrane at a crystaline state and fluid state


glycolipids, and gangliosides (phospholipids being the most common). There are also different types of phospholipids, with either glycerol or serine as the backbone, with the former being the most abundant type. Glycerol has three hydroxyl groups attached to its three carbons and one of them has a phosphate group, to which an ethanolamine or serine is attached. The phosphate moiety has a strong negative charge, thus making this end of the molecule the highly hydrophilic head group.

The other two hydroxyl groups of glycerol are linked to two fatty acids through an ester bond. Typically, one of those two fatty acids is saturated and the other is unsaturated, with a cis double bond in-between C14 and C24. The degree of unsaturation affects the packing of the lipid bilayer. Saturated fatty acids allow more dense packing, while the double bonds in the unsaturated fatty acids create kinks, which reducepackingandincreasethemembranefluidity.

A lipid bilayer can be in a gel state or in a liquid crystalline state depending on the temperature and degree of hydration. A lipid bilayer’s phase transition temperature is affected by its composition of fatty acid and cholesterol. As temperature decreases, the lipid bilayer changes from a liquid-crystalline state to crystalline (or gel) state. A higher content of shorter, unsaturated fatty acids increases the fluidity of the lipid bilayerand decreases its phase transition temperature.

Another molecule playing a key role in the membrane properties of animal cells is cholesterol. Cholesterol has a small polar head group linked to a rigid planar region of steroid rings that are further linked to a more flexible non-polar tail. Cholesterol interactswith phospholipids to stabilize the region closer to the head group and to make the lipid bilayer less inclined to become crystalline. Overall, cholesterol increases the membrane permeability to small compounds and makes the membrane less fluid. Cholesterolcontent varies in different lipid bilayer membranes. Its level in the cytoplasmic membrane is higher, but

Characteristics:• One saturated, one cis-unsaturated (C14-C24)

typically constitute the tail of the phospholipid.

• Fatty Acids (the tail group) on the lipid affect the packing of lipids in bilayer membrane . Saturated fatty acids allow more dense packing; double bonds in unsaturated fatty acids creates kinks, reduce packing, increase fluidity.

• Cholesterol has a small head polar group linked to a rigid planar region of steroid rings followed by a more flexible non-polar tail. They interact with phospholipids to stabilize the region closer to the head group as well as to make the lipid bilayer less inclined to become crystalline . Overall, they increase the membrane permeability to small compounds, and make the membrane less fluid.

• Depending on temperature and the degree of hydration, lipid bilayer is in gel state or in liquid crystalline state . The temperature of bilayer phase transition from the crystalline lipid bilayers to fluid bilayers is affected by fatty acid and cholesterol composition.

Cholesterol in a Lipid Bilayer

Fig. 2.3: Schematic drawing of a cholesterol molecule interacting with two phospholipid molecules in one leaflet of a lipid bilayer


Membrane Proteins in the membrane of many organelles it is very low.

A typical biological membrane has ~50% lipids and ~50% proteins, by mass. In terms of molecules, however, the lipid:protein ratio is actually about 50:1, since proteins have much higher molecular weights than lipids. The protein content of a membrane is greatly affected by the tissue of origin and by the membrane’s function in the cell. The mitochondrial membrane, through which many molecules (e.g., amino acids, pyruvate, various ions andmanyotherproteins)passatahigh flux,hasahigh protein content of about 75%, by mass. On the other hand, the myelin membrane, which serves as a protective sheath between the nerve cell and its surroundings, has a low protein content of about 25%.

Lipid bilayer membranes separate cellular content from their surroundings and divide the organelles from the cytosol. Not only do they create a barrier for the physical retention of a cell’s contents, but they create a rather different chemical environment across membranes. For example, cells maintain about an 80 mV electric potential across the plasma membrane and about 140 mV across the mitochondrial membrane. The ER membrane separates an oxidative environment (inside the ER) from a reduced one (in the cytosol).

The maintenance of various chemical, electrical, and redox potentials across a membrane is accomplished by various membrane proteins. Rat small intestinal enterocyte has about 150,000 Na+ pumps per cell, which collectively allow each cell to transport about 4.5 billion Na+ ions out of the cell, each minute. The sodium and potassium membrane gradients generated by those pumps, as well as the electric potential across cytoplasmic and mitochondrial membranes, are fundamental to cellular bioenergetics.

• A typical biological membrane has ~50% proteins by mass; in terms of molecules, lipid:protein = 50:1

• Metabolically active mitochondrion has 75% protein in its membrane .

• Na+/K+ ATPase acts as a pump, using ATP to pump 3Na+ out and 2K+ into the cell .

• The electric protential across the plasma membrane is about -80mV .

Table 4. Biochemical Composition of Hepatocyte Plasma Membrane

Total Lipids

Total Protein

Protein/Lipid mass ratio

Cholesterol/Phospholipid molar ratio

Cholesterol in total lipids

Phospholipids in total lipids

30-40% (by

mass)

50 -60% (by mass) 1 - 2 0.4 - 0.8 12 - 20% 50 - 70%

Adapted from The Liver: Biology & Pathology, 4th Ed., p. 78 (2001)


Membrane Dynamics The cellular membrane is in a dynamic state; membrane constituents are continuously being added and removed. This is not only for membrane expansion and cell growth, but also for turnover and forvesicletrafficking.Likeothercellularcomponents,the turnover of the cellular membrane is necessary to replace lipid molecules that have been oxidized or damaged, or to allow cells to change their membrane composition to adapt to new environments.

The turnover rate of a cell membrane varies widely. Phospholipids are said to have a half-life of three hours, while the half-life of cholesterol is about two hours. Cellular membrane proteins are also turned over. Their half-life ranges from a couple of minutes to a couple of days, whereas macrophage membrane proteins are turned extremely rapidly.

Inter-organelletraffickingandthesecretionofproteinsinto the extracellular environment also contribute to a membrane’s dynamic state. Protein molecules that are destined for export are carried from organelles to the cytoplasmic membrane by vesicles. Upon reaching the inner surface of the cytoplasmic membrane, those vesicles fuse with the cytoplasmic membrane and release their contents outside of the cell.

In the liver, each hepatocyte synthesizes ~120 x 103 albumin molecules per min (translating to about 15 pg/cell/day). All of those molecules are wrapped in 280 – 400 nm of vesicles and delivered to the basal plasma membrane of the cell. The infusion of those membrane vesicles would cause the membrane surface to expand at a rate of 0.5%/min. However, since hepatocytes are typically in a G0 state (i.e., not dividing), the size of their cytoplasmic membrane does not need to increase to accommodate cell growth. Therefore, the lipid molecules that are added to the cytoplasmic membrane must be recycled back into the intracellular organelle (Golgi bodies) to maintain the cytoplasmic membrane in a homeostatic state.

Similarly, cells active in endocytosis can internalize up to 0.8%/min of a plasma membrane. The loss of lipids from membrane caused by endocytosis must be replenished to maintain the size of cell’s outer envelope.

Cellular membrane is in a dynamic state contributed by:

• Lipid turn-over

• Inter-organelle shift of membrane vesicles

• Secretion, endocytosis

Homeostasis of cellular membranes• Professional secretory cells in the body can add

0 .5% per minute of their plasma membrane due to the fusion of secretory vesicles with the plasma membrane; they must be recycled to maintain a balance

• Phospholipids in the membrane are subject to turnover


Cytoplasm and OrganellesThe cytoplasm and nucleus are both enclosed by cytoplasmic membrane in the cell. The cytoplasm can be largely divided into two groups: various organelles and the highly-viscous cytosol. The cytosol has a very high concentration of proteins (100 – 300 mg/mL). For comparison, the protein content in blood plasma is only 90 mg/mL. The cytosol also contains the inorganic solutes, building blocks, and intermediates and metabolites of metabolic reactions.

The cytosol is not only full of soluble components. It also contains large assemblies (or aggregates) of particles. The ribosome is the main machinery for making proteins; it is a complex particle consisting of many ribosomal proteins and ribosomal RNAs (rRNA). Each cell contains thousands of ribosomes of ~30 nm in size. Many ribosomes are located on the cytosolic surface of the endoplasmic membrane and appear as a black spot, when viewed under an electron microscope. Some enzymes also form large complexes that can be seen under electron microscope, such as pyruvate dehydrogenase complexes.

Alsorichinthecytosolarethefiber-likestructuresofthe cytoskeleton. These large protein particles, enzyme complexes, cytoskeletal proteins, and organelles make the cytoplasm of a cell very crowded and render its solution phase very dense in mass. Under light microscopy, an animal cell appears to be primarily cytoplasm, wrapped in a membrane, with a nucleus sitting near the center spanning over half of the cell’s diameter. Other than the nucleus, various organelles include the mitochondria, the endoplasmic reticulum, the Golgi apparatus, peroxisomes, endosomes, etc., and are visible only by electron microscopy.

• Cytoplasm is not a simple solution

• Some protein complexes (like pyruvate dehydrogenase and ribosomes) are aggregated

• Cytoskeletal network is interspersed in cytosol

Total protein concentration in cytoplasm

150 g / L (~4 μM)

Total protein concentration in plasma

90 g / L (1.2 μM)

Albumin (MW 69,000) 45 g / L (0.65 μM)Globulins (MW 140,000) 25 g / L (0.18 μM)

Fibrinogen (MW 400,00) 103 g / L (0.0075 μM)


NucleusIn bacterial cells, DNA molecules are roughly localized at the center of the cell. Both DNA and RNA synthesis occur in the nucleoid region that is adjacent to the chromosome formed by the large DNA molecule. The nucleoid occupies a distinctive part of the cytoplasm and the DNA is tightly coiled and is bound by many proteins. If completely extended, a DNA molecule of an E. coli cell is nearly 1-mm long.

In contrast, a eukaryotic cell’s genome is separated into a number of DNA molecules, which each form a chromosome. Then, the DNA molecules are segregated into nuclear compartments. The average genome of a mammalian cell is about three orders of magnitude larger than that of E. coli. If stretched, it extends to about 1-m in length. This large amount of DNA is packed into a small space by forming DNA-protein (histone) complexes.

DNA/RNA synthesis and ribosome assembly occur in the nuclear compartment and are segregated from the metabolic processes and protein synthesis in the cytoplasm. Ribosomes are assembled in nucleoli and are subsequently exported into the cytoplasm to participate in protein synthesis. The complex tasks of sorting out which segments of DNA, or which genes, are to be transcribed into RNA at a given moment occur in the nucleus. A large array of transcription factors and other transcription regulators are synthesized in the cytoplasm and then importedintothenucleuswheretheybindtospecificgenetic loci to perform their role in transcription.

Thus,thereisa largevolumeofmaterialtraffickingbetween the nucleus and the cytoplasm. Components of the ribosome, nucleotides/deoxynucleotides, nuclear structural proteins, and transcription factors need to be imported into the nucleus. The RNAs (mRNA, tRNA, and some non-coding RNA) are exported into the cytosol for protein synthesis.

A double-layered membrane separates the cytosol and the nucleoplasm. The nucleus and the mitochondrion are two organelles in the cell that have double membranes, instead of only a lipid-bilayer membrane. Much of the trafficking occurs

Fig. 2.4: Organelles in an animal cell

endosomeendocytosis

lysosome

smooth endoplasmic reticulum

nuclear envelope (nuclear membrane)

nucleus nucleolus mitochondria

plasma membrane

chromatin

secretory vessel

golgi apparatus

rough endoplasmic reticulum


through nuclear pores on the surface of the nuclear membrane (also known as nuclear envelope).

MitochondriaThe mitochondrian is the most common organelle in a cell. With about 1,700 per cell, they make up 20% of the cell’s volume. Mitochondria are about the size of bacteria and are thought to have originated from bacteria-like structures that were acquired by primitive eukaryotes.

Mitochondria serve as the cell’s power plants. The most reactive reactions in the cell (e.g., oxidizing nutrients and generating energy through electron transfer and oxidative phosphorylation) take place in the mitochondria. Cells with different energy needs have different numbers of these power plants. In a high-energy demanding cell, there can be as many as 3,000 mitochondria.

The main ATP-generating process occurs via electron transfer, across the inner mitochondrial membrane. The total surface area of all mitochondrial inner membranes in a cell is greater than that of the cytoplasmic membrane. At the mitochondrial inner membrane, reactive electrons in electron transfer react with oxygen to form H2O. Mitochondria are thus rich in potentially damaging free radical species. By confiningthesereactionstothemitochondria,thecellcan potentially reduce unintended cellular damage.

The mitochondrion resembles a bacterium, not only in size but also by having its own genome in the form of a circular DNA molecule. Each mammalian mitochondrion contains one or more mitochondrial genomes of about 18 kbp. The control of mitochondrial DNA replication is separate from the regulation of genomic DNA replication. The biogenesis (i.e., the replication) of mitochondria is independent of cell division.

An active respiring mitochondrion has a negative 140 mV electric potential and pH of 1.0 across its inner membrane. The pH inside a mitochondrion is higher, as the H+ ion concentration is lower inside, so pH is pumped against the concentration gradient. The pH gradient and the electric potential are critical

Mitochondria are....

• The most abundant organelle in a cell (about 1,700 per cell)

• Take up to 20% of cell volume

• In the catabolism of glucose to carbon dioxide, the oxygen atom in CO2 is contributed from water molecules . The oxygen reacts with H in NADH, FADH2, to form water in mitochondria

• Active mitochondrion has a negative 140 mV electric potential across its inner membrane, and 1 .0 units of pH gradient (inside mitochondria pH is higher [H+ concentration is lower] and pH is pumped against concentration gradient)

• The membrane potential cannot be charged up too much . Therfore the homeostasis of mitochondria is critical.

• Cells meet long-term energetic needs by biogenesis of mitochondria .


for cells. The electric potential and pH gradient are created by pumping protons out of mitochondria. This occurs while transferring electrons at a high energetic state in NADH to a low energetic state that can be received by oxygen to form water. In other words, the chemical potential energy in the high energetic electron is transformed and “stored” in the electric potential and proton gradient.

The membrane potential cannot become too high; it can burst the organelle. It is important to maintain a homeostatic condition in mitochondria. When the energetic need of a cell is high over a long period, cells respond by increasing their number of mitochondria. The flux of energy (primarilypyruvate) into mitochondria is tightly controlled.

Mitochondria (along with other organelles) cannot be generated merely from the genetic content in the nucleus. A cell must have mitochondria at its origin in order to make more mitochondria as it proliferates. The mitochondrial genome encodes a number of mitochondrial proteins and RNA molecules, while other mitochondrial components are encoded by cellular genomic DNA and imported into the mitochondria.

Fig. 2.5: Proton and electric potential (charge) gradient across mitochondrial inner membrane. The direction of fluxes of major species are indicated by and arrow. Note NADH oxidation coupled electron transfer pumps protons against proton and charge gradient, while the movement of proton to drive ATP synthesis is in the direction of proton and charge gradient.


Endoplasmic Reticulum Theendoplasmicreticulum(ER)islargelyclassifiedinto the smooth ER and the rough ER, based on morphology. The smooth ER is rich in enzymes involved in chemical transformation reactions. In liver cells, the smooth ER takes on the role of detoxification; in the ovaries and testes, it makeshormones. The rough ER is the site of folding and processing of proteins destined for some organelles, integral membranes, and secretion. It gains its name through the attachment of a large number of ribosomes to its cytosolic domain surface, thus appearing to be rough under the transmission electron microscope.

Professional secretors in the body have abundant ER, such as the pancreatic beta cells that secrete insulin and the antibody-secreting plasma cells. As B cells (non-antibody secreting) differentiate to become plasma cells, the ER and Golgi apparatus expand drastically, more than 15 fold.

Hepatocytes secrete many proteins, including albumin, which are coagulation factors that constitute many of the blood’s protein components. In the liver, some hepatocytes specialize in protein secretion, while others play major roles in oxidative detoxification.These hepatocytes have distinctive ERs. Those involved in protein secretion have an abundance of rough ER, while those more specialized in xenobiotic metabolism have an abundance of smooth ER.

ER is also a major site of protein post-translational modifications, and is involved in Ca+2 homeostasis and cholesterol synthesis. The ER lumen is rich in proteins that facilitate protein folding and catalyze the formation of intermolecular disulfide bonds.

Smooth ER

• Function varies with tissue, in liver cells it detoxifies; in ovary and testes it makes hormones

Rough ER

• Proteins for some organelles, integral membrane proteins and secreted proteins are folded in ER

• Professional secretors in the body, such as pancreatic beta cell, hepatocyte and antibody secreting plasma cell, all have abundant ER. As B cells differentiate to become plasma cell, ER and Golgi apparatus expand drastically, at least by 15 fold

Some characteristics of ER

• In hepatocyte, surface of ER is about 63,000 μm2 per cell, or about 40 times of plasma membrane

• ER lumen very viscous, gel-like. Diffusion coefficient of fluorescent probe is 9 - 18 times lower than in water

• ER has much higher oxidative environment than cytoplasm, appropriate for disulfide bond formation

• High free Ca2+ environment

• Many proteins are present at very high concentrations (PDI, GRP94, GRP74)

• A major site of protein folding, other post-translational processing, Ca++ homeostasis, cholesterol synthesis

Protein processing occurs in ER

• Cleavage of signal peptide

• Addition of high mannose core oligosaccharide to Asn-x-Ser / Thr N-linked glycosylation site

• Trimming of terminal glucose and mannose residues from initial glycan

• Fatty acid addition

• Disulfide bond formation


The classical view of the Golgi apparatus is as a stack of flattened sacks. More recently, the Golgiapparatus is viewed as a dynamic region where many key reactions occur. Many proteins are modifiedin Golgi bodies after they are folded in the ER.

The Golgi apparatus is loosely divided into four compartments: cis, medial, trans, and the trans-Golgi network (TGN). The protein cargo from the ER is transferred to the cis Golgi and then to other Golgi compartments through membrane vesicles. The enzymes in the four Golgi compartments are not identical. Thus, different reactions may take place in different compartments.

Protein Processing Occurs in Golgi• Addition glycoform modification

• Sulfation of tyrosines or carbohydrates

• glycosylation

• Peptide proteolytic cleavage

• Gamma-carboxylation of glutamic acid

• Beta-hydroxylation of aspartic acid

Golgi Apparatus and Protein Post-Translational Modification

Protein Secretion Through ER and Golgi Apparatus For a high-producing industrial cell line, the secreted

recombinant product constitutes a very large fraction of all of the total protein synthesized. Cells devote a large portion of their protein processing capacity to the secreted protein product. It is therefore useful to review this process of protein secretion.

In a professional secretor, approximately 30% of all cellular proteins are destined for organelles, membranes, and secretion and are processed through the ER. Although some proteins are translocated into the ER post-translationally, most (including typical recombinant DNA protein molecules) are translocated as nascent protein molecules.

Proteins destined for secretion have a leader sequence at the amino terminus that serves as the signal peptide. After translation initiation, the signal peptide of the nascent protein is recognized by signal recognition particles (SRPs). This halts translation and docks the nascent protein (which has only the beginning segment of the entire sequence) to a receptor on the ER membrane. It thus prevents the protein molecule from being elongated in the cytosol. The nascent polypeptide is then transferred to a translocon on the ER membrane. Subsequently, translation elongation resumes and the elongating polypeptide passes through the channel of the translocon into the ER lumen.

Folding of the polypeptide starts immediately upon

Some Characteristics of Golgi• Compartmentalized into functionally distinct regions:

Golgi stack (consisting of cis, medial and trans cisternae), and trans Golgi Network (TGN)

• Proteins, lipids are sorted in Golgi for delivery to different cellular locations

• From here proteins go to secretion (exocytosis) or other organelles

• During mitosis the Golgi apparatus breaks down and reassembles after mitosis

• Different molecules of the same secretory protein spend different amounts of time inside the cell, i .e . there is a distribution of “holding time” in the cell

• Translation of a protein molecules takes only seconds, but the secretion process takes tens of minutes to hours


translocation into the ER lumen. The signal peptide on the elongating polypeptide in the ER lumen is cleaved upon entry into the ER. The protein concentration in the ER is estimated to be 100 mg/mL, a concentration at which proteins would otherwise aggregate and fall out of solution. A class of ER chaperones and other proteins that facilitate protein folding act on the nascent protein molecules to prevent aggregation and assist in folding. Their actions require cellular energy (ATP). An important member of the ER luminal chaperones, BiP, is also a component of the translocon complex. In addition to BiP (also known as GRP78) major ER luminal chaperones include calnexin, calreticulin, and protein disulfide isomerase (PDI).

As will be discussed later, extensive glycolyation occurs along the secretion process, starting in the ER and continuing into Golgi bodies. In the ER, glycosylation also serves as an indicator of correct protein folding.

Protein molecules that have completed the folding process are exported from the ER by inclusion in membrane vesicles. Vesicle fusion, fission, andtraffickingarethemainformsofmoleculartransferfrom the ER to different organelles. Secretory proteins in the vesicles are taken from the ER to the cis-Golgi, which, along with the trans-Golgi, comprises an array of tubules and vesicles on the opposite side of the medial-Golgi. The medial-Golgi, typically containing three to seven stacks of cisternae, is the main site of glycan elongation for glycoproteins.

There are two different views on how protein cargos are transported outward towards the TGN and eventually to other organelles or secreted out of the cell. The vesicle diffusion model hypothesis states that the cargo from an earlier compartment is transported to the next compartment by the membrane vesicles. The cisternae maturation model views the cargo as stationary inside the stack, once they enter the compartment. The cisternae (including the cargo) then moves outward, with its enzyme constituents changing along the way, and the cargo protein molecules becoming “mature”.

Eventually, the cargo at TGN is transported through thevesiclestoitsfinaldestination,beittheplasmamembrane (for secretion) or to other organelles. As

The Becoming of Plasma Cells - A Hint to the Creation of a Super Secretor• Overloading of secretory protein molecules induces

unfolded protein response (UPR), which triggers the differentiation process of B cells to become non-dividing plasma cells .

• B cells differentiate into plasma cells (or memory cells) upon antigen stimulation, along with helper T cells, increasing their size significantly and their ER by at least 15 fold (in 4 days) . They also increase metabolic machinery significantly.

• Xbp1 codes for XBP-1 (55KDa) . XBP-1 is post-transcriptionally modified upon UPR, and activates transcription of many ER proteins. Increase in XBP-1 coincides with an increase in antibody production (day 4 ), but lags in ER expansion .

• ER appears to expand by increasing its abundance, not merely selectively increasing some ER proteins. The expansion starts before mass antibody production. ER is a very oxidized environment (unlike cytoplasm that is highly reduced). Disulfide bridges are formed in ER and catalyzed by protein disulfide isomerase (PDI) . PDI and four oxidoreductase level increases in ER as the B cells differentiate. Many of the redox balance enzymes in cytosol and mitochondria are also upregulated . There is also evidence to show that Golgi increases along with ER .

Note: the drastically increased antibody secretion is through biogenesis of ER and other protein secretion machinery, NOT merely by faster “throughput.”

Fig. 2.6: Physiological changes incurred during the transition from B cell to plasma cell


the contents of the early compartment translocate to the later compartment, they need to be recycled after the cargo is delivered. Thus, there are vesicles for retrograde transfer, in addition to anterograde transfer. The ER, Golgi, lysosomes, and endosomes are all part of the secretory network. They communicate throughthedynamictraffickingofmembranevesicles.

An estimated 100 to 200 glycosyltransferases, transporters of various nucleotide-sugars, are membrane proteins that constitute the majority of Golgi enzymes.

Aftertranslation, it takesa finiteamountoftimetoprocess protein molecules before they are excreted. For an average protein of about 350 amino acids in length, the translation takes only tens of seconds. However, the time required for synthesized proteins to be secreted depends on the nature of the protein, and can thus range from 30 minutes to afewhours.Forexample,theα1-proteaseinhibitoris among the fastest secreted proteins, with a half-life of about 28 min. Transferrin, in contrast, takes around two hours to be secreted. Even for the same protein, the secretion time is not uniform for all molecules. Rather, we observe distribution between shorter and longer “holding times”.

Table 5. Secretion time of liver proteinsHalf-life in ER

(min)Half-life in Golgi

(min)Transferrin 110 45Ceruloplasmin 80 30Anti-trypsin 30 - 40 10IgG Total ~120

• Secretory proteins spend different amounts of time in the ER and Golgi apparatus and in different proportions

Fig. 2.7: Secretion time of IgG heavy chain. Intracellular proteins were completely labeled with C14N15 - arginine, then switched to unlabeled medium. ~50% heavy chain is secreted in two hours .


Protein Secretion• Nascent protein molecules destined for ER have a special ER signal sequence being synthesized in

organized polysome. They are recognized by SRP (signal recognition particle), a ribonucleoprotein.

• SRP binding transiently arrests elongation, directing the ribosome/nascent polypeptide complex (RNC)to the receptor on ER membrane and transfer the growing polypeptide to translocon.

• SRP is released from the ribosome/nascent polypeptide complex.

• The nascent polypeptide begins to pass through translocon and elongate into ER lumen.

• Signal peptide on the elongating polypeptide is cleaved.

• Protein folding and post-translation modification begins as polypeptide continues to elongate.

• Major ER luminal chaperons: BiP, calnexin, calreticulin and PDI .

• Ribosome is released once the translation is complete.

• Folded protein (with inner core of glycan if it is a glycoprotein) concentrate at exit site of ER and is thought to bud into vesicles and translocate to Golgi as pre-Golgi intermediates .

• Golgi apparatus is in a dynamic state. There is also retrograde transport (its own proteins need to be recycled) and anterograde transport .

• After reaching trans Golgi network (TGN), secretory proteins are packaged into post Golgi vesicles and move along cytoskeletal network through cytoplasm to fuse with plasma membrane and be secreted .

• Different molecules of the same secretory protein spend different amounts of time inside the cell, i .e . there is a distribution of “holding time” in the cell .

• Translation of a protein molecules takes only seconds, but the secretion process takes tens of minutes to hours .

AAAAA

AAAAA

AA

AA

A

Nucleus

Endoplasmicreticulum

Endocytosis

Endosome

Anterogradetransport

TGN

Trans Golgi

Medial Golgi

Cis Golgi

Retrogradetransport

Bip

Translocon

SRP

SRP

Signalpeptide

AAAAA

Fig. 2.8: Synthesis and secretion of proteins to an extracellular environment


Other Organelles In addition to the nucleus, the mitochondria, the ER and the Golgi apparatus, a number of other organelles are also in the cytoplasm.

A lysosome is an organelle with a low pH in its interior. It is the site of degradation of ingested materials or cellular materials that are no longer needed by the cell. Most cellular materials have a useful life span, regardless of whether they are catalyzing chemical reactions or playing structural or mechanical roles.

Occasionally, a catalyzing enzyme can be improperly “locked up” in its transition state, resulting in an amino acid being modified to lose its catalytic capability.Even in the cellular environment, some amino acids in the protein may get oxidized. The accumulation of such “damages” may render a protein non-functional.Thus,mostproteinshavefinitelifespan.

Proteins that need to be turned over are tagged by ubiquitin and sent to the proteosome for degradation. Proteosomes are a complex of protealytic enzymes that are capable of degrading proteins.

Through a process known as endocytosis, eukaryotic cells can take up external particles by wrapping the particles within cellular membranes and taking them up as vesicles enclosed in lipid bilayers. This process is not seen in prokaryotes. Some cells in higher organisms are specialized “scavengers” that engulf foreign particles or dead cells. Lysosomes are the sites within those cells where engulfed particles are degraded. Lysosomes contain a large number of digestive enzymes. They have proton pumps in their membrane to maintain a low interior pH (pH = 5.0).

In fat cells, the catabolism of lipid occurs in peroxisomes. The reactions involved in such metabolism generate large amounts of reactive oxygen and, thus, need to be contained within these specialized organelles.

Lysosome:• Low pH

• Site of degradation of cellular materials destined for degradation and endocytosed material

• Part of secretory pathway

Peroxisome:• Site of fatty acid oxidation

• Rich in oxidative enzymes

Endosome: • In endocytosis the invaginated plasma membrane

forms small organelles

• They move inward along the microtubule network

• There is extensive cargo distribution and sorting

• Some material sent to lysome

• Some recycle to plasma membrane


Cytoskeleton

Microtubules A microtubule is an assemblage of hollow tube structures formedbypolymerizedα-andβ-tubulinmolecules. The polymerization and de-polymerization occurs at the ends of the microtubules, allowing them to extend and shrink their length rapidly.

The hollow organization enables cells to use a smaller amount of material to give a longer protrusion with a high structural rigidity, like the hollow legs of aluminum ladders sold in hardware stores. If the same amount of material were made into solid legs, the legs would be rather thin and would easily deform when subjected to stress. Being tubes, microtubules are relatively straight and do not bend in sharp angles or high curvatures.

Microtubules can be extended to protrude from some region of the cell, and can rapidly shrink to retract a part of the cell. They are also used as a “train track” in the cytoplasm to transport “cargos”, such organelles and membrane vesicles, to different parts of the cell.

When two ends of a microtubule are attached to different objects, they can also pull them together or push them apart. For example, during mitosis, multiple microtubules work in coordination to separate a pair of chromosomes, thus allowing the daughter cells to each receive one copy.

• Long, hollow tubes of polymerized subunit tubulin (MW 50 kD), about 25 nm diameter, more rigid than actin filaments .

• Typically long and straight, many have one end (-) attached to a single microtubule organizing center (centrosome) .

• Form and break down rapidly.

• α- and β-tubuline (GTP) form heterodimers; the dimer assembles in a head-to-tail fashion into chains called protofilaments, 13 of which make up the microtubule wall .

• Microtubules also play a key role in intracellular organelles and small vesicle transport .

• For secretory protein, the movement of post-Golgi vesicle to plasma membrane is mediated by microtubules .

Fig. 2.9: Structure of a microtubule molecule and its cellular organization

As discussed earlier in this chapter, cells are not merely a droplet-like structure with its constituents enclosed by a lipid bilayer. They form various shapes and sustain mechanical forces by transmitting and responding to mechanical force stimuli within, and also between, cells. A class of proteins makes up the cytoskeleton, accounting for their shape and their ability to transmit and exert force. The three major components of cytoskeleton are microtubules, actin fibers, and intermediate filaments.


Intermediate Filaments Themainroleofintermediatefilamentsistotransmitmechanical force, like the cable holding a suspended bridgeinplace.Eachfiberismadeofsubunitproteinsoriented in the same direction. The tail end of a subunit is locked into the head of the next subunit. Each intermediatefilamentfiberismadeofmultiplefibrils,which are in turn made of a series of subunit proteins.

To increase the structural integrity, the tail-head “lock” positionsofdifferentfibrilsareatdifferentlocationsinthemulti-fibrilfilament.Theseintermediatefilamentfibers are flexible, capable of absorbing energyexerted by external force and transmitting it to other regions of the cell. In a tissue or in interconnected cells in culture, intermediate filaments alsohelp totransmit forces between cells. Their deformable nature allows them to act like a shock absorber and to reduce the deformation of cells upon stress.

• Play a structural role, stable, transmit mechanical force

• The subunit is not a globular protein, but fibril, different from the tublin and actin

• 10 nm diameter, has a head, a tail and a α-helical rod; can be made of a wide variety of proteins in various tissues.

Actins are two- or three-stranded filaments thatoften form web-like bundles underneath the plasma membrane. Like ropes that are woven into a net, actin fibers are twisted strings of filaments.This geometry enhances their structural integrity while maintaining a high degree of flexibility.

The actin-rich region immediately underneath the cell’s plasma membrane often has a high concentration of actin, where it forms a gel-like structure, called the cortex. They are like a mesh of thin nets underneath the lipid bilayer membrane. Theyactasthefirstabsorberofexternalmechanicalperturbations and give the lipid bilayer local shape.

Cellsextendtheirbodyandspreadflatonasurface,both in tissues and in culture. The edge of an adherent cell has an irregular shape, much like a fried egg. In the protruded regions, actin fibers localize in thelamellipodia and filopodia. In stationary cells, the

Actin Filaments

Fig. 2.10: Structure of an intermediate filament molecule and its cellular organization

Fig. 2.11: Structure of an actin filament molecule and its cellular organization


actin fibers form stress fibers throughout the cell,but in moving cells, these visible fiber structurestend to localize at the moving ends of the cell.

Actin filaments, along with the other twomajor components of the cytoskeleton, require many other component proteins to be present for their polymerization, dissociation, cargo translocation, and other functions.

• Two stranded filaments (F actin) of helical polymer of actin (G actin)(50 kD)

• 5-9 nm flexible structures, organized into linear bundles, 2-D networks and 3-D gels

• Distributed all over the cell, but concentrated in the cortex beneath the plasma membrane

• The subunit, G actin, is a globular protein

• Projections from cells, like microvilli, lamellipodia, microspikes and filopodia are maintained by rigid bundles of actin filaments

• In non-motile cells, actin filaments form bundles called stress fibers, loose meshwork of filaments underlies cell membrane .

• In actively moving cells, stress fibers disappear and actin filaments concentrate at the leading edge.

• There are many actin-related-proteins which affect the polymerization and motor functions of actin filaments

• Actin is involved in motor functions. Best example is muscle contraction.

Transport Mechanisms

Fig. 2.12: Order of magnitude estimation of the permeability of various molecules across lipid membrane

The lipid bilayer membrane separates cells from their environment. It presents a barrier to keep most compounds outside the cell and to prevent those inside from leaking out. It has a very low permeability for large molecules, like proteins and polysaccharides. Even polar or charged small molecules cannot pass through easily.

Among the nutrients and metabolites, only oxygen, fatty acid, and ethanol pass through the membrane at a fast enough rate to meet growth requirements. Specialized transport mechanisms mediate the movement of the vast majority of nutrients and the excretion of metabolites. Cells have a large number of transporters (sometimes called permeases) that allow molecules to cross the cytoplasmic membrane and membranes of various organelles.

Such transporter-mediated transport is used to pass small molecular weight compounds (up to about 1 kDa) across the cell membrane (e.g., sugar, oligosaccharides, amino acids, oligopeptides, nucleotides, cholesterol, ions, organic acids, etc.). Macromolecules are transported across membranes by membrane fusion (in the secretion process through the ER and Golgi apparatus), pinocytosis,


Major Mechanism of Transport

The cellular transport of solutes (as opposed to macromolecules) is grossly divided into two categories: transport along or against the concentration gradient of the solute. The former is thermodynamically favorable, whereas the latter (called active transport) requires energy input to make the process possible.

The energy source of active transport can be derived from coupling to a chemical reaction, such as the hydrolysis of ATP. Active transport of a species may also be coupled to the diffusion of another solute along its concentration gradient. Thus, the driving force to transfer the second solute along its gradient is used to “push” the firstsolute to move up against concentration gradient.

Transport along the concentration gradient can be mediated by carrier (transporter) proteins or by channel proteins.

Channel proteins open into a duct-like structure across themembrane that is specific for a specific solute,such as water, Na+ or K+. Channel proteins exist either in an open state or a closed state. Once the channel is open, the transfer is very fast in the direction of theconcentrationgradient.Thefluxisaffectedbythenumber of channel protein molecules on the membrane and by the time period that the channel is open.

Carrier-mediated transport is also called facilitated diffusion. It entails, first, the diffusion of solutefrom the high concentration side of the membrane into the transporter, followed by the translocation of the solute to the low concentration side of the membrane. Once on the low concentration side, the solute is free to diffuse away.

Under normal culture conditions, amino acids and glucose are transported by facilitated diffusion. A ubiquitous transporter for glucose is the glucose transporter 1 (GLUT1). The rate of transport by a transporter is dependent on the concentration of the solute. More precisely, it depends on the concentration

Three Classes of Transport Processes• Channel-mediated diffusion:

Molecules or ion specific; once channel is open, very fast flux.

• Facilitated difussion: Provides molecule specific opening in the membrane; barrier for molecular diffusion.

• Active transport: Moves molecules up against a concentration gradient.Requires ATP or ion gradients of ion (H+, Na+) as an energy source to drive the transport .

or exocytosis. Specific receptors, such LDL andtransferrin receptors, may be involved in pinocytosis.


difference of the solute across the membrane. Its mechanism is similar to a typical enzyme-catalyzed conversion of a substrate to a product.

The dependence of transport rate to solute concentration can be described by Michaelis-Menton enzyme kinetics. At low concentrations, the transport rate is in proportion to the solute concentration, while at high concentrations, the rate is constant as the transporter becomes saturated.

The half-saturation constant (km) for GLUT1 is about 0.1 mM. In the 0.01 – 0.1 mM range, the glucose import rate of the cell increases with increasing glucose concentration. In the range typically used in cell culture media (1 – 10 g/L, or 5.5 mM to 55 mM), the rate is not affected by glucose concentration at all.

Transporters for facilitated diffusion and active transport can also be categorized according to the number of solutes each carries and the direction of solute flow.

Uniporters transfer a single solute from a high concentration side to a low concentration side, for example the GLUT1 transporter for glucose and the GLUT5 transporter for fructose.

Symporters and antiporters transfer two solutes, simultaneously. If the two solutes move in the same direction, the transporter is called symporter. Conversely, antiporters transfer two solutes in opposite directions.

Collectively, symporters and antiporters are called co-transporters. Co-transporters are often used to transport charged organic molecules. Dissociable solutes exit with a counter ion to maintain electric charge neutrality. When a charged solute moves from one side of the membrane to the other, the charge neutrality must be maintained. Otherwise, the net charge will accumulate across the membrane and create impudence for further transfer of the solute.

For example, lactic acid exists as lactate in an aqueous solution. If it is removed from the cell, a negative charge will be moved along with it. After a while, the cell membrane will be negatively charged outside, creating a negative voltage. The negatively charged outside will prevent further

General Types of Transporters• Uniporter: Transfers a single molecule (e.g. glucose,

fructose), usually uncharged .

• Bispecies-transporter (co-transporters): Requires stoichiometric exchange of two species simultaneously; important in change balance .

• Symporter:Two species transported in the same direction.

• Antiporter:Two species transported in the opposite direction.

Fig. 2.13: Three types of transport processes across the cell membrane


excretion of the negatively charged lactate.

In order to prevent the buildup of a charge across the membrane, charged solutes are transported by co-transporters. Two mechanisms are commonly seen: 1) co-transport with a counter ion (such as H+ for lactate) by a symporter, and 2) co-transport with an ion of the same positive or negative charge, but in the opposite direction (such as Cl- for HCO3

-).

In co-transporter mediated transfer, the transport rate is not only affected by the concentration difference of the solute, but also by the concentration gradient of the co-transported ion. Thus, the transport of lactate by the monocarboxylate transporter (MCT) is not only affected by the concentration of lactate, but also by pH.

Co-transporters may also be involved in active transport. One such case involves an ion species being transported along its concentration gradient. The tendency of the ion to “push” across the transporter is used to “drive” the transport of a solute against its gradient.

For example, the Na+/glucose transporter in the epithelium of the intestine can take up glucose from the digestive track, even when glucose is lower than in the cell. This is accomplished by using the Na+/glucose transporter to transport two Na+ atoms from the lumen of the intestine into the cell, where the Na+ level is very low. As the sodium ion is transported, a glucose molecule also binds to the transporter and is transported simultaneously. The propensity of Na to move across the membrane is so high that it can drive glucose to move against a large concentration gradient.

Fig. 2.14: Three types of transporters categorized by the solute being transported


Transport of NutrientsMajor nutrients like glucose, other sugars, amino acids and oligopeptides, are taken up by cells through facilitated transport. The excretion of lactate, ammonium, and some non-essential amino acids are also transported by the same mechanism.

A large number of glucose and amino acid transporters are present in the mammalian genome. Different glucose transporters are expressed in different tissues for different cellular needs, but GLUT1 is present in all cells. GLUT4 is responsive to insulin and is expressed only in some tissues. Upon insulin stimulation, the intracellular GLUT4 molecules are translocated to the plasma membrane to take up glucose.

The number of amino acid transporters in a mammalian genome is also large. Some transport entire classes of amino acids that share a common property, such as the neutral amino acid transporter for uncharged amino acids. Others are specific for one or a small number of amino acids.

The monocarboxylic acid transporter (MCT), the transporter for lactate, also transports pyruvate. A number of MCTs are expressed in different tissues. In a cell, different MCTs are located at the cytoplasmic membrane and others at the membranes of some organelles.

Another class of transporters for active transport is the ATP-binding cassette (ABC) transporter, which transports some hydrophobic compounds by utilizing ATP. After prolonged exposure to methotrexate, some cancer cells develop drug resistance by pumping the chemical out of cells using ABC transporters.

• There are 12 different facilitative glucose transporters (GluT) in animal cells. Different transporters are expressed in different tissues.

• GluT 1: the major transporter in all cells

• Amino acid transporters have overlapping amino acid specificity. Some amino acids compete for the same transporter. Many have alternative transporters.

• MCTs transport lactate, pyruvate together with H+

Ion Transport Bulk ion species (H+, Na+, K+, PO4-3, Cl-) are present

at very different concentrations across the cell membrane. For instance, Na+ and K+ have opposite directions in their concentration gradients across the plasma membrane. The intracellular concentration of K+ is 20 – 50 times higher inside than outside the cell, while the Na+ concentration is 10 – 15 times higher outside the cell versus inside the cell. Along with the concentration gradients of major ions,


cells also maintain an electric potential gradient of -80 mV across their plasma membrane. This electric potential is fundamental to the transport of many compounds across the membrane.

Because of this concentration gradient across the membrane, sodium ions have a natural tendency to flowintothecell,wherevertheyareallowedtopass.The -80 mV membrane potential further enhances the propensity of Na+toinflux,asthenegativechargeinthe inner surface of the membrane draws Na+ to move across the membrane. The combined concentration and electric potential gradients are used as a driving force in the Na+-dependent glucose transporter, which will transfer glucose from the lumen of the digestive track into intestinal epithelial cells, moving against a glucose concentration gradient.

The Na+/K+ ATPase transporter, which is present in the cytoplasmic membranes of all animal cells, is important for establishing sodium and potassium gradients across the plasma membrane. ATPase is an integral cell membrane protein that has multiple subunits. It simultaneously transports two K+ ions into the cell and three Na+ out of the cell.

Although the cytoplasmic membrane is relatively impermeabletoions,itdoesallowasmallbutfinitediffusion of Na+ and K+ along their concentration gradient(i.e.,anetinfluxofNa+andanetoutfluxofK+). It should be noted that the membrane permeability for K+ is higher than Na+. Intracellular K+ also leaks out through potassium channels. Overall, through diffusion across the membrane and transport by channel proteins, there is a net movement of ions into the environment from the cytosol. This balance is maintained by the action of the Na+/K+ ATPase.

Na+/K+ ATPase has three binding sites for Na+ and two for K+. The Na+ binding sites on the cytosolic side of the ATPase have a Km for Na+ in the range of sub-millimolar concentrations. Because the cytosolic Na+ concentration is about 10 mM, virtually all three Na+ binding sites exposed to cytosol will be occupied. Na+/K+ ATPase also has a binding site for ATP. Hydrolysis of ATP results in the phosphorylation of the protein subunit and release of ADP. This allows two K+ ions to bind to the protein on the extracellular

Fig. 2.15: Transporters involving the transfer of ions and ABC transporter


side, while simultaneously exposing the Na+ binding sites to the extra-cellular solution. At the external side of the enzyme, the Km of Na+ is at a higher value. As a result, Na+ is released to the outside environment. After the release of Na+, the phosphate is released from the protein and K+ releases to the intracellular environment. The Na+/K+ ATPase then resets, ready for another round of reactions.

The net result is that ATPase uses ATP to pump three Na+ ions out and two K+ ions into the cell. With its 3:2 stoichiometric ratio of sodium to potassium, a prolonged operation of ATPase without any balancing action will inevitably generate a large electric potential across the membrane. To counter this, cells also have chloride ion pumps to pump negatively charged Cl- out of the cell. In some cells, Na+ and Cl-

channel proteins also facilitate the maintenance of the membrane electric potential in the correct range.

Extracellular Matrices and Cell MovementECM

• The concentrations of other major ion species (H+, Na+, K+, Ca2+, PO4

-3, Cl-) across membrane are polarized . Na+ and K+ have opposite direction in their concentration gradient across the plasma membrane, which is about ten fold difference. For Ca2+ the intracellular concentration is so low that the gradient is extremely steep .

• Na+-K+ ATPases play a major role in maintaining Na+ and K+ gradients. ATPase utilizes the hydrolysis of ATP as the energy source .

• Iron is extremely reactive and participates in many redox reactions. In biological systems, it exists as “bound” form. In its free form, it catalyses the formation of peroxide and peroxidizes unsaturated fatty acids. In cell culture, it is supplied as transferrin-bound or bound by other chelators and taken up via transferrin receptors .

• Another class of ions are transported into the cells via binding to proteins and internalized through specific transportors . One example is iron transport by transferrin. Iron is extremely reactive, participates in many redox reactions. In biological systems, it exists as “bound” form. In its free form, it catalyses the generation of peroxide and peroxidize unsaturated fatty acids. In cell culture, it is supplied as transferrin bound or bound by other chelators and taken up via transferrin receptor .

The vast majority of cells in the body are embedded in tissues in an acelluar tissue structure. When cultured in vitro to plastic or glass surfaces, they secrete materials onto the surface after adhering. Those excreted materials to which cells attach are collectively called the extracellular matrix (ECM).

The ECM is made of proteins, proteoglycans, and glucosaminoglycans. Different types of cells excrete somewhat different ECM components. Among the prominent members of ECM proteins are collagens, laminins, and fibronectin.

The role of ECM is not merely to provide a surface appropriate for cell adhesion. It is also important for cell-surface signaling and growth control. Receptors on the cell membrane establish cell adhesion complexes with ECM components and a tension force is then transmitted through cytoskeletal fibers and


Cell Movement

the cell’s internal signaling pathway to allow cell cycle to proceed. The cell adhesion receptors exhibit a wide range of diversity. For example, integrin receptors haveavarietyofαandβcomponentsandformalargenumber of combinations of integrin complexes that havedifferentaffinityfordifferentECMcomponents.As a result, different cell types often have different ECM requirements for adhesion and growth.

Many ECM components are highly negatively charged. This allows many protein growth factors to be adsorbed to the ECM and released to surrounding cells, perhaps even serving as chemoattractants. Therefore, they also play a role in providing cues for cell migration and differentiation.

The vast majority of cells are capable of movement on surfaces. In general, cell movement can be the result of an attraction to chemicals, or unidirectional random movement.

Cell movement is a coordinated series not unlike walking. It involves a restructuring of the cytoskeleton, a protrusion of the membrane, the establishment of surface adhesions on one side of the cell, and the detachment of the cell membrane from adhesion complexes in the rear end of the cell.

The actin fibers and plasma membrane of movingcells extend the cell to become more elongated in one direction. The extended regions form lamellipodia, which may also contain microspikes, or filopodia,which are active even within a few minutes.

Cells moving in an “open” surface (i.e., not crowded) move randomly. They exhibit locomotion contact inhibition, meaning when two moving cells encounter each other, both will move away in opposite directions. Furthermore, the two daughter cells of a dividing mother cell move away from each other when cell division is complete.

Cell migration is a regulated by many factors, including growth factors. For example, epithelial cells

• The filopodia extend as a result of microtubules growing at the cell front, and establish a “grip” on the surface

• The subsequent dissociation of cell-substrate contact at the rear of the cell allows the cell’s center of mass to move forward

ECM Proteins • Collagen

• Laminin

• Firbronectin

Proteoglycan • Chondroitin sulfate

Glucosaminoglycan • Heparin

• Hyaluronic acid

• The extracellular matrix is rich in electrocharges, allowing growth factors, cytokines etc. to be “stored” inside them

• The extracellular matrices are the substrate for adhesion of many cell types, and provide cues for growth, differentiation and development


Growth, Death and Senescence

respond to hepatocyte growth factor by moving away from each other and become more scattered, instead of forming cell clusters typical of epithelial cells.

Cell migration is not intentionally controlled or manipulated in cell bioprocessing. However, in some cases, when seeding cells into three-dimensional matrices for tissue engineering applications,efficientcellmigrationintotheinteriorof matrices is important for subsequent growth.

Cell Cycle and Growth Control

Positive and Negative CuesCell growth is the manifestation of a delicate balance between positive and negative regulations that respond to signals both outside and inside the cell. Positive signals stimulate cell growth and proliferation and suppress the cell death mechanism, while negative signals suppress those events and promote cell death. External signals from the environment tell cells the availability or absence of nutrients necessary for DNA replication and biomass synthesis. External signals from other part of the body allow cells to coordinate their response to the need of the organism. The internal signals modulate cellular programs to increase cellular component content, to divide, or to die.

Eukaryotic cells progress through four stages in their procession to grow in cell number: G1, S, G2, and M phases. G1 and G2 refer to the gap phase. S and M phases derive their designation from DNA synthesis and mitosis, respectively. The four stages constitute a cell cycle and this cycle is repeated every time a single cell becomes two daughter cells. Cells that are in a long period of quiescence, such as terminally differentiated cells,divertfromG1andenterG0stageindefinitely.

Checkpoints are present between the different stages of cell cycle control. After mitosis, cells increase in size and mass. They only enter the S phase from G1 if cellular conditions are right. Similarly, they only enter mitosis from G2 if cellular components are ready.

Cell Cycle• G1, S, G2 and M constitute four consecutive phases

of cell cycle . S stands for DNA synthesis and M for mitosis . They are separated by two gaps .

• The duration of S and M phases is relatively constant. Cells grow at different rates and spend differnt amounts of time in G1 and G2. For mammalian cells, the duration of S phase is 5-8 hours and that of M phase is 1 hour .

• The progression from G1 to S, and from G2 to M phase is tightly controlled. Only when a cell is “ready”, will it proceed to the next phase .

• Before M phase, chromosomes and all organelles, ribosomes and other cellular contents are all duplicated from time 0 (immediately after cell division) .

Fig. 2.16: A cell moving on its substrate. Lamellipodia extend in the leading edge on the right side. A few filopodia are extruding out.


Additionally, the decision to enter the S phase is subjected to the regulation of external positive mitogenic factors, such as insulin, insulin-like growth factors, and fibroblast growth factors.Anchorage-dependent cells also receive growth stimuli by establishing contacts between the surface receptors and the ECM, and thereby maintaining tension in the cytoskeletal network.

Countering the actions of the mitogenic factors are those factors that provide signals to cause growth arrest. Cell-cell contact, for instance after reaching a confluent state, causes growth to cease. Suchcontact inhibition of growth has been noted for more than five decades. Only recently have researchersfound that it is the interference of the adherent junctions between cells that disrupts a cell’s internal signaling networks to cause growth arrest.

Whether cells divide and grow or self-destruct is the outcome of a balancing act of a network of external and internal positive and negative factors. Loosening the controls may lead to unscheduled proliferation and transformation of cells to their malignant derivatives.

Cyclins and CDKs The progression through each of the four phases of the cell cycle (G1, S, G2 and M) is positively regulated by cyclins and cyclin-dependent kinases (CDKs) and negatively controlled by CDK inhibitors (CDI), which deactivate cyclin-CDK complexes.

Each of these regulatory proteins displays a characteristic periodic dynamic profilethroughout the cell cycle. Each protein’s profileis the result of interactions of the other cell cycle regulatory components with their expression, activation, inactivation, or degradation corresponding to a specific time frame.

An important cell-cycle checkpoint occurs along the transition from the G1 to S phase. The pivotal player in the G1/S phase transition is the CDK4/6-cyclin D complex. The activated CDK4/6-cyclin D complex can phosphorylate the regulatory protein retinoblastoma

Fig. 2.17: Phases of cell cycle and approximate duration of each phase


(pRB). pRB, in its unphosphorylated state, binds to and inhibits the transcription factor E2F. Upon phosphorylation, pRB dissociates from E2F, leading to the activation of cyclin E transcription by E2F. E2F activation positively regulates the transcription of genes involved in cell cycle progression.

Inputs from growth factor signaling and cell adhesion-mediated signaling are prerequisites to the G1 phase. These two pathways are not independent of each other. On the contrary, they have rather extensive crosstalk. In normal untransformed cells, all the important growth factor signal transduction cascades are regulated by integrin-mediated cell adhesion. As a result, adherent cells rely on attachment to the ECM for growth.

Except for vaccine production, where normal diploid human fibroblasts are employed, virtuallyall cells used for protein production are continuous cell lines, including CHO, BHK, HEK 293, and mouse myeloma cells, such as NS0 and Sp2/0. All of these cell lines have lost their normal growth control. Their cell cycle checkpoint controls have been compromised and their entry into a quiescent state in the absence of mitogen has been relaxed.

Growth Control• Growth, the increase in biomass and its contents (i.e.

organelles and cytosol) as well as the increase in cell number of almost all cells (except those which have been adapted in vitro) is regulated by growth factors, cytokines .

• The regulation balances positive factor (mitogenic) and negative factors. which prevent cell death or apoptosis .

• The most common growth factors for cells of bioprocess interest are insulin or insulin-like growth factors (IGF) .

• IGF and insulin have different receptors on cell surface . Insulin regulates glucose metabolism and has a mitogenic effect. IGF, which is used in much lower concentrations and also has a mitogenic effect.


Fig. 2.18: Schematic representation of the interaction between cell cycle and apoptosis pathways. CDK Cell cycle-dependent kinase, IRF-1 interferon-regulatory factor 1, pRb phosphorylated retinoblastoma, ERK extracellular signal regulated kinase, FADD Fas associated death domain protein, FLIP FLICE-inhibitory protein, Cdc42 cell division cycle 41, EIF4E eukaryotic translation ini-tiation factor 4E, Cyc Cyclin, XIAP cross-linkied inhibitor of apoptosis proteins, Apaf-1 apoptotic peptidase activating factor – 1, BAX Bcl-2 associated X protein, BAK Bcl-2 homologous antagonist/killer, Ub ubiquitination, cyt C cytochrome C


In many developmental events, individual cells servetheirfunctiononlyforafiniteperiodoftime.Beyond this period, their existence may interfere with, or even imperil, the well being of the organism. In those cases, cells are built to die after their functional duration by endowing an individual cell’s survival to depend on the presence of positive factors or the absence of negative effectors.

In the event that a cell survival signal is absent or a cell death signal is present, a cell undergoes self-destruction and ceases to serve its function. Developmentally related apoptosis is largely regulated by death receptors on the cell surface. The death receptor pathway is mediated by binding, or a lack of binding, of ligands to death receptors. For example, immature neurons die in large numbers during early brain development because neuronal cells require positive survival signals. The lack of such positive survival signals leads to neurodegenerative disorders.

Ligand binding to death receptors initiates the recruitment of an adaptor molecule, Fas-associated death domain (FADD), to the cytoplasmic end of the receptors. The presence of FADD causes caspase 8 or 10 to associate with the receptor, forming a death inducing signaling complex. The caspase is then proteolytically activated, triggering

Death Receptor Pathway

Apoptosis is the process of regulated cell death in response to developmental cues or to accumulating non-lethal stresses, such as nutrient depletion, growth factor deprivation, virus infection, and/or metabolite accumulation.

This process of programmed death is marked by specific cell morphological changes: DNAcondensation, chromatin shrinkage, and membrane bulging(alsocalledblebbing).Thefinalintracellularevent involves a series of cascades leading to cellular destruction. These final acts of self-destructionare similar in all apoptosis mechanisms; however, the initiating “signal” can differ. The two major apoptosis signaling pathways are the death receptor pathway and the mitochondrial pathway.

Apoptosis• Cells, under some conditions, commit suicide, undergo

programmed cell death; this is different from cell death caused by injuries (necrosis) .

• Necrosis may entail cell swelling, rupture and leakage of cellular materials . In vivo, necrosis may cause inflammation of surrounding tissues after encountering cell debris .

• Apoptosis entails cell shrinkage, mitochondria breakage and release of cytochrome C, DNA fragmentation, and release of phosphatidylserine from phospholipids which causes phagocytotic cells to engulf the cell fragments .

• Apoptosis can be caused by the lack of positive signal/growth factors (e.g. withdrawal of IGF) or the imposition of negative signals.

• Two general pathways for apoptosis: one, induced by specific signals, occurs during cell development; another, induced by a variety of stress conditions.


the activation of a series of downstream effector caspases (3, 6 and 7). The activation of these effector caspasesleadstothefinalstagesofcelldestruction.

In addition to its role in energy metabolism, mitochondria also sequester pro-apoptotic proteins in the space between their outer and inner membranes. These pro-apoptotic factors are released in a controlled manner in stressed cells to initiate apoptosis. Mitochondrial cytochrome C, a major component of electron transfer, is also a signal for apoptosis.

As in the control of cell growth, the components of the mitochondrial apoptosis pathway also involve positive proapoptotic and negative anti-apoptotic factors. The Bcl-2 family that consists of over 20 pro- or anti-apoptotic proteins is a major player in the mitochondrial apoptosis pathway. The pro-apoptotic subfamily includes Bax, Bak, and Bok, which all contain BH1, 2, and 3 homology domains.

Upon exposure to death signals, Bax undergoes conformational changes and translocates to the mitochondria, where it inserts into the outer mitochondrial membrane and forms channels. These channels allow the leakage of cytochrome C and other pro-apoptotic molecules.

Cytochrome C proceeds to form a complex with Apf-1, pro-caspase 9, and dATP, known collectively as the apoptosome. In the apoptosome, the inactive pro-caspase 9 is activated and the active enzyme subsequently activates downstream caspases.

Two anti-apoptotic proteins, Bcl-2 and Bcl-xL, counter the actions of the pro-apoptotic components. Bcl-2 is localized on the mitochondrial membrane and inhibits the release of pro-apoptotic molecules from the mitochondria by maintaining membrane integrity. Bcl-xL is localized in the cytoplasm and binds to pro-apoptosis members of the Bcl-2 family.

The involvement of multiple protagonist and antagonist factors ensures tight control of apoptotic event. This scheme also provides an amplification

Mitochondrial Apoptotic PathwayNon-Apoptotic State: The balance of anti-apoptotic and pro-apoptotic factors

• At non-apoptotic state anti-apoptotic factors hold pro-apoptotic factors in check

• Apoptogenic factors are held in inter membrane space of mitochondria

Apoptotic State:Upon insult of apoptotic inducing agents or environment factors

• Anti-apoptotic factor(s) had conformational change (exposing BH3)

• Membrane disruption releases cytochrome C and other pro-apoptotic and apoptogenic factors

• The releases of procaspase3 form apoptosome with the adaptor (Apaf -1), becomes capase-3 .

• Caspase-3 converts procaspase-9 to caspase-9 which becomes the executioner caspase and starts the apoptotic cellular event

Mitochondrial Pathway


The vast majority of animal cells isolated from tissues require surface adhesion in order to multiply, since they are anchorage-dependent cells. Cells are typically isolated from tissues by an enzymatic dissociation of the tissue. After dissociation and the removal of large chunks of undissociated debris, cells are plated on a compatible surface overlaid with media. Under the microscope, cells in media suspension can be seen to attach to the surface or out-grow from the remaining tissue chunks. Subsequently, they extend their body length, spread and begin to multiply. Those cells derived from normal tissues generally possess two sets of chromosomes and are diploid cells.

Eukaryotic cells enter an exponential growth phase, similar to microbial cells. The growth rate slows as they begin to cover the entire surface area to form a “monolayer”. Upon reaching confluence, they stopdividing. While the cell bodies of neighboring cells may cross each other, their nuclei never overlap. This is called contact inhibition of cell growth.

Cells can be dissociated from a surface after being treated with trypsin (i.e., trypsinized) or with other proteases. They can then be plated on a larger surface area for continued growth. This process can then be repeated to expand the population. Each round of detachment and expansion is called a “passage”.

Non-immortalized cells cannot be grown in culture indefinitely by simple repeated passage in culture.Normal diploid cells from animals (except stem

Senescence and Telomeres

of the desired signal. An excellent example of this is the cascade of caspases. When cell destruction is needed, the signal is greatly amplified througha series of steps where one caspase activates another caspase in an exponential fashion.

Fig. 2.19: Mitochondrial pathway mediated apoptosis

Fig. 2.20: “life” of normal cells from isolation to senescence and occurrence of cell line


cells) have a limited life span in culture. Fibroblasts (a cell type from connective tissue) isolated from a mouse embryo can be cultured in vitro for about 60 doublings. As that limit approaches, the cells begin to fail to reach confluence. Eventually, highpassage cells will cease to grow. This is referred to as having reached “crisis”. Such a limit in the proliferating potential is called “Hayflick’sphenomenon”. It is a common phenomenon for all normal diploid cells obtained from vertebrates.

In a historical experiment carried out half a century ago, the continued passaging of mouse fibroblastcells beyond crisis gave rise to a small fraction of survivors. These cells eventually grew, expanded, and could be cultured continuously in vitro without a limited life span. They were given the name 3T3 because the cells were passaged every three days by expanding the surface area three times more. These cells appear normal and are subjected to contact inhibition of growth under typical culture conditions.

However, although the parental mouse fibroblastcells had diploid set of chromosomes before reaching crisis, 3T3 cells have abnormal number of chromosomes. Cells that succumb to Hayflick’sconstraint (e.g., those that are diploid and have a limited life span) are called “cell strains.” The cells that reestablish after crisis and can grow in culture indefinitely,butareaneuploid(donothaveanormalset of chromosomes), are referred to as “cell lines.”

Cells derived from cancer also give rise to cell lines. Cell lines can also be established by “immortalization” through viral or oncogene transformation. These transformed cells are also often aneuploid. They are capable of growing beyond the monolayer, since they are not subject to contact inhibition of growth.

Normal diploid cells, thus, appear to “count” their number of doublings. They achieve this by using their telomeres. Telomeres are special repetitive sequences at the end of chromosomes. They are not replicated by DNA polymerase during DNA replication, but are synthesized by telomerase. The reaction is not precise for accurately reproducing

tissue dissociationPlating in nutrient medium

Growth

Contact inhibition

Cell Dissociation

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Fig. 2.21: Anchorage dependency and contact inhibition of cultured normal diploid cells

Fig. 22: Telomere at the end of a chromosome


Concluding RemarksIn this long chapter we provided a condensed overview of knowledge in cell biology that is essential for biotechnologists to practice cell culture bioprocessess. The structure and make-up of cells that gives them their functional versatility, also constrains their capability. In practicing biotechnology, while exploiting their biological versatility, we also must understand cell’s structural and functional constraints and biological limits. In the meantime, we must also keep in mind that our objectives are frequently different from scientists studying the biology of the cell. To fully harness a cell’s biological potential, we do not necessarily need

to be bound by the nature of cells; rather we should employ means to “adapt” them to serve our goals better. For example, most cells used for biologics production were anchorage-dependent originally and are now cultivated not only in suspension, but alsoinhighlyturbulentflowconditions.Mostoftheadaptation processes in the past two decades were conducted empirically. At a molecular level, what causes those cells to have the adapted behavior is poorly understood. By equipping ourselves with a better knowledge of cell’s capability and limits, we will be able to push the technological boundary further.

the number of tandem repeats of the sequence, thus there can be much variation in telomere length among cells. As the number of passages increases, telomeres decrease their length unless they are repaired by telomerase. For instance, in stem cells, telomerase activity is high to maintain the telomere length. Unlike cell strains, stem cells do not exhibit senescence.


Cell Physiology for Process Engineering

CELL PHYSIOLOGY FOR PROCESS ENGINEERING | 57

Overview of Central Metabolism of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58Glucose and Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60

Oxidation of Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Pentose Phosphate Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Lactate Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Carbon Flow and Reaction Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67NADH Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Role of Transport and Transporters in Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71Glucose Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Lactate Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Transport Across Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Regulation of Glucose Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74Isozymes and Differential Allosteric Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 75Signaling Pathways and Regulation by Growth Control . . . . . . . . . . . . . . . . . . . . 77

Metabolic Homeostasis and Lactate Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78Glutamine and Its Relation to Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 80

Amino Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83

Lipid Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Fatty Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85Acetyl CoA shuttle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Cholesterol & Its Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Glycan Biosynthesis and Protein Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .89Importance of Glycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Protein Folding and Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Glycan Extension in Golgi Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Glycan Types and Microheterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Synthesis and Transport of Nucleotide Sugar Precursor . . . . . . . . . . . . . . . . . . . . 93Glycan Diversity Among Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96


Cells in culture take up sugar, amino acids, lipids, and nucleosides from their growth media. They metabolize these components to derive energy and use them as building blocks to generate more cell mass, create more cells, and produce products. The processes of making more cell mass and protein product are very energy intensive. Proteins constitute over 50% of the dry mass in a typical cell; they are essentially amino acids connected by peptide bonds. The synthesis of each peptide bond costs at least 3 ATP, which is nearly 1/10 of the amount of energy that can be obtained by oxidizing one glucose molecule. One high-producing recombinant cell produces over 40 pg per day of IgG protein. Since an average cell has about 400 pg of cell mass (or about 200 pg of cellular proteins) it is easy to see that producing the protein product is a major energetic load for cells.

A classical cell culture medium contains 1 – 5 g/L of glucose, and somewhat lower levels of amino acids (about 0.8 mM, or 1 g/L). The sum of the nutrients, together, typically generates only about 1 – 3 x 109 cells/L, or approximately 0.1 – 0.3 g/L of cell drymass. The efficiency of producing cellmass from glucose and other nutrients is rather low.

Glucose is the most important source of energy for most cells. Even when another sugar such as galactose or fructose is used as the sole carbohydrate source, it still enters the metabolic pathway that has evolved for glucose (called glycolysis) to get catabolized.

The complete oxidation of one glucose molecule consumes six O2 and generates six H2O and six CO2. For cells in culture, however, the majority of consumed glucose is not completely oxidized; it is converted to lactate and excreted. By converting to lactate instead of completely oxidizing to CO2, much less energy is derived from each mole of glucose. This is the root causeofthelowefficiencyinconversionfromglucoseto cell mass. For some cells, especially transformed cell lines, each mole of consumed glucose produces almost two moles of lactate, which is the theoretical maximum of the glucose to lactate conversion.

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Glucose Oxidation

Glucose Anaerobic Metabolism

Glutamine Oxidation

Overview of Central Metabolism of Cultured Cells

Fig. 3.1: Lactate and ammonium profiles in manufacturing runs with high product titers (blue) and low product titers (red). Lactate profile correlates to productivity, but not ammonium.


This type of “wasteful” metabolism is common to almost all vertebrate cells in culture. For bioprocessing, the accumulation of these byproducts inhibits cell growth and impedes productivity.

Cells invariably produce lactate from glucose when growing rapidly. However, under some conditions, such as the stationary phase of fed-batch culture, lactate may also be consumed. It is not unusual that under the same operating conditions, different culture runs have different metabolic outcomes. In some runs, lactate production in the rapid growth phase continues into the stationary phase. In others, the transition from lactate production to lactate consumption occurs in the stationary phase.

When production data from a manufacturing plant were analyzed, it was found that the top productivity runs switched from lactate production to lactate consumption, while low productivity runs remained in lactate production mode throughout the culture. This is an indication that cell metabolism plays a key role in determining productivity.

Glucose, Glutamine Major Carbon Source• Both glucose and glutamine consumed in excess to

what are need to grow biomass• Most glucose consumed, converts to lactate• Excess glutamine consumption, results in ammonium

excretion

Lactate Acid and Fedbatch Culture Productivity• Lactate is produced in exponential growth phase• In stationary phase, some cultures continue to produce

lactate, others switch to lactate consumption• Lactate consumption correlate to sustained higher

viability and high productivity


Glucose is mainly catabolized through three pathways: glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid cycle (TCA) cycle. In glycolysis, one mole of glucose is converted to two moles of pyruvate. In this segment of catabolism, only a small fraction of the chemical potential energy of glucose is converted to the “usable” form of chemical potential energy in the cell, i.e., ATP. Two moles of ATP are generated per one mole of glucose. Pyruvate may enter the TCA cycle for further oxidation, or it may become a shunted product as lactic acid (at a neutral pH it exists as lactate). Through the TCA cycle, the carbon skeletonofglucoseisfinallybrokendowntoCO2 and H2O. The PPP is a shunt from glycolysis. It generates five-carbon sugars for nucleoside synthesisand supplies NADPH for many biosynthesis reactions and to maintain a redox state in the cell.

In eukaryotic cells, glycolysis and PPP take place in the cytosol, while the further oxidation of pyruvate to CO2 occurs in the mitochondria. It is in the mitochondria that the majority of the chemical potential energy of glucose is converted to ATP for use in cellular synthesis and other energy-dependent cellular processes.

Glucose Oxidation• Main metabolic pathways in energy metabolism:

• Glycolysis

• TCA cycle (tricarboxylic acid cycle)(Kreb cycle)

• Pentose phosphate pathway (PPP)

Glucose and Energy Metabolism

In glycolysis, glucose is broken down to two pyruvates and generates two ATP and two NADH. Pyruvate may then be further converted to lactate as a final product, instead of enteringthe TCA cycle. Thus, when glucose metabolism is terminated at glycolysis, it produces two lactate molecules and two ATP, but no additional NADH.

Although overall glycolysis generates high-energy compounds(e.g.,ATPandNADH),itsfirstsegmentactually consumes ATP. Two ATP are used to add a phosphate group to each end of the glucose molecule. The two phosphate groups pull their surrounding electron clouds toward the two ends of the molecule, thereby making the carbon-carbon bond

Oxidation of Glucose


in the middle susceptible to enzymatic cleavage.

After cleavage, the six-carbon skeleton becomes two three-carbon compounds: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP). These two compounds are interconvertable through a reversible reaction. The continued reaction of glyceraldehyde-3-phosphate (G3P) effectively draws DHAP toward G3P and moves it further downstream in glycolysis. The conversion of two G3P to the end product of two pyruvates also converts two NAD+ and four ADP to two NADH and four ATP. The net energetic consequence of the conversion of glucose to two pyruvate in glycolysis is the generation of two ATP (because two ATP are consumed to activate glucose) and two NADH.

The further oxidation of pyruvate takes place in the mitochondria,where it is first converted to acetylCoA, releasing one CO2 and generating one NADH. Acetyl CoA is then fed into the TCA cycle where it is broken down to two CO2. The pathway is cyclic, with four- to six-carbon skeletons cycling in a loop. At the beginning of the cycle, the four-carbon oxaloacetate (OAA) takes in acetyl CoA to become citrate. Citrate has three carboxylic acid groups; hence the name “tricarboxylic acid cycle”. The TCA cycle is also known as the citric acid cycle and the Krebs cycle.

As noted before, molecular oxygen does not react with the carbon compounds in the reactions from the TCA cycle. CO2 is released, through decarboxylation reactions, from the carbon skeleton without the participation of molecular oxygen. In two of the reactions catalyzed by pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, the energyfrom the breakup of the C-C bond is preserved in the high-energy compounds acyl CoA (acetyl CoA and succinyl CoA, respectively) and NADH. In the other case, one of the three carboxylic acid groups in citrate is released and one NADH is generated.

If the carbon-carbon bond is broken by directly reacting with oxygen, such as the case for combustion, a very high temperature is necessary to provide the activation energy. As we all know, it takes a fire to burn wood. Furthermore, the

Glycolysis - Each Mole of Glucose (6 Carbons)• Consumes two moles ATP (to activate to fructose, 1,

6-bisphosphate)• Produces two moles NADH, 4 moles ATP• Net: Becomes 2 PyruvateProduces 2ATP, 2NADH

TCA cycle• Pyruvate enters mitochondrion• Pyruvate loses CO2, becomes acetyl CoA (2 carbons)• Acetyl CoA enters TCA cycle,

• Becomes 2 CO2• Produces NADH, FADH2, which stores energy

• Never reacts with oxygen directly

NADH, FADH2 enter electron transfer pathwaypasses its high energy electron down, also its proton• As electron passes on energetic ladder, it pumps

protons out of mitochondrion• create a high pH inside mitochondrion• also a negative charge of ~120 mV across

mitochondrion inner membrane• The electron and proton, at the bottom of energetic

ladder, react with oxygen to form water .

Oxidative Phosphorylation Pathway• The higher concentration of proton in cytosol and the

negative charge inside mitochondrion drives proton to move into mitochondria

• Those protons moves into mitochondrion by passing through ATP synthase; as they pass through ATP synthase, ADP is converted to ATP inside mitochondrion

• There are a lot of fluxes across mitochondrial membrane, inclusind

• going in: pyruvate, ADP, phosphate, H+, • going out: ATP, CO2


Symbols of metabolites in energy metabolism: Glc: Glucose; g6p:Glucose 6-phosphate; f6p:Fructose 6-phosphate; f16bp (f16p2): Fructose 1,6-bisphosphate; f26bp (f26p2): Fructose 2,6-bisphosphate; gap: Glyceraldehyde 3-phosphate; Dhap: Dihydroxyacetone phosphate; 1,3bpg: 1,3-bisphosphoglycerate; 3pg: 3-phosphoglycerate; 2pg: 2-phosphoglycerate; pep: Phosphoenolpyruvate; pyr: Pyruvate; lac: Lactate; NADH: Nicotinamide adenine dinucleotide (reduced); NAD: Nicotinamide adenine dinucleotide (oxidized); NADPH: Nicotinamide adenine dinucleotide phosphate (reduced); NAPD: Nicotinamide adenine dinucleotide phosphate (oxidized); Gln: glutamine; Glu: glutamate; Asp: aspartate; ala: alanine; Mal: malate; αKG: α-ketoglutarate; OAA: oxaloacetate; SucCoA: succinyl CoA; 6pg: 6-phosphogluconate; ru5p: ribulose 5-phosphate; r5p: ribose 5-phosphate; xyl5p: xylulose 5-phosphate; e4p: erythrose 4-phosphate; s7p:sedoheptulose 7-phosphate

Symbols of enzymes and transporters in energy metabolism: GLUT: Glucose transporter; HK: Hexokinase; GPI: Glucose phosphate isomerase; PFK: Phosphofructokinase; PFKFB: 6-phosphofructo-2-kinase/fructose-2,6 bisphosphatase; ALDO: Aldolase; TPI; Triosephosphate isomerase; GAPD: Glyceraldehyde 3-phosphate dehydrogenase; PGK: Phosphoglycerate kinase; PGM: Phosphoglycerate mutase; ENO: Enolase; PK: Pyruvate kinase; LDH: Lactate dehydrogenase; PYRH: Pyruvate mitochondrial transporter; G6PD: Glucose 6-phosphate dehydrogenase; 6PGD: 6-Phosphogluconate dehydrogenase; RPE: Ribulose phosphate epimerase; RPI: Ribose phosphate isomerase; TK: TransketolaseTA: Transaldolase; PRPPS: Phosphoribosylpyrophosphate synthetase; PYRH: Pyruvate mitochondrial transporter; MCT: monocarboxylate transporter

Figure 3.2. Major pathways in energy metabolism. Glucose and glutamine uptake, glycolysis, lactate excretion, TCA cycle, and oxidative phosphorylation.


chemical potential energy would have been released as heat. Cells utilize decarboxylation reactions to form CO2 and to preserve energy in NADH.

Oxygen is then used to extract chemical potential energy from NADH and FADH2, in order to generate ATP that can be used in cellular work. The participation of oxygen in the oxidation of NADH/FADH2 generates the six O2 molecules required to oxidize one glucose, as shown in the stoichiometric equation of glucose oxidation.

Extraction of the chemical potential energy of NADH and FADH2 takes place through an electron transfer chain residing in the mitochondrial inner membrane. The high-energy electrons of NADH and FADH2 enters the electron transfer chain down the energy ladder, mediated by electron carriers such cytochrome C. The energy released is then used to trigger a proton pump to drive H+ out of the mitochondrial inner membrane. In the last step of the electron transfer chain, the electron reacts with oxygen and H+ to form water.

The export of H+ from the mitochondria creates a single unit pH difference across the membrane, as well as about -120 mV of electric potential. Because of the higher pH (lower proton concentration) and excess negative charge inside the mitochondrial membrane, there is a propensity for the proton ions outside the mitochondria to cross the mitochondrial membrane. They enter the mitochondria through an ATP synthase embedded in the inner membrane of mitochondria. Through the process, the act of a proton passing through ATP synthase brings an ADP and a phosphate together to synthesize ATP. The electron transfer and the generation of ATP are often referred to as “oxidative phosphorylation”.

The amount of ATP generated per mole of glucose varies somewhat among different species because of their variable expression of ATP synthase. However, in general, the number of ATP generated per mole of glucose is about 30 – 32 for mammals. The older literature tends to list the number as 36 moles of ATP / mole of glucose. Under some physiological conditions, the electron transfer chain

Fig. 3.3: Structure of key compounds in glycolysis.

Fig. 3.4: Structure of key compounds in TCA cycle and glutamine metabolism.


and oxidative phosphorylation are uncoupled. Instead of generating ATP, the energy from NADH is released as heat to maintain body temperature.

The amount of energy, two ATP and two NADH (or the equivalent of six ATP, since one NADH in the cytosol can be roughly considered to be two ATP), produced from splitting glucose into two moles of pyruvate is only about 1/6 of what can be generated from completely oxidizing glucose to CO2 and H2O. In glucose oxidation, the majority of energy conversion in glucose metabolism therefore occurs in the hundreds of mitochondria in the cell and not in the cytosol.

Pentose Phosphate Pathway

PPP• Two segments: • Oxidative: remove 1 CO2 from glucose-6-

phosphate, • Generate 5 carbon sugar phosphate for

synthesis of nucleotides and other compounds • produce 2 NADPH • Molecular transformation • Interconverts 5 carbon sugar phosphate to 3

carbon and 6 carbon • To allow NADPH and 5 carbon sugar to be

produced at different ratios• NADPH is important in biosynthesis and in

neutralizing ROS

PPP is an important shunt from glycolysis that supplies five carbon sugars and NADPH. PPP isdivided into two segments: an oxidative segment and a monosaccharide transformation pool.

In the first segment, glucose-6-phosphate fromglycolysis is oxidized and then decarboxylated to formthefive-carbonribulose-5-phosphateandtwoNADPH. The five-carbon sugar phosphate is usedin nucleotide (such as ATP and dATP) synthesis to supply the building units for RNA and DNA.

Cells use two different nicotinamide-adenine dinucleotides as reductive chemical potential energy carriers: NADH and NADPH. NADH is used to store chemical potential energy in glycolysis, the TCA cycle, and lipid catabolism. Eventually, NADH is used to derive ATP in the mitochondria.

NADPH, on the other hand, carries a chemical potential that is used in biosynthetic reactions, such as in the synthesis of lipids, nucleotides, etc. NADPH is also used to reduce oxidized glutathione and to regenerate it. The reduced form of glutathione is important in maintaining the cell’s reductive environment and in the suppression of reactive oxygen species (ROS).

The second segment of PPP is a molecular conversion pool that allows a two-carbon aldehyde unit or three-carbon keto units to be translocated among a number of three-carbon to five-carbon aldoses.


This allows the interconversion of carbohydrate molecules that are three to seven carbons in length. This “mixing pool” enables five-carbon sugarsfrom the first segment of PPP to be connected toglycolysis through six-carbon fructose-6-phosphate or three-carbon glyceraldehyde-3-phosphate.

The first segment of PPP generates five-carbonribulose and NADPH at a molecular ratio of 1:2. However, cells do not always need those two compounds at a 1:2 proportion. The molecular conversioninthesecondsegmentallowsthenetfluxfrom glycolysis to PPP to vary to meet the cellular demand of ribose and NADPH at different proportions.

Aerobic Glycolysis• Cultured cells and cancer cells undergo glycolysis

and produce lactate even at high oxygen concentration

• This propensity toward lactate production is not for lack of oxygen (anaerobic glycolysis)

• At high glycolysis flux, not all NADH can be oxidized by electron transfer in mitochondria

• Lactate production serves to regenerate NAD• continuous supply of NAD is crucial for glycolysis

to proceed

Under anaerobic conditions, some bacteria and yeast produce ethanol or lactate. In the absence of oxygen, the electron transfer chain does not operate because no oxygen is available to receive the electron from NADH. When NAD is not regenerated by NADH oxidation, the TCA cycle ceases to operate. The accumulated pyruvate from glycolysis is then excreted as lactate or ethanol. (Note that we denote NAD+ as NAD in text without the superscript “+” associated with its positive charge).

Mammalian cells in culture take only a small portion of pyruvate generated in glycolysis into their mitochondria, for further oxidation to CO2. They appear to have a limited capacity to translocate pyruvate into the mitochondria. The rest of glucose is converted to lactate. This occurs in spite of the presenceofsufficientoxygen.Thephenomenon is,thus, different from anaerobic fermentation in bacteria or yeast, and is referred to as “aerobic glycolysis”.

Not all cells in our body convert a large portion of the glucose they take up into lactate. The vast majority of cells in our body are in a quiescent (non-proliferating) state. They consume less glucose than proliferating cells. The contrast in cellular glucose metabolism,knownastheWarburgeffect,wasfirstobserved between normal tissues and cancer cells. Whilenormalcellshavealowerglucoseflux,cancerand proliferating cells consume a larger amount of

Lactate Formation


glucose and convert much of the glucose to lactate.

Lactate synthesis is catalyzed by lactate dehydrogenase. This reversible reaction takes one pyruvate and one NADH to become one lactate and one NAD.

In glycolysis, two ATP and two NADH are generated, along with two pyruvate. Continued glucose metabolism through glycolysis requires continued supplies of both ADP and NAD as reactants. ATP is used by cells to perform many tasks, such as synthesis, maintaining osmotic balance, etc. It is continually being consumed in various cellular reactions and converted back to ADP to resupply the reactant for glycolysis.

NADH is converted back to NAD through the electron transfer chain in mitochondria. To be regenerated in theelectrontransferchain,cytosolicNADHmustfirstenter the mitochondria and the regenerated NAD must be exported out of the mitochondria. A reaction with lactate dehydrogenase allows NAD regeneration from NADH to be carried out into the cytosol, therebyenablingglycolysistocontinueatahighflux.

Cells in culture convert about 90% of their glucose to lactate. Most of the other 10% of glucose is converted to CO2. At the completion of glycolysis, two ATP are generated while about 30 ATP are generated, following complete oxidation. The 90% of glucose converted to lactate generate 1.8 ATP (2 ATP x 0.9), while the other 10% generate 3 ATP (30 ATP x 0.1). Aerobic glycolysis of proliferating cells generates a significantamount of total energy to allow for proliferation.

2 2 2 2

2 2

cosGlu e ADP P NAD Pyruvate

ATP NADH

i+ + +

+ +

+

Pyruvate NADH Lactate NAD2 2 2 2+ + +

cosGlu e ADP P Lactate ATP2 2 2 2i+ + +

Energetic Yield of Aerobic Glycolysis1 . Oxidation

2. Reduction [oxidizing pyruvate to lactate (or ethanol as in yeast)]

Net Reaction:


Carbon Flow and Reaction Intermediates

Pyruvate is a controlling node• Generation rate of pyruvate is balanced by entry into

the mitochondrion and conversion to lactate

• Reduction of pyruvate to lactate recycles NAD+ for glycolysis to continue

• Lactate dehydrogenase (LDH) is reversible

• Transport of lactate by monocarboxylate transporter which is coupled to proton gradient

Pyruvate NADH Lactate NAD

Lactate dehydrogenase reaction+ + +

Among all of the pathways in the cellular metabolic reaction network, glycolysis has the highest fluxin terms of moles of substrate flowing throughit. For cells in culture, carbon flux (based onthe number of moles of carbon atoms, or the number of carbons in the compound multiplied by the number of moles of the compound) or molar flux(basedonthenumberofmolesofeachcompound) of glycolysis is normally a few times higher thanthatof theTCAcycle.PPP fluxusuallyconstitutes only about up to 5% of glucose intake. Glycolysis and the TCA cycle also supply precursors to build cellular components. Culture media do not necessarily supply cells with the right balance of all of the components that they need to synthesize cell mass. The three main pathways for energy metabolism also serve as key distribution centers for carbon skeletons needed for other cellular functions. Glycolysis supplies glycerol phosphate, which is for the synthesis of phospholipids.

Glucose-6-phosphate and fructose-6-phosphate are both a source of nucleotide sugars for glycan synthesis, such as UDP-galactose, UDP-glucose, and GTP-mannose. Except for liver cells (hepatocytes), cells in culture have little gluconeogenesis activity; that is, they cannot make hexose from lactate or amino acids. So, even if cells can derive energy from lactate and amino acids, they will still need hexose to synthesize ribose and glycans.

Cells in culture take up a large quantity of amino acids, especially glutamine. The intake of amino acids exceeds what is needed to make cell mass and product. The surplus of nitrogen is either excreted as ammonia or transferred to pyruvate to form alanine, and excreted. Since alanine is much less growth inhibitory than ammonium, pyruvate has some moderating effect on ammonium toxicity.


Glucose 6 Phosphate

NAD

NADH

PyruvateLactateNAD

NAD

NAD

Pyruvate

cytosol

mitochondrionreducing equivalent

oxidized

malate aspartate shuttle

NADH- Pyruvate Balance

NADH NAD

Election transfer chain

Equal moles of Pyruvate and NADH reducing equiavlent enters mitochondria

• While glucose is oxidized in Glycolysis and TCA cycle, its carbons never react directly with O2

• The energy is preserved to NAPH/FADH2, which is then reacted with O2 in oxidative phosphorylation in mitochondria to generate ATP

• Altogether 12 reducing equivalents (NADH/FADH2) are generated to react with 6O2, generating 6H2O

• 2 of the 12 reducing equivalents, two are generated in cytosol (in glycolysis) 10 in mitochondria

A total of 12 moles of reducing equivalent (10 NADH and 2 FADH2) are produced when 1 mole of glucose is oxidized to CO2. The 12 mole reducing equivalents consume 6 moles of O2 in oxidative phosphorylation, consistent with the stoichiometry of glucose oxidation (1 glucose/6 O2). Among the 12 NADH/FADH2, 10 are produced in the mitochondria and the other 2 NADH are produced in cytosolic glycolysis. The two reducing equivalents produced in the cytosol must then be transported into the mitochondria where they drive the reaction that consumes the 6th molecule of O2.

NADH does not pass through the inner membrane of mitochondria. Rather, it passes its reducing potential through a carrier system called the malate-aspartate shuttle. This system takes the reducing equivalent into mitochondria through an exchange of molecules between the mitochondria and the cytosol. On the cytosolic side, NADH is oxidized to NAD and transfers its reducing equivalent to malate by reducing oxaloacetate (OAA). Malate is then transported across the mitochondrial membrane. Once inside the mitochondria, the reducing equivalent in malate is transferred back to NADH by being oxidized to again become OAA.

The net result of the transfer must be that only one NADH in the cytosol can become one NADH in the mitochondria. To maintain that balance, cells employ two transporters: one for transporting malate and the other for transporting aspartate (hence the name “malate-aspartate shuttle”). To maintain the charge balance in the transport process, each of the two transporters transfers a pair of compounds in opposite directions (malate and α-ketoglutarate;aspartate and glutamate). In this fashion, there is no net change in total carbon or nitrogen on either side of the mitochondrial membrane.

On each side of the membrane, the same glutamate-OAA to α-ketoglutarate-aspartate amino-transferreaction occurs, but it is in the opposite direction. The transfer of the reducing equivalent of NADH into

NADH Balance

Fig. 3.5: NADH/NAD balance in cytoplasm. NADH generated in glycolysis is recycled back to NAD via lactate production and malate-aspartate shuttle to transfer the reducing equivalent into mitochondria.


Glucose Glyceraldehyde3-Phosphate

1,2-Bisphosphoglycerate

Pyruvate

Lactate

NAD+ NADH

NAD+QAA

MalateAspartate Glu αKG

Malate-AspartateShuttle

GluAspartate αKG MalateMitochondria

OAA

NADH

NAD+

Oxidative Phosphorylation

ATP

Pyruvate

• Each enzyme reaction/co-transportation is given in the same color• Direction of reaction in NHDH reducing equivalent transport is indicated by an arrow• Net transport of one reducing equivalent-1 NADH in cytosol, and +1 NADH in mitochondria• Transport of 1 malate + 1 glutamate into mitochondria and 1 αKG + asparate in cytosol

Fig. 3.6: Detailed reactions of malate-aspartate shuttle.

Fig. 3.7: Transfer of NADH reducing equivalent from cytoplasm into mitochondria requires continuous transport of four malate, aspartate, glutamate and α-ketoglutarate.

the mitochondria is therefore not only dependent on the NADH concentration but also linked to amino acid metabolism and the activities of the TCA cycle, through the concentrations of associated carriers.

Glycolysis and the TCA cycle are thus connected by a cytosolicbalanceofpyruvateandNADH.Thefluxofboth pyruvate and NADH are stochiometrically linked in glycolysis, as well as through the LDH reaction. As aresult, thefluxesofpyruvateandNADHenteringthe mitochondria are also stoichiometrically related. Regardless of whether cells are at a lactate-producing or -consuming state, the molar ratio of pyruvate flux toNADH flux is always one.


Fig. 3.8: Biochemical reactions in glycolysis, TCA cycle and Pentose phosphate pathway

Energetic Yield of Oxidation of GlucoseCytosol

Mitochondrion

• The overall energetic yield is ~30ATP, considering the cost of ATP transport out of mitochondria

• The NADH generated in cytosol (Glycolysis) is recycled back to NAD+, either through reduction of pyruvate to lactate, or by NADH/NAD+ shuttle into mitochondria for oxidation.

2 2 2 2 2pyruvate acetylCOA NADH ATP CO

acetylCoA NADH FADH GTP CO2 6 2 2 4

2

2 2

+ + +

+ + +

cosglu e pyruvate NADH ATP2 2 2+ +


Glucose Transporter Isoform

• GLUT1 is highly expressed in all cells• Km is small for GLUT1. At culture glucose concentration

it operates at maximum rate .

Fig. 3.9: Michaelis-Menten kinetics plot for GLUT1 transporter

Role of Transport and Transporters in MetabolismEnergy metabolism takes places in multiple compartments, separated by lipid bilayer membranes. First, glucose must cross the cytoplasmic membrane to undergo glycolysis in the cytosol. The products from glycolysis (pyruvate and NADH, in the form of reducing equivalents) are transported into the mitochondria. This exchange of molecules across the membrane is mediated by membrane transporters.

Glucose Transporters Glucose transporters mediate the influxof glucose across the cytosolic membrane. Generally speaking, there are two types of glucose transporters: GLUT and SGLT.

The GLUT transporters are uniporters for facilitated transport, allowing glucose to move along its concentration gradient. They have twelve transmembrane regions and intracellular carboxyl and amino termini. According to common sequence motifs, they are divided into three subclasses.

GLUT1 is ubiquitous, found in almost all cells. It can transport glucose and galactose in a concentration-dependent manner that is described by Michalis-Menten kinetics. The Km for glucose is very low (1 – 2 mM). At the glucose concentration used in culture medium, the flux ofGLUT1 is at itsmaximum. Insome cells, GLUT1 is under the regulation of the transcription factor HIF-1 (hypoxia inducible factor). Under hypoxic conditions, the expression of GLUT1 is up regulated to increase the uptake rate of glucose.

The Km of GLUT1 for galactose is rather high. When galactose is used as the only sugar, even at a concentration of 25 mM, the uptake rate is so low that only a little lactate is produced.

A few other notable GLUT transporters are: insulin responsive GLUT4 and fructose transporting GLUT5. In addition to GLUT1, cells in culture and in different tissues may express other GLUT transporters at different proportions. The expression of different transporters will give them different responses to the concentration of glucose or other sugars.

Two Main Types of Glucose Transporters• GLUT transporters mediate facilitative diffusion across

plasma membrane .

• SGLT, the sodium dependent glucose co-transporters are expressed primarily in small intestinal absorptive cells or renal proximal tubular cells . They use Na+-K+ ATPase pump for active transport of glucose .


Lactate Transport Lactate and pyruvate are transported by monocarboxylate transporters. These transporters exist in two forms: one on the cytoplasmic membrane and one on the mitochondrial inner membrane. Lactate and pyruvate are both negatively charged. Their movement across the cellular membrane will cause a charge unbalance and create an electric potential, unless measures are taken to counter that imbalance. The monocarboxylate transporters (MCT), which are responsible for their transport, are a family of co-transporters that couple the transport of lactate or pyruvate to the transport of a hydrogen ion in the same direction to neutralize the charge transfer. MCT is thus a symporter; its mechanism of transport is facilitated diffusion. Lactate transport is enhanced by a large difference in lactate concentration between intracellular and extracellular environments. pH also affect the flux of lactate throughMCT, however,whether the

Fig. 3.10: Monocarboxylate transporter for lactate and pyruvate

Table 1: Glucose TransportersTissue

Expression Affinity

Class 1

GLUT1 ubiquitous glucose, K m = 1 - 2 mM

GLUT2 Liver, pancreas, intestine, kidney

glucose, K m = 16 - 20 mMglucosamine K m = 0.8 mM

GLUT3 brain, neurons glucose, K m = 0.8 mM

GLUT4 heart, muscle, adipose glucose, K m = 5 mM

Class II

GLUT5 intestine, testis fructose K m = 10 - 13 mM

GLUT7 intestine, testis glucose, Km = 0.3 mMfructose K m = 0.1 mM

GLUT9 kidney, liver fructose K m = ? mM

GLUT11 heart, muscle fructose K m = ? mM

Class III

GLUT6 brain, spleen, leukocytes glucose, K m = 5 mM

GLUT8 testis, brain, liver glucose, K m = 6 mMGLUT10 liver, pancreas glucose, K m = 0.3 mM

GLUT12 heart, muscle, prostate not well known

The second type of glucose transporter, SGLT, is a co-transporter with Na+. It transports two sodium ions and one glucose molecule into the cell. The Na+ concentration is low intracellularly but is high inthemediumandinbodyfluid.Thelargesodiumconcentration difference and negative electric potential across the cytoplasmic membrane gives rise to a high propensity of Na+ to enter the cell. Thus, the chemical potential energy of the sodium gradient and electric potential is used to drive the uptake of glucose against a concentration gradient.

SGLT transport is abundant in intestinal epithelial cells and is responsible for moving glucose from the gut into the intestinal epithelial cells. The glucose is then exported into the blood stream on the other side of the cellular barrier.


effect is enhancing or retarding is dependent on the direction of the proton gradient. MCT allows for lactate transport in both directions, for excretion as well as uptake. Keeping medium pH at a lower level reduces lactate production during rapid growth period, but enhanced lactate consumption in the stationary phase.

Cells in culture typically channel about 1/20 of the carbons from their glucose intake to the TCA cycle and oxidize them to CO2.Themolarfluxofpyruvateinto the mitochondria is thus about 1/10 of that of the glucose consumption rate. Each mole of pyruvate entering the TCA cycle via acetyl CoA generates about 15 moles of ATP. These are exported to the cytosol and require the import of equal moles of ADP and PO4

3- for their synthesis. Each mole of pyruvate generated from glycolysis or lactate consumption is also accompanied by one mole of NADH, whose reducing equivalent is transferred into the mitochondria through the malate-aspartate shuttle. Additionally, cells in culture consume glutamine at a high rate (approximately 1/5 to 1/10 of glucose in molar ratio). Nearly half of the glutamine enters the TCA via α-ketoglutarate. CO2 produced in the TCA cycle is then exported out of the mitochondria.

Besides these major species, many other molecules (including amino acids and nucleotides) are transported into the mitochondria for DNA, RNA, and protein synthesis. As will be described later, the precursor for fatty acid and cholesterol synthesis, acetyl CoA, is generated in the mitochondria, while fatty acid and cholesterol synthesis occurs outside the mitochondria. Acetyl CoA is very reactive and does not get transported directly across the inner membrane of the mitochondria. Rather, it is transported out of the mitochondria as citrate. After cleaving off acetyl CoA, the remaining four carbons are returned to the mitochondria as pyruvate or malate. Thus, the citrate and malate flux across the mitochondrial membrane is alsosubstantial to sustain lipid and cholesterol synthesis.

The transport across the mitochondrial inner

Transport Across Mitochondria

• The chemical potential energy generated by pyruvate oxidation/TCA cycle, i.e. NADH and FADH2, is not necessarily all converted to ATP . The process can be decoupled to generate heat instead of ATP, as occurs in hibernating mammals.

• The mitochondrion is also the main site of molecular interconversion and degradation of amino acids and the main source of acetyl CoA . The excess glutamine consumed enters the TCA cycle through α-ketoglutarate. For some cells, asparagine acts as a sink (in addition to alanine), which is formed through oxaloacetate and aspartic acid.


membrane is dynamic and complex. Many compounds crossing the membrane are charged, yet their transport must not perturb the proton and electric potential gradient. The transport across the mitochondrial membrane must be tightly regulated. Our understanding of its regulation is still rather limited.

Underphysiologicalconditions,thefluxofglycolysisand the TCA cycle is not controlled by one or a small numberof “rate-limiting” enzymes.Glucose flux isthe result of mutual constraints of many enzymes in the pathway, through their feed-forward and feedback inhibition and activation. A large number of pathways are highly inter-connected and crosstalk with each other through shared common substrates or regulators. In mammals, different tissues serve different metabolic roles to maintain the overall homeostatic state of the organism. The partition of metabolic roles is largely accomplished by giving different tissues a different set of isozymes. Different isozyme sets allow cells to respond to environmental fluctuations or cellular cues differently.

Regulation of Glucose Metabolism


• Cells express different isozymes in different tissues, under different conditions

• different isozymes have different kinetics and regulation

Fig. 3.11: Allosteric regulations in glycolysis. Note strong activation of glycolysis flux by F16BP and inhibition by lactate.

Different isoforms (isozymes) of glycolytic enzymes catalyze the same reaction step, but have very different kinetic properties and are often subjected to contrasting regulations. Isozymes of four glycolysis enzymes, hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and phosphofructokinase/fructose biphosphatase (PFKFB), play key roles in controlling glycolysis flux. Their different allosteric regulations givethem very distinct reaction characteristics.

The isoforms of those enzymes making up glycolysis are different in proliferating and quiescent cells. They are thought to be responsible for endowing thehighglycolysisfluxandhighlactateproductionin proliferating and cancer cells and are thought to be related to oncogenic transformation. They also give tissues their metabolic capabilities.

PFK is pivotal in modulating the overall rate of glycolysis and is a key node in energy metabolism. Its activity is subjected to allosteric inhibition by ATP and citrate, and is activated by AMP. PFK has three isozymes: liver (PFKL), muscle (PFKM), and platelet (PFKP). Among the three isoforms of PFK, the muscle and the liver isozymes are activated allosterically by fructose 1,6-bisphosphate (F16BP). Muscle phosphofructokinase is inhibited by lactate, a characteristic which may facilitate reduced glycolysis flux at high lactate levels.

Fructose-2,6-phosphate is a shunted glycolytic intermediate that plays a key regulatory role in rapidly modulating the activity of PFK. All three PFK isozymes are activated by fructose 2,6-bisphosphate (F26BP). F26BP activates PFK1 by allosterically increasing its affinityforF6P,eveninpresenceofinhibitorssuchas ATP, lactate, etc. The synthesis and degradation of F26P is catalyzed by a bi-functional enzyme, PFKFB (also known as PFK2); its kinase activity catalyzes the synthesis of F26P and its phosphatase activity catalyzes the hydrolysis of F26P to F6P.

PFKFB has four isozymes, each with different kinase and phosphatase activities, allowing each to

Isozymes and Differential Allosteric Regulation


• F2,6P plays a key regulatory role• Key regulatory enzyme of glycolysis

• PFK (PFK1)• PFK2• PKM

• Cancer cells (fast growing cells) and quiescent cells have different isozymes

respond differently to regulators. The brain isoform, PFKFB3, has the highest kinase to phosphatase activity and is expressed in several tumor cells. This suggests that PFKFB3 can be accountable for the glycolytic phenotype of reported cancerous cell lines by allowing them to have high cellular F26P levels.

The enzyme catalyzing the penultimate step of glycolysis, pyruvate kinase, has three isozymes in mammalian systems. The muscle isozyme is expressed as either of the two splice variants, M1 or M2. The M1 isoform is mostly expressed in adult tissues whereas the M2 isoform is expressed exclusively in rapidly growing tissues, such as fetal and tumor tissues, and also is thought to be a critical player in the transformation leading to cancer. In addition, the M2 isoform is known to be under positive feed forward regulation by F16P.

Isozymes are often named after the tissue in which they are the dominant isoform. However, it is important to remember that the expression of isozymes is not limited to one form in a cell type. Different isoforms are often expressed in the same tissue or the same cell. Different combinations of isozymes give rise to different kinetics and regulatory behaviors that may meet different physiological needs.

With the available genomic tools, we can easily determine the relative expression of different isoforms of the key enzymes of glycolysis, and further evaluate how to influence cellular metabolism.


Glucose

Fructose-6-Phosphate

Pyruvate

Fructose-1,6-Bisphosphate


PFK1PFK2

Glucose-6-Phosphate

NADPHPPPTIGAR

p53

ROS

H2O

PGMp53

NADH

ATP

COXp53

Glucose

GLUT1,4

SCO2

Transcriptional InhibitionTranscriptional ActivationAllosteric Activation

Mitochondria

Lactate

Regulation of Metabolism

Akt and Myc Regulation of Metabolism

Glucose

Fructose-6-Phosphate

Pyruvate



PFK1

PFK2

Glucose-6-Phosphate

Akt

Glucose

GLUT1Activation by phosphorylationor localization

Transcriptional ActivationAllosteric Activation

Mitochondria

HK

GlutamateMalate Glutamine

Lactate

Glutamine (Extraacell

ular)TCA

ASCT2 SN2GLS

LDH

Myc

Fig. 3.12: Tumor suppressor p53 negatively regulates glycolysis flux.

Fig. 3.13: Signaling kinase, AKT and transcription factor, Myc, positively regulate energy metabolism.

Signaling Pathways and Regulation by Growth ControlSignaling pathway/growth rate control of Glycolysis• Insulin signaling

• positively regulating growth rate

• through AKT regulate glucose, amino acid metabolism

• P53 (tumor suppressor) supresses glucose uptake and glycolysis

• cMyc (proto-oncogene) stimulates glycolysis

• Fast growing (tumor) cells have fast glycolysis (and lactate production)

The regulation of cellular metabolism is tightly linked to the control of cell growth. The signaling pathways that regulate growth rate (involving key players like p53 and cMyc) also play regulatory roles in regulating glucose metabolism. Transformation that causes cells to switch from a quiescent state to a proliferating state also triggers metabolic changes toincreasetheirglucoseuptakeandglycolysisflux.

p53 is a major tumor suppressor that plays key roles in cell cycle arrest, senescence, and apoptosis. Its role in the regulation of glucose metabolism and oxidative phosphorylation was not well understood until only recently. It induces the overexpression of TIGAR under mild oxidative stress conditions. TIGAR contains a fructose-2,6-bisphosphatase catalytic activity domain, which mediates the degradation of fructose 2,6-phosphate, leading to a decrease in PFK1 activity and the attenuation of the glycolytic flux.

p53 can also modulate glycolytic rate by regulating the activity of PGM, GLUT1, and GLUT4 transporters. Furthermore it up-regulates mitochondrial oxidative phosphorylation by upregulating the expression of SCO2 (synthesis of cytochrome c oxidase 2), which mediates the assembly and activity of the cytochrome c oxidase complex.

Pro-oncogenic genes may also provide the link between increased glycolytic flux and oncogenictransformation. Akt is a pro-oncogene which, when deregulated, affects many cellular functions including metabolism, cell proliferation, and protein synthesis. In tumor cells, constitutively activated AKTissufficient toshift thecells fromaprimarilyoxidative state to a primarily glycolytic state.

Myc is another proto-oncogene whose pleiotropic regulatory roles include energy metabolism. Glycolytic enzymes have Myc canonical E-boxes in their promoter and are deregulated when Myc is overexpressed.


Metabolic Homeostasis and Lactate ConsumptionCells in culture, including those used in bioprocessing, have all been selected for their capability to proliferate. Their particular set of glycolytic isozymes directs theirmetabolism toahighglucose fluxandahighrate of lactate production. However, even the exact composition of isozymes is not monolithic among different cells. Most cells express multiple isoforms of the same enzyme in glycolysis and their proportion is not identical. Consequently, even though the metabolism of all cultured cells share the common traitofahighglucosefluxandhighlactateproduction,their metabolic behaviors are not all identical.

Lactate accumulation in culture inhibits cell growth and hastens the decline of cell viability in the stationary phase. A key to achieving a high cell concentration and high productivity is to direct cell metabolism to minimize the accumulation of lactate. One may aim to alter the cells’ metabolism to reduce or eliminate lactate production. However, it is important to keep in mind that virtually all rapidly proliferating cells produce lactate. Such a metabolic state might be a default state that is “essential” for cell growth and cannot be easily altered over a long period without affecting growth behavior. It is also important to keep in mind that through in the course of culturing cells for manufacturing, the duration of the actual production period is only a very small fraction of the total process lifetime. The long duration of the process time is mostly for expanding cell numbers to reach a production scale. Altering the metabolism from the cell’s “default” state may have an unforeseen effect on cells.

Another approach for alleviating lactate inhibition is to tamper with the cell metabolism only in the productionscaleorinthefinalstagesofproduction.Increasing evidences show that the switch from lactate production during the growth stage to lactate consumption in late stages is correlated with a high productivity. One may seek to alter cell metabolism to change lactate consumption in the last stage to be more reproducible.

• Metabolism is a balancing act of many constraining reaction nodes

• Glycolysis flux influenced by:

• Regulation (feed back and growth)

• Pyruvate/NADH flux into mitochondria

• LDH (NAD recycle rate)

• Lactate export

• Glucose intake


Fig. 3.14: Metabolic fluxes in lactate production and consump-tion metabolism.

The LDH reaction that catalyzes pyruvate to lactate is reversible. The direction of the flux dependson the concentrations of NAD, NADH, pyruvate, and lactate. Those concentrations, in turn, are related to fluxes of reactions that produce orconsume them, including pyruvate entry into mitochondria and lactate export (or import).

Further observations related to lactate consumption:

Lactate consumption occurs in post-rapid stages of growth, when the glucose consumption rate is low. The pyruvate flux into themitochondria is ratherlimited.Whencellsareatahighglucoseflux,theexcesspyruvate must be converted into lactate to regenerate NAD+.Therefore,ahighglucosefluxstateisalwayslinked to a lactate production state. Furthermore, rapid proliferation is generally associated with high glucose flux. Lactate consumption occursonly when the growth rate is slow. Experimental observation has demonstrated that the absence of rapid growth and a low glucose consumption are necessary conditions for lactate consumption.

Glucose is still being consumed while cells consume lactate. While lactate is being taken up by cells, its specific consumption rate isrelatively small. Lactate is converted to pyruvate, and then it enters the mitochondria for further oxidation and energy generation. Even though energy is being derived through lactate oxidation, cells still need many constituents derived from glucose for glycan synthesis, including NADPH (from PPP), glucosamine, and galactose. These compounds cannot be derived from pyruvate in most culture cells, since they lack key enzymes for gluconeogenesis. They must be supplied through glucose. The glucose consumption rate in the lactate consumption stage is small, but it is never zero.

The conversion of lactate to pyruvate by LDH does not occur in isolation. It is coupled to the reduction of NAD to NADH (also catalyzed by LDH), and linked to the reverse transport of lactate and H+ by MCT from the medium to the cytosol. The propensity and rate of lactate consumption is affected by the pH of the media. In addition to transporting pyruvate

Cycle

Cycle


formed from lactate into mitochondria, NADH has to be transported from the malate-aspartate shuttle into the mitochondria for oxidation.

• Supply TCA cycle intermediates

• causes NH3 release

• used in nucleotide and protein synthesis

For most cultured cells, glutamine is the second highest consumed nutrient. Its molar consumption rate is about 1/5 to 1/10 of that of glucose for most cells. Glutamine is a major amino acid constituent of cellular proteins. It supplies amino groups for the synthesis of purine and pyrimidine bases, which are the backbone of nucleic acids. However, the amount of glutamine consumed by cells far exceeds what is needed for synthesizing cellular components.

Glutamine is not an essential amino acid for mammals; it is only essential for cultured cells. Cells in some tissues, like the liver, synthesize glutamine by the incorporation of an ammonium into glutamate at the expense of an ATP using glutamine synthase. The transcript level of glutamine synthase in cultured cells is low. It actually decreases by nearly two orders of magnitude when liver cells are put into culture.

A large portion of glutamine is converted to glutamate byglutaminase,andthentoα-ketoglutaratebeforeentering the TCA cycle. Through α-ketoglutarate,glutamine is a major contributor to central metabolic flux. There is increasing evidence that glutamineis needed to drive the TCA cycle for faster growth.

In the course of being converted to glutamate and α-ketoglutarate, the nitrogen is releasedas ammonium or transferred to an amino acid, such as alanine or asparagine, and excreted. The ammonium that is released from glutamine contributes to the waste metabolite accumulation.

Glutamine and Its Relation to Energy Metabolism

Glutamine• Consumed by growing cells at a high rate

• Non-essential in vivo. Glutamine synthesase decreases upon in vitro culture

Fig. 3.15: Entry of glutamine into TCA cycle. Glutamine is converted to glutamate by glutaminase. Glutamate is then convert to α-ketoglutarate by either transamination reaction (transfer its α-amino group to pyruvate or oxaloacetate) or via oxidation by glutamate dehydrogenase (converting NADP to NADPH and releasing ammonium).


Amino Acid Degradation • Amino acids are consumed at a rate of three to five

times what is needed for making biomass and product.

• Excess amino acid consumed must be excreted .

• The nitrogen (amino group) is removed from the carbon skeleton by transamination, oxidative or non-oxidative deamination. The excess nitrogen is excreted as ammonium ion or as excreted amino acids (e .g . alanine, proline, asparagine)

• The carbon skeleton mostly enters the TCA cycle to be converted to excreted non-essential amino acids or to be converted to pyruvate and lactate, or to contribute to acetyl CoA in cytosol through citrate .

• Large excess of glutamine consumed by cells in culture is converted to glutamate in the cytosol, or is transported into the mitochondria and then converted to glutamate . Glutamate is then converted to α-ketoglutarate and enters carbon metabolism .

• Glutamine (amide group) is used as an amino group donor in adenosine (AMP), guanosine (GMP) and Cytosine (CTP) biosynthesis .

• Aspartic acid and glycine are also used in nucleic acid synthesis

• Methionine is methyl group donor . Tryptophan is used in NAD synthesis

• Glutamate participates in a large number of reactions. The flux of its synthesis or supply is expected to be high.

Amino Acid MetabolismMammals can synthesize only fewer than 10 of the 20 amino acids used in the translational synthesis of proteins. 11 or 12 (depending on the species) essential amino acids that they cannot synthesize must be acquired through diet.

Cells in culture have more essential amino acids requirements than the organism; a number of non-essential amino acids become essential for in vitro culture, including glutamine, tyrosine, serine under some growth conditions, proline for some cells. All essential amino acids must be supplied in medium, while non-essential amino acids can be derived from other amino acids.

Amino acids are taken up by cells through a large number of amino acid transporters. Most amino acid transporters transfer a family of amino acids with similar characteristics, such large neutral (uncharged side chain) amino acids, cationic or anionic amino acids. The rate of transfer for particular amino acids is thus not only dependent on the concentration difference of itself between intracellular environment and extracellular medium, but also on the competition with other amino acids for the same transporter. One amino acid may be taken up via more than one transporter, albeitwith different affinity.

The amino acids taken up by cells are not likely to be in the “right” stoichiometric ratios to meet their need. Excess amount of amino acids is converted to the non-essential amino acids which are not supplied in sufficient quantities,or is degraded. The interconversion and degradation of amino acids takes place through the intermediates of glycolysis and TCA cycle.

Before the carbon skeleton of an amino acid enters carbon metabolism pathways its amino group or other nitrogen containing functional groups is first removed. Glutamine and glutamate becomeα-ketoglutarate, asparagine and aspartate becomeoxaloacetate; all are catabolized through the TCA cycle. Alanine becomes pyruvate, while leucine, isoleucine and others enter carbon catabolic pathway through

• Amino acids are transported with overlapping transporters .

• All IgG antibodies produced by mammalian cells are glycoproteins, with an N-linked oligosaccharide attached to each heavy chain in the hinge region at Asn-297

Fig. 3.16: Major amino acid transporters


acetyl-CoA. Although the degradation of amino acids yields energy, it is not a major energy source.

The excess nitrogen from amino acid degradation is excreted as ammonia or as the amino group of non-essential amino acid, especially as alanine or proline. In some cell culture processes ammonium accumulates to growth inhibitory levels. Although the adverse effect of ammonium on cell growth is usually not as severe as lactate accumulation.

α-Ketoglutarate

Succinyl-CoA

CO2

CO2

Isocitrate

Citrate

Oxaloacetate

Fumarate

Acetyl-CoA

Pyruvate

CO2Acetoacetate

Citric acid cycle

AlanineCysteineGlycineSerineThreonineTryptophan

AsparagineAspartate

AspartatePhenylalanineTyrosine

IsoleucineMethionineValine

ArginineGlutamateGlutamineHistidineProline

IsoleucineLeucineLysineThreonine

IsoleucineLeucineLysineThreonine

Fig. 3.17: Entry of amino acids into catabolism


Lipid MetabolismLipids play key roles in many physiological functions critical to cellular properties of particular interest to bioprocessing. In addition to forming the bilayer membrane, lipids are also involved in cell signaling. Lipid content in bilayer membrane affects membrane fluidity and permeability. Lipid bilayermembranepartitions various organelles from the cytosol. Secretedrecombinantproteinsareprocessedfirstinendoplasmic reticulum (ER) and the Golgi apparatus. They are excreted via membrane vesicles. For optimal protein secretion capacity, the membrane homeostasis and biogenesis among organelles, secretory vesicles and plasma membrane is critical.

Not all lipid bilayer membranes are the same. The lipid composition of ER, mitochondrial and plasma membrane differ from each other. The plasma membrane of hepatocytes is enriched in cholesterol; however, the amount of cholesterol in the rough and smooth inner ER is lower. There is very little cholesterol in the inner mitochondrial membrane.

Functions of Lipids• Contributes to the membrane fluidity

• Storage of precursors metabolized to second messengers (diacylglycerol, inositol triphosphate)

• Lipids (such as, polyphosphoinositide) involved in protein traffic and membrane fusion events

• Anionic lipids (such as, PS) involved in attachment of cytoskeletal proteins to membranes

• Cholesterol and sphingolipids form microdomains or ‘rafts’: enriched in specific subsets of membrane proteins

Subcellular Localization of Lipid Metabolism in Animal Cells Cytosol

• NADHP synthesis (pentose phosphate pathway, malic enzyme)

• Isoprenoid and early cholesterol synthesis

• Fatty acid synthesis

Mitochondria:• Fatty acid oxidation

• Acetyl-CoA synthesis

• Ketone body synthesis

• Fatty acid elongation

Endoplasmic Reticulum:• Phospholipid synthesis

• Cholesterol synthesis (late stage)

• Fatty acid elongation

• Fatty acid desaturation

Peroxisome:• Cholesterol precursors on synthesis

• Final steps of cholesterol synthesis also occurs in peroxisome


Although some cells can be cultured in lipid-free medium, most cell culture media contain some fatty acids and lipids. Cells readily take up fatty acids, phospholipids, and cholesterol from the medium and incorporate them into cellular lipids.

Lipids can be supplied as serum lipoproteins in the form of a complex with albumin or liposomes, or as solubilized conjugates, such as sorbitol-fatty acid esters. The cellular uptake of fatty acids is a passive, non-energy dependent process. After being taken up by cells, fatty acids quickly become esters; the intracellular levels of free fatty acids are quite low.

Cholesterol is complexed to low density lipoprotein (LDL) in the body and is taken up by cells through the LDL receptor. For cells in culture, cholesterol is often supplied as a conjugate with serum albumin, or as complexes with cyclodextrin.

Lipid TransportLipid Transport ProcessesIntramembrane Transport: Transbilayer movement of lipid molecule

• Transbilayer movement at eukaryotic ER: relatively non-specific, ATP-independent process

• Three classes of transport:

• Aminophospholipid translocases (also called flippases)

• Bidirectional transporter or scramblases

• ATP Binding Cassette (ABC) pumpsIntermembrane Transport: Movement of lipid molecules from one distinct membrane domain to another

Possible mechanisms:• Monomer solubility and diffusion (for molecules such as

free fatty acids)

• Soluble carriers such as lipid transfer proteins

• Carrier vesicles

• Membrane apposition and transfer

• Membrane fusion


Fatty Acid Metabolism Most cells have the capability of synthesizing various fatty acids. Under starvation conditions, cells also perform β-oxidation to degrade fattyacids into acetyl CoA in the mitochondria or peroxisomes. Acetyl CoA then enters the TCA cycle to generate energy. Typically, cells in culture do not need to derive energy from fatty acid oxidation.

Fatty acids are synthesized from acetyl CoA in the cytosol. The first step of fatty acid synthesisinvolves adding a CO2 to acetyl CoA to form malonyl CoA, which then reacts with acetyl CoA to become a four-carbon fatty acyl CoA. It is noted that even though CO2 is a catabolic product, it is also an essential nutrient for biosynthesis. Fatty acid synthesis, thus, involves the step-wise elongation processes of using three-carbon malonyl CoA to add a two-carbon unit to fatty acyl-CoA in each cycle. NADPH is also consumed in this process. There are a number of fatty acid synthetases that can synthesize fatty acids to different lengths.

The fatty acid products from elongation reactions are all saturated fatty acids. Double bonds are then synthesized by unsaturation reactions after saturated fatty acids are made.


Acetyl CoA is the building block of fatty acids. It is generated primarily through the oxidative decarboxylation of pyruvate in the mitochondria. However, fatty acid synthesis takes place in the cytosol. Acetyl CoA does not pass through the bilayer membrane. Rather, it is exported to the cytosol via an indirect process. Citrate, formed by condensation of OAA and acetyl CoA in the TCA cycle, is then transported into then cytosol, where it is split into oxaloacetate and acetyl CoA. Oxaloacetate gets reduced to malate at the expense of one NADH. Malate is then converted to pyruvate, losing one carbon and consuming one NADPH. Pyruvate then recycles into the mitochondria. The process of making fatty acids using acetyl CoA is thus energetically expensive.

Acetyl CoA shuttle

Acetyl CoA shuttleOn mitochondria inner membrane• Citrate transporter: transport citrate into cytosol

• Malate, α-ketoglutarate transportor: transport into mitochondria

• Pyruvate transporter: transport into mitochondria

In Cytosol

• Citrate lyase: citrate +CoA+ATPOAA+acetylcoA

• Malate dehydrogenase: OAA+NADH+Malate +NADH+

• Malic enzyme: malate+NADP Pyruvate +NADPH+CO2

In mitochondria matrix• Pyruvate carboxylase: Pyruvate+CO2+ATPOAA+ADP

• Malate dehydrogenase: malate+NAD+OAA+NADH+

• Citrate synthase: OAA+acetylCoACitrate +CoA

Cholesterol & Its Biosynthesis Cholesterol is a 27-carbon molecule. Mammals require cholesterol as a constituent of membranes and as a precursor for the synthesis of steroid hormones, bile acids, and lipoproteins. Cholesterol is relatively insoluble and resides exclusively in various cell membranes. Its regulation is particularly important since excess cholesterol forms solid crystals, leading to cell death.

Proper cell function requires that cellular membranes have the appropriate composition,

Citrate

αKG

SucFum

Malate

OAA

Glu

NHg

Glu Gln

NH3

Citrateacetyl CoA

CO2Pyr

Pyr

CoA

acetyl CoAADP+Pi

ATP CoA

OAA

Fatty acid cholesterol synthesis

NADHNAD

MalateNADPH

CO2

NADP

Fig. 3.18: Citrate and acetyl CoA shuttle.

Fig. 3.19: The structure of cholesterol


including cholesterol, in order to maintain bilayer fluidity, impermeability and other characteristicsspecific to different organelles. Cholesterolconstitutes ~10% of the dry weight of plasma membranes, and plasma membrane cholesterol accounts for 65% to 80% of total cellular cholesterol. Cells in culture obtain cholesterol either by de novo synthesis or through receptor-mediated uptake of exogenous low-density lipoproteins.

Cholesterol is synthesized from acetyl CoA, which is condensed by 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) to form HMG-CoA. HMG-CoA is converted to mevalonate by HMG-CoA reductase (HMGCR). This enzyme is the target of statins, the class of drugs that suppress cholesterol synthesis in patients.

Further synthesis of mevalonate to farnesyl-diphosphate takes place in peroxisomes. Subsequent condensation of two molecules of farnesyl diphosphate to form squalene, lanosterol, lathosteroland finallycholesteroloccur in theER.

Out of the 18 key enzymes taking part in cholesterol biosynthesis, 5 enzymes reside in the peroxisome and 13 reside in the ER. HMGCS is upstream of HMGCR and is found in the cytosol. Thus, there are at least three different sub-cellular compartments involved in cholesterol biosynthesis.

Although cholesterol in mammals is synthesized primarily in the liver, most cells have the capability of synthesizing cholesterol for their own growth requirements. NS0 cells lack an enzyme, 17-β-hydroxysteroid dehydrogenase,which converts lanosterol to lathosterol. In NS0 cells, 17-β-hydroxysteroid dehydrogenaseis silenced through methylation of a CpG island upstream of its promoter, leading to the cell line’s dependency on cholesterol for growth.

• Cholesterol metabolism is a tightly regulated pathway subjected to stoppage of cholesterol biosynthesis in the presence of excess cholesterol .

• Animal cells obtain cholesterol from both de novo biosynthesis and receptor mediated uptake .

Cholesterol• Component for membrane biogenesis and precursors

for the synthesis of steroid hormones, bile acids and lipoproteins .

• Cholesterol resides exclusively in cell membranes and its regulation is particularly important since excess cho-lesterol forms solid crystals leading to cell death .

• Cholesterol is ~10% of dry weight of plasma mem-branes . Precise lipid composition of the plasma mem-brane has been controversial since it is difficult to iso-late high yield of pure samples from tissues or cultured cells .

• Cells obtain cholesterol by de novo synthesis or through receptor-mediated uptake of plasma lipoproteins .


Cholesterol Biosynthtic Pathway• Cholesterol is synthesized from acetyl CoA, which is condensed by 3-hydroxy- 3-methylglutaryl (HMG)-CoA

synthase (HMGCS) to form HMG-CoA .

• HMGCS exists in a mitochondrial form in hepatic tissue and is present in cytosol as 53kD protein for other tissues.

• HMG-CoA is converted to mevalonate by HMG-CoA reductase (HMGCR)

• HMGCR exists as a 97-kDa glycoprotein in the endoplasmic reticulum (ER) and is exemplified as the rate determining the enzyme of the biosynthesis pathway .

• Mevalonate is further metabolized to farnesyl-diphosphate by a series of peroxisomal enzymes as shown in the figure.

• First committed step to cholesterol byosynthesis is typified by condensation of two molecules of farnesyl diphosphate to form squanlene by farnesyl diphosphate farnesyl transferase-1 (Fdft1), or (squalene synthase) a 47-kDa protein residing in the ER

• Squalene is converted to the first terol, lanosterol by the action of squalene epoxidase.

• Conversion of lanosterol to lathosterol involves a series of oxidations, reductions and demethylations. It is a 17

Fig. 3.20: Cholesterol biosynthesis takes place in mitochondrion, peroxisome and endoplasmic reticulum.


A vast majority of recombinant therapeutic proteins are glycoproteins. The extent of glycosylation and the structure of the glycans on those glycoproteins may have a profound effect on their activities and circulatory half-life. Glycans are classified asO-linked or N-linked glycans. O-glycans attach to the polypeptide through the -OH group of serine or threonine. N-glycans link to protein through the amide group of asparagine. For N-linked glycan, the asparagine is in an Asn-X-Thr/Ser recognition sequence, where X indicates no specificity.

N- and O-glycans attached to proteins are structurally heterogeneous. The glycans attaching to the same attachment site of different glycoprotein molecules are often structurally different. Such heterogeneity is called microheterogeneity.

Multiple glycosylation sites are often present on a protein molecule. Not all glycan attachment sites on a protein molecule may be occupied. Different protein molecules may have different combinations of occupied and free sites; such difference in the occupancy on different attachment sites is called macroheterogeneity.

Glycan Biosynthesis and Protein Glycosylation

N-linked Glycosylationattachment of oligosaccharide to the protein through the amine group of an asparagine

O-linked glycosylationattachment of oligosaccharide to the protein through the hydroxyl group of a serine or threonine

All IgG antibodies produced by mammalian cells are glycoproteins, with an N-linked oligosaccharide attached to each heavy chain in the hinge region at Asn-297

Importance of Glycan

Effect of Glycan • Facilitate protein folding in the ER• Increase solubility• Affect biological activities

• Fucose for ADCC activity• Affect half-life in circulation and pharmacokinetics

Heterogeneity In Glycoforms• Macroheterogeneity: when multiple sites of

glycosylation are present in a protein, the occupancy on different sites differs on different molecules

• Microheterogeneity: the structure of the glycan occupying the same site differs among different molecules

Glycosylation starts while the protein is still being translated and undergoing protein folding in the ER. The presence of glycans affects the solubility, aggregation, and the stability of the folding protein.

The glycan structure affects the half-life of blood circulation and immunogenicity of a therapeutic protein. The presence of carbohydrates delays clearance from blood, as demonstrated by a comparison of glycosylated protein and its non-glycosylated counter-part. Higher sialic acid content increases the circulation half-life of erythropoietin (EPO). Under-sialylated glycoproteins are thought to be cleared by a higher liver uptake via the hepatic asialoglycoprotein binding protein. It was also postulated that the glycosylated recombinant proteins are better trapped by the extra-cellular matrix, thus having a longer bioavailability in vivo than their unglycosylated variant.


Glycan on glycoprotein may also affect its biological activities. Many therapeutic antibody IgG molecules facilitate killing of target cells through antibody dependent cellular cytotoxicity (ADCC). ADCC activities of those antibodies have been reported to be affected by its glycan structure. Molecules without a fucose on itsmannose core have a fiftyfold higher ACDD activity than those with a fucose.

Protein Folding and Glycosylation O-glycosylation initiates in either the ER or the Golgi. Overall, our understanding of O-glycosylation is far less than N-glycosylation. N-glycosylation is initiated by the transfer of a preassembled oligosaccharide (Glc3Man9GlcNAc2, an oligosaccharide of three glucose, nine mannose, and two N-acetylglucosamine) to an asparagine in the recognition sequence of a nascent protein in the ER lumen. The addition of glycan occurs during the process of protein synthesis, prior to the completion of protein folding.

The first part of N-glycan synthesis involvesthe assembly of a high mannose backbone on a membrane anchored dolicol molecule, on the outside surface of the ER. The glycan is linked to the dolicol carrier through a pyrophosphate group in an activated form. After the seven-sugar backbone is formed (with five mannose and two N-acetylglucosamine),itflipsovertotheinterioroftheER.No transporters are needed for the transport of backbone glycan; rather, a flippase catalyzes theirtranslocation into the ER lumen. Inside the ER, the backbone acquires an additional four mannose and three glucose to become a mature core.

The mature core is then transferred to a binding site (Asn-X-Thr/Ser) on a nascent protein molecule. However, not all glycan binding sites may be occupied. This could be due to competition between protein folding and core glycan transfer. Hence, different permutations of site occupancies in different protein molecules exist, giving rise to a macroheterogeneity of glycosylation patterns. After being synthesized, chaperones surround protein molecules to facilitate their folding process. The three glucose molecules on the glycan core serve as a quality control signal for the proper folding of these glycoprotein molecules. Upon

Fig. 3.21: Glycosylation of secretory proteins

Fig. 3.22: N-glycan processing of glycoproteins in endoplasmic reticulum


completion of folding, the three glucose molecules are removed from glycan, signaling the protein’s readiness to be transitioned to the Golgi apparatus. Proteins that are not folded properly, with glucose still in their glycan, may be recycled through the calnexin pathway for refolding. Some unfolded proteins are sent to the proteosome for degradation.

The well-folded glycoprotein molecules are enclosed in membrane vesicles of ER and then bud from the ER and translocate to the Golgi apparatus. Once there, they fuse with the Golgi body membrane and the glycoprotein cargos are released into the lumen of the Golgi apparatus.

Glycan Extension in Golgi Apparatus Inside the Golgi, mannose is trimmed from the N-glycan core, effectively reducing the number of mannoses from nine to three, before extension takes place. Three main carbohydrate molecules constitute most of the extended glycan: N-acetyl glucosamine, galactose, and sialic acid. Different glycosyltransferases add a different monosaccharaides to the growing core glycan on the protein. The incoming monosaccharide provides the activated carbonyl group and the receiving carbohydrate moiety on the growing glycan on the protein may have more than one hydroxyl group available for extension. The glycosyltransferases recognize different incoming monosaccharaides and catalyze different glycosidic bond formations. However, a number of glycosyltransferases do allow for some flexibility in glycosidic bond formation.

Intermediate glycans on the protein have more than one sugar that can be extended and each sugar may have more than one hydroxyl group that is available for extension. Importantly, the extension reaction does not take place on all of the available reaction sites.

Each growing glycan, thus, has multiple available reaction paths for extension. The glycans can grow into different numbers of branches, creating structures such as biantennary and triantennary glycans. In some cases, the reactions of adding those sugars to different branches of the glycan may occur in different sequential orders, but lead to the same product. In other cases, the

Fig. 3.23: N-glycan extension in Golgi apparatus


addition of a particular glycosidic linkage may hinder the reaction of the others; thus, the reaction, itself, leads to different glycan structures.

Consequently, a very large number of glycan structures can be formed in the N-glycosylation pathway; however, the number of glycosyltransferases for the extension reactions expressed in a cell or tissue is relatively small (only a dozen or so). However, diverse patterns of glycosylation are often seen throughout many glycoproteins.

The web of glycan extension reactions forms a complex network which, when drawn out graphically, indeed resembles a network of diverging and converging paths leading to a number of different fully-extended N-glycan structures.

Glycan Types and Microheterogeneity N-glycan structures are generally classified intothree principal categories: high mannose, complex, and hybrid. All of them share a common tri-mannosyl (Man3GlcNAc2) core structure. The high mannoseglycanshavefivetoninemannose(Man5-9GlcNAc2) sugars. Those with two GlcNAc’s attached to the tri-mannosyl core are called “complex”. As its name implies, the hybrid types are a combination of high mannose and complex glycans, and have at least three mannose sugars but only one GlcNAc on one nonreducing mannose. This diversity of glycan sugar composition on each glycosylation site is referred to as microheterogeneity.

N-glycan microheterogeneity arises through alternative reaction paths of extension in the Golgi apparatus, as described above. The Golgi apparatus consists of stacks of membranous compartments commonly grouped into cis, medial, trans, and trans-Golgi network (TGN) cisternae. These cisternae are not biochemically homogeneous. As the secretory glycoproteins traverse through these Golgi compartments, the glycan extension reactions are catalyzed by varying the composition of glycosylation enzymes in each compartment. Adding to this diversity, not all protein molecules spend an equal length of time in different Golgi compartments; some exit early while others linger. Some glycans

Fig. 3.24: Different types of N-glycans in glycoproteins


Glycosidic bond formation is mediated by nucleotide sugars. The nucleotide (NTP) reacts with an activated sugar (glucose-1-phosphate, or glalatose-1-phosphate, N-acetyl-glucosamine-phosphate, mannose-1-phosphate) to form an NDP-sugar. Uracil is used for glucose- and galactose-based sugars (e.g., UDP-glucose and UDP-galactose), guarnyl for mannose and fucose, and cytidyl for sialic acid.

Mannose, galactose, and fructose are synthesized in branches of the glycolysis pathway. All three sugars are activated at their first carbon. Therefore, theylink to glycans through the formation of (1→n)glycosidic bonds. For example, UDP-NAcGlc is added to a growing core by forming an N-acetylglucosamine β(1→n)mannosebond;therecanbeanumberofpossible positions on mannose (e.g., 2, 3, 4, or 6).

For sialic acid, the second carbon is activated; thus CMP-2-sialic acid will form a sialyl (2→n) bondwith galactose. The synthesis of all the precursor sugars occurs in the cytosol, including a nine-carbon sialic acid and N-acetyl neuraminic acid. Similarly, all nucleotide sugars are formed in this way, except for sialic acid. The activation of sialic acid to CMP-sialic acid occurs in the nucleus.

The backbone of N-linked glycan is synthesized on the cytosolic side of the ER membrane through the membrane-anchored dolicol. The nucleotide sugars used in the formation of the backbone, GDP-mannose, UDP-N-acetyl glucosamine, and GDP-glucose are synthesized in the cytosol and directly react with dolicol or with the growing glycan backbone.Theassembledbackboneisthen“flipped”into the ER and the subsequent reactions occur inside the ER. Transporters for these nucleotide sugars have been reported to be present in the ER.

The nucleotide sugars for the extension reactions in the Golgi apparatus are also transported through transporters. These include CMP-sialic acid, GDP-fucose, UDP-N-acetyl glucosamine,

Synthesis and Transport of Nucleotide Sugar Precursor

do not acquire a terminal sialic acid, while others have multiple sialic acid molecules on them.

Fig. 3.25: Transport of nucleotide-sugar precursors into organelles


UDP-galactose, and the activated sulfate donor (3’-phosphoadenosine, 5’-phosphosulfate). All of these transporters are antiporters, meaning that a stoichiometric exchange of nucleotide sugars with NMPs is responsible for the charge balance.

• Glycan synthesis use nucleotide sugar as monomer unit

• Nucleotide sugar highly charged, require specific transporter into organelles

• Sialic acid synthesis occurs in cytosol, activation to CMP-Sialic acid to places in the nucleus .

Fig. 3.26: Biosynthesis of precursors of glycans


Glycosylation• Enzymes are somewhat different among species

• Possible immunogeneity (e .g . high mannose glycan from most yeast

Glycan Diversity Among Species Unless intended for vaccination, the immunogenicity elicited by recombinant proteins is a concern. An antibody elicited by and against the protein therapeutic can result in neutralization of the therapeutic protein and may result in an unintended drop in efficacy,thus causing serious adverse clinical effects.

The potential immunogenicity of recombinant therapeutics may arise from the aglycosylated protein core or from the glycan associated with them. There are at least two mechanisms by which glycans on a protein may affect the immunogenicity of a human therapeutic: 1) by being a foreign glycan structure, or 2) by shielding a segment of the protein that is otherwise antibody inductive.

Different recombinant human therapeutic proteins that are produced in different organisms are differently glycosylated (such as those from CHO versus yeast) or aglycosylated (such as from CHO versus E. coli). Comparison of those proteins indicates that the “shielding” effect of minimizing immunogenicity is affected by the nature of the protein, as well as by the source of the protein. The concern about the immunogenicity of different glycoforms of the rDNA proteins produced in insect cells and in transgenic plants has hindered those technologies’ application for rDNA therapeutic protein production. Glycosylated proteins produced in CHO and mouse myeloma cells are minimally immunogenic.

The glycosylation pathway is highly conserved in mammals. Nevertheless, a divergence among different species does occur. Host cells derived from other species may possess a set of glycosylation enzymes that are different from humans. Human glycans have terminal N-acetylneuraminic acid (NANA), whereas other mammals have N-acetylglycolylneuraminic acid (NGNA). NGNA is the hydrolytic product of CMP-sialic acid hydroxylase, which is present in nearly all mammals but absent in humans. Thus, glycoproteins produced in CHO have some NGNA present among all sialylated glycans. Similarly, glycoproteins expressed in CHO


CHO Glycan• Has NGNA instead of human NANA

• Has only α(2,6) sialic acid, human has both α(2,3) and, α(2,6).

cells have only terminal α(2,3)-linked sialic acids,in contrast to α(2,6) and α(2,3) seen in humans,due to a varied composition of sialyltransferases. Such differences in glycan composition have posed a concern; however, the antigenicity of recombinant proteins directly caused by glycans is still scant.

Concluding RemarksIn this chapter, we presented a brief overview of broad areas of cellular metabolism. We explored the core of energy metabolism, the process of glucose utilization through glycolysis, PPP and the TCA cycle, and how all of these affect cell growth behavior productivity. Through interconnected pathways, the central corridor of energy metabolism also influencesthesynthesis,andeventheglycosylation,of the product proteins. The excessive consumption of glucose and glutamine and the corresponding accumulation of lactate and ammonium in culture all contribute to growth inhibition and low productivity. Lactate consumption in the late stage culture has been positively associated with a high productivity. There are, therefore, ample incentives

to better understand cell metabolism and to possibly finddifferentwaysofmanipulatingcellmetabolismto better redirect the process. In recent years, we have developed a better understanding of the link between glycolytic regulation and growth control. We have also established better tools to probe the relationship between metabolic flux distributionandotheraspectsofphysiologythatinfluencebothproductivity and product quality.With the benefitof global physiological perspectives, we continue to gain a deeper understanding of metabolism. Global viewsatasystemiclevelwillsignificantlyenhanceour capacity to manipulate cell metabolism, and thus increase productivity and product quality.

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Optimization of Cell Growth Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97A Guide for Medium Design—Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Types of Media and Classes of Medium Components . . . . . . . . . . . . . . . . . . . . . . . . . . .101Types of Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Basic Components of Cell Culture Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103Classes of Medium Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Components of Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106Sugars and Energy Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109Nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109Optimal Concentration of Organic Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110Fatty Acids and Lipid Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111Non-Nutritional Medium Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

High Molecular Weight and Complex Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . .117Medium for the Industrial Production Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125

Optimization of Cell Growth Environment

Medium Design for Cell Culture Processing

Medium, like the food we eat, exerts a fundamental influence on the well-being of cultured cells,profoundly affecting their growth, metabolic activities, and other biological capability. The question of how best to devise culture medium emerges whenever new in vitro cultivation-based science or technology is on the horizon, as occurred three decades ago driven by the revolution in recombinant mammalian cell based biotechnology, and as is occurring now concomitant with the emergence of stem cell science.

Cells are the heart of cell technology; however, without proper medium, cell cultivation cannot accomplish process goals. Most cell types share common basic nutritional requirements although their needs for growth factors and cytokines may

Industrial Cell Culture Process

1 . Cell expansion

2 . Production/differentiation

• Cell expansion stages last much longer than production

• Medium design for both stages

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differ.Inthenearlyfivedecadessincescientistsbeganto isolate and cultivate cells, the focus of medium design has been to optimize cell growth, maintain growth potential and sustain the differentiated properties in cultures of differentiated cells.

With the growing importance of biologics, the focus of medium design has been extended to enhancing production characteristics, such as productivity or product quality. However, even with the focus shifting to production, optimizing medium for cell growth remains important. In fact, the time that cells spend in the production stage in a manufacturing reactor is a comparatively small portion of their life span. A cell spends the majority of its lifespan in growth, yielding progeny togeneratea sufficiently largenumberof cells forproducing product. Providing cells with an optimal medium during the expansion stage is critical.

Recent advances in stem cell science have spurred renewed interest in elucidating the nutritional needs of cells in culture. Although the fundamental aspects of nutritional requirements of a stem cell are not different from other cell types, their requirements for growth factors, surface matrices, and other microenvironmental factors makes medium design for stem cells far more complex than that for any cell lines used in protein biologics production. Furthermore, stem cell applications require that the stem cell progeny be directed to differentiate to specific lineages, for which the growth factorrequirements pose an even greater challenge.

Regardless of traditional biologics-based cell technology or stem cell bioprocessing, the culture process will involve both cell expansion and product formation or differentiation. Medium optimization strategy for cell expansion and for production may be rather different. For expansion, the long term healthy state of a cell while proliferating must be safeguarded. In contrast, for production of biologics, the cells are approachingtheirfinalstageofutility,andafterallproducts have been released into the medium, cells and product molecules must be separated. Hence, even conditions that might ordinarily hamper

• Classical Medium Design - Optimize Cell Growth

• Optimization for production - squeeze cell’s last productivity out

• Resurgence of media research in stem cell culture

• opportunities in growth factors, antogonists, and signaling pathway manipulation

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growth or harm cells, such as reduced temperature or increased osmolality, are sometimes used.

For stem cell processing, or for other cell therapy, the distinction between cell expansion and produc-tion isnot as significant. Even though cells arenolonger being expanded during the differentiation stage or other final stage of preparation for clini-cal applications, cells must not to be subjected to deleterious conditions. Since the final product iscell mass itself, their survivability and functional capability after the cell culture process is critical.

In the following sections we will focus on nutritional needsforcellexpansionfirst,asthatisbestknown.We will then discuss how “optimal” medium for pro-duction compares with that required for growth.


To design an optimal medium for cell growth, it is instructive to examine the chemical environment of their natural niche. The ultimate objectives of medium design are cell expansion, differentiation, and production, not necessarily to reproduce their niche. Understanding their native chemical environment provides us with a starting point from which to devise an environment suited to process goals.

The vast majority of cells in the body are not in direct contact with blood, but are surrounded by interstitial fluid. The chemical composition of interstitialfluid, especially the protein and hormone content,varies with tissues. However, the general chemical composition of small molecular weight solutes in interstitialfluidandinintracellularfluidissimilar.

The low molecular weight solute composition of interstitial fluid bears a few importantcharacteristics. The total osmolarity is around 280-300 mM (or mOsm). A couple of percentage points of error notwithstanding, the osmolarity can be taken as the sum of molarity of all dissolved species in the fluid.Thelargestcontributortothefinalosmolarityis Na+, followed by Cl–. In addition to Na+, other inorganic species are present; notable are K+, Mg2+, and Ca2+. However, those positively charged ions are all present at low concentrations. Since the net charge in a solution must be neutral, the total molarity of positively and negatively charge ions must be equal. In general Cl– concentration is lower than Na+ because bicarbonate (HCO3

–) is also present at ~30 mM contributing to the negative charge in the solution.

For many ion species the interstitial concentration and intracellular concentrations are strikingly different. Both Na+ and Cl– are present outside the cell at a ten-fold higher concentration than inside the cell, as is Ca2+. In contrast, K+, Mg2+ and PO4

3– concentrations are much higher on the intracellular side.

Cells can tolerate deviations from “optimal” conditions for some period of time. The estimated range of non-lethal physiological concentrations of key compounds vary considerably. Keep in mind

Table 1 . Cellular Chemical Environment in vivoApproximate Concentrations in Cellular Environment

Interstitial (mM)

Intracellular (mM)

Na+ 140 14K+ 4 .0 140Ca 2+ 1 .2 0 .01

Mg2+ 0 .7 20CI-- 108 4HCO3

- 28 .3 10HPO4

3-, H2PO42- 2 11

SO43- 0 .5 1

Amino Acids 2 8Lactate 1 .2 1 .5Glucose 56Protein 02 4Total Chemical Species(mmole/L) 301 .8 302 .2Corrected osmolar activity (mM) 281 .3 281 .3

A Guide for Medium Design—Body Fluids

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that the non-lethal range for the human body can be rather different from that for cultured cells. For example, cells in culture can tolerate low (80 mM) or high (140 mM) sodium concentrations, or high osmolarity (400 mOsm), for a period of days, whereas these extremes would not be tolerated by most aquatic animals for more than a few hours.

Normal RangeApproximate Nonle-

thal LimitsOxygen 35 – 45 10 – 1,000 mm HgCarbon dioxide 35 – 45 5 – 80 mm Hg

Sodium ion 138 – 146 115 – 175 mmol / L

Potassium ion 3 .8 – 5 .0 1 .5 – 9 .0mmol / LCalcium ion 1 .0 – 1 .4 0 .5 – 2 .0mmol / LChloride ion 103 – 112 70 – 130mmol / LGlucose 75 – 95 20 – 1500 mg / dL

Body temperature

98 – 98 .8 (37 .0)

65 – 110 (18 .3 – 43 .3) F° (C°)

pH 7 .3 – 7 .5 6 .9 – 8 .0

Types of Media and Classes of Medium ComponentsA complete cell culture medium often has two major categories of components: basal medium and growth supplements. The basal medium is the nutrient mixture consisting of the small molecular weight components including sugar, amino acids, vitamins, various salts, etc. The basal medium does not merely provide a nutritional source for deriving energy and making new cell mass and product, it also provides balanced salt concentrations and osmolarity to allow for cell growth.

However, most cells will not grow if provided with basal medium alone, as basal medium does not contain growth factors or other factors necessary for “optimal” growth conditions. Growth supplements that may be added to basal medium include growth factors, phospholipids, soy hydrolysate, serum, etc. These supplements may promote cell growth by providing constituent componentsforspecificsignalingpathways,ormaysupply special nutritional needs (such as delivering cholesterol), and may direct cellular differentiation

Basal Medium

• Sugar

• Amino acids

• Fatty acids, lipid precursors

• Vitamins, nucleosides

• Bulk salts, trace elements

• pH buffer

Supplements

• Serum, hydrolysates

• Growth factors

• Carrier proteins

Table 2. Non-Lethal Range of Medium Constituents


or maintain cells at a particular differentiation state.

There are numerous formulations of basal media that are made into powder, packaged and sold for commercial or routine use. Sterile filtration is the standard means of sterilizingbasal medium, but heat sterilization (as is used for microbial media) is also possible.

Types of Medium Traditional cell culture medium contains up to 15% animal serum in addition to basal medium. Serum is a highly complex fluid in terms of itschemical composition. Such a medium, containing a largely undefined chemical composition, iscalled a complex medium. Many supplements commonly used in industrial processes, e.g., plant hydrolysates, soy phospholipids, also fall into this category. Their use renders the chemical composition of the medium undefined.

A chemically defined medium contains onlycomponents whose chemical composition is known and characterized, and has all of its chemical species specified. It does not containany mixture of components with unknown or undefined composition. For example, “lipids” or“phospholipids” are not well defined compounds,but are mixtures of a class of compounds and are not chemically specified. A chemicallydefined medium often contains growth factors,cytokines, and carrier proteins. Thus, a chemically defined medium is not necessarily protein-free.

Chemically Defined Media• Progress in this area will likely be accelerated by:

• an urgency to demonstrate control over all aspects of production and downstream processing for licensing by the FDA

• the availability of recombinantly produced (in E . coli) tissue culture supplements (i.e. insulin, EGF)

• genetic engineering of cells to produce their own growth factors

• development of small, synthetic peptides that can mimic the action of the larger, naturally occurring protein (i .e ., RDG sequence)

• acceptance of continuous bioreactor systems and the operation of these systems in a “maintenance” mode

Complex Medium vs. Chemically Defined Medium

Serum-Free and Animal Component-Free Medium

A large number of industrial production processes of rDNA proteins has eliminated serum from the medium in the past decade. The use of serum-free and animal-component-free medium has become the industrial manufacturing norm, with the intent to minimize animal virus or prion contamination. Recently, serum-free medium has been increasingly used even during the cell line development stage to eliminate exposure of cells to serum and animal components throughout the

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entire cell banking and manufacturing process.

Nonetheless, the use of serum-free medium is not yet universally practiced. For example, bovine serum is still widely used in manufacturing viral vaccines. As well, it is often not an easy task to eliminate animal serum from processes involving the cultivation of primary differentiated cells.

Serum-Free Media• Serum-free medium consists of nutritionally

complete basal medium supplemented with an empirically determined mixture of hormones, growth factors, attachment factors, attachment proteins and binding proteins .

• Many serum-free media contain a complex mixture of undefined components, such as soy-meal hydrolysate, peptone or beef hydrolysate, or other plant hydrolysate .

Protein-Free Medium By definition, protein-free medium contains noprotein. Most protein-free media are, as well, chemicallydefined.However,aprotein-freemediummaycontainundefinedlipidsorfattyacids,andthus,maynotbechemicallydefined.Cellsthatgrowwellinchemicallydefinedmediumarelikelytobehighlyadapted or transformed, which eliminates any dependence on mitogenic molecules or lipid sources.

Basic Components of Cell Culture Medium

Classes of Medium ComponentsStoichiometric vs. Habitation-Conducive Basal Medium Components

Medium serves two important roles: to provide a chemical environment in which cells can grow, and to supply the components cells need to generate energy and convert to cell mass and products. Some medium components are taken up by cells and “utilized” to make more cells and products; other components provide the chemical environment but are not appreciably taken up by cells. The majority of medium components, including glucose, amino acids, lipids, and vitamins, are consumed stoichiometrically. The more cells are made, the more of those components are consumed.

In a most precise sense, nearly all medium components are utilized to generate more biomass. Even water is taken up by cells; as the cell volume expands water must be taken up along with other components that constitute cellular materials. However, in practical terms, many medium

• Stoichiometric: glucose, amino acids, vitamins, nucleotides, lipids, fatty acids, some growth factor, some salts

• Habitation conducive: Bulk salts, such as sodium, chloride, and some proteins, albumin, some growth factors .


components are consumed in such small quantities that their consumption may not be measurable.

Stoichiometric medium components must be supplied in sufficient quantities to reach thecell, and must meet the product concentration target of the production. To reach a higher goal, more stoichiometric components must be supplemented. For the unconsumed components of the medium—components whose role is only to provide a conducive environment—the key issue is to maintain their concentration.

Examples of conducive, unconsumed medium components include chloride ion, sodium ion, and even phosphate ion. Under ordinary conditions, only a negligible amount of these components are taken up by cells. However, these normally unconsumed salts may be taken up in substantial quantities in fed-batch cultures where fortified feeding isused to add more glucose, amino acids, and other nutrients to grow cells to a higher concentration. In that case, these will also need to be replenished. Thus, unconsumed medium components may become stoichiometric components under high density cultivation conditions.

Example

• PO4-3 concentration in medium is typically 1 mM, while its cellular concentration is 11 mM. The

intracellular ATP/ADP concentration is about 2mM, which represents around 6mM of phosphate. Total DNA and RNA contain another 35 mM equivalent of phosphate . A culture with cells of an average volume of 2 x 10-12 L and at a cell concentration of 1010 cells / L take up about [(11+6+35) mmole / L x 2 x 10-12 L / cell x 1010 cell / J= ~ 1 mmole / L of PO4

-3 .• Cell growth will certainly cause PO4

-3 concentration in the medium to decrease drastically at such a cell concentration.

• For instance, phosphate and trace metals are consumed in very small amounts, although their depletion may not lead to stoppage of growth immediately, they must still be supplied in stoichiometric quantities.

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• Some high M .W . medium components are interalized and consumed

• Others are not internalized when exerting their functions and are degraded only slowly

Cell culture medium often contains macromolecules such as insulin, fibroblast growth factor (FGF),serum albumin, etc. These constituents serve a variety of purposes; some are carrier proteins carrying ligands into the cell, while others are growth factors that bind to receptors on the cell surface. Molecules that are taken up by cells may need to be replenished to maintain their concentration, whereas molecules that transmit signals by binding to cell surface receptors may not need to be replenished as frequently during cultivation.

Many macromolecules are internalized, and some are degraded (consumed), while others are recycled. For example, the ferric ion carrier, transferrin, binds to transferrin receptor and is internalized. After being internalized, transferrin is translocated to lysosome, and after releasing ferric ion in the low pH environment of the lysosome, transferrin is recycled to the extracellular medium. As long as ferric ions are available (i.e., replenished), transferrin can continue its role as ferric ion carrier.

Insulin, another commonly used growth factor, binds to the insulin receptor and triggers a signaling event that does not involve its own internalization. However, when present at a high concentration (a few microgram per ml), insulin is rapidly internalized by hepatocytes and degraded. Therefore, even though as a growth factor it is not consumed, its concentration does decrease.

An example of a consumable macromolecule is low-density lipoprotein (LDL). After an LDL particle binds to an LDL receptor on the plasma membrane, the receptor-ligand complex is internalized in a clathrin-coated pit that pinches off intracellularly to become a coated vesicle. The clathrin coat then depolymerizes, resulting in an uncoated (smooth surfaced) vesicle, often called an endosome. The endosome then fuses with an uncoupling vesicle that has an internal pH of about 5.0, which causes the LDL particles to dissociate from the LDL receptors.

Stoichiometric vs. Catalytic Macromolecular Components


Components of Basal MediumWater

Sugars and Energy Source Glucose and glutamine are the primary nutrients that supply a cell’s energy needs in culture. The physiological concentration of glucose in blood is 0.8 g / L. In culture, glucose is typically present from 1 g / L (5.5 mM) to 5 g / L (27.5 mM). In the production reactor, sometimes a high level of glucose, as much as 15 g / L (82.5 mM), is used. In this case, glucose is a large contributor to the osmolarity of the medium, and adjustment of the composition of the medium must be made (by reducing sodium and chloride concentration) to

Mammalian cells are exceedingly sensitive to the quality of water used for media preparation. City water, the usual source of water for medium preparation contains particulates, bacteria which are the source of endotoxin, trace organics, and various inorganic ions including harmful heavy metals. Typical water preparation processes include deionization through ionexchange,microfiltrationtoremoveparticulatesandbacteria,andfinallyreverseosmosistoreduceconductivity(orincreaseresistance)to>20MΩcm.

In some cell therapy applications, because the product(i.e.,cells)issubjectedtolittlepurificationprocess before administration to the patient, cell culture medium may be prepared using water for injection (WFI) to avoid the entry of any pyrogenic contaminants. WFI is prepared by low evaporation rate distillation, minimizing the chance of any water droplet in the evaporating stem from carrying over any solute or particle from the water.

• Types of Contaminants in City Water:

• Inorganics—heavy metals, iron, calcium, chlorine

• Organics—by-products of plant decay, detergents

• Bacteria—endotoxin or pyrogen

• Particles—colloids or particles

• A typical water preparation process involves filtration, Reverse Osmosis

• WFI used to be employed, now mostly pure water .

The LDL receptors are then recycled back to the plasma membrane. The vesicles containing the LDL particles fuse with lysosomes, in which the cholesterol esters are hydrolyzed to fatty acids and cholesterol. Cholesterol is incorporated into cell membranes.

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maintain osmolarity in a growth permissible range.

All cultured cells express the GLUT1 transporter at a significant level, and take up glucose readily.GLUT1 also transports galactose. Thus, galactose can be used as an alternative sugar to glucose. The galactose km for uptake is higher than for glucose. In the concentration range used for glucose, galactose is taken up by cells at a lower rate, resulting in lower lactic acid production in the culture.

Fructose is also transported by the GLUT5 transporter. The Km for fructose transport by GLUT5 is also high. Thus, similar to galactose, the uptake rate for fructose is lower than for glucose unless a high concentration of fructose is used. At a low uptake rate, the use of fructose also results in lower lactic acid production compared to glucose.

Glutamine is an essential amino acid for cells in culture. Most cells in tissues express glutamine synthase, and make glutamine from glutamic acid. In culture, they consume glutamine at roughly 1/5 to 1/10 of the consumption rate of glucose. Glutamine supplies the amino group for nucleotide biosynthesis. It also supplies the carbon backbone of TCA cycle intermediates by converting to α-ketoglutarate. Through carbon13 tracer experiments, it has been shown that glutamine contributes to lactate production during cellular energy metabolism in culture.

Glutamine spontaneously degrades in aqueous solution, and releases ammonium. Consequently, to avoid degradation, glutamine is typically added to culture medium immediately prior to use.

Six-Carbon Sugars • Glucose:1-5 g / L,

• Fructose, galactose, may also be used .

• Galactose and glucose can both be transported by the GLUT1 transporter, which is present in most cells .

• Fructose is transported by a different transporter (GLUT5); unless the transporter is expressed, the cell may not be able to use fructose efficiently.

• The alternative sugar is often taken up by cells at a slower rate, which may reduce lactate production and be good for cell maintenance .

• Pyruvate and ribose are sometimes supplied in small quantities, insufficient to supply cell's energy needs.

Glutamine Serves Two Roles: • source of amino acid for protein

• nucleoside synthesis, energy source .

• Glutamine is consumed in large quantities, approximately 1/5 to 1/10 of glucose in molar amount .

• Glutamine is spontaneously degraded in aqueous solution.


Amino Acids Aminoacidsareclassifiedasessentialornonessentialbased on nutritional studies using animals or tissue culture cells. Cells lack the biosynthetic pathways for making essential amino acids, and rely on exogenous supply to meet growth needs. Some amino acids are essential only for cells in culture, but not for animals.

In animals, different tissues may cross feed each other; amino acids synthesized in one tissue (e.g., liver) may be transported to cells in other tissues. Some enzymes involved in amino acid biosynthesis are expressed at lower levels in cultured cells in vitro than in cells from their tissue of origin. Glutamine is non-essential for animals; it is synthesized from glutamic acid through glutamine synthetase. The expression level of glutamine synthetase decreases when cells are cultured in vitro. There is also a CpG island in the promoter region of glutamine synthetase, which may be methylated to cause glutamine synthetase to be silenced in some cultured cells.

Upon cell isolation for in vitro culture, expression of some amino acid synthesis enzymes is suppressed, but expression of some amino acid transporters is elevated, which allows for a faster transfer rate to meet growth needs.

Cell culture media developed in the 1960s and 1970s contained at least the 14 essential amino acids. Those media were designed to be used with serum supplementation, which also supplies some amino acids. Media designed for serum-free culture include all amino acids.

In medium preparation, amino acid mixtures are often prepared as concentrated stock solutions organized into groups: neutral, acidic, basic, etc. Although the solubility characteristics of amino acids indicates that solubilizing them is feasible, the kinetics of their dissolution is slow. Some stock solution preparation methods rely on pH adjustment using acid or base to increase the rate of dissolution. Those stock solutions also introduce additional salts into the culture medium. Thus, when using such stock solutions, it is important to assess the osmolarity of culture.

Table 3. Essential and Non-Essential Amino Acids

Essential amino acids† Non-essential amino acids

L-arginine* L-alanineL-cysteine* L-asparagineL-histidine L-asperatic acidL-isoleucine L-glutamic acidL-leucine L-glycineL-lysine L-prolineL-methionine L-serineL-phenylalanineL-threonineL-tryptophanL-tyrosine*L-valine L-glutamine**Essential for cells in culture, not for animals

• Most culture media contain all twenty amino acids .

• proline is required by mutant CHO cells;

• serine is frequently required at clonal densities;

• asparagine is required by certain malignant cells;

• glycine sometimes needed in case of borderline folic acid deficiency or in the presence of folate analogues (methotrexate and aminopterin)

• Small peptides can serve the same function as amino acids--some of these are more stable (e .g ., glycine-glutamine) or are transported more readily than their free amino acids counterparts .

• Some non-essential amino acids are excreted into cul-ture medium; alanine is most commonly seen . Aspara-gine and proline may also accumulate in medium .

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Vitamins The biological activities of vitamins vary. Even though they are classified as a common class ofnutrient, their biological roles are diverse. They all share the common feature of being essential for the vitality of humans, and are needed only in minute quantities compared to glucose and amino acids. Some vitamins are cofactors involved in biochemical reactions, and are required by all cells. These include biotin, thiamine pyrophosphate (or its precursor), riboflavin and cobalamin. Some vitamins such asvitamin D, vitamin K, vitamin A, and vitamin C are required by only certain differentiated cell types.

• Ascorbic acid may be beneficial for cells that synthesize collagen .

• Vitamin A can have a pronounced effect on growth and differentiation of some cell types.

• Vitamin K is required for gamma-carboxylation and correct processing of vitamin K dependent proteins .

• Vitamin E functions as an antioxidant.

• Vitamin D regulates Ca+2 and is regarded by many as a hormone rather than a vitamin . Most toxic of all vita-mins when present in excess .

• Thiamine pyrophosphate catalyses the transfer of car-boxyl group, transketolase, transaldolase .

• Pyridoxal phosphate (pyridoxine vitamin B6) catalyses transamination.

• Biotin is a carrier of activated CO2, and is involved in pyruvate dehydrogenase, pyruvate carboxylase, and fatty acid synthesis.

• Cobalmin (B12) is involved in free radical reactions of intramolecular C-C bond rearrangement, methylation, and conversion of ribonucleotides to deoxyribosenu-cleotides.

Nucleosides Nucleosides are not included as essential components of basal media when serum is supplemented. Inclusion of small quantities of nucleosides is common in serum-free medium. The small quantities included reflect the low nucleoside content incell mass: nucleic acids (RNA and DNA together) constitute only about 5% of dry cell mass. A purine source (adenosine or hypoxanthine) together with thymidineisbeneficialwhenfolicacidisinlimitedsupply (e.g., in the case of methotrexate selection).

Table 4 . Nucleosides in Basal MediumRNA DNA

Adenosine Thymidine

Cytidine 2’deoxyadenosine

Guanosine 2’deoxycytidine

Uridine 2’deoxyguanosine

Fatty Acids and Lipid Precursors Lipidsconstituteasignificantportionofbiomassofmammalian cells. Lipid bilayer membrane forms vesicles which play key roles in protein secretion and virus replication. The composition of lipids in membrane affects its fluidity. However, the effectof lipid supply in medium is usually rather subtle.


Unlike the supply of essential amino acids or sugar, whose deficiency cause observable effects on cellgrowth almost immediately (with only a delay of one doubling time), the effect of supplementing or removing lipid is not as easy to assess.

Mammalian cells can synthesize almost all lipids required for their growth, including fatty acids, phospholipids and cholesterol except for auxotrophic mutants. Cells can make fatty acids of all different carbon length in membrane, including most unsaturated fatty acids, which constitutes almost half of the fatty acids in phospholipid. However, mammals do not introduce double bonds beyondC9 into fattyacids. Linoleate(18:2cis-∆9,∆12)andlinolenate(18:3cis-∆9,∆12∆15)arethusessential fatty acids. Hence complete medium for serum-free culture usually contains oleic acid, linoleic acid, sometimes also arachedonic acid. However, the removal of essential fatty acids from culture medium, does not cause immediate cessation of growth, subtle changes in membrane properties is often masked by its residual amount in the cell and visible effect may take a long time to emerge.

Ideally supplementing cells with phospholipids is a good practice. However, non-animal source of phospholipids with reproducible quality is not easily available. Often precursors of phospholipids (choline, ethanolamine, inositol) are supplied. Cholesterol is supplied to its auxotrophic mutants, such as NS0 cells.

Cholesterol and fatty acids have very low solubility. Fatty acids are conjugated in serum albumin when serum supplement is used. In albumin-free medium fatty acids may mixed in with other hydrophobic supplements. Cholesterol can be supplied as derivatized ester with a organic acid attached to its hydroxyl group to increase its solibility, or can be supplied as cyclodextrin conjugated complex.

Fatty Acids• Cells can synthesize fatty acid, but can’t introduce

double bond beyond C9 .• Some cell lines benefit from cis-unsaturated

fatty acids, such as oleic acid, linoleic acid and arachidonic acid (a precursor for prostaglandin formation)

Phospholipid Precursors• Choline• Ethanolamine

Inositol—precursor for phosphatidyl-inositol biosynthesis

• Cholesterol—required by some cell lines (e .g . NS-0 myeloma)

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Optimal Concentration of Organic Nutrients

Suboptimal

Gro

wth

Rat

e

Optimal

Inhibitory

Nutrient Concentration

Optimal range typically spans over 10 fold or

more

Starvation

Bulk Salts and Trace ElementsMineral elements are also essential components of cell mass. Phosphate constitutes part of nucleotides and nucleic acids; magnesium is present in high concentrations in the cell as it is conjugated to ATP, which is present in the mM range; calcium, which is

Fig. 4.1: Optimal range of nutrients for cell growth

Upon determining the essential and beneficialcomponents of medium, it is necessary to decide on the optimal concentration of those nutrients. The experimental determination of concentration range is complicated by cell growth and consumption that causes medium composition to change. Furthermore, metabolites accumulate as cell grow, making culture conditions change as time goes by.

This problem was resolved by using clonal growth as originally demonstrated with ells of human, chicken and other species.. Cells were seeded in small petri dishes at very low density of about 10-50 cells/cm2 as opposed to 104 cell/ cm2 At such a low cell concentration, the amount of nutrients consumed is negligible compared to the amount present in the medium. It was further assumed that nutrient utilization is completely independent;

thereby allowing the effect of one component to be tested when all the other components are present in their “optimal” concentrations determined by the point of experimentation. After plating cells are dispersed as single cells on the surface. A couple weeks later, the extent of growth from all the cells initially plated is estimated by the total surface area covered by cells after cells on the dish is stained with a dye. The optimal concentration is one which gives largest cell growth area.

The optimal concentration for almost all nutrients (amino acids and other small molecular weight molecules) span over a wide range of at least ten fold. One can thus conclude that for the purpose of cell cultivation, most cells have a wide range of optimal concentration for most organic basal medium components.


Table 6 . Trace Elements in MCDB 104 (serum-free medium for hu-man diploid cells) (mM)

CuSO4·5H2O 1 .0 x 10-6

FeSO4·7H2O 5 .0 x 10-3

MnSO4·5H2O 1 .0 x 10-6

(NH4)6Mo7O24·4H2O 1 .0 x 10-6

NiCl2·6H2O 5 .0 x 10-7

SeO2 3 .0 x 10-5

Na2SiO3·9H2O 5 .0 x 10-4

SnCl2·2H2O 5 .0 x 10-7

NH4VO3 5 .0 x 10-6

ZnSO4·7H2O 5 .0 x 10-4

Role of Bulk Ion• Maintenance of membrane potential (Na+, K+)

• Osmotic balance (Na+, Cl-, HCO3-etc .) contribute most

of the osmolality of fresh medium. Optimal range is 280 - 310 mOsm / kg .

• Biological roles:

Mg+ conjugate with ATP

Ca2+, Mg2+ for cell adhesion

PO43- for nucleotides

• Signaling (Ca2+ )

• Buffering (HCO-3, HPO3

-4)

Table 5. Concentrations of bulk ions in basal medium (mM)

DMEM/F12 (1:1)

William’s E

DMEM RPMI F12

Na+ 150 .31 143 .71 155 .12 137 .74 144 .03

K+ 4 .18 5 .37 5 .37 5 .37 3 .00

Mg2+ 0 .71 0 .81 0 .81 0 .41 0 .60

Ca 2+ 1 .05 1 .80 1 .80 0 .42 0 .30

Cl- 126 .66 125 .33 118 .48 108 .03 134 .83

PO43- 1 .02 1 .17 0 .78 5 .63 1 .17

HCO3- 29 .02 26 .19 44 .04 23 .81 14 .00

SO42- 0 .41 0 .81 0 .81 0 .41 0 .00

NO3- 0 .85 0

Total 313 302 327 282 288

essential for signaling in some differentiated cells, is present in high concentrations in endoplasmic reticulum (ER) and in some organelles. Calcium and magnesium are also involved in cell-cell and cell-substrate adhesion. It must be noted that sodium and potassium, through their reverse abundance in cytosol and medium, balance membrane potential, which is fundamental to life.

Many trace metals play key biological roles. Ferrous ion plays a key role in electron transfer complexes, as does copper ion. Zinc is present at high concentration in pancreas and is conjugated with insulin. Selenate serves as an antioxidant. These elements are required by cells in minute quantities; however, their long term deprivation is detrimental to cells.

There is a wide range of concentrations of bulk ions that is conducive to cell growth. What is apparently most important for growth is the balance of osmolarity. The most important contributors to osmolarity are sodium ions, chloride ions, and bicarbonate; while the concentrations of these can be varied, the total osmolarity must be maintained in the range of 270-330 mOsm.

When bicarbonate is not used in culture medium its contribution to osmolarity must be replaced by other salts. It is common practice to add a mixture of NaCl and KCl to maintain their molar ratio at about 30.

There is a wide range of conducible concentration for bulk ions. What is apparently most important is the balance of osmolarity during growth stage. The most important contributors to osmolarity is sodium and chloride ions, the concentration of both can be varied, so is bicarbonate. However, the total osmolarity must be maintain in the range of 270-330 mOsm.

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Non-Nutritional Medium Components

Some components of medium are additives that make it operationally easier to grow cells. They can be removed from medium without harmful effect, and do not appear to be taken up by cells. However, their presence in the medium, especially under process conditions, can minimize operational deviations.

Sodium Bicarbonate Buffer Sodium bicarbonate provides a pH buffer in our body fluid, andwas used to buffer cell culturemediumin the early days of in vitro culture development. However, the pKa of bicarbonate is 6.1 making it less than ideal as a buffer for neutral pH. The buffering capacity of bicarbonate derives from the equilibrium with soluble carbon dioxide as shown in the inset. The buffering action of bicarbonate requires the presence of CO2. With a given bicarbonate concentration, the pH is inversely proportional to the CO2 level; thus, as gas phase CO2 goes up, pH goes down.

Typical cell culture medium contains 14-44 mM NaHCO3, which at equilibrium requires 10% CO2 to maintain pH at 7.4. As pH decreases due to lactate production, the CO2 level in the gas phase can be reduced, which takes the proton to the left hand side of the equilibrium equation, to maintain pH. Conversely, as cells begin to consume lactate in the late stage of a fed-batch culture, the CO2 concentration in the gas phase is increased to maintain pH.

Trace Elements• Those clearly required by cultured cells are: iron, man-ganese, zinc, molybdenum, selenium, vanadium, copper .

• Trace elements are ubiquitous contaminants of chemi-cals and supplements used in preparation of medium.

• Some media contain rare trace elements such as rubid-ium, cobalt, zirconium, germanium, molybdenum, nickel, tin and chromium; may be needed for long-term growth in protein-free medium .

• What are the roles of heavy metal ions? The tables provide a reference of optimal starting

When bicarbonate is not used in culture medium its contribution to osmolarity must be replaced by other salts. It is common practice to add a mixture of NaCl and KCl to keep their molar ratio at about 30.


Bicarbonate Concentration in medium44mM in DMEM, 14 mM in F12, 26 mM in circulating blood.

• It is necessary to use 5 – 10% CO2 in the incubation chambers; media that contain bicarbonate become alkaline very rapidly due to loss of CO2 when removed from the incubator .

• The low pKa of bicarbonate (6.1) results in suboptimal buffering throughout the physiological pH range.

• NaHCO3 buffer requires appropriate CO2 concentrations in the gas phase. The reactions are:

CO2 dissolves in aqueous solutions.

The CO2 concentration in liquid is described by Henry’s Law.

H: Henry’s law constant CO2 in an aqueous solution forms a bicarbonate ion .

The equilibrium is described as:

From the definition of pH and pKa

The pH of the solution is affected by PCO2 and HCO3- .

From the equation, one can plot the relationship among HCO3-, PCO2 and pH .

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Under some conditions, bicarbonate buffer is insufficient for maintaining pH—for instance,when a CO2 incubator is not available. There are a number of buffers known to sustain cell growth. Some of these buffers have a pKa in the vicinity of 7.0, and are more suitable for sustaining neutral pH than bicarbonate. Unfortunately, most of them have a lower maximum concentration that can be used without a negative effect on cell growth, which limits the buffering capacity they can provide.

It should be remembered that CO2 is a metabolite, a medium component for buffer, and is also a nutrient required for some biochemical reactions, such as carboxylation reactions in fatty acid synthesis. Normally, the role of CO2 as a nutrient is not apparent because, as cells respire, the CO2 produced is sufficient to supplyCO2 needed for carrying out carboxylation reactions in the cell. However, when cultivating cells in a bicarbonate-free medium at a low cell concentration, CO2 may become limiting for cell growth. If continuous air is continuous flownover the culture and thus stripping of CO2 occurs, CO2 produced by cells may be stripped away and maynotbesufficientforsupportinggrowth.Underconditions such as these, using an air supply with a small supplement of CO2 (0.2-0.5%)will suffice.

• Sodium beta-glycero-phosphate (20 mM) also functions as a detoxifier of ferric chloride hydroxo compounds (i .e ., Fe3+ chelator)

• Zwitterionic buffers: HEPES (N-2-hydroxyethylpiperazine-N-2-ethane) is used between 10 – 50mM .

• Alternative buffers can be growth inhibitory at high concentrations (>25 mM)

• Requires balance of osmolality (by adjusting bulk salt levels, e .g . if 2 mM is used and 40 mM of NaHCO3 is eliminated, the osmolality must be balanced with NaCl / KCl . Need to maintain Na+ / K+ ratio (~30:1).

Table 7. Cell Culture Tested Biological Buffers

Description pKa valueat 37° C

AnhydrousMol. Wt.

Working Concentration

(mM)

Glycine 1 .0M 9 .53 75 .0 50 - 200

Glycylglycine 7 .95 132 .1 10 - 20

HEPES 7 .31 238 .3 10 - 28

MOPS 7 .01 209 .3 10 - 20

Sodium Bicarbonate 6 .28 84 .0 2 - 26

TRICINE 7 .80 179 .2 <50

Alternative Buffer

How do bicarbonate and CO2 work together as a pH buffer?

The equilibrium equation, with pKa = 6.36 can be used to plot the relationship between pH and bicarbonate concentration. The relationship is also dependent on CO2(g). Two lines are shown for two different CO2(g)

concentrations. Each point on the line represents the corresponding pH and carbonate concentration. To buffer the medium at a given pH one has to select a combination of bicarbonate and CO2(g) concentrations.

Figure 4.2: Relationship between sodium bicarbonate concentration, atmospheric carbon dioxide and pH


Reactive oxygen species (ROS) arising from biochemical reactions cause transient presence of superoxide radical (O2

-) and hydrogen peroxide (H2O2). Their reactions with lipids, proteins, and DNA cause damage to cells and to media components. Physiologically, cells deal with ROS through reducing reactions that use cellular reducing “agents” such as glutathione. Conventionally, the issue is dealt with through medium design either by providing antioxidant compounds to cells to facilitate their capacity to cope with ROS, or by supplying components to minimize oxidation of labile medium components in culture fluid.

Except for mercaptoethanol, few other antioxidants are routinely used in cell culture. However, for the cultivation of differentiated cells or for the cultivation of cells under stress conditions, some additional antioxidants are included to prolong the period before irreparable damage to cells occurs.

Physiologically Relevant Antioxidants• Vitamin E• Taurine• Beta-carotene• Transferrin• Ceruloplasmin • Selenium - Selenium-deficient cells are more

sensitive to oxygen toxicity (selenium is a cofactor for glutathione peroxidase, an enzyme which helps remove peroxidases from cells) .

• Catalase • Superoxide Dismutase • Reduced glutathione• ß-mercaptoethanol

Antioxidants

Some medium components are added with the intention of modulating the physiochemical environment of the cell. In addition to osmolarity, viscosity was speculated to be important for sustaining cell growth in culture in the early days of serum free culture medium development.

Most process cell culture media contain a protective agent, Pluronic F68, which was firstemployed in the 1960s in growing BHK cells in a stirred tank bioreactor. Many Pluronic surfactants are available; all are block copolymers of polyoxyethylene and polyoxypropylene. The larger the POE (polyoxyethylene) group, the more hydrophilic the molecule is and the greater its detergent-like activity and cell cushioning effects. The larger the POP (polyoxypropylene) group, the greater the toxicity and the anti-foaming ability.

Presently, Pluronic F68, within a concentration range of 0.01–0.1%, provides adequate cell cushioning, but the degree of foaming is high. Therefore, it is desirable to determine if a suitable

Table 8. Synthetic Protective Agents Used in Cell Culture

Common name Chemical identity

Pluronic F68 or F88

Block copolymer glycols of poly(oxyethylene) and poly(oxypropylene) (M .W . ~8350)

PEGPoly(oxyethylene) glycol (or polyeth-ylene glycol) (M .W . ~20,000)

PVA Polyvinyl alcohol (M .W . ~20,000)

MCMethylcelluloses (15 cps methocel) 0 .1- 0 .2%

CMC, Edifas B50 Sodium carboxymethylcellulose

HES Hydroxyethyl starchPVP Polyvinylpyrrolidone

Dextran Dextran (M .W . ~78,5000, 20-60 g / L)

Mechanical Damage Protective Agents

MEDIUM DESIGN | 117

replacement is available. The only Pluronics other than F68 known to provide suitable growth/productivity are F88 and F77. Both have somewhat different micelle formation characteristics than F68.

Polyethylene glycol or carboxymethyl cellulose have been used as protective agents, but are seldom used now.

Antibiotics The use of antibiotics in cell culture is unevenly practiced. For manufacturing processes, antibiotics are rarely used, although in vaccine production, the use of neomycin, polymyxin B, streptomycin, and gentamicin are sometimes seen. For the cultivation of primary cells, which are not subjected to a long duration of quality control prior to processing, the use of gentamycin reduces the rate of contamination significantly. Some antibioticsare only moderately more inhibitory to bacteria than to cultured cells. Toxicity testing is necessary.

Table 9. Antibiotics for Cell Culture

Antibiotic Concentration Antibiotic Spectrum

Chlortetracycline5 mg / L

Gram-positive and Gram-negative bacteria

Gentamicin sulfate

50 mg / L

Gram-positive, Gram-negative bacteria and mycoplasma

Nystatin 50 mg / L (or 100 U / mL) Fungi and yeasts

Penicillin G 100 U / mL Gram-positive bacteria

Spectinomycin20 mg / L

Gram-positive and Gram-negative bacteria

High Molecular Weight and Complex SupplementsSome calls such as NS0, some hybridoma/myeloma cells, and hepG2 cells are capable of rapid growth using only basal medium without any further supplement. However, such extraordinary capability is the exception rather than the norm of cells in culture. Most cells in culture require supplementation of at least a number of growth factors and carrier proteins. Many stem cells and other normal diploid cell lines need high concentrations of serum to grow. In fact, for isolation of cells from tissue, serum is still commonly used, at least in the early stage of cell cultivation. In discussing the role of various supplements it is instructive to start by examining the role of animal serum.


Serumisthebloodfluidleftbehindaftercoagulation;it is free of blood cells and most coagulation proteins. Serum is an extremely complex mixture that contains nutrient substances, metabolites, hormones, plasma proteins, substances released from damaged cells (e.g., hemoglobin and growth factors from platelets), antibody molecules against various antigens to which the animals have been exposed, and may even harbor infectious agents such as viruses carried by the animal.

Fetal bovine serum (FBS) is the most widely used serum in animal cell culture because it contains higher concentrations of growth stimulatory factors and lower concentrations of growth inhibitory factors. Other commonly used sera are human, bovine calf, newborn bovine, donor bovine, and donor horse.

Serum serves many different and important roles in cell culture. In addition to providing nutrients not sufficiently present in basal medium (e.g.,cholesterol), serum provides factors for cell-substrateattachment(e.g.,vitronectin,fibronectin)for adherent cells, modulates colloid osmolarity, a physiological property of medium with respect to viscosity. Serum contains protease inhibitors, and neutralizes trypsin used in cell detachment as well as other enzymes released by dead cells. Serum contains the carrier proteins transferrin and serum albumin. Carrier proteins function to chaperone components that are in very low concentration or are otherwise poorly soluble, such as fatty acids (carried by albumin), or are unstable such as ferric ion (carried by transferrin).

Serum is rich in “bulk” proteins (e.g., serum albumin) that canpreventnon-specific adsorptionof critical factors to culture vessels. Serum also plays an important role as a scavenger. In the course of cell cultivation, various contaminants may arise from numerous sources. For example, this could result from leaching of minute chemical components from reactor parts, from the filterused in medium preparation, or even from medium

Functions of serum in cell culture medium:• Protease inhibitors (alpha 2 macroglobin) neutralize

proteases used in trypsinization or produced by cells.

• Provides hormones and growth factors .

• Provides carrier proteins

• for low molecular weight substances (e .g .,transferrin)

• for nutrients which dissolve poorly (e.g., fatty acids, cholesterol, apolipoprotein)

• Binds compounds which are toxic when present in excessive amounts, and releases slowly

• Binds and/or neutralizes toxic substances (e .g . detergents) .

Serum or Biological Fluids

MEDIUM DESIGN | 119

supplements. Some of these chemicals may be detrimental to cell growth or may have other negative effects on cells. In the presence of serum in the medium, those compounds may be sequestered by adsorption to serum proteins before they can act on cells, thus minimizing potential damage.

Animal serum, however, has numerous disadvantages inaddition tocostand thedifficultyof controllingconsistent quality. The most serious concern is the possibility of contamination with animal viruses or prions. The presence of serum in culture medium also makes downstream processing more complicated, andmakesthetaskoffinalproductcharacterizationmore complex. Serum carries antibodies, some of which may be against viruses that the animal donors had been exposed to. For virus production processes, if serum antibodies cross-react with the product virus, the production will be drastically affected.

Disadvantages of serum in cell culture medium• potentially introduce animal viruses into cell culture

and other undesirable contaminants (e.g. adventitious agents, antibiotics, proteases).

• Availability of high quality serum

• high running costs and unnecessary capital outlay.

• Normally purchased in large lot sizes and costly storage .

• Serum lot testing tedious and costly.

• Increase complexity of downstream processing and final product characterization.


Insulin plays a key role in regulating glucose uptake by many cell types. In addition to modulating glucose metabolism, insulin also exhibits mitogenic effects, and stimulates cell growth through an overlapping pathway with IGF-1. IGF-1 has an acute effect on protein and carbohydrate anabolism by increasing cellular uptake of amino acids and glucose, and by stimulating glycogen and protein synthesis. IGF-1 also affects cell proliferation, differentiation, and apoptosis. It is a potent mitogen acting to increase DNA synthesis and to stimulate the expression of cyclin D in a wide variety of cells.

Both insulin and IGF-1 bind to the insulin receptor (IR) and IGF receptor (IGF1R) but with different affinities. After insulin binding, IR orIGF1R is phosphorylated, leading to activation of an insulin receptor substrate (IRS). There are multiple isoforms of IRS that are distributed differently in cells of different tissues. The signal is then relayed to downstream signaling pathways.

The response of the cell to insulin and IGF is dependent on the abundance level of the different IRS isoforms. Most cells, including CHO cells, express both IR and IGFR. However, NS0 cells express only IGFR. Differential binding to IR and IGF1R, as well as differential activation of various IRS isoforms leads to different responses to insulin and IGF1.

Insulin is used in cell culture at concentrations that are nearly a hundred-fold higher than found in blood. At such high concentrations insulin can trigger a mitogenic response. IGF has a much stronger affinity for IGF1R and stimulates cellgrowth at much lower concentrations than insulin.

• Insulin stimulates glucose uptake by adipocyte and other cells, also has a mitogenic effect at high concentrations

• Insulin is used in culture at 1 - 10 µg /ml range . Blood insulin level is 4µU/ml or 1 .3 µg / ml (1 µU=0.33 µg) ; IGF1 level is is 100 to 200 ng / mL.

• Insulin and IGF have overlapping signaling pathway through IR and 1GF1R

• IGF1 regulates cell growth, IGF2 invovles in development . IGF1 can replace insulin in cell culture at a much lower concentration

Insulin and Insulin-like growth factor (IGF-1)

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Transferrin is the iron carrier glycoprotein in mammals. Ferric ion is highly oxidative, and exists primarily bound to heme and other proteins with iron centers. In circulation, ferric ion is bound to transferrin. Transferrin has a very high binding constant for iron, but dissociates readily at low pH. Transferrin receptor-bound iron is taken up by cells and translocated to lysosomes, where iron is released for incorporation into cellular proteins.

For industrial processing, it is desirable that all growth factors supplemented to culture medium be derived from recombinant DNA expression rather than having been isolated from human or animal sources. Although recombinant insulin and other growth factors have long been available, recombinant transferrin became available only in recent years.

The specificity of transferrin binding to cross-species transferrin receptor is not universally high. The recombinant transferrin used commercially is primarily of human origin. The concentration required for cells of different species may differ and must be empirically determined.

Transferrin can be replaced by iron chelating agents, including citrate. Most chelating agents have a much lower binding constant for iron than does transferrin, and they are used in higher concentrations than are needed for transferrin.

• Typical concentration: 1 – 30 µg / mL (MW 80kd, 10 µg / mL=0.1 µM)

• 80 kDa glycoprotein with homologous N-terminal and C-terminal iron-binding domains . Binds to iron very strongly with a dissociation constant of approximately 1022M-1 .

• Low interspecific potency; for human and rodent cell lines can be replaced by other iron binding protein (i .e . hemoglobin),

• May be replaced by an iron-chelating agent, such as citrate .

Table 10 . Some Iron Chelators as Transferrin ReplacementsFerric citrate 0 .1 - 0 .5 mMFerric iminodiacetic acid 0 .001 µMFerric ammonium citrate andTropolone 2 - 10 µM

Transferrin

Industrial NS0 cells or other myeloma lines are grown without insulin supplementation, and many CHO cells have been adapted to grow without insulin. It is likely that the signal transduction pathways have been altered downstream of IR or IGFR in those adapted cell lines.

Insulinwasoneofthefirstrecombinantproteinstobe made available for therapeutic use. Recombinant insulin has long been used in cell culture. IGF1 is also used in cell culture, often in a commercial form.

in cell culture, often in the commercial form.


Anchorage dependent cells are employed in the vaccine industry and in emerging stem cell and cell therapy-based technologies. In this section we will address adhesion of cells only to conventional stationary surface cultivation, not microcarriers. Although stainless steel or chemically modifiedpolymer surfaces are still used, the vast majority of cells are grown on conventional plastic or glass surfaces. Many suspension-adapted cells revert to attachment when grown on adhesive (positively

Cell Adhesion Molecules

The most abundant protein in serum is albumin. Albumin is a versatile molecule and is a carrier for many compounds that may have low solubility in aqueous solution. Most notably, albumin is a carrier for fatty acids, which can be toxic when present in free form at high concentrations. Albumin also binds bilirubin, heavy metal ions, and other agents that may harm cells. Albumin is probably the most important protein that mediates scavenger functions of serum in cell culture medium.

Recombinant forms of human albumin are also available. However, unlike other recombinant proteins, which are easily characterized for use in achemicallydefinedmedium, thediversebindingcapacity of albumin makes its complete chemical properties harder to define. Not all albuminpreparations are the same—albumin from different preparations may be bound to different amounts and varieties of fatty acids or other compounds.

In addition to transferrin and serum albumin, serum also contains other carrier proteins as shown in the table. Most are rarely used in culture.

Serum Albumin• used at 0 .1 – 5 mg / mL

• High interspecific potency

• Fatty acid composition and content depend on method of preparation and species.

• Most defined medium use fatty acid-free albumin coupled to specific fatty acids, particularly oleic acid or linoleic acid .

Table 11 . Transport and Carrier ProteinsTransport proteins

Source Structure Effects

Serum albumin Plasma 1-chain (MW=68000)

Supplies free fatty acids Detoxifyer contains trace elements

Transferrin Plasma 1-chain (MW=77000)

Supplies iron detoxifyer

High density lipoprotein (HDL)

Plasma

Particle (multiple protein subunit)

Accepts and transports cholesterol and cholesterol esters

Low density lipoprotein (LDL)

Plasma Particle (Apo B)

Transports cholesterol and cholesterol esters

Trenscobalamin Plasma Binds vitamin B12

Ceruloplasmin Plasma1-chain (MW= 135000)

Binds copper

Hemoglobin Red cells

4 subunits (MW ~65000)

Transports O2

Serum albumin and other carrier proteins

MEDIUM DESIGN | 123

charged) surfaces. Adhesion can be enhanced in the presence of serum or by coating surfaces with adhesion molecules. For the cultivation of industrial cell lines, there is, in general, no need for coating the surface with adhesion molecules.

Adhesion molecules are used to promote adhesion of various stem cells, differentiated cells, or some highly transformed cells that do not attach well to tissue culture flask surfaces.Commonly used adhesion molecules, as shown in the table, include biological molecules (fibronectin, laminin), ECM extract (matrigel,which is rich in laminin), and synthetic molecules (poly-L-lysine or RGD peptide (arg-gly-asp).

These adhesion molecules may be used to revert suspension cells to an adherent state for cell cloning. Under adherent conditions, cells form colonies on the surface of culture dishes and can be easily isolated using a cloning ring.

In some cases, one might want to prevent cell adhesion to a surface. Inclusion of heparin, heparin sulfate, or Pluronic in the medium, along with use of a non-adhesive surface can minimize cell adhesion.

Table 12 . Adhesion Molecules Used for Cell CultureAdhesion proteins Source Effects

Fibronectin Plasma, cell lines Promotes attachment growth of mesenchymelly derived cells

Laminin Extracellular matrix Promotes attachment and growth of ectodermally and endo-dermally derived cells

Collagens (I-IV) Skin, extracellular matrix, placenta

Promotes attachment and growth either directly or through the binding of other adhesion proteins

Vitronectin Plasma Promotes attachment and growth of a variety of cell typesFetuin FBS Promotes attachment of cells to glass and plastic

Poly-d-lysine Synthetic Polymer Promotes attachment of many cell types (even in the presence of serum)

• Cells clumping and cells sticking to vessel surfaces can be partially corrected by adding to medium:

• Pluronic F68 (0 .01 – 0 .1%)

• Heparin (10–100 ug / mL)


Protein Hydrolysates Hydrolysates or extracts from animal or plant tissues were commonly used in cell culture processes to reduce the dependence on serum. A beef extract, Ex-Cyte, was an excellent source of phospholipids; however, the use of animal extracts has been largely discontinued, and has been replaced by hydrolysates from soy, rice, and other plants derived by enzymatic or acid hydrolysis. Lot to lot variation among such extracts is huge. In a transcriptome analysis of CHO cell samples grown under different reactors, pH, andotherconditions,itwasfoundthatthespecificlot of hydrolysate overrode other experimental variables in sample clustering. In a data mining experiment encompassing data from more than 100 manufacturing runs, it was found that hydrolysate lot had a strong correlation with productivity.

The roles of hydrolysates are not completely understood. Hydrolysates are complex mixtures of amino acids, peptides, derivatized peptides, carbohydrates, and some lipids. They provide some nutrients and minerals and may also act as scavengers through undefined molecular interactions withpossible contaminants. Hydrolysates may have some growth-stimulating or anti-apoptotic activities. Given that synthetic peptides have been found to interfere with signaling pathways by binding to signaling intermediates, the possibility cannot be excluded that some hydrolysates have similar effects.

• Soybean hydrolysate and peptone are commonly used

• Peptones derived from acid or enzyme hydrolysates of casein, gelatin, meat, soy, egg and lactalbumin have been used as supplements in cell culture

• Contain a mixture of amino acids, small peptides, inorganic ions, carbohydrates and vitamins

• Possible roles of hydrolysate

• Source of amino acids in the form of oligopeptides

• Some oligopeptides may mimic analogues of signaling molecules by having non-specific binding various cell surface receptors

• Such effects may be growth-promoting or apoptosis-retarding

MEDIUM DESIGN | 125

Medium design for industrial cell culture processes encompasses two aspects, cell expansion and production. For cell expansion the focus is on optimal growth and sustained viability; for production, the objective is rapid growth to production cell density, sustained viability, and productivity at high cell density. For the production of non-cell products including recombinant proteins and viruses, the viability of the culture at the end of production may not need to be high; thus an extreme composition that favors production at the expense of viability may be used.

In the past decade it has become a common practice tousehighosmolarityinthefinalstageofproductionculture. Glucose concentrations as high as 15 g/L (83 mM) may be used at the initiation of production culture. At such a high level, glucose contributes significantly to overall osmolarity; thus, theconcentration of NaCl is often reduced to compensate for the additional glucose. High osmolarity affects the growth rate of most cells, although a high glucose concentration does not appear to affect growth of some CHO and myeloma cells. As discussed in the section on Fed-Batch cultures, most industrial production is initiated at about 70% of reactor volume, and concentrated nutrient mixtures of amino acids, glucose, etc. are added during cultivation. The concentrated nutrient mixtures usually carry extra salts used to dissolve amino

Medium for the Industrial Production Culture

Lipids are sometimes added to serum-free culture medium, although they must be used with caution. Most lipids in circulation are associated with carriers. Too much free lipid in culture medium is not desirable. Phosphatidyl choline, phosphatidyl ethanolamine, and/or sphingomyelin may be added to cell culture medium in the form of liposomes or may be dissolved in DMSO. Reconstituted lipid supplements containing a defined composition ofphospholipids are commercially available for use in chemicallydefinedmedium.

Lipid Supplements


acids, which additionally increase the osmolarity. As a consequence, it is not unusual to reach an osmolarity of 400 mOsm by the end of production.

In recent decades, the concept of industrial medium design has undergone a major shift. The focus on providing cells with optimal growth medium is now restricted to applications involving cell line isolation, banking, and expansion. A new focal point is the design of medium for production cultures. In some cases, even in the expansion of the seed train toward a production scale, the medium used has been altered from traditional “optimal” formulations; for example, sometimes high glucose concentrations are used. It has not been shown that such conditions provide any advantage for cell expansion, but new points of view have replaced the notion that “optimal” growth conditions are likely to be found by recreating the native niche of a particular tissue of origin.

For industrial processes, the purpose of medium design is not to provide conditions that cells like, but to devise conditions that can be imposed on cells to maximize their productivity. Significant advancestoward that objective have been accomplished.

Methods and Strategies in Cell Line Development

METHODS AND STRATEGIES IN CELL LINE DEVELOPMENT | 127

Host Cells and Recombinant Protein Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127Transient Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Stable Cell Line Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .129What Makes a Hyper-Producing Cell Line? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129Basic Steps for Generating a High-Producing Cell Line . . . . . . . . . . . . . . . . . . . 131Basic Elements on a DNA Plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138Cell Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142Stability of Selected Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Automation and High Throughput Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146

Cell Line DevelopmentWith Contributions From Gargi Seth

Host Cells and Recombinant Protein ProductionAnimal cells are powerful vehicles for the production of therapeutic recombinant proteins. Therapeutic proteins, along with viral vaccines and cell therapy products, are generally called biologics to differentiate from small-molecule drugs. The process of transforming the host cell line is critical to producing cells of high productivity. Cells must be screened for those that contain a superior level of production and growth characteristics. This chapter discusses the process of generating cell lines suitable for the large-scale production of recombinant proteins.

Industrial expression of recombinant proteins is generally categorized into two types: transient or stable. Transient expression is frequently used for the generation of small quantities of protein (up to gram quantities), for protein characterization


The overexpression of exogenous gene(s) in target cells is an important technique used to understand the functional significance of an unknown gene.Often, the desired effects can be observed with a transient expression of the protein, so in these cases, stable transduction is often un-warranted and un-necessary. Transient expression is also frequently used to produce and isolate protein products encoded by a transgene. The aim, in this instance, is to simply obtain a sufficientquantity of theproductproteinin a short amount of time. The scale of production in these cases is often small, but could also be fairly large (e.g., up to tens of liters of reactor volume).

For protein production through transient expression, the host cells are loaded with a very large amount of exogenous DNA, such as a plasmid. In transduction, only a small fraction of exogenous DNA is actually taken up by cells and even smaller fractions actually enter the nucleus where the transgene gets transcribed. Thus, to see high levels of exogenous protein expression, researchers employ the use of a strong promoter to drive gene

Transient Expression Protein Production• Transgene in plasmid DNA or virus (advenovirus or

vaccinia virus) is introduced into the cell, the nucleus where it exists as an extrachromosomal unit .

• Cells are heavily loaded with vectors to increase the production

• The heterologous DNA is not integrated into the host genome .

• plasmids generally do not replicate

• viral vector may be maintained inside the cell as an autonomous replicon

or animal testing. This process does not generate a clonally derived cell line; it temporarily transduces an existing cell line (e.g. HEK293 or COS) with plasmids encoding the product protein.

Conversely, stable cell lines are generated for the industrial production of therapeutic proteins. These cell lines must be capable of producing product of the same quality in different batches, and in different locations, throughout the years. Once a production line is transduced and selected, cells are expanded to establish a master cell bank, which is stockpiled. Cells from a mater cell bank are then expanded further to create a working cell bank from which frozen vials of cells are taken for use in the production. The banked cells, typically stored in liquid nitrogen, are used for manufacturing purposes throughout the production cycle. In generating the production line, both the choice of the host cell and the choice of the vector carrying the product’s gene are pivotal.

Transient Expression:

• Not suitable for long term production or commercial manufacturing

• Useful for rapid production of small quantities of research materials:

• Drug candidate identification and in-vivo evaluation

• Assay development (e .g . binding assays)

• Structural studies

• Toxicology studies

Transient Transfection


transcription and transduce large quantities of starting material. However, if too successful, the overexpression of some genes can be toxic to cells.

When transiently expressed, the exogenous vector does not get integrated into the genome. After transfection, they exist as extra-chromosomal elements. Most plasmid vectors, except for some episomal vectors, do not self-replicate and are gradually lost in about a week’s time. Even some viral vectors, such as adenoviral vectors, do not persist over a long time and are largely lost in a month. This is fitting for the overproduction ofprotein, where the aim is to generate a short and intense, rather than a long and sustained, burst of protein production. Thus, in transient situations, the percentage of cells actually taking up and expressing exogenous DNA (i.e., the transfection efficiency)cansignificantlyaffecttheoverallexpressionlevel.

HEK-293 (293; human) and COS (African green monkey) cells are frequently used for the transient expression of proteins, but product quality parameters,suchasglycoformprofile,maydifferformaterials derived from different species (e.g., human versus Chinese hamster). However, CHO cells are unique. Transient material produced in CHO cells is more representative of the material produced from stable CHO cell lines, so there has been an increasing use of CHO cells for stable expression. Unfortunately, the transient transfection productivity in CHO is relatively low compared to that of 293 host cells.

Host Cells Most Frequently Used for Transient Expression:• HEK-293 (human embryonic kidney fibroblasts) • COS (from green monkey kidney cells)• BHK cells

• CHO cells (to a lesser extent)

Production Life of the Transient Expression System is Usually Limited by:• Loss of DNA from the cell with time

• Deleterious effect of foreign DNA on cell viability

• Typical titers ranges from >1 to 100mg / l in 5 - 10 days (~ 0 .1-1 pg / cell / day)

Stable Cell Line DevelopmentWhat Makes a Hyper-Producing Cell Line?

Two types of cells are most commonly used as host cells for the generation of stable production cell lines: CHO cells and myeloma cells. Virtually all therapeutic proteins made with these cell lines are secreted out of the cell and can be harvested from their culture fluid. CHO cells, which do notsecrete any protein in an appreciable amount in their native state, must be made to develop the capability of protein secretion while becoming producers. In contrast, myeloma cells have well-


developed protein secretion machinery, left over from their original purpose of secreting antibodies.

Many scientists have studied the changes that occur during the maturation of B-cell to plasma cell, both at the proteome and transcriptome levels. The cellular alterations appear to include elevated energy metabolism, higher protein secretion and glycosylation capacity, as well as an increased redox balance, to counter the effects of reactive oxygen species.

B-cells and plasma cells have only one functional immunoglobulin gene in their genome. In the developmental maturation process, one of the two gene copies in the diploid alleles is inactivated, thus preventing the production of two immunoglobulin molecules in a single cell (i.e. “allele exclusion”). Even a single copy of a transgene is sufficient for a cell to become asuper secretor, like the plasma cell. Therefore, to transform a myeloma cell into a high producer of protein, one needs only to introduce a single copy of the product gene into the appropriate locus of the genome. The pre-existing cellular machinery is already tuned to secrete a high level of protein.

CHO cells, contrary to myeloma cells, must have modifications to enhance their protein secretionmachinery and to acquire characteristics of high-producing cells. In addition to bolstering the protein secretion machinery, a high-performing cell line should also have superior growth and metabolic characteristics. Manufacturing conditions differ profoundly from laboratory cultivation. For example, the prolonged stationary phase in a fedbatch culture, in the presence of lactic acid, allows for a longer period of protein production at a stage when the cell concentration is high, thus leading to a maximal product titer. The important question, in this case, is how to identify candidate cell lines that harbor those desirable traits.

In the past decade, numerous transcriptome and proteome studies have been conducted to examine the traits leading to hyper-productivity. Biotechnologists now realize that there is

Host Cell Protein Secretion

CHO DG44 serum dependent, adherent, DHFR-/- nilCHO DXB11 serum dependent, adherent, DHFR+/- nilCHO K1 serum dependent, adherent, proline

dependentnil

SP2/0 serum dependent, suspension plasma cell

Myeloma NS0

serum dependent, suspension, cholesterol-dependent

plasma cell

Typical Characteristics of Producing Lines• Serum independent

• Suspension growth

• Highly secretory, associate physiological change

• Energetic

• Secretory pathway

• ROS-redox balance

• Sustained growth in stationary phase

• Ready switch to lactate consumption

• Glycosylation capacity


probably no single master regulator that can be “turned on” to make a CHO or NS0 cell a hyper-producer. Hyper-productivity is the culmination of multiple changes in multiple cellular pathways, such as metabolism, secretion, redox balance, and growth/death control. The acquisition of hyper-productivity is more likely to involve diminutive gene expression changes on a vast scale, rather than larger alterations in only a few master switches.

Potentially a large combination may give rise to similar productivity.

‘Best’ Producer

Energy Metabolism

Secretion Redox Growth/Death Control

Different changes may lead to the same incremental improvement of cell characteristics necessary for high productivity

Cumulative effect of combination of different changes may not be the same, some may not be additive

Fig. 5.1: Hyperproductivity of recombinant protein in producing cells contributed by multiple cellular functions. Many alternative combinations of superior traits may lead to high-productivity.

Basic Steps for Generating a High-Producing Cell Line

A few basic steps are generally followed to make a host cell become a high producer of the desired product. First, a transgene coding for the product protein is typically introduced to the host cell using a plasmid. In addition to the transgene, the plasmid carries a gene that confers a selectable trait, such as antibiotic resistance, so that after transfection, a selective pressure can be applied to enrich for those cells which have internalized the plasmid. The plasmid does not replicate in mammalian cells and would otherwise be gradually lost as cells multiply. By applying selective pressure over an extended period, all of the cells that are selected for would have the plasmid integrated into the chromosome.

After a stable cell population is obtained, preliminary in vitro screening is then performed to screen for clones with the highest production levels.Thisisoftenfulfilledbyassayingtheproductconcentration in the supernatant of a 96-well plate. Another common practice is to grow cell clones as small colonies on soft agar and then perform immunoassays in situ to identify those with a larger immunoprecipitation zone around the colony.

Thenext step, theamplificationof transgene copynumber, is practiced in some instances (such as when CHO cells are used), but not all (such as when myeloma cells are used). To do this, the stable cells are subjected to a high concentration of an inhibitor

Steps in Cell Line Creation• Transfection

• Selection

• Amplification

• Single cell cloning

• Screening

• Adaption


totheamplificationmarker,whichthecellsrequirefor survival. This high concentration of inhibitor kills the vast majority of cells, except those that have multiple copies of the marker and resistance genes.

Astheamplificationmarkermultiplies,theadjacentintegrated transgene(s) are also co-replicated. The number of copies may increase dramatically in different regions of the genome, thereby giving rise to high levels of transcription and translation. Through this process, in some high-producing cells, the transcript level of recombinant IgG heavy chain becomes the most highly expressed transcript in the cell. An excessive expression of protein can overwhelm cell’s protein folding capacity and lead to an unfolded protein response in the endoplasmic reticulum and, thereby, induce apoptosis. Consequently, cells which have not developed appropriate machinery to handle the increased production may not survive.

Afteramplification,theselectedcellsnotonlyhavemultiple copies of the transgene and a high level expression of resistance and product genes, but they also have developed the secretory capacity to allow for enhanced protein secretion. These clones have a high propensity to become high producers, but not all of them do. Hyper-productivity also requires many other traits, such as the capability to quickly grow to a high cell density and the capability to sustain a high viability over a long duration in the stationary phase.

Subsequently, single-cell cloning is performed on those surviving cells, typically by sorting single cells into culture wells by flow cytometry.Cloning can also be performed by dispensing cells (approximately 0.2 cell per well) into multi-welled culture plates, so that the probability of having more than one cell in each well is very low. Thus, all cells that arise from a given well all originated from the same cell. Single cell cloning is necessary because a pool of stable cells would have vastly different genetic backgrounds, perhaps in the loci of exogenous gene integration, and potentially have different mutations caused by those integration and other unknown aberrations. Such a mixture of cells of genetic heterogeneity is called a “cell pool”.

Fig. 5.2: Typical steps in introducing a transgene for generating high producing cell lines for manufacturing.


After single cell cloning, the productivity of each clone is then assessed and those with high productivity are isolated. The selected clones are further expanded for stock preparation, growth characterization and product quality assessment. In some cases, the producing cells are then adapted to growth conditions more amenable to manufacturing conditions.

Single cell cloning is considered critical for establishing a production cell line, as the cells arising from a single cell are genetically homogenous. It is practiced regardless of whether there is an amplificationstepornot.Althoughthehostcellsusedfor establishing production lines are all aneuploid and can be prone to further genome reorganization and epigenetic reprograming, a culture of cloned cells are much more homogenous than cell pools. From a single cell at the beginning of single cell clone to the end of a production run, the production cell may have gone through more than 60 doublings. If that is extended to the entire production lifetime, the number of cell doublings may be greater than 80 doublings. It is important to minimize the outgrowth of mutated cells in the original pool, during the course of cell expansion, by single cell cloning during the generation of production cell lines.

A number of different methods are commonly used to introduce expression vectors into host cells. The choice of method is dependent on cell type, the available quantity of cells and plasmids, and the experience of the lab practicing it. Although different methods depend on different mechanisms of plasmid uptake by cells, all methods require the cytoplasmicmembrane to first becomepermeableto plasmids. Plasmid delivery by calcium phosphate precipitation, cationic polymers, and liposomes relies on direct interactions of the particles, or lipid vesicles containing plasmid DNA, with the cellular membrane through an endocytosis-like of mechanism. In electroporation and microinjection, physical force is used to introduce openings in the cell membrane for DNA entry.

The “Methods of Gene Transfer” table lists

DNA-Calcium Phosphate Co-PrecipitationDNA and calcium chloride is added dropwise into a HEPES buffer with sodium phosphate (1 mM). A fine precipitate forms in 5-30 min, and is added directly to the cells (~2-40μg/106) .

ElectroporationExpose cells to a high-voltage electropulse in the presence of DNA solution. This introduces pores in the plasma membrane, allowing entry of DNA. Duration of pulse and strength of electric field varies with cell type .

Lipofection/Lipid Mediated Gene TransferA mixture of DNA with amphipathic compound (DOTMA, DOPE, etc), that simultaneously interacts with DNA and hydrophobic portions of the membrane, allowing passage of DNA into the cell .

Methods of Gene Transfer


commonly used methods of DNA introduction. All methods use a very high plasmid-to-cell ratio, but only a moderate DNA concentration. While up to a thousand copies of the plasmid can enter cells, only a small proportion actually translocate to the nucleus and are transcribed to allow for the expression of selectable marker resistance gene.

DNA-Calcium Phosphate Co-PrecipitationDNA and calcium chloride is added dropwise into a HEPES buffer with sodium phosphate (1 mM). A fine precipitate forms in 5 - 30 min, and is added directly to the cells (~2-40μg/106) .

ElectroporationExpose cells to a high-voltage electropulse in the presence of DNA solution. This introduces pores in the plasma membrane, allowing entry of DNA. Duration of pulse and strength of electric field varies with cell type .

Fig. 5.3: A typical vector for introducing transgene into host cells for recombinant protein production.

Basic Elements on a DNA PlasmidPlasmids facilitate the introduction of specificgenes into mammalian cells. They also contain elements that enhance the transcription and translation of the product gene. Both viral and bacterial plasmid vectors are commonly used for introducing transgenes into mammalian cells for research. However, the prevailing vehicle used for introducing product gene for recombinant protein production is a plasmid vector rather than a viral vector. Although viral vectors are used often in gene therapy, they are rarely employed for establishing production cell lines for therapeutic proteins.

The expression of the product gene may be driven by a constitutive, inducible, or conditional promoter.Conditionalpromoters,drivenbyspecificendogenous factors or events, are frequently used in the research of differentiation and development. It allows a reporter gene or selectable gene to be expressed after a particular differentiation event. It enables the selection or sorting of differentiated cells

Table 2. Estimates of Number of DNA Molecules per Cell for Commonly Used Non-Viral Gene Transfer Methods .

Method DNA concentration (μg/mL)/pM

Cell concentration (106 cells/mL)

Number of DNA

molecules per cell

Calcium phosphate

50 / 15 5 1 .8*106

DEAE dextran 10 / 3 5 3 .6*105

Lipofection 40 / 12 5 1 .5*106

Electroporation 40 / 12 10 7 .3*105


In the course of introducing DNA into host cells, only a fraction of them actually express the plasmid. Although the number of plasmids entering each cell is likely to be very high (probably in the order of hundreds to thousands of plasmids), the probability of their entry into the nucleus and subsequent integration onto the genome is very low. These plasmids are not capable of self-replication; they only replicate after integrating into the host chromosome. Free plasmids in the cell are, thus, gradually degraded or otherwise lost.

After transfection a very large fraction cell population are untransfected. To identify and select

in a population. For example, the albumin promoter isexpressedandactivatedspecificallyinlivercells.When driving the expression of green fluorescentprotein (GFP), cells of the liver lineage become green.

For protein production, the vast majority of vectors employ a constitutive, and very strong, promoter. Traditional viral promoters, such as SV40 late promoter or the CMV promoter, are frequently used. In the past few years, constitutive promoters, such as the promoters of elongation factor 1 (EF-1) and glyceraldehyde dehydrogenase (GAPDH), from CHO, have been isolated and used in product gene expression.

In addition to a strong promoter, enhancer elements can also be included in the intron of the transgene construct, to ensure a high level of transcription. It is rather common to see that at least one intron is included in the product gene construct. Furthermore, the DNA sequence of the product gene is often “codon optimized” to match the efficiency of translation machineryof the host cell. Through codon optimization, one can replace the codon for a rare tRNA with a more abundant tRNA, to avoid the translation rate being limited by the supply of the rare tRNA.

In addition to the promoter, enhancer, and the product gene, the vector must also has a selectable marker, and if amplification isto be involved, the amplification marker.

Elements in a Vector• Promoter• Coding sequence (often with introns) of gene of

interest• poly A signal• Selectable marker• Other elements of plasmid (cloning site, origin of

replication)

Target Gene• Positional cloning: screen library for chromosomal

markers known to flank the gene of interest, and then “walk”, testing individual genes identified in the region.

• PCR amplify

Promoter• Constitutive• Conditional

• Inducible• Native dynamic regulated


those that have the transgene, a selectable marker is included in the plasmid. Cells that have at least one copy of plasmid integrated into the chromosome and express the selectable marker gene will survive in the presence of selectable marker.

Selectable markers are classified into twocategories: dominant and recessive. A recessive marker resides in cells with a particular genetic background and causes a growth deficiency.Then, the introduction of a compensatory gene leads to overcoming the deficiency. For example,a cell line without a functional dihydrofolate reductase (DHFR) gene requires the additional supplementation of thymidine and glycine in the culture medium for cell growth. The introduction of a functional DHFR enables it to grow without the supplements. Therefore, after the transfection of plasmid containing DHFR, only the transfectants will grow in the absence of thymidine/glycine. Similarly thymidine kinase (TK)-defective mutants require thymidine to be included to the culture medium. The introduction of a functional TK gene allows for cell growth in the absence of thymidine.

Conversely, the presence of a dominant selective agent is lethal to the cell. By introducing the selectable marker gene to the cell, the cell is endowed with a resistance to the selective agent. The most frequently used selectable markers, and their mode of action in mammalian cells, is listed in the adjacent table. Each resistancegeneencodesanenzyme,whichmodifiesthe selective chemical agent to destroy its activity.

The phosphorylation and acetylation reactions employed for inactivating antibiotics require intracellular reactants (ATP, acetyl group donor). Those enzymes are, thus, only effective in destroying the selective agents intra-cellularly. The hydrolysis enzyme, on the other hand, may be active even when released into medium after cell lysis. In any case, in the selection process, the concentration of the selective chemical agent decreases with time and the rate of decrease is dependent on the concentration of transfected cells. Thus, the optimal concentration for selection for each agent is not only dependent

Recessive Selection • DHFR (dihydrofolate reductase)

• On DHFR deficient background• TK (thymidine kinase)

• On TK deficient background

Dominant Selection• Antibiotic resistance

• Neomycin• Hygromycin• MDR (multi-drug resistance)• DHFR

Reporter Gene• gfp family• β-galactosidase• luciferase• secreted alkaline phosphatase


on cell line but also on its concentration. Clonal selection and population selection may have rather different optimal concentration of selective agent.

Another class of selective agents interferes with the uptake of a toxic selective agent. The multidrug resistance gene (MDR) confers cells with resistance by increasing their ability to pump toxic substances, such as colchicine, out of cells. Its overexpression allows for selection from a background of cells not expressing MDR.

Table 2. Commonly Used Drugs for Selection of Stably Transfected Mammalian CellsAntibiotic Family Mode of Action Resistance gene Mode of

resistanceGene

size (bp)Drug

concentration range

(μg / mL)Geneticin (G418)

Aminoglycoside Block protein synthesis by inhibiting elongation

Neomycin phosphotransferase (npt)

Phosphorylation of Geneticin

795 100 - 800

Hygromycin B Aminocyclitol Inhibit protein synthesis by disrupting translocation and promoting mistranslation

Hygromycin phosphotransferase (hpt)

Phosphorylation of Hygromycin B

1011 10 - 400

Puromycin Aminonucleoside Block protein synthesis by causing pre-mature chain termination

Puromycin N-acetyltransferase (pac)

Acetylation of Puromycin

603 0 .5 - 10

Blasticidin S Peptidylnucleoside Inhibit protein synthesis by interfering with peptide bond formation

Blasticidin S deaminase (bsr)

Deamination of Blasticidin S

396 1 - 10

Zeocin Bleomycin (Glucopeoptide)

Intercalate into and cleave DNA

Bleomycin resistance protein (ble)

Bind stoichiometrically and prevent Zeocin from binding DNA

375 0 .1 - 50


Amplification Two systems are commonly used for gene amplification in mammalian cells: DHFR andglutamine synthetase (GS). The most commonly usedgeneamplificationsystemisbasedontheDHFRgene, whose chemical antagonist, methotrexate (MTX)canbeusedtodrivegeneamplification.DHFRis an enzyme which catalyzes the conversion of folate to tetrahydrofolate, a compound required for the biosynthesis of glycine, thymidine monophosphate and purine. Methotrexate, a folate analogue, binds and inhibits DHFR, thereby leading to cell death in the absence of thymidine and purine in the medium.

When cells are selected for growth in methotrexate, the surviving population contains increased levels of DHFR which results from an amplificationof the DHFR gene. This is also accompanied by amplification of 10 – 10,000 kilobases of DNAsurrounding the site of integration. Therefore, by introducing a gene of interest (i.e. protein product gene) alongside the DHFR gene, co-amplificationof theproductgenecanbeachieved.

The amplification process can be perform asa single step of MTX exposure over one to two weeks, or in multiple step-wise increases in methotrexate concentration. As MTX concentration is increased, surviving cells with higher degrees of DHFR gene amplification are obtained.Highly methotrexate resistant cells may contain several thousand copies of the DHFR gene.

DHFR based amplification is more efficient in aDHFR defective genetic background. Otherwise, endogenous DHFR may get amplified withoutconcurrent amplification of the product gene.Chinese hamster ovary cells deficient in DHFRwere isolated after ethyl methanesulfonate- and UV irradiation-induced mutagenesis. These DHFR-deficient cells require the addition of thymidine,glycine, and hypoxanthine to the media. These cells do not grow in the absence of added nucleosides unless they acquire a functional DHFR gene.

The glutamine synthetase (GS) selection system is based on the biosynthetic pathway of glutamine

• The strategy is to use a mutated form of the enzyme that has a lower catalytic activity or to use an enzyme inhibitor

• DHFR with methotrexate (MTX)

• DHFR: dihydrofolate+NADPH → tetrahydrofolate+NADP

• GS with methionine sulphoximine (MSX)

• The metabolic enzyme (amplifiable marker) needs to be amplified in order to supply sufficient reaction rate for cell survival

• Glutamine Synthetase:Glutamate + ATP + NH3 → Glutamine + ADP + Pἱ


from the substrates glutamate and ammonia. Most mammalian cell lines require glutamine supplementation in their culture media to grow since their endogenous GS activity is low. The promoter region of GS in CHO cells is rich in CpG and evidences indicate that GS in CHO is silenced possibly by methylation of cytidine.

The GS expression vector contains a glutamine synthetase gene along with the gene of interest, allowing for selection by growth in glutamine-free cell culture media. The GS gene is usually driven by a weaker promoter, typically the SV40 promoter. With a high concentration of the glutamine synthetase inhibitor, methionine sulphoximine (MSX), it is possible to select for transfectants with gene amplification.

Several variations on the systems described above have been developed for increasing achievable expression levels. DHFR is used in conjunction with an impaired neomycin resistance gene. After transgene induction and under G418 selection only cells with the vector integrated in a transcriptionally active region will express neomycin resistant transcripts at high enough level to survive. Since the locus of integration is transcriptionally active, the high expressing clones isolated after amplificationhaveonlyafewintegratedgenecopies.

Afteramplification,lastingforaweektotwoweekstypically, the concentration of antagonist selective chemical agent is reduced. With a lower level of selective pressure the number of copies of transgenes may also decrease and resulting in a decrease in its transcript level and productivity. The propensity for losing transgenes is probably affected by the loci of integration on the chromosome, with those near the distal end of the chromosome arm being more prone to dislodge from genome. Usually a clone becomes more stable after the initial drop of copy number and product titer upon the reduction of selection pressure. In most cases the remaining transgenes are stable in subsequent cell cultivation. Nevertheless a low level of selection pressure is often maintained to suppress any possible deleterious mutants which may have a lower copy number and faster growth rate.

DHFR Amplification

• More efficient in DHFR- background

• CHO DXB11; one DHFR is deleted, the other has missense mutation

• CHO DG44: both DHFR deleted

• With sufficiently high levels of MTX, amplification can be carried out in DHFR+ background


Classical DHFR Amplifiable Vector

Fig. 5.5: A gene construct for introducing heavy chain and light chain molecules of immunoglobulin gene.

Fig. 5.4: A vector for DHFR based amplification of transgene.

Classical DHFR transfections employ a plasmid in which DHFR is tethered downstream to the gene of interest driven by a promoter (e .g ., SV40 enhancer/promoter) . The plasmid is used to transfect CHO cells (such DXB11) deficient in DHFR. The transfected cells are selected in nucleotide-free medium, in the presence of methotrexate (MTX). Subsequently, MTX concentrations are increased to enrich for cells that have multiple copies of DHFR and, concomitantly, the gene of interest .

There have been a number of variations of this method. Some methods use another resistance marker, such hygromycin or neomycin resistance, for the first selection of cells co-transfected with DHFR and the gene of interest, followed by amplification by selection of amplified cells in the presence of high concentration of MTX.

The DHFR/MTX system has been widely used for the generation of antibody-producing cells. The example shown uses a two-plasmid system . DHFR resides on the plasmid containing the gene encoding the light chain . Note that instead of cDNA, the gene of interest is interspersed between two intron sequences . The heavy chain gene, on another plasmid, has Neo as the drug resistance marker . The two plasmids are co-transfected into CHO cells, often at a stoichiometric ratio that slightly favors the heavy chain plasmid . The transfected cells (which co-express DHFR/light chain plasmid and the heavy chain plasmid) are then enriched using MTX treatment .


Glutamine synthetase (GS) is selectable marker in most mammalian cells, as they require glutamine for growth in culture . It is used frequently in my-eloma and CHO cells . The transfectants are selected by growth in a glutamine-free medium . Vector am-plification can subsequently be achieved by using the inhibitor of GS, methionine sulphoximine (MSX) .

Glutamine Synthetase (GS) as Selectable Amplifiable Marker

The locus of transgene integration influences the expression level, due to a position effect on gene expression. There are strategies to select clones whose transgenes are more likely to have integrated into transcriptionally active region (hot spots).

An impaired neomycin phosphotransferase, which confers neomycin G418 resistance, has been used successfully for hot spot integration. In the impaired enzyme, the translation initiation site has been mutated to reduce its translation initiation efficiency; thus more transcripts are needed to synthesize the same levels of proteins that confer G418 resistance .

Moreover, an artificial intron is introduced to decrease the level functionally active Neo transcripts, by increasing the frequency of unsuccessful splicing. As a result, only those clones which have the Neo inserted into a transcriptionally active region of the chromosome will have a high enough expression level to overcome drug selection.

Most of the selected resistance clones have only one copy of the transgene . The selected clones can be further amplified using the traditional DHFR system. With the increased probability of transcription, fewer clones will need to be screened to obtain high producers, and most obtained clones have low levels of DHFR amplification. Furthermore enhancement is obtained using a single promoter (CMV) for heterologous protein and selectable (amplifiable) marker, while having an IRES between the heterologous gene and selectable marker gene.

Directing Integration to a Transcriptionally Active Region

Fig. 5.6: A vector with GS selectable marker.


Cell Adaptation In a typical cell line development process, a large number of product-secreting clones are selected and subjected to further screening of their growth characteristics, product titer, and product quality, glycosylation patterns, and other post-translational modifications.Insomecases,thecellsdonotgrowwell in the culture environment used in production (e.g., high agitation rates or altered medium composition). They often have to be “adapted” to new culture conditions by long-term cultivation with a gradual change of environmental factors. Over time, the cells gradually develop the ability to grow under the new chemical and physical environment.

The presence or absence of a physical surface for cell attachment is probably the most drastic difference in cultivation conditions. Most normal diploid cells used for virus production are strictly anchorage dependent. Some cells, like myelomas and hybridomas, are suspension cells that can be readily grown in a mixing vessel. Many cell lines commonly used for recombinant protein production, including CHO, BHK, and HEK293 cells, are derived from adherent cells. Although, not being strictly anchorage dependent, they often prefer adherent growth given a compatible surface.

When cultivated in suspension, the unadapted cells either fail to grow or form large aggregates with extensive cell-cell contacts and intercellular adhesion. They can be adapted to grow in suspension bybeingculturedinshakerflasksorspinnerflasks.The initial growth rate is slow, and dispersing agents like heparin sulfate, reduced serum (if any), or calcium incorporation may be necessary to prevent adhesion to the wall of the culture vessel in the early stage of adaptation. Gradually, the growth rate is increased and the cells eventually adapt and appear indistinguishable from regular suspension cells.

The adaptation of cells to a new nutrient environment is also commonly practiced in cell line development. For example, the requirement of complex lipid additives and growth factors may be reduced or even eliminated through adaptation, although sometimes these adapted cells exhibit lower productivity.

• From transfection to working bank takes about 4 - 6 months .

Hostcell

Transfection

Targetgene

• adaptation to suspension growth• anti-apoptotic gene• glycosylation modulation

Hostcell

Transfection

Targetgene

• adaptation to suspension growth• anti-apoptotic gene• glycosylation modulation

Cell pool,selection,cell clone

Selection ofhigher and

stable producer

Adaptation tosuspension

growth

Initial processdevelopment

Adaptation toserum-free/

Animal-component-freegrowths

Initial drug testing(chemistry, biological,

and animal)

Working cell banking

Process development & Scale-up• feed batch/perfusion• metabolic shift

Fig. 5.7: Typical steps in cell line development.


Stability of Selected ClonesMutations and epigenetic alterations occur in cultured cells at relatively high frequencies. Some of these events are affected by culture conditions. For example, many types of stem cells are prone to differentiation, even by changes in cell density, oxygen tension, growth factor concentration, and cell aggregation state, depending on the particular cell type.

Cells used for human vaccine production are mostly diploid and are considered to be stable under established cultured conditions. These cells do not exhibit any visible phenotypic alterations or macroscopic chromosomal abnormalities within the accepted range of doubling, or until they reach senescence. The stability of these cells is not a general concern in bioprocessing.

In contrast, the extensively selected, hyper-producing recombinant cell harboring transfected, and often amplified,transgeneshaveahigherpropensitytolosetheir high productivity. To begin with, the aneuploid host cells used to generate those hyper-producing cells are less stable than their diploid counterparts. In addition to having abnormal chromosomal counts, many of the chromosomes also have macroscopic structural aberrations. Such chromosomal alterations accumulate over time, generating cells of divergent karyotypes from the same parental line.

Divergent karyotype and chromosomal abnormality

• From thawing (2x108 cell) to production (20m3) needs at least 16 doublings .

• Stability is tested over 40 doublings

• Normal diploid cells used in vaccine production are “stable” within the accepted population doublings

• Continuous cell lines have variable karyotypes in culture

Fig. 5.8: A timeline for generating an industrial producing cell line.


Cell Line Stability Issues• Productivity decrease over time

• Product quality changes over time

• Karyotype changes over time

• Microscopic chromosomal aberration occurs over time

• The last two issues may not be critical for protein biologics, but are of concern for cell therapy applications

Possible Causes of Instability in Recombinant Protein Productivity• Mutation, especially in intergenic region in pivotal

product gene loci

• a producing cell may contain many copies of product genes, but maybe only a dominating few contribute to most product transcript

• Epigenetic silencing of pivotal genes, at DNA level or at histone reprogramming level

• Loss of copies of product gene due to deletion or chromosomal rearrangement

is, thus, an inherent nature of producing cells derived from continuous cell lines. Even though production lines may not be stable (in relation to ploidy), they must maintain their production characteristics related to growth, productivity, and product quality over the number of population doublings required for creating sufficient cell banks. They must alsobe hardy throughout the thawing process, as well.

Assuming that a product life time of 10,000 runs at10,000literscale,withafinalcellconcentrationof 1010 cells/L, the selected cell clone will have to double nearly 80 times. This number of doublings greatly exceeds that required of a fertilized egg to grow into a hamster, a mouse, or even a human adult. In that long duration of time, the occurrence of mutations, epigenetic changes, and chromosomal rearrangement in some cells of the population is unavoidable and probably cannot be eliminated with today’s technology.

In discussing cell line stability, we decided to focus on property changes that affect productivity and product quality. The critical component for sustaining productivity over time is to prevent any cell with a lower productivity from overtaking the population, or to prevent a very high rate of productivity loss in the majority population.

A gross and rapid change of productivity in a large fraction of cells may occur upon the removal or reduction of selective pressure after transgene amplification. This problem is alleviated byemploying a lower degree of amplification and byestablishing the clone only after the copy number of transgene has stabilized. For long-term stability, one could carry out serial cultures for 40 to 60 doublings and examine the productivity, as well as the transgene copy number. From a thawed liquid-nitrogen frozen cell bag of 1010 cells to a production reactor, cells may undergo 15 doublings, so a 40-doubling test of stability will certainly give sufficient margin.

Thestabilityofproductquality ismoredifficult toassess. Mutations in genes affecting product quality, such glycosyl-transferases, may occur and lead to a subpopulation of cells that produce inferior


Automation and High Throughput TechnologyDeveloping a cell line for production purpose is a very labor-intensive process. To increase the probability of obtaining a very high producer, a large number of cells need to be isolated at every step, which involves productivity variation from successive rounds of selection and amplification.In the past decades, lab automation and high-throughput technology have become an integral part of bioprocess development. Liquid and cell handling, both cell pool and cell clones, quantification ofproduct titer, data acquisition, data processing and analysis, and archiving have all becoming automated.

Many of the automated systems are based on culture plates, or wells, and resemble other liquid handling systems for high throughput chemical screening. The difference is that a incubation system, with temperature and atmospheric control for gas mixture and humidity, is necessary. Robotic arms are often used to move plates onto the working “stage” and allow multiple manipulations to be performed on multiple plates without human interference. In many cases, the system is installed inside a clean room or clean hood to minimize microbial contamination.

The culture handling system is usually integrated with an assay system to assess product titer and cell growth. Multi-step assays and screening protocols can be performed by transferring culture fluidinto automated assay systems. The results can be directly integrated into culture handling systems to further expand wells or plates selected for

PerkenElmer - Basic Model

Tecan -- more automated with multi-plate capabilities and computer interface

Fig. 5.9: Examples of high throughput cell clone screening system.

product. Mutations causing protein sequence alteration may occur in one or more copies of product genes in all, or a subpopulation of, cells. Since not all copies of product genes in the cells are transcribed and translated at equal efficiency,not all mutations of product genes in a production cell population will be manifested to the same degree. With deep sequencing technology, one may be able to detect such mutations, even at minute level, in the consequent producing cell population.

• Production cell lines are tested for their ability to retain the product gene in the genome and produces the product

• The focus is production stability, not cell/genome stability


of stability is still labor intensive. The use of host cell lines,whichhavebeenadaptedormodifiedtoharbor all desirable growth characteristics, have greatly reduced the need of adaptation. There is an increasing movement toward miniaturizing cell culture while still simulating large-scale reactors, although this progress is still limited.

The advance in genomics has brought about a fundamental change in the way we can study the process of cell line development and brightened the prospects that we can gain mechanistic insight into hyper-productivity. This knowledge may allow us to quickly select the “right” clone by examining the transcriptome or genome of the candidate cells. With more tools for genome engineering becoming available, it may also become feasible to impart on the cells favorable genome-wide modifications.

Concluding Remarks

further investigation. An integrated microscopic imaging system can provide the added capability of determining anything from clonal colony growth to colony morphology to ensuring that cells picked from each well are single-celled clones.

Another type of automated system integrates cell cloning with product titer assessment by performing cell screening on agar plates. In this case, the secreted product molecules (mostly antibodies) are entrapped in agar that contains antibodies against the product. A halo ring of immunoprecipitation zone is formed around the colony. The size of the halo ring reflects the amount of product secreted.Image analysis is then used to extract the data for selecting high producing clones to pick.

• Basic liquid handling manipulations

• Distribution of liquid to 96 - 384 well plates

• Sampling/removal of liquid

• Transfer from plate to plate

• Cherry picking – transfer from well to well

More complex models are capable of:

• Cherry picking

• Multiple plate handing (i.e. movement of plates from a “hotel” to the pipetting stage)

• Multiple “steps” performed sequentially (e.g. DNA preparation protocols)

• Other “add-ons” like PCR machines, incubators, spectrophotometers, etc

Under best culture conditions, a hyperproducing industrial cell line derived from CHO or myeloma cells can secrete 50-100 pg protein/cell-day, a level which rivals professional secretors in vivo. Such remarkable cell lines are created through the combination of optimized genetic constructs, selection, amplification, cell clone screening, and the insightofpicking the “right” clones.Although the specificproductivity of the producing cells has increased by about one order of magnitude, the methodology of generating high producing cells has remained largely the same in the past three decades. The entire process is still empirical and very labor intensive.

High-throughput cell handling and screening systems allow for the screening of a large number of potential high producing clones in the early stages of cell line development. However, subsequent steps of adaptation, growth characterization, and testing

STOICHIOMETRY AND KINETICS | 147

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147Cell Mass and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148

Cell Mass and Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148Material Balance on Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149

Variation in Cell Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Amino Acid Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Intracellular Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Growth of Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154Quantitative Description of Cell Growth & Product Formation . . . . . . . . . . . . . . . . . .156

Stoichiometric Ratio and Yield Coefficient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158Integral Cell Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Kinetic Model of Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162A Model Describing Growth and Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Monod Model and its Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .164Environment, Kinetics and Stoichiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .165Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166

Stoichiometry and Kinetics of Cell Cultivation

IntroductionCultured cells take up nutrients to generate energy and to make more cell mass and products. For manufacturing,itisimportanttosupplyasufficientquantity of nutrients. These nutrients allow cells to grow and produce product while minimizing the formation of waste product. To make the manufacturing process efficient one must alsoproduce the targeted product quantity within a given period. Therefore, one must not only know how much nutrients to supply, but often also how fast to deliver in order to sustain the production environment. We use stoichiometric principles to determine how to supply the correct quantity of nutrients; and use kinetic principles to guide the process along a desired path. Awareness of these concepts is key to an overall understanding of how to culture cells. This chapter discusses stoichiometry,

STOICHIOMETRY AND KINETICS | 149STOICHIOMETRY AND KINETICS | 148

Cell Mass and Composition

Cell Mass and Size Both microbial and mammalian cells vary widely in size. In general, the dry biomass of bacteria, yeast, and animal cells is in the order of 10-12, 10-11, and 5 x 10-10 g per cell, respectively. The most abundant chemical species in a cell is water, accounting for 90% of the volume of plant cells, 80 – 85% of animal cells, and 70% of bacterial cells. Since cell cultivation is carried out in aqueous environments and the amount of water taken up by cells during growthisextremelydifficulttoassess,thematerialbalance on cell culture is typically performed only on “dry” matter, excluding water. Our discussion on stoichiometry will be largely based on dry biomass of cell number, as commonly practiced in cell culture.

Macroscopically, cells are made of a few classes of macromolecules (protein, DNA, and RNA) or macromolecular assemblies (primarily lipid bilayer membranes). These organic matters constitute the vast majority of the dry mass in a cell. Protein molecules constitute the largest portion among them, providing the machinery for DNA, RNA, and protein synthesis. Protein molecules also serve as the structural components of the cell and execute all of the catalytic, transport, and communication functions. The lipid content of an animal cell is greater than in a bacterium. The abundance of organelles contributes to their higher content and their lipid bilayer membrane in an animal cell.

Intracellular carbohydrates exist as oligosaccharides on many proteins and lipids. Carbohydrate also exists as ribose in DNA, RNA, and nucleotides (e.g., ATP, GTP, etc). Only a small fraction exists in a free (or phosphorylated) form. The cellular content of carbohydrate is harder to estimate, because it usually exists as a part of other molecules.

Table 2. Average Composition of an Animal Cell

Pg/Cell Range Percentage % of Dry Biomass

Wet weight 3500 3000 - 8000Dry weight 600 300 - 1200Protein 250 200 - 300 10 - 15 ~50 - 70Carbohydrate 150 40 - 200 ~1 - 5 ~5Lipid 120 100 - 200 ~1 - 2 ~5DNA 10 8 - 17 ~0 .3 ~2RNA 25 20 - 40 ~0 .7 ~4Water 55 - 80Volume 4x10-9cm3

Table 1 . Typical Dry Weight of Cells

Bacterial 10-12 g / cellYeast 10-11 g / cellAverage Animal Cell 3-6 x10-10 g / cell

kinetics of cell growth, and also gives the typical values of stoichiometric and kinetic parameters commonly encountered in cell culture processes.


Material Balance on Cell GrowthThe principle of material balance holds true in cell culture. The total mass of inputs and outputs, and the amount accumulated in a system, is always in balance. The two most common, and most abundant, nutrients in cell culture are glucose and glutamine (although some cultures do not require glutamine). They serve both as constituents of cell mass and as sources of energy. Other common inputs to cell culture processes include lipids, lipid precursors, vitamins, and salts; these will be discussed in the Medium Design chapter, as these nutrients supply key cellular constituents, but contribute less to generating energy.

To generate energy, glucose is converted to lactate, CO2, and H2O through glycolysis, the TCA cycle, and the pentose phosphate pathway. Glutamine is deaminated, releasing NH3, before its carbon skeleton is used for energy metabolism. Consequently, the accumulation of metabolites, including lactate, NH3, CO2, and H2O, is commonly seen in cell culture. In many cases, the amino group from the metabolized glutamine and other amino acids are exported as non-essential amino acids (such as alanine, asparagine, and proline), in addition to being excreted as NH3.

Energymetabolismsatisfiestheenergeticneedsofmaking biomass, through the biosynthesis of DNA, RNA, protein, and organelles. Other important energy-intensive aspects of cellular events are the uptake of nutrients and the sustained balance of cellular osmosis and membrane potential. As will be discussed later, the cell’s cytoplasmic and mitochondrial membranes have a negative electric potential that must be maintained, at the expense of energy, to sustain cell viability

The process of growing cells and producing a product can be formulated into an “overall biomass synthesis” equation. This equation can be viewed as, essentially, the “apparent” composite of all reactions involved in generating energy and synthesizing biomass. At the center of the reaction will be biomass, so a formula for the cell mass

Growth of Biomass Involves:• Consumption of nutrients

• Production of new biomass

• Excretion of products

GlucoseAmino acidsOther

macronutrients (lipid, nucleotides, etc.)

Micro-organic nutrients (vitamins)

Bulk saltsTrace mineralsOxygen

→ CELLS →

BiomassProductCarbon dioxideWaterLactate, NH3Excreted amino

acids

C6H12O6 (glucose) + 0 .15C6 .14H12 .36N1 .50O2 .08 (weighted average of amino acids) + 0 .34C5H10N2O2 (glutamine) + 1 .39O2

→2 .37CH1 .97N0 .26O0 .49 (cell mass) + 0 .0058CH1 .83N0 .14O2 .06 (antibody) + 1 .53CO2 + 1 .28H2O + 1 .44C3H6O3 (lactate) + 0 .16NH3 + 0 .13C4H7NO2 (alanine)

Example of the “equation” for hybridoma growth:

Such a formula is used when one performs metabolic flux analysis.


Variation in Cell Volume The volume of a typical animal cell is a few pico liters (about 1,000 times larger than bacteria). The average cellular diameter ranges from 10 to 20 µm. Even for the same cell line, one can detect great size variations over a range, since cells immediately, before and after mitosis are about two times different in their size. At a given time in a growing culture, cells are in different stages of the cell cycle and their size distribution is somewhat larger than two fold. For aneuploid cells, the distribution of size is typically greater than normal diploid cells.

The distribution of cell size changes with the growth stage. Rapidly growing and quiescent cells may have different sizes. Furthermore, in

Table 3 . Size of Animal Cells

Cell Type Volume (μm3) Diameter (μm)Hybridomas 12 - 20

Endothelial Cells 1400 - 2500 17Trypsinized and reattached before spreading occurs 2000

Chinese Hamster Ovary cells (suspension) 1200 - 1800 ~14

Chinese Hamster Ovary Cells (anchored) 1300 - 1800

Human foreskin fibroblasts (FS-4) 7000

can then be established, based on its elemental composition. Usually, we use a formula that neglects all elements except for carbon (C), hydrogen (H), nitrogen (N), and oxygen (O). This formula is only useful for describing the ratio of those elements. One can arbitrarily assign the stoichiometric numbers to give them different “formula weights”. In the example shown, the stoichiometric number of carbon is chosen to be 1. Others may prefer to assign the formula mass to be 100.

The inputs in a cell growth process include all nutrients consumed by cells to proliferate. Since only C, H, N, and O are considered in the stoichiometric equation, we consider only glucose, glutamine, and other amino acids. Other minute media components containing C and N (such as vitamins or nucleotides) are neglected. Instead of writing down all amino acids separately, one may also use a weighted average, according to the stoichiometric ratio of their consumption.

The outputs include “new” cell mass that has been generated as the result of nutrient consumption, as well as the metabolites and product that have been excreted. One can write a molecular formula describing the formation of the protein product, by knowing its composition. Typically, the metabolites excreted also include lactate and ammonia, as well as some non-essential amino acids.


a culture, cells that lose viability often become visibly smaller, as measured by flow cytometry.

Cell size varies amongst different cell types. Many types of stem cells are fairly small. Their nucleus spans more than 70% of the cell diameter and their cytoplasm is relatively small. Liver cells and antibody-secreting plasma cells are at the other end of thecell size spectrum,andcontaina significantamount of cytoplasm for protein secretion.

It is instructive to remember that the volume of a sphere (which is a reasonable approximation of a cell) is proportional to its diameter raised to the third power. Therefore, cells that are twice as large in diameter are eight times larger in cell volume. Although cell number is traditionally used for the quantification of cellconcentration, it may not sufficiently capturethe difference when comparing different processes, in which cell sizes are very different.

• Cell Volume exhibits a distribution at any point in time

• Dead cells are often smaller

• Varying with culture stage

Fig. 6.1: Cell size change during a batch culture


Amino Acid Composition Microbial and plant cells often grow on simple carbon sources supplemented with an inorganic nitrogen source, such as ammonium or urea. The cells convert inorganic nitrogen to all 20 natural amino acids that are used to make proteins. Animal cells lack the capability to make 11 to 12 of those 20 natural amino acids. These essential amino acids must be supplied for animal cell culture, to enable them to grow and make products. Thus, knowing the amino acid composition of cells, and of the protein product, is important.

The protein content and composition of cells change under different growth conditions; however, they are seldom measured. Nevertheless, literature values of some cells are available, as well as the amino acid composition product, IgG. Given target levels of biomass and product to be produced, a stoichiometric amount of all essential amino acids must be supplied.

In addition to essential amino acids, which must be provided, non-essential amino acids are usually also supplied. However, these can be derived by metabolic transformation from other amino acids and can be considered “substitutable”.

Table 4. Amino Acid Composition of Cells and IgG

Cell CompositionMean

Standard deviation

IgG composition

ALA 9 .03 0 .32 5 .31ARG 4 .74 0 .32 2 .43

ASN10 .08 0 .59

3 .49ASP 3 .95CYS 0 .26 0 .04 2 .43GLN

12 .62 0 .635 .01

GLU 5 .16GLY 9 .14 0 .57 6 .98HIS 2 .22 0 .07 1 .67ILE 5 .73 0 .35 2 .43LEU 9 .00 0 .68 6 .83LYS 6 .85 0 .49 6 .98MET 2 .27 0 .14 1 .37PHE 3 .73 0 .31 3 .49PRO 5 .51 0 .58 7 .13SER 6 .19 0 .16 12 .90THR 5 .42 0 .22 7 .74TYR 2 .73 0 .14 4 .10

VAL 6 .54 0 .27 9 .10


Intracellular Fluid Water constitutes 70 – 80% of total cell volume. The soluble components in water also make up a large fraction of the biomass. The intracellular fluid contains electrolytes, carbohydrates,amino acids, metabolism reaction intermediates, nucleotides (ATP, ADP, etc.), and many other components. Most amino acids are present at the 0.05 – 0.5mM range in the intracellular fluid.The vast majority of intracellular amino acids reside in cellular proteins and only a tiny fraction exists as free amino acids in intracellular fluid.

Typical concentrations of other major soluble components in intracellular fluids are listed inTables 5 and 6, along with the typical extracellular environment conditions, in vivo. Potassium, magnesium, and phosphate are present at high concentrations. A large fraction of Mg2 + is associated with ATP, which is typically present at the 1 – 3 mM range. In addition to free phosphate, phosphate is also present in phosphorylated compounds (DNA, RNA, nucleotides, phosphorylated sugars, lipids, etc).

Many inorganics, including phosphate, potassium, and magnesium, are present at much higher concentrations intracellularly, compared to the extracellularfluidorculturemedium.Ascellsgrow,they take up nutrients at large enough quantities that they accumulate in the intracelluar fluid. Itis important to ensure those nutrients are also supplied in sufficient quantities. By knowing theirintracellular content, the stoichiometric amount required to produce the biomass can be estimated.

In addition to the major inorganic species (K+, Na+, phosphate, Mg2+, and Cl-), many minute inorganic elements are also constituents of cellular components, including iron, copper, selenium, zinc, cobalt, etc. Many primarily exist as a prosthetic group of proteins. These elements must also be supplied in enough quantities to generate biomass. Unfortunately, the cellular content of those elements are seldom reported and may vary widely among different cell types or even under different culture conditions. For example, the zinc content is much higher in pancreatic cells than in other cells. Similarly,

Table 5. Intracellular Concentrations of Amino Acids

mM mM

Ala 0 .2 - 2 .0 Lys 0 .1 - 0 .6

Ang < .05 Met 0 .01

Asp 0 .4 - 0 .8 Ornithine 0 .120

Asn 0 .4 - 0 .8 Phe 0 .3 - 0 .5

Asp 0 .306 Proline 0 .137

Citrulline 0 .036 Ser 0 .149

Glu 0 .3 - 12 Thr 0 .1 - 4

Gln 0 .05 - 4 Tyrosine 0 .059

His <0 .05 - 0 .09 Valine 0 .171

Ile 0 .3 - 0 .5

Leu 0 .1 - 0 .4

Table 6. Approximate Concentrations in Cellular Environment

Interstitial (mM)

Intracellular (mM)

Na+ 140 6 - 14K+ 4 .0 100 - 140Ca2+ 1 .2 0 .01Mg2+ 0 .7 3 - 20Cl- 108 4HCO3

- 28 .3 10HPO4

2- H2PO4- 2 11

SO42- 0 .5 1

PhosphocreatineCamosine 14Amino acids 2 8Creatine 0 .2 9Lactate 1 .2 1 .5Adenosine triphosphate 1 .5Glucose 56 0 .05Protein 0 .2 3-4


Growth of Mammalian CellsA cell culture process can be described by its cell growth curve, nutrient consumption curves, and product concentration profile. Cell growth, ingeneral, is divided into different growth stages: lag phase, exponential growth phase, stationary phase, and death (or the decline phase). The exponential growth phase is characterized by a linear increase in cell concentration on a semi-logarithmic plot over time and is easy to determine. In many cases, the extent of cell expansion in a culture is small (only 3 – 5 fold increase in cell concentration). Also, the time point for the transition from lag phase to exponential phase and from the exponential phase to stationary phase may not be clearly depicted in a growth curve.

Some cultures may experience a slow, or no-growth, period ranging anywhere from a few hours to a few days. This may be caused by using the inoculum from cultures that had already reached a stationary or decline phase, or by inoculating cells into vastly different media or culture conditions. Low inoculum cell concentrations may also cause poor initial growth,duetoinsufficientconditioningfactorsintheculture medium. In general, anchorage-dependent cells should be inoculated at a minimum of 105

cells/mL or 104 cells/cm2; while suspension cell cultures are generally started at about 105 cells/mL.

The exponential phase is marked by a constant cell growth rate, or doubling time. Doubling times of different cells span over a wide range, even under favorable culture conditions. For instance, while mouse embryonic stem cells divide every 11 – 12 hours, cells commonly used in bioproduct production double every 15 – 30 hours, depending on medium composition. Some human fibroblastic cells takenearly two days to double their number, even under optimal conditions with a high serum concentration.

iron is rich in muscle and red blood cells. Without quantitative data on the cellular contents of those elements, one can only resort to titration experiments underdefinedcultureconditionstoseewhetherthesupply of those elements is limiting the cell growth.

Table 7 . Doubling Time of Some Culture Cells

Cell type/contitions Doubling Time

Mouse embryonic stem cell ~12 hr

Human Diploid Fibroblast (10% FBS) 24 - 40 hr

Human Diploid Fibroblast (2% FBS) 40 - 60 hr

CHO K1 (5% FBS or in rich medium) 16 - 24 hr

NSO cell 16 - 24 hr

Fig. 6.2: Growth phases in a batch culture


After a period of rapid cell growth in the exponential phase, the cell growth rate reduces and the culture enters the stationary phase. Many reasons may cause this transition, including the exhaustion or suboptimal supplementation of key nutrients in the culture, and/or the accumulation of growth-inhibitorymetabolites. A flat cell growth curve inthe stationary phase may indicate that a culture that has ceased to proliferate. It may also reflecta balance between cell growth and death. In the latter case, the growth curve is characterized by a constant viable cell concentration, along with an increasing concentration of dead cells.

In the decline, or death phase, viable and total cell concentrations decline due to an exhaustion of key nutrients, the accumulation of metabolites to an inhibitory level, or adverse culture conditions (such as high osmolality). The growth behavior at a late stage of culture often varies, depending on whether cells are adherent or in suspension. Many anchorage-dependent cells grow substantially slower once celldensityon thesurfaceapproachesconfluence.Theycanalsosustainaconfluentcelldensity,overa period of days, without entering the death phase. Conversely, cells grown in suspension, especially those with an inherently strong receptor-mediated apoptotic mechanism, often enter the death phase soon after the viable cell concentration peaks.

In a production process, the rate of product formation is at its highest when cell concentration is maximal. A good production practice is to reach the target cell concentration quickly, while sustaining the culture as long as possible at that point.


Quantitative Description of Cell Growth & Product FormationA cell culture process can be characterized by its cell growth, nutrient consumption, and product accumulationprofiles.Toquantitativelydescribecellculture kinetics, three classes of quantities are used: concentrations of components (e.g., cells, nutrients, metabolites, and product), activity parameters (e.g., specific rates of growth, nutrient consumption,and product formation), and stoichiometric ratios.

After obtaining the concentration profiles of keyprocess variables, the next step is to calculate the rate of change of cell, nutrient, and product concentrations. These are referred to as volumetric rates because they are normalized to culture volume. A quickly-changing culture has a high volumetric rate, which may be the result of having more cells, or by having cells that are more active.

Usually, cell volume constitutes only a very small fraction of the volume in a suspension cell culture. The volumetric rate is essentially based on the liquid volume. In some cases, such as with high-density solid microcarrier culture, the volume occupied by solid beads is large; thus liquid volume and culture volume are not equal. Usually the concentrations of nutrients, metabolites, and product are measured based on liquid volume, not on culture or reactor volume. In such cases, the “volume” used for different kinetic parameters must be clearly stated.

Another set of descriptors, called the specificrate, is normalized to a per cell basis. These activity parameters describe how active each unit of the cell is for activities like making new biomass (specific growth rate), consumingglucose (specific glucose consumption rate), orproducinglactate(specificlactateproductionrate).

Cell growth is autocatalytic, meaning the rate is dependent on the cell concentration. Because cell concentration may be measured in different ways, such cell mass, cell number, or even cellular DNA or protein, the specific growth rate mayalso be based on different cell measurements.

Specific Rates

Growth rate (G) (change in cell concentration per unit time, number of cells/L-hr; or gm of cells/L-hr)

x: viable cell concentration

Specific Growth Rate (μ) (number of cells/cell-hr or gm cell/gm cell-hr)

Doubling Time

Specific Nutrient Consumption Rate (gm nutrient/gm cell-hr)

s: substrate (nutrient) concentration

Specific Product Formation Rate

p: product concentration

Gdtdx= (Eq . 1)

dtdx x

x dtdx1

n

n

=

=(Eq . 2)

(Eq . 3)( )ln

xx t t1

22 1n= -

.lnt 2 0 693d

n n= =

(Eq . 4)q

x dtds1

s = -

(Eq . 5)q

x dtdp1

p =


Quantitative Description of Cell Growth & Product FormationDifferent measurements may give somewhat different specific growth rates, because cell size,cell mass, and cellular content are not necessarily proportional to each other at all culture stages.

It is important to note the difference between volumetric cell growth rate (the number of cells/L/hr) and the specific cell growth rate (μ,number of cells/cell/hr or hr-1). The two are related quantities; one describes how fast the culture is increasing in cell concentration, the other describes how active cells are proliferating.

The cell doubling time is easily obtained from experimentally-determined growth curves. By separating the variables x and t into two sides of the differential equation and integrating with respect to time and cell concentration, respectively, one obtains the relationship between cell concentration andtime,givenaconstantspecificgrowthrate(Eq.3). The doubling time is the time period it takes to develop a two-fold increase in cell concentration.

In the case that non-viable cells are present in a significantportionofthecellconcentration,aspecificdeathrate,α,canbeincorporatedintotheequationfor cell balance, with xd representing the dead cell concentration (Eqs. 6, 7). In some rare cases, cell lysis occurs in culture. Lysed cells are not observable by cell counting. To describe the growth kinetics of such a culture, one can also include a cell lysis term.

Thespecificnutrientconsumptionrateisobtainedbydividing the volumetric nutrient consumption rate (ds/dt)bythecellconcentration.Thespecificproductformationrateisdefined,similarly.Inallcases,thecellconcentrationusedtocalculatethespecificrateis the viable cell concentration. In other words, one assumes that dead cells are metabolically inactive.

Experimentally,thespecificratescanbecalculatedfrom the changes in concentration over time. These specific rates are important for describingthe dynamics of various activities in cultures.

(Eq . 6)

(Eq . 7)

(Eq . 8)

(Eq . 9)

(Eq . 10)

(Eq . 11)

dtdx x x

dtdx x

x x x

qx dt

ds

x dtdx

x dtdx

1

1

1

vv v

dv

t v d

s

v

v

t

v

d

n d

d

n

d

= -

=

= +

= -

=

=

dtdx x xv x

dtdx x x

vv v v

dv v d

n d m

d m

= - -

= -

(Eq . 12)

(Eq . 13)

λv: Dead Cell Lysis Rate

A Model Considering Cell Death

With Cell Lysis


Stoichiometric Ratio and Yield Coefficient

In evaluating a process, we need to assess the efficiency of material conversion; that is, how much of a raw material is actually converted to the product or to cells. If we know a theoretical maximum of the conversion efficiency, or know its historical value, we can then assess how much the current process can be further optimized.

A yield coefficient is the ratio of the quantity of the product or cell produced, to that of the raw materials used. A yield coefficient for a cell can be based on different input materials, e.g., glucose, ammonium, or other nutrients.

In microbial processes, yield coefficient is frequently used to evaluate material utilization efficiency. The yield coefficient is given a symbol Yx/s or Yp/s, for cell mass or product, respectively, and is based on the consumption of a particular substrate. These variables are key indicators of process efficiency, because substrate cost is often a major portion of the total product cost. The efficiency of substrate utilization is essential.

Yield coefficient is rarely used in animal cell culture processes. Cell concentration in a typical animal cell culture process is rather low and is seldom measured in mass. Furthermore, bulk materials that are used by cells, i.e., glucose and amino acids, are not the major contributors to the cost of goods.

The stoichiometric ratio of various nutrients and metabolic products is more frequently used in cell culture processes. Under different metabolic conditions or in different growth stages, cells utilize various nutrients differently and change the amount of different metabolites produced. The stoichiometric ratios of various nutrients and metabolites are indicative of such alterations in metabolism.

For example, the stoichiometric ratio of lactate to glucose, e.g., the ratio of the amount of lactate produced to that of glucose consumed, is a strong indication of energy metabolism being at a highly glycolytic or highly oxidative state. If most glucose is channeled through glycolysis to

Yield of Biomass on Substrate

Yield of Product on Substrate

Stoichiometric ratio of lactate to glucose

Stoichiometric ratio of product to substrate

(Eq . 14),Ysx

dsdx

xs DD=

,Ys

pdsdp

ps D

D=

(Eq . 15)

,GL

dGdLa

DD= (Eq . 16)

(Eq . 17)

Description of the “State” of the Cultures

• Physiological state

• There is no unique or universal definition for physiological state. In general, the kinetic parameters described above are sufficient to describe the cell’s physiological state

• Metabolic state

• The stoichiometric ratios can be used to de-scribe metabolic state


Integral Cell Concentration Product accumulation rate in a culture can be described by multiplying the specific productformation rate by cell concentration (Eq. 18).

Integrate over the culture period from t0 to tf, to obtain the product concentration at tf .

If qp is constant, one can take it out of integral. Onecanseethatthefinalproductconcentrationisproportional to the integral of cell concentration. In a plot of cell concentration (x) vs. time, the integral is the area under the curve of the x curve. This is often called the integral cell concentration.

With the assumption that qp is constant, integral cell concentration is thus proportional to product concentration, and can be used as a firstapproximation estimate of product concentration.

(Eq . 19)

(Eq . 18)

Integral Cell Number is the Area Under the Growth Curve

lactate, the ratio is close to two moles of lactate per mole of glucose. Conversely, if most glucose is directed toward the TCA cycle for aerobic oxidation, the ratio will be close to zero, while the stoichiometric ratio of oxygen to glucose will be closer to six moles of oxygen per mole of glucose.

The stoichiometric ratio and yield coefficient, based on a given pair of compounds, can be expressed in different units, e.g., mol/mol or g/g.


Fig. 6.4: Plots of process data of a typical cell culture

Example of Experimental Data Processing and Plotting of a Fedbatch Culture

Typical process data include those from on-line and off-line measurements. On-line data are often continuously recorded except that some control actions (e.g. turning on base pump or oxygen valve) may be discrete in time. Off-line measurements are invariable only on discrete time points. The measured data should be plotted to discern inconsistency and outliers . Then the calculated (or derived) data are also displayed . The example data shows that the oxygen uptake rate (OUR) peaks slightly before cell concentration, since OUR is a more sensitive indicator of cell’s activity. O2 flow rate often reflects OUR. We also see that even though specific rates may change over time, some stoichiometric ratios remain constant.


• Stoichiometric ratios change under different metabolism

• In a fedbatch culture with glucose or glucoflutamine level control, the stoichiometric ratio change can reflect metabolic shift

• A combination of stoichiometric ratios can reflect the metabolic states of cells .

Myeloma cells were grown in batch or fedbatch cultures. In the fedbatch cultures, glucose and glutamine were at lower concentrations, initially. Once the concentration reached the set point, concentrated glucose or glucose/glutamine was fed continuously via computer control to maintain the concentration(s) around the set point. The final product concentration, the amount of glucose, glutamine, and oxygenconsumed, and the total amount of lactate and ammonium produced were used to calculate the stoichiometric ratios. The duration of fedbatch culture was substantially longer than the batch culture.

It is notable that more glucose, glutamine, and oxygen were consumed in the fedbatch culture, because the culture lasted longer and the cell concentration was higher. However, from the stoichiometric ratio, one can see that the lactate to glucose ratio decreased from the initial value, similar to the batch culture, indicating a metabolic change by limiting glucose at a low level. This also indicates that by controlling glutamine, one can affectlactateproduction.Thismetabolicchangeisconfirmedbytheoxygen/glucoseratio.Thelowerlactate/glucose ratio was accompanied by a higher oxygen/glucose ratio, suggesting that more glucose was channeled into the TCA cycle for oxidation. Glutamine control also resulted in a lower production of ammonium.

Table 8. Typical stoichiometric ratios of hybridoma cells under different metabolism in batch and fed-batch cul-tures

Batch Fedbatch with Glucose Control

Fedbatch with Glucose Glutamine Control

Growth Data Initial glucose conc. (mM) 17 1 .4 1 .4

Initial glutamine conc. (mM) 4 0 .3 0 .3

Glucose conc ./set point (mM) 0 .55 0 .55

Glutamine conc ./set point (mM) 4 ~0 .5 0 .2

Maximal viable cell conc . (106 cells / mL) 2 .4 12 10 .5

Antibody conc. (mg / L) 8 60 200

Consumption/Production (mmole/mmole)

Glucose consumption 12 46 .5 27

Glutamine consumption 4 17 10 .1

Oxygen consumption 24 143 122

Lactate production 3 37 .4 17

Ammonium production 3 9 .7 4 .5

Stoichiometric Ratio (mmole/mmole)

Lactate/glucose 1 .5 1.60 → 0.16 0.90 → 0.05

Oxygen/Glucose 1 .85 1.9 → 6.0 2.7 → 4.7

Ammonium/Glutamine 0 .75 0 .5 0.31 → 0.10

Alanine/Glutamine 0 .35 0.34 → 1.35 0.08 → 0.42

Osmolality (mosm / kg) ~350 410 400

Example of Stoichiometric Ratios as Indicators of Metabolic States


Kinetic Model of Cell Growth

A Model Describing Growth and Production

If we consider only a cell, a nutrient (e.g., glucose) and a product (e.g., a recombinant antibody) as the three most important variables in a culture system, the mathematical model will consist of three material balance equations for the concentrations of the three species. The change of cell concentration will be due to growth, which is described by the multiplication of specific growth rate and cell concentration.Similarly, the rate of change of substrate and product concentrations is described by multiplication of

A mathematical model can be used for different purposes:

(1) To summarize a large volume of experimental data. Data, or plots of data, are extremely difficult to describe in words. If data are fit to a mathematical model, regardless of whether the model is empirical or mechanistic, the behavior of the data can then be recreated, given the model and value of the parameters.

(2) To explore concepts and test hypotheses. When we study a physical system and its behavior (such as growing cells consuming glucose), we first develop a verbal description of the system and the behavior. We may then propose a hypothesis, also in verbal form. The verbal description can be translated into a mathematic form. The mathematical model can then be used to explore the system’s possible behavior under different conditions.

(3) To predict the behavior of the systems, given a model with sufficient complexity. The model can be used to predict regions of parameter space that have not been experimentally tested, previously. It can also be used to optimize or control the dynamics of the system.

With an increasing emphasis on the notion of Quality by Design, the application of the mathematical model will gain importance. Proper applications of well-posed models will help explore system behaviors and define optimal operating regions, in a potentially vast design space.

Utility of Mathematical Models

1 . Summarizing experimental data

2 . Probing concepts and testing hypothesis

3 . Predicting and optimizing processes


Kinetic Model of Cell Growth the respective specific rate and cell concentration.

These three equations describe only the balance of those three species. To describe the “dynamics” of the system, we will need to have description of howtheparameter(orthespecificrate)changesforimportant variable(s). For anchorage-dependent cells, the growth rate is dependent on the extent of cell “confluence”onthesurface.Modelsforanchorage-dependent cells, thus, attempt to describe the dependenceofthespecificgrowthrateoncelldensity.

For process design or optimization purposes, one first needs to identify the variable that mostaffects the process outcome and then develop a relationshipbetweenspecificgrowthrateand thisvariable. For example, if the glucose concentration is an important variable, then one can develop a model describing the relationship between growth rate and glucose concentration. Then, as the glucose concentrationchanges,sodoesthespecificgrowthrate. On the other hand, if the most important factor affecting the cell is lactate concentration, then one will need a balanced equation for the production of lactate and a model relating cell growth (µ), and possibly also specific glucose consumption andproduct formation rates, to lactate concentration.

Once a model is available, one would still need experimental data to identify the parameter values in the equations. Then, with appropriate initial conditions (i.e., the initial concentrations of cell mass, glucose, lactate, and product), the system behavior can be explored by simulation of the model.

A number of mathematical models have been tested for describing animal cell growth in culture. Most are based on Monod-type models that were traditionally used to describe microbial growth. Some employed more complicated structured or segregated models. These models are empirical in nature. However, using these models to predict growth conditions outside the range of conditions under which the kinetic parameters are obtained may not give reliable prediction.

What is needed to develop a mathematical description of a cell culture processs?

• A description for cell growth (and death), product formation, nutrient utilization

• Growth model - dependence of specific consumption rate on the “controlling variable” (growth rate, nutrient concentration, etc.)

• Product formation - dependence of specific production rate on “controlling variable”

• Experimental data - the model will have some parameters (such as half saturation constants). The experimental data are used to determine the value of those parameters

• Material balance equations for “state variables” (the concentrations of cell, nutrients, product, inhibitors, etc .)

Balance Equations for a Batch Culture Growth Kinetics

Cell balance

Glucose balance

Product balance

Lactate balance

Models describing growth rate or production rate dependence on environmental factors

Cell density dependence

Nutrient dependence

Inhibitor dependence

(Eq . 20)

(Eq . 18)

(Eq . 21)

(Eq . 22)

(Eq . 23)

(Eq . 24)

(Eq . 25)


A Monod model employs two parameters, μmaxand ks, to define the relationship between thespecific growth rate, μ, and the limiting substrateconcentration,s.Thespecificgrowthrateisaffectednot only by substrate concentration, but is also a function of pH, temperature, the status of other nutrients or growth supplements (e.g., serum), and the presence of waste products. In applying a Monod model, one assumes only substrate s is limiting and that all factors are supplied in enough quantities.

The term-limiting substrates have been used in two different contexts: 1) a stoichiometric-limiting nutrient refers to the nutrient that is first depleted in a batch culture and whosedepletion causes the cessation of growth; or 2) a rate-limiting nutrient is the nutrient whose concentration restricts the growth rate of cells.

The equation gives a saturation type of kinetic behavior; meaning μ increases with increasingsubstrate concentration, until it reaches a maximal value. At low concentrations, increasing the rate limitingnutrientconcentrationincreasesthespecificgrowth rate, linearly. At very high concentrations, the specific growth rate is constant at μmax.

A Monod model can be incorporated into the balance equations describing the batch culture growth kinetics to provide a relationship between the equations for cell growth and substrate. Indeed, the resulting equation can be used to describe batch growth when glucose is used as the limiting substrate. Starting at a high concentration of substrate, cells grow at μmax; as the substrateconcentration decreases so is the growth rate. The simulated growth curve will show the entry into stationary phase as the substrate decreases to a level where μ is substantially reduced and, eventually,growth ceases when the substrate is depleted.

However, animal cells require multiple nutrients for proliferation. In addition to glucose, glutamine is also a major nutrient. Many amino acids and other

If s » Ks, then μ → μmax

• Two parameters, (μmax and ks), define the relationship between μ and limiting substrate concentration s .

• μ is also a function of pH, temperature, nutritional status, (i .e . serum), waste products; it may also depend upon cell density for anchorage-dependent cells which are subject to contact inhibition.

Notes• These models are all empirical models that can be used

for first order approximation of cell growth

• Under most process conditions growth is not limited by nutrient availability

• For stem cells and primary cells, growth factors have more profound effects than nutrients

K ssmax

s

nn

=+

(Eq . 26)Modod Model

Fig. 6.5: Growth rate dependence on rate limiting substrate based on Monod model

Monod Model and its Derivatives


nutrients are also required, although often most are provided in excess and are not limiting. Monod modelsaremodifiedtodescribethedependenceofgrowth rate on multiple nutrients. Many include multiplicative terms to incorporate multiple substrate utilization and product inhibition.

In the multiplicative model for cell growth, two substrates, S1 and S2, are considered to be growth rate limiting. Each substrate has its corresponding half saturation constant for saturation kinetics. In most common applications, the two substrates are glucose and glutamine. The model can be extended to consider metabolite inhibition. For example, in the case where A and B are inhibitory metabolites. Most models consider lactate and ammonia as inhibitors.

We have discussed balance equations for cells, substrates, products, and the model relating growth rate to limiting substrate. Together, they can be used to describe cell growth in culture. For simple microbial systems, this is often sufficient.However, for animal cells, the specific substrateconsumption and production rates are profoundly affected by their environment. One, thus, also seeks to describe the relationship between qx, qp, and key variables affecting their behavior.

The kinetics of product formation in microbial systems are frequently categorized, according to their relationships to growth rate. qp is considered to beinfluencedbytwofactors:a“growth-associated”term, α, which describes dependence on specificgrowth rate; and a “non-growth associated” term, β. Depending on the relativemagnitude of α andβ, the production can be growth associated, non-growth associated, or mixed growth associated.

Such classifications of production kinetics areuseful for microbial fermentation of amino acids, organic acids, and antibiotics. However, their applicability to animal systems are limited. In general,thespecificproductionratesofanimalcellculture products are less sensitive to growth rate. For some recombinant proteins, the productivity

Specific Product Formation Dependence on Growth

pq αµ β= + (Eq . 29)

(Eq . 27)K ss

K ss

max

1 1

2

2 2

2: :n n=+ +

(Eq . 28)

Multiplicative Saturation Kinetics Model

Monod Model and its Derivatives

Environment, Kinetics and Stoichiometry


is somewhat higher in the stationary phase of fedbatch cultures. At that stage, many factors, including osmolality, lactate, and CO2 concentration, all have deviated from optimal growth conditions. It is possible that some of those factors exerted a stronger effect on productivity than growth rate.

Another complication in applying a Monod-type of model is that cell growth is rarely limited by substrate concentration in cell culture bioprocess. Cells enter the stationary phase often due to the accumulation of metabolites (e.g., lactate and ammonium) or the accumulation of salts (Na), due to base addition or CO2. Lactate and ammonium are produced from glucose and amino acid (primarily glutamine) metabolism, while Na accumulation arises from base addition to neutralize lactate and maintain pH. CO2 comes from both cell metabolism and pH control actions.

Stoichiometric and kinetic relationships among process variables are important in describing different cultures. From an experimental perspective, these parameters and variables provide a basis for quantitatively comparing different processes. They also allow the simulation of a culture’s kinetic behavior under different growth conditions. A high productivity process often includes a cell growth phase and a prolonged stationary phase, in which cells are kept at a high concentration and high productivity state. Growing evidences suggest that a switch of a cell’s metabolism from high

glucose consumption and high lactate production to low glucose and lactate consumption is the key to a high product accumulation. The simple models described in this chapter can be made more effective models by including a mechanistic description of how a cell’s metabolic shift occurs in response to environmental factors. Incorporation of a mechanistic model for describing cell culture bioprocesses will enhance our physiological understanding of cell metabolism and increase the utility of models for optimizing the process.

Concluding Remarks

CELL CULTURE DATA ANALYSIS | 167

Process Data Analysis and PAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .167Data Processing Pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .168

Standardized Templates for Data Logging and Processing . . . . . . . . . . . . . . . . . 168Cell Culture Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169A Typical Spreadsheet for Analysis of Cell Culture Data . . . . . . . . . . . . . . . . . . . 170Data Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172Mapping Data to Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .174

Process Data Analysis and PAT

Cell Culture Data Analysis

Process analytical technology (PAT) is gaining greater attention for its potential role in enhancing bioprocess robustness. PAT encompasses: 1) the acquisition of data pertaining to process and product attributes, related to both productivity and product quality, through on-line and off-line measurements; 2) the analysis of process data in each run; 3) developing and employing mathematical models to relate process variables to process outcome for a better control; and 4) the data mining and recognition of hidden patterns of behavior in historical data for further process enhancement. Every element of PAT has been used in process development for decades. The new emphasis is on integrating each individual element to create a more comprehensive understanding of the process and to generate useful information.

Modern production plants are thoroughly electronically monitored and controlled. They


Data Processing Pipeline

The importance of having a standardized data format for data collection and archiving cannot be overstated.

After collection, data is processed to remove faulty values (such as data resulting from wrong entry or sensor failure). The raw data obtained from different sources, such as from different analytical instruments, may have different units or be formatted differently and are homogenized. The data are then further used to calculate derived variables and are plotted for visualization. Finally, the data are archived and sometimes employed in data mining, for generating more process insights. Throughout this transformation, the data are transferred between many different software platforms.

Many of those data processing steps are often repeated over and over; not only in manufacturing settings but also in research environments. They are often performed by different workers at different times and at different manufacturing sites. Thus, setting a standardized data format and uniform data processing protocol is critical to increase efficiency and minimize frustration.

What exactly should be standardized? Highest on the list are the terminology, nomenclature, units of quantities, and data format. The same object should be named the same way, consistently across all data sets, and should use the same symbol, units,

Use Standardized Template in Data Processing• Speed up routine data analysis

• Automatically perform calculations• Automatically generate standardized plots • Automate data regression and calculation of

estimated values.

• Ensure consistency and accuracy• Uniform terminology, units, graphs• Built-in formulas minimizes mistakes in calculations• Up-to-date set of key plots eases communication

and data interpretation within teams.

• Facilitate data archiving• Develop a format which allows easy transfer

of data to other programs for analysis and visualization

• Incorporate other important parameters, such as passage number, medium composition, and operating information.

• Additional Consideration of Spreadsheet Templates• Metabolic flux analysis, visualization process

modeling or modeling .• Data archiving:• Searching• Experimental / run details• Standardized upload / download platforms

Standardized Templates for Data Logging and Processing

employ instrumentation in the reactor for process monitoring, as well as to measure raw material and product quality. A significantchallenge in the application of PAT is the accumulation of the vast amount of data, which can potentially impede process understanding.

The key to successfully implementing PAT is, thus, to produce a better pipeline for data processing and to have a better appreciation of methodologies for bioprocess data analysis.


andequalnumbersofsignificant figures invalues.

When treating data, the formula must be uniformly applied to different data sets. However, inadvertent mistakes may occur through incidents, such as errors from formula entry. To facilitate the workflow,theuseofauniformdataformatandtheminimization of inadvertent errors are advisable to set up data entry and process templates for various programs. The same templates should be used by all colleagues performing similar experiments.

By setting up a template, various tasks can be automated. For example, upon the entry of experimental data into the spreadsheet template, the related specific rate and stoichiometricratios can be automatically calculated, and their profiles over time can be plotted instantaneously.With templates for different software programs in place, even data transfer from one software program to another can be automated.

Cell Culture Data Processing The first level of bioprocessdata is rawdata thathas been acquired from various measurements, including the concentrations of cells, nutrients and metabolites, pH, and oxygen. Subsequently, stoichiometricratiosandspecificratesarecalculatedover a time course to discern the trend of metabolic and productivity changes in the entire culture. The stoichiometric ratio can then be determined from the amount of nutrients consumed over a time period,andisanintegralquantityinnature.Specificrate is the rate of change of the quantity, further divided by cell concentration. It is determined from the slope of the curve of the quantity produced or consumed over time. Specific rates are,therefore, quantities determined by differentiation.

Except in simple batch cultures, the concentration profiles of nutrients and products are often notmonotonic functions. Even culture volume may not be constant. Feed medium is added occasionally, resulting in step changes in nutrient and metabolite levels. Typical culture profiles, thus, often entaila step increase in nutrient levels after feeding and a step decrease in metabolite levels, due

Three Types of Data

• Measurement Data• Cell concentration

• Nutrient and metabolite concentrations

• Process parameters: pH, DO, temperature, etc.

• Calculated Integrated Data• Cumulative nutrient consumption and metabolite

production

• Integral viable cell concentration (IVCC)

• Calculated Differentiated Data• Specific rates

• Stoichiometric ratios

Two Types of Calculations

• In-process calculations• Process monitoring

• Troubleshooting and diagnosis

• Post-process calculations

• Data smoothing for analysis


to a dilution caused by the volume of the feed.

Thefirststepindataanalysisisthecompilationofcumulative data pertaining to nutrient consumption and metabolite production/accumulation. Insteadof using concentrationprofiles toperformcalculations, the cumulative amounts of nutrients consumed and the amount of product produced are calculated. The cumulative curves are mostly monotonic, except for those few nutrients or metabolites that are consumed at one time and producedatanother. Aconcentrationprofilewithmany step changes cannot be easily subjected to regression. Cumulative curves have no step changes, thus allowing a regression to be performed on the entire profile. The regression equation is thenused to calculate the slope for the determination of stoichiometric ratios and specific rates.

Cell culture processes often extend over a long duration, from 5 to 15 days up to a couple of months. Data processing starts while the process is still in progress and continues after its completion. In-process data analysis allows for potential outliers and faulty conditions to be detected and corrected. Post-process analysis often involves additional chemical analyses to provide more process insight. A template spreadsheet can be used for the first stage of data analysis.

A Typical Spreadsheet for Analysis of Cell Culture Data

A spreadsheet template shall contain columns of raw data entry followed by columns of cumulative data for all measurement data for nutrients which are consumed and products. Any time point of row that incurs feed addition will incur a total mass balance that takes volume change into consideration.

A simple way to account for volume change is to perform calculations in total mass balance (concentration of nutrient: x volume) instead of on concentrations alone. In this case the amount consumed between two time points is simply StVt- St+1Vt+1)=St cumulative consumption is then the sum of St over time. Forgetting to account for volume change in material balance is a


common mistake in fedbatch culture data analysis.

The calculation of cumulative data can be automated once the measurement data are entered. The next setof columnsare specific rates. Specific ratesarebest calculated for cumulative data by regression. The regression of cumulative data can be automated. Usuallyathirdorderpolynomialfittingworkswellfor most data. However, inspection is necessary to ensure a good fit. The measurement data,cumulative consumption/production and specificrate data shall be all automatically for visualization.

Upon the calculation of cumulative data stoichiometric ratios are also automatically plotted. This allows for detection of metabolic changes in culture.Ifastoichiometricratiodeviatessignificantlyfrom historical data, it may also serve as a diagnosis alert for checking pasable process abnormality.

An add-on to the spreadsheet template is an algorithm for metabolic flux analysis. If the measurementsinclude all the major carbon compounds, glucose, lactate, glutamine and other amino acids (if not all, the majority), ammonium, then material balance can be performed on the nitrogen balance. Carbon balance will require the measurement of CO2 produced in metabolism and is not easily done without isotope labeling. If oxygen consumption data is available, one can assume that R.Q. being 1.0 and set CO2 production to be same as oxygen consumption.

From the extent of carbon, nitrogen balance one can assess the reliability of some stoichiometric ratio data. If the carbon and nitrogen is reasonably closed the data can then be further subjected to metabolic flux analysis.MFA algorithm is typicallyin MatLab or other mathematical solvers. The Excel template can build in an exportable table for ready transferofthespecificratedatatothoseprograms.

• Two-point specific growth rate calculations and specific nutrient calculation for fedbatch culture

qx V dt

dSx V x V t t

S S1

2

12 2 1 1 2 1

2 1s $

$$ $

$.=+ -

-

• Slope calculation from curve of regression data

Specific Rates

Cumulative Data

IVC V dt IVC v vt

t

t t t t t

0

1 1 1$ $ $ $.\ \ \= + -- - -#

S q x V dt V s V s V s, , 0 , ,i t i t

t

t i t t i t f

k

f

00

0 k k$ $ $ $ $ $= = - +/#Si,t: Cumulative amount (mMole or g) of nutrient i

consumed or produced at time tV: Volume of culturesi: Concentration of component i in the culture brothVf: Volume of feed medium addedsf: Concentration of component i in the feed mediumk: Total number of feed medium additions up until time tIVC: Integral Viable Cell number

ai,j: Stoichiometric ratio for nutrient i with respect to nutrient j at time t

S SS S

SS

, ,

, ,

j t j t

i t i t

j

i

t2 1

2 1

TT

--

= c m

Stoichiometric Ratio

Fig. 7.1: Plots of cumulative consumption data


Multidimensional/Interactive Data Exploration

Data Visualization Visualizing the data is critical for developing a deeper understanding of the effect of various parameters on process performance. Each cell culture run usually entails many measurements over multiple time points. Instead of browsing data through tables, we plotted each quantity as a concentration profile over time. We alsoplotted data of one variable against another variable, to specifically examine the ratio ofspecific rates or the stoichiometric ratios.

As data accumulate over time, it is even more important to plot data of multiple runs together, so that different runs under the same or different experimental conditions can be compared easily. In such analyses, mathematical and statistical tools are important; however, the importance of data visualization in the analysis of multiple runs cannot be overemphasized.

When working with a large set of data from multiple runs, visualization software is very useful. Quick access to data, the rearrangement of data into different combinations of dimensions, or the filteringofdatabydifferentprocessperformanceor other criteria can greatly facilitate deeper insight. In the plot shown, lactate concentration profiles from over 250 runs are colored by thefinalproducttiter.Onecanseethathigh-titerrunsmostly consume lactate in the late stage of culture, while the low-titer runs produce lactate. The trend is easily seen when all data are plotted together.

We then plot the specific rates of glucoseconsumption and lactate production at different time points, for all runs. It can be seen that lactate consumption (negative values) occurs only when glucose consumption is also low. One can further see that even when the cells are consuming lactate, the glucose consumption rate is still significant.

With the aid of a visualization tool, data from all of these runs can be easily plotted in different ways. To take advantage of the data trove from large number of runs, a means for quick visualization is very important.

• Process data are intrinsically multidimensional and should be examined in multiple dimensions (e.g. time course and stoichiometric ratios) to provide different insights

• Data from multiple cultures can be consolidated and examined for trends

• Software for visualization interactive for multiple dimensional viewing analysis – E.g.: Spotfire DecisionSite .

Fig. 7.2: Plots of archived historical data for discerning process patterns


Mapping Data to PathwaysPathway related data is another type that its analysis can greatly benefit from a visualizationtool. An example is the fluxes through differentreaction steps in various pathways obtained from metabolicfluxanalysis(MFA).Anotheristhegeneexpression transcript level data from microarray analysis. Presentation of the flux data on apathway map makes it easy to compare metabolic change over time or under different conditions.

An example below shows flux distribution incells grown under three different conditions that

also exhibit three different glucose consumption rates. By plotting fluxes on to a metabolic map,it can be seen that high glucose consumption rate leads to high lactate production, high glutamine consumption and high TCA cycle flux.

Such visualization will become almost essential when dealing with very complex pathways like glycan biosynthesis on proteins passing through Golgi apparatus before being secreted.

Metabolic Flux Analysis/Pathway Mapping

Metabolic flux analysis uses culture data to estimate fluxes of intracellular species through metabolic pathways . It is useful to transfer these numbers onto a map of central metabolism to visualize the differences among calculated values .

Mapping of data into formats that incorporate physiological information facilitates interpretation of complex datasets .

This example compares fluxes of lactate production state and lactate consumption state .

Fig. 7.3: Metabolic chart for visualizing metabolic fluxes


Concluding Remarks

Visualization of Glycan Profiles• The Pathways leading to N-glycans form a

complex network• A small number of enzymes are involved to

form a large number of glycans• Most enzymes are used multiple times• Most glycans, intermediate glycans and late

glycans may all appear in the final product• Visualizing the distribution of glycans can

help deduce the plausible paths taken to from each species

Comparison of Intracellar and Culture Medium N-Glycan

Fig. 7.4: Visualization of glycosylation fluxes

Data analysis holds the key to understand and improve the process. A large amount of software for data processing, analysis and visualization is available. Setting up a consistent and efficientway of processing and plotting the data can be hugely beneficial. A set of templates areincluded in the template folder, along with some

sample data to demonstrate the calculation and plotting. They can be used as a starting point for modificationtomeet individualneeds. Theuseoftemplate will also make it easier to compile and analyze historical data accumulated over time. A good practice of data processing and analysis is fundamental to process analytical technology.

METABOLIC FLUX ANALYSIS| 175

Overall Material Balance for Reaction Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175Chemical Reaction Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

MFA on a Cellular System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181Utility and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181General Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183


Overall Material Balance for Reaction Systems

Metabolic Flux Analysis in Cell Culture Systems

In cultivating cells, it is sometimes necessary to know where and through which pathways the absorbed nutrients have traveled. This allows one to manipulate the distribution of nutrients and to optimize the process. One useful tool for performing such tasks is material balance analysis. When such balance is used to analyze the distribution of materials in biochemical systems (either on whole cells or on specific pathways),it is called metabolic flux analysis (MFA).

Metabolic reactions are chemical reactions. They are governed by stoichiometric principles. However, there are special characteristics in cellular systems that set them apart from chemical reaction systems. In a test tube or in a chemical reactor, the reactions (i.e., the conversion of reactants to products)occurdirectlyinthefluidphase.Forcellsin culture or in tissue, the reactants (i.e., nutrients) are delivered to individual cells. Products are also

METABOLIC FLUX ANALYSIS | 176

formed in and excreted by individual cells into the fluid phase. The reactions take place not ina continuous phase, but in many discrete cells.

By applying MFA to cells in culture, one could theoretically combine all of the cells’ mass into one entity and could consider all cells as one biotic phase andthefluidasoneabioticphase.Allofthenutrientstaken up by cells (not just the nutrients added to the culture) and products excreted into the fluid areconsidered to be inputs and outputs, respectively.

Sometimes, MFA is applied to a pathway or a group of pathways. In this case, the balance is applied to a system, which does not have a physical boundary, like a cell or even a group of cells. The pathway of interest (for example, glycolysis or mitochondrial reactions) occurs in many different locations within the cell mass. And yet, we would treat all reactions of interest in ALL cells as a system and perform a balance on it.

Another unique characteristic of the cell system is that cells are growing and expanding in biomass, as well as in cell volume. The output of cellular reactions, thus, includes cell biomass. Furthermore, because the volume of cells is expanding, it has a dilution effect on the concentration of cellular materials.

In this chapter, we will first use a familiarchemical reaction system to emphasize the notion that the fundamental concept of MFA is material balance. This will then be followed by a discussion of the basic steps in MFA.

Chemical Reaction SystemsConsider a chemical reaction system, in which multiple reactions are taking place simultaneously andwewanttodeterminetheextent,ortheflux,ofeachreaction. We can solve this problem by performing measurements on the concentrations of compounds in the inflow and outflow streams of the systemand their relative flow rates. Then, we can applymaterial balance on the stoichiometric equations for all reactions known to occur in the system.

In the example shown below, we aim to determine


Material Balance on Reaction Systems

Example: Incomplete combustion of CH4 to CO2, CO and H2O in a batch reactor. Two reactions can possibly occur. We don’t directly measure the extent of each reaction, but we can measure the inputs and outputs at the beginning and the end of reaction, and see how much has been consumed (i.e. the amount of reactants left at the end subtracted from the amount put in initially), and how much has been produced. From these balanceswecancalculatethefluxofeachreaction.

Known Reactions Output4 2CH O+ → 4 2 2 2

14 2 2

2 2

2 3 2 4

CH O CO H OCH O CO H O

ξ

ξ−

+ → +

+ → +2 2

CO CO H O→ + +

If all the inputs and outputs (i.e., the amount of CH4, O2 consumed and that of CO2, CO, H2O produced) are completely balanced, the fraction of CH4 going to each reaction can be determined. On the other hand, if the material balance is not closed, there will be uncertainty about the solution, and the distribution of materials can only be estimated.

Case I. 4 moles of CH4 and 7 moles of O2 are combusted to produce 2 moles each of CO2 and CO and 8 moles of H2O.

In this case, all three elements involved—C, H and O—are completely balanced. The only unknown ξ can be easily calculated using a balance on any element, e.g., use C balance:

ξ = 0.5

ξ = 2 mole CO2

44

mole C4 mole CH 1mole CH

×

2

mole C1mole CO

×

InCaseI,inprincipal,onedoesnotneedtoknowthequantitiesofallinputsandoutputstofindthesolution.From three elemental balances (C, H, O), we can have three linear equations. As long as there are only three unknowns,wecanalsosolveforξ.Forexample,evenifthequantitiesofCOandH2O are not measured, the solution will be the same.However, in the real world, there is always a high degree of uncertainty about the accuracy of measurement; the overall balance is always an important check of the validity of the results.

Case II: 4 moles of CH4 and 7 moles of O2 are combusted to form 1.5 moles of CO2 and 1.6 moles of CO2. The amount of H2O produced is not measured. Determine the fraction of methane completely combusted.

In this case, the input and output materials are not balanced. There are a total of 4 moles of C combusted but only 3.1 moles are accounted for in the products. This presents a problem regardless of whether H2O is measured.

The cause of input and output materials not being balanced is not always known. It may be due to measurement error, or possibly other reactions have occurred but are not accounted for are not accounted for. In this case, we can only get a plausible answer of how much CH4 goes to complete and incomplete combustion. If we know the extent of various measurement errors and the amount of H2O, we can get a more reliable estimate.


We will use the methane combustion in a furnace as an example to illustrate how to set up equations for MFA. Although the problem is so simple that it can be solved using a piece of paper and a pencil, the approach shown is more systematic and will be suitable for calculating fluxes for alarge reaction system, such cellular metabolism.

Setting Up Material Balance Equations We will perform material balance on the materials consumed and produced, in order to determine the fluxes forthereactions involved. First,wedefinethe system, including its inputs and outputs (i.e., net consumption and production), as well as the chemical (or metabolic) reactions involved. In the example shown, two reactions are listed, for which the chemical formula of the compounds and the stoichiometric coefficients of the reactions are all known.

The rate of consumption and production is given

thefluxesofmethanefortworeactions,whichoccursimultaneously in the furnace, using measurements of compounds in the inlet and outlet. The concentration differences between inlet and outlet of the furnace give the value of net consumption and net production. They are the inputs and outputs into the reaction system. In case 1, the carbon, hydrogen and oxygen entering the system are all accounted for in the products. One can easily determine how methane distributes itself between the two reactions.

On the other hand, as in many cases, the material balance from the measurement is not closed. This happens frequently because of measurement errors. Sometimes, it can be caused by compounds in inputs or outputs that are not accounted for. For instance, there might have been some unknown reaction that occurred, leading to the emergence of products that are not measured and included in the output. Under such a situation, uncertainty on the outcome of material balance inevitably occurs. One then has to rely on the knowledge of the reaction system to make appropriate assumptions, or to perform further measurements.

A Systematic Way to Solve Material Balance Problems


thesymbol“q”.InMFA,q’sarealwaysthespecificrates. Consumption is designated as a negative value and production as a positive value. We expressthefluxofeachreactionas“J”.Bydenotingthe flux of reaction 1 as J1, the fluxes ofmethane,oxygen, carbon monoxide, and water reacted in reaction 1 are -2J1, -3J1, +2J1 and 4J1 respectively. Their fluxes are related by the stoichiometriccoefficients (-2, -3, 2, and 4) of the reaction.

Next, we set up a material balance equation for each species, which is present in the system. Each compound involved in the system is given a differential equation describing the change of its concentration in the reactor as the balance of its consumption, production, and other input and output, if any. The balance equation for CH4, thus, reflects that the rate of change of CH4 is the sum of its input (qcH4) and its reaction fluxes (-2J1).

Step 1 .Write material balance equations for every species

Quasi-Steady StateThe equations set up above are differential equations. For a short duration of time, the concentration of all species in the reactor (or in the cell) may not be changing very rapidly. In that case, the left-hand side of the differential equations can be assumed to be negligible (e.g., the system is assumed to be at a pseudo-steady state). With this assumption, the left-hand side of all differential equations becomes zero. The equations, thus, become a simple system of linear algebraic equations.

Stoichiometric Matrix, Flux Vectors and Solution

Tosolvealargesetofequations,itismoreefficientto perform matrix operations. In these cases, the matrix is called the stoichiometric matrix. The stoichiometric matrix is the collection of stoichiometriccoefficientsorganizedbyreactionsinone dimension, and by species involved in reactions in the other dimension. In the example shown, there are two vectors for J1 and J2, respectively. The two columns in the matrix correspond to the stoichiometriccoefficientsofreactions1and2.Eachrow in the matrix represents the stoichiometric

Step 2 .Pseudo-Steady State Assumption


coefficients in the balance of a compound in thesystem. For example, the balance of CH4 is the net of -2J1 (+0J2) and the output from the furnace -qCH4. If a compound exists only in the reactor but is not part of the input or output, then its value in the vector on the right hand side is zero. In applying MFA to cell systems, many reaction intermediates exist only intra-cellularly (such as glucose-6-phosphate and fructose-6-phosphate), so their q is zero.

Undermostconditions,thefluxesareunknown.Theinputs, outputs, and q’s can usually be measured. Insettingup fluxanalyses,onetypicallyconsidersonly the branching points (called nodes) of the reactions, in which one reactant is split into two or more reactions, or two reactions lead to a common product. In a linear pathway, the material flowis easily related by a stoichiometric relationship along the path. For example, the flux of glycolysisis a linear pathway, but only if the small diversion of glyceraldehyde-3-phosphate to glycerol-3-phosphate for lipid biosynthesis is ignored. At steady state, hexokinase flux is half of that of pyruvatekinasefluxand,onamolarbasis,isdefinedbythe1:2proportionof their stoichiometric coefficients.There is no need to set up balances for reactions along the pathway between the two reactions.

Next, the equations that are not independent are removed from the system of equations. The resulting system of equationsmay be overspecified (in thecase of having more independent equations than unknowns), underspecified (in the case of havingmore unknowns than independent equations), or have a unique solution (in the case of having the same number of independent equations as unknowns). For underspecifiedsystems,oneseekstoperformmoremeasurements.Foroverspecifiedsystems,onehastousehis/herknowledgetoremovealesssignificantreactionorfindasetofsolutionsthatgivesrisetominimal errors. In general, for biological metabolic reactions, the system is always overspecified.

Step 3 .Write the equations in the matrix form

Matrix Form

Specific rates vector r:• Positive : production• Negative : uptake

Stochiometric Matrix A:• Each column is a reaction• Each row is a species

• Solve simultaneous equations AX=Q• Over specified system: Least-squares method


MFA on a Cellular System

Utility and Limitations Metabolicfluxanalysisistheapplicationofmaterialbalances on metabolic systems. The principle and approach are not different from the example shown for a chemical reaction system. When MFA is applied to a cellular system, usually the inputs and outputs into cells are measured and known. One needs to enlist all of the reactions involved and set up material balance equations for all intracellular components that are considered, and then proceed to solving the resulting system of equations. In most cases, the effect of cell expansion and the effect of dilution due to volume enlargement are ignored. This is regarded as acceptable because the timeframe considered in the analysis is usually much shorter than the doubling time of cells.

MFA provides an additional dimension to analyze experimental data. Instead of merely examining various specific rates, MFA provides a bird eye’sview of the distribution of nutrients to different metabolic pathways. This insight is especially useful when comparing different metabolic behaviors under different culture conditions or growth stages. By combining MFA with transcriptome data, which provides a global view of the changes of transcripts in cells, one can possibly gain much more insight into the dynamics of cell metabolism, which is not easily seen otherwise.

However, like material balance in chemical reactions systems, the accuracy of MFA is limited by the knowledge of reactions involved in the system and the accounting of material balance. Further complicating the analysis is the fact that cellular metabolism is compartmentalized in organelles and cytosol. This poses additional constraints on the balancing of many molecules playing key roles in inter-organelle communications.

Experimentally, a complete accounting of materials in a cell culture system is challenging. Material balance in a reaction system should be balanced on every elemental species, or at least for the major ones


(C, N, H and O). The consumption of major nutrients, including glucose and all amino acids, can be easily accomplished. Oxygen consumption can also be measured. The products, including lactate, some non-essential amino acids, and the protein product, canalsobequantified.However,amajormetabolicproduct, carbon dioxide, is not easily measured.

A typical cell culture medium contains a rather high concentration of bicarbonate, as pH buffer, makingitverydifficulttoquantifyCO2 produced by cells, unless isotope is used. One way to estimate carbon dioxide production takes into account the fact that the respiratory quotient of most cells under typical culture conditions is about 1.0. By measuring oxygen consumption rate and assuming RQ is 1.0, one can obtain the CO2 production rate and use the value for closing the carbon balance.

Nitrogen balance can be obtained by measuring the consumption of amino acids and accounting for the production of cellular protein and product. However, with measurement errors and uncertainty in estimating biomass composition, it is common to see the total nitrogen in inputs and outputs differ by more than 10%. Still, this is often better than the extent of closure one often obtains for carbon balance.

Some cell culture media contain complex components, such as serum or plant hydrolysate. Those components provide peptides, lipids, and fatty acids for growth. The extent of their consumption is hardly measured, making it even more difficult to close the material balance.

MFA can give much insight into the metabolic characteristics of cell culture processes, but one should be extra diligent in the execution of experiments and collection of measurements, to ensure the results of MFA are reliable.

A Generalized Approach to MFA

Measure Specific Rates

Check Carbon/Nitrogen Balance

Calculate Fluxes J

Visualize Fluxes on Metabolic Map

Examine Calculated Specific Rates

Examine Calculated Carbon/Nitrogen Balance

Stoichiometric Matrix

Fig. 8.1: A flow chart for metabolic flux analysis


General Approach A typical mammalian cell expresses about 15,000 genes at a given time. Only a fraction of the genes are actually involved inmaterial flow (or fluxes);others are involved in cellular structure or in signal flow.MFAprimarilyconcernstheflowofmaterials.For all practical purposes, we can account for only carbon- and nitrogen-containing species. The flowof ionic species (e.g., sodium,potassium,phosphate, etc) are not considered in MFA.

Even considering only the flow of carbon- andnitrogen-containing compounds, the number of reactions is over a thousand. However, only a portion of these reactions has a significant flux.Overall, the glycolysis pathway has the highest flux for cultured cells. There are only a hundred,or so, reactions whose flux is >5% of that ofglycolysis. The vast majority of the reactions have a flux in the order of 1% of glycolysis, or lower.

Considering the errors in carbon and nitrogen balances,MFAisbestappliedtoanalyzethefluxofmajorarteries incarbonflow. Itdoesnotprovideaccurate estimates for minor reactions, such as glycan distribution in the product protein. To applyMFA for the reactionswithminor fluxes, anaccurate quantification of the specific formationrate of key reaction intermediates is necessary.

The first step in applying MFA to a cell systemis to reduce whole cell reaction networks to a manageable and meaningful subset of pathways. In general, those pathways include glucose and amino acid metabolism, and the biosynthesis of building blocks for biomass and product formation. In the course of simplifying the reaction network, different assumptions are made and often a different set of reactions are included in the analysis. The selection of pathways will impact the flux distribution.

Selecting Reactions for Analysis

Applying MFA to entire cell metabolism

• a vastly large number of reactions involving material flux

• most of them have very small fluxxes, much smaller than errors in closing balance of materials

• select the reactions and pathways which are relevant and sufficiently large

• to zoom in on pathways with very small fluxxes, use a root type tracer to examine fluxxes of local reactions

Fig. 8.2: Simplification of metabolic pathways for metabolic flux analysis.


Compartmentalization Cells are compartmentalized to segregate various reactions; genome replication and RNA synthesis occurs in the nucleus and glycolysis takes place in cytosol. Among the major metabolic reactions, TCA cycle, oxidative phosphorylation, and fatty acid oxidation occur in the mitochondria. Across the boundary of cytosol and mitochondria, pyruvate, glutamine, and components of the malate-aspartate shuttlepassathighfluxes. Thefluxofthemalate-aspartate shuttle transports the reducing equivalent of NADH into the mitochondria, at the same level as pyruvate. The fluxof thecitrateshuttlegeneratesacetyl CoA in the cytosol and is typically lower, but under some growth conditions it can be rather large.

These shuttles and transfers across the mitochondrial membrane pose further constraints onmaterialflow.ImposingthoseconstrainsonMFAisimportantforobtainingagoodestimateoffluxesof energy metabolism. The regulation of fluxesacross the cytosol and the mitochondria may play a roleinshiftofmetabolism.Therefore,buildingafluxanalysis model based on two compartments of the cytosol and mitochondrion is a worthwhile effort.

Biomass Equations With the exception of cell mass, all outputs of a cell system have a defined chemical composition.Their production rates are measured and readily used in MFA. In contrast, the cell concentration is often measured by the cell number, and not biomass. Furthermore, the composition of the cell mass is seldom characterized.

Under most culture conditions, the vast majority of carbon taken up by cells as nutrients is converted to lactate and carbon dioxide. Only a small fraction is actually incorporated into new cell mass. Because this amount is such a small fraction of the total carbon consumed, the error in the estimation of biomassdoesnotsignificantlyaffecttheMFAresults.

However, under an oxidative metabolic state (characterized by reduced specific glucoseconsumption and very little lactate production or consumption), the amount of carbon and nitrogen channeled to biomass becomes a significant

• Carbohydrate metabolism takes place in cytosol and mitochondria

• lipid metabolism involves even more compartments

• intercompartmental traffic of pyruvate and malate-aspartate shuttle also involve amino acids and TCA cycle intermediates

• flux balance must be met for the intercompartmental traffic

• biomass equation is very difficult to construct and is subject to errors

• under fast growing and lactate production conditions, biomass formation constitutes only a small fraction of total carbon


portion of the overall material flow. Since thecomponents of the biomass are drawn from fluxes leading to building blocks, the compositionof cell mass affects the results of flux analysis.

Despitetheirpotentialinfluenceontheestimationof fluxes, the stoichiometric equations of biomassformation are not often addressed. Based on elemental analysis of C, N, O, and H of a mouse hybridoma cell line, a general compositional formula was given as: CH1.975N0.2605O0.489 . The general range of cellular composition of lipids, proteins, nucleic acids, and polysaccharides is also available. The amino acid composition of cellular proteins has been reported for a number of cell lines. Overall, the available data is very limited. One should be aware of this potential error when applying the literature to the biomass equation.

BIOMASSProteins

DNA/RNALipids/Carbohydrates, etc.

Amino acidsGlucose

Biomass Equation Derived from Cellular Components

Macromolecule pg per cell

Protein 300 DNA/RNA 15/30Lipids/Carbohydrates 55Total dry weight 400

Elemental composition of cell

C N H O

1 0.2605 1.975 0.489

Solution and Analysis Asimplifiedcellularreactionsystemmayconsideronly the metabolism of glucose and amino acids, while simultaneously lumping lipid and nucleotide synthesis into a biomass formation equation. The resulting reaction network consists ofabout fifty fluxes involvinga similarnumberofcompounds. Such a system can be solved using software such as Mathmatica and MatLab, using the least square method to minimize residue.

Thesolutiongivesasetofvaluestounknownfluxes,as well as to the specific rates whose measuredvalues are already known. One should compare the values given by the solution and the measured values. It is also prudent to check material balance based on the solution values. If major deviations are seen, the results need to be reevaluated.

The results of MFA involve tens of variables depicting the fluxes in the reaction network.Such data are difficult to comprehend, without aproper graphic presentation. To gain insight, it is helpful to link the results of MFA to a metabolic map for visualization. Such visual images greatly enhance our ability to discern shifts in metabolism.

Fig. 8.3: Determining elemental composition for biomass stoichiometric formula


An Example For an example of MFA, a MatLab algorithm is presented and its solution is included. The reactions considered in the reaction network are listed in the accompanying table. These include reactions for glycolysis, TCA cycle, amino acid degradation pathways, biomass, and antibody synthesis. The metabolic network is compartmentalized into the mitochondria and the cytosol. Reactions in the malate-aspartate shuttle (also known as the NADH shuttle) are included to account for the transfer of the reduction potential of NADH generated in cytosol into the mitochondria, thereby regenerating the levels of cytosolic NAD. Further, three enzymes, including mitochondrial malic enzyme, cytosolic malic enzyme, and pyruvate carboxylase, are also included in the reaction network. The respiratory quotient is assumed to be 1.0; in other words, the carbon dioxide production rate is assumed to be the same as the oxygen uptake rate.

The specific rates determined for two metabolicstates of NS0 cells: one produces lactate in the exponential growth phase and the other consumes lactate in late growth stage. These rates are used to solvethefluxesineachsituation.Uponthesolution,the results are displayed in a metabolic chart.

The contrast of the two metabolic states is seen, not only in the specific rates of glucose andglutamine, but also in the internal distribution.


# Reaction Compartment Pathway

1 GLCc + 2NADc → 2PYRc + 2NADHc + H2O Cytosol Glycolysis

2 PYRc + NADHc → LACc + NADc Cytosol Lactate Dehydrogenase Reaction

3 PYRm + NADm → AcCoAm + NADHm + CO2 Mitochondria Krebs Cycle

4 OAAm + AcCoAm → CITm Mitochondria Krebs Cycle

5 CITm + NADm + H2O → AKGm + NADHm + CO2 Mitochondria Krebs Cycle

6 AKGm + NADm → SUCCoAm + NADHm + CO2 Mitochondria Krebs Cycle

7 SUCCoAm + H2O → FUMm Mitochondria Krebs Cycle

8 FUMm + H2O → MALm Mitochondria Krebs Cycle

9 MALm + NADm → OAAm + NADHm Mitochondria Krebs Cycle

10 GLNc → GLUc + NH3 Cytosol Glutaminolysis

11 GLUm → AKGm + NH3 Mitochondria Glutaminolysis

12 PYRc + GLUc → ALAc + AKGc Cytosol Alanine Synthesis

13 SERc → PYRc + NH3 Cytosol Amino Acid Degradation

14 GLYc → SERc Cytosol Amino Acid Degradation

15 CYSm → PYRm + NH3 Mitochondria Amino Acid Degradation

16 ASNc → ASPc + NH3 Cytosol Amino Acid Degradation

17 HISm → GLUm + NH3 Mitochondria Amino Acid Degradation

18 ARGm + AKGm → 2GLUm Mitochondria Amino Acid Degradation

19 PROm → GLUm Mitochondria Amino Acid Degradation

20 ILEm + AKGm → SucCoAm + AcCoAm + GLUm Mitochondria Amino Acid Degradation

21 VALm + AKGm → GLUm + CO2 + SucCoAm Mitochondria Amino Acid Degradation

22 METm → SucCoAm Mitochondria Amino Acid Degradation

23 THRm → SucCoAm + NH3 Mitochondria Amino Acid Degradation

24 PHEm → TYRm Mitochondria Amino Acid Degradation

25 TYRm + AKGm → GLUm + FUMm + 2AcCoAm Mitochondria Amino Acid Degradation

26 LYSm + 2AKG → 2GLUm + 2 CO2 + 2AcCoAm Mitochondria Amino Acid Degradation

27 LEUm + AKGm → GLUm + 3AcCoAm Mitochondria Amino Acid Degradation

28 0 .0104GLNc + 0 .0110ALAc + 0 .0050ARGm + 0 .0072ASNc + 0 .0082ASPc + 0 .005CYSm + 0 .0107GLUc + 0 .0145GLYc + 0 .0035HISm + 0 .0050ILEm + 0 .0142LEUm + 0 .0145LYSm + 0 .0028METm + 0 .0072PHEm + 0 .0148PROm + 0 .0267SERc + 0 .0160THRm + 0 .0085TYRm + 0 .0189VALm → CH1 .539N0 .2645O0 .314

Cytosol/Mitochondria Antibody Synthesis

29 0 .208GLCc + 0 .0377GLNc + 0 .0133ALAc + 0 .0070ARGm + 0 .0ASNc + 0 .0261ASPc + 0 .0004CYSm + 0 .0006GLUc + 0 .0165GLYc + 0 .0033HISm + 0 .0084ILEm + 0 .0133LEUm + 0 .0101LYSm + 0 .0033METm + 0 .005PHEm + 0 .0081PROm + 0 .0099SERc + 0 .0080THRm + 0 .0040TYRm + 0 .0096VALm → CH1 .975N0 .2605O0 .489

Cytosol/Mitochondria Biomass Synthesis

30 CITm + MALc → CITc + MALm Cytosol/Mitochondria Fatty Acid Synthesis

31 CITc → AcCoAc + OAAc Cytosol Fatty Acid Synthesis

32 MALc → MALm Cytosol/Mitochondria Glutaminolysis

33 GLUc → GLUm Cytosol/Mitochondria Glutaminolysis

34 OAAm + GLUm → AKGm + ASPm Mitochondria Malate Aspartate Shuttle

35 OAAc + NADHc → MALc +NADc Cytosol Malate Aspartate Shuttle

36 AKGc + ASPc → OAAc + GLUc Cytosol Malate Aspartate Shuttle

37 ASPm + GLUc → ASPc + GLUm Cytosol/Mitochondria Malate Aspartate Shuttle

38 MALc + AKGm → MALm + AKGc Cytosol/Mitochondria Malate Aspartate Shuttle

39 MALm + NADm → PYRm + CO2 + NADHm Mitochondria Malate Decarboxylation (Malic Enzyme)

40 MALc → PYRc + CO2 Cytosol Malate Decarboxylation (Malic Enzyme)

41 2NADHm + O2 → 2NADm Mitochondria Oxidative Phosphorylation

42 2FADH2 + O2 → 2FAD Mitochondria Oxidative Phosphorylation

43 PYRm + CO2 → OAAm Mitochondria Pyuvate Caboxylation (Pyruvate Carbox-ylase)

Table 1. List of Energy Catabolism Reactions for MFA


Abbreviation NameAB AntibodyAcCoA Acetyl Coenzyme AAKG A-KetoglutarateALA AlanineARG ArginineASN AsparagineASP AspartateBIOMASS BiomassCYS CysteineCO2 Carbon DioxideFUM FumarateGLC GlucoseGLN GlutamineGLU GlutamateGLY GlycineHIS HistidineILE IsoleucineLAC LactateLEU LeucineLYS LysineMAL MalateMET MethionineNH3 AmmoniaOAA OxaloacetatePHE PhenylalaninePRO ProlinePYR PyruvateSER SerineSucCoA Succinate Coenzyme ATHR TheonineTYR TyrosineVAL ValineO2 OxygenNADH Nicotinamide Adenine Dinucleotide

(Reduced)NAD Nicotinamide Adenine Dinucleotide

(Oxidized)FADH2 Flavin Adenine Dinucleotide (Reduced)FAD Flavin Adenine Dinucleotide (Oxidized)

Subscript Compartmentc Cytosolm Mitochondria

Table 2. Abbreviation of Intermediates of Reaction Modes


Concluding Remarks

MFA is an important analytic technique of quantitative physiology. It can provide insight into process optimization and metabolic engineering. A fluxbalancecanbewritten foreachmetabolite,within a cellular or metabolic system, to yield the dynamic mass balance equations that interconnect various metabolites. With the knowledge of stoichiometry and steady state assumption, one can obtain the flux estimate for individual reactions.

MFA is a powerful tool, but it is invariably based on a simplified reaction network. Theassumptions made in simplifying the reaction network will affect the results of the analysis. It is best used for a first approximation to obtainsome insights for further exploration of ideas.


Cell Culture Bioreactors

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192Basic Types of Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .193

Stirred Tank (Well Mixed) Vs . Tubular Reactor (Plug Flow) . . . . . . . . . . . . . . . 193CSTR and PFR with Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Implication When Growth or Reaction Occurs in the Reactor . . . . . . . . . . . . . 195Heterogeneous Reactor- High Solid Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Operating Mode of Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .196Batch and Continuous Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196Batch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

Tissue Culture and Disposable Cell Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . .199Disposable Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Cell Support Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .202Suspension Culture vs . Adherent Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202Microcarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Cell Culture Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .206Simple Stirred Tank Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206Airlift Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Fluidized Bed Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Membrane Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208Multiple Membrane Plate Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

Other Bioreactor Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209Microsphere Induced Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Agarose Cell Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210Membrane Stirred Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210Spin Filter Stirred Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210Vibromixer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212


CELL CULTURE BIOREACTORS | 191


IntroductionIn the past decade, industrial mammalian cell culture has become the production vehicle of choice inthefastgrowingfieldofmedicalbiologics.Theseproducts now extend from therapeutic proteins and viral vaccines to cells and viruses for cellular and gene therapy. Before the arrival of recombinant DNA technology, industrial mammalian cell culture was used only in the production of viral vaccines, and was confined to relatively smallscale operations, involving the culture of adherent cells in tissue culture flasks. The arrival of rDNAtechnologyledtosignificantchanges.Now,thehostmammalian cells are invariably continuous cell lines, and the scale of operations has skyrocketed.

The transition from cultivation of adherent cells in flasks to a truly industrial scale has transformedbioprocess. The most dramatic changes have been in the process, rather than in the basic design of the reactor. In the 1980s and early 1990s, research and commercial development focused on exploring possibilities for new bioreactors. Researchers pushed to overcome a number of real and perceived hurdles, e.g., the notion that mechanical agitation and air sparging had detrimental effects on mammalian cells, the fact that the culture systems supported only low cell numbers and resultant low product concentrations, and that the accumulation of metabolites caused growth inhibition limiting potential production.

What was unexpected was the extraordinary capability of cells to adapt to suspension growth, and to adapt to serum-free, and even protein-free, conditions. Over time, the bioreactors used for industrial cell culture have evolved into a few basic configurations.

This chapter will discuss the fundamentals of bioreactors and highlight specific bioreactorsdeveloped in the 1980s. These examples are useful for their conceptual novelty and help to illustrate the limitations on their use as industrial bioreactors. Although these reactors are not commonly used in current processes, they might


Basic Types of Bioreactors

find specialized applications in other areas, suchas tissue engineering, or gene and cellular therapy.

Stirred Tank (Well Mixed) Vs. Tubular Reactor (Plug Flow)

The distinction between a well-mixed continuous stirred tank reactor (CSTR) andplug flow reactor(PFR) is best illustrated by a comparison of their behavior after a step change in feed concentration. Consider a continuous reactor that has an inlet stream (feed) and an outlet stream that are equal in volumetricflowrate;thus,thevolumeofthereactorisconstant.Initiallyboththefluidinthereactorandthe feed stream are colorless. At time=0, the feed stream is switched toa fluidcontaininga reddyeat a concentration of C0. We then observe how the concentration of the dye has changed at the outlet.

In the case that the reactor is well mixed (i.e., a CSTR) as soon as the feed stream is switched, the color is distributed uniformly in the reactor. Because the dye is uniformly distributed, regardless

x

oC

F

V

FC

Continuous Stirred Tank Bioreactor

Bioreactors can be generally categorized according to their mixing characteristics. The two extremes of mixing are, at one pole complete, instantaneous mixing and, at the opposite pole, a solid-like complete absence of mixing. The differences are best illustrated using a continuous flow reactor,although many bioreactors are used in batch fashion.

The two extreme mixing patterns characterize two types of idealized continuous reactors: well-mixed stirred tank and plug-flow (tubular)reactor. In an ideal well-mixed bioreactor, the mixing is assumed to be intensive such that the fluid is homogeneous throughout the reactor.When a new solution is added to the reactor, the solute is instantaneously, uniformly distributed.

A tubular reactor is at the other extreme of an idealized bioreactor; it has absolutely no mixing. It is also called a plug-flow reactor, or piston-flowbioreactor. As its name implies, a solution entering the bioreactor will move downstream like a wall or a plug, and steadily forward until it reaches the exit.

Stirred Tank Bioreactor

Introduction

Tubular Bioreactor

Idealized Bioreactor• Plug flow reactor (no mixing)• Well mixed reactor (instantaneously perfectly mixed)

Fig. 9.1: Schematic of a tubular reactor

Fig. 9.2: Schematic of a well-mixed stirred tank reactor.

Fig. 9.3: A continuous flow stirred tank bioreactor with a switch for tracer injection.

Initial condition: t = 0, C = 0


ofwheretheeffluentstreamisdrawnfrom, itwillalso acquire the red dye at the same concentration as in the reactor. Of course, for the purpose of our discussion, we neglect the time delay caused by flow in the pipe lead into and out of the reactor.As more colored feed comes into the reactor, the concentration of the color seen in the outlet will gradually increase. The balance equation for the concentration of the dye that gives the color is shown,whereFistheflowrate,Vthereactorvolume,C the dye concentration, and subscript o denotes the feed. Note that in this case, the concentration in the outlet flow is the same as in the reactor,because of the assumption of being well mixed.

By setting initial condition C=0, one can solve the equation, and plot the profile of the normalizedconcentration of the dye (C/Ci) over time. V/F has units oftimeandrepresentstheholdingtime(θ),thetimeit takes one reactor volume of feed to pass through the reactor. One can see that after a holding time, the concentration of the dye in the reactor is 0.63 of that inthefeed.Ittakesthreeholdingtimes(3θ)fortheconcentration to approach (~98%) that in the feed.

In a PFR, the red colored dye moves downstream like a sharp band, since there is no backmixing or diffusion to blur the sharp boundary between the color and colorless streams. After switching the feed stream to the red color, it takes one holding time for the red color to appear at the exit. When the color appears at the exit, its concentration is identical to the feed concentration.

CSTR and PFR with Reaction The discussion above considers only mixing behavior in the reactor, essentially just mixing tanks. In a bioreactor, consumption (of nutrients) and generation (product formation and growth) take place; these processes (reaction) will cause the concentration profile to differ from that in asimple mixing tank. For CSTR, the assumption that the concentration at the outlet is identical to that in the reactor holds. The effect of the reaction is dealt with by adding a term to the material balance equation to account for the reaction. One equation

The same reaction carried out in PFR or CSTR gives different kinetic behavior:

• In PRF, as the feed moves downstream, the reactant(s) is consumed and the product(s) is produced. Different locations in the reactor have different concentrations.

• In CSTR, the concentration of reactant(s) and product(s) is homogeneous. The concentration at the exit is the same as in the fluid in the reactor.

Fig. 9.4: The concentration profile of the tracer after injection into a CSTR

Fig. 9.5: A plug-flow reactor with a switch for tracer injection.

Fig. 9.6: Concentration of tracer detected at the end of a PFR after tracer injection.


Implication When Growth or Reaction Occurs in the Reactor

Plug-flowbioreactorsareintrinsicallymoredifficultto scale up than mixing vessels, as the concentration gradient of essential nutrients, oxygen in particular, will inevitably become limiting in the downstream region of the reactor. One way to overcome the limitation is to increase the nutrient supply rate by using a higher concentration in the feed, or by operatingatahigherflowrate.Thereare,however,practical limits on both nutrient concentration and flow rate. For example, nutrient concentration islimited by its solubility, and a high flow rate willrequire a higher capacity pump to overcome a higher pressure drop across the reactor. These kinds of limitations are especially true for growth of aerobic organisms, because oxygen solubility in water is very low and is quickly depleted unless supplied continuously. Thus, the size and scalability of a PFR

is written for each of the reactants and products.

The equation describing the overall balance of the dye in a PFR differs from that for a CSTR. In a PFR, the concentration is no longer constant throughout the reactor, but is dependent on the position within the reactor. Because the concentration of both reactants andproductschangesalongtheflowdirectioninthereactor, the size (i.e., the length) of the reactor, or the holding time in the reactor, affects their values. To account for the effect of position, we do a material balance on the dye over a very small section along the reactor. The equation has both time and position as independent variables. Once a steady state is reached, the dependence of time can be eliminated, and the equation becomes an ordinary differential equation with the position as the only independent variable.

It can be seen that in the presence of reaction, the concentration of the reactant decreases as the feed stream moves downstream, while the product concentration increases. For bioprocess applications using a PFR, the nutrient level will decrease while metabolite concentration increases along the bioreactor. At some position, the nutrients will be depleted, and a reactor longer than that length becomes unproductive.


Heterogeneous Reactor- High Solid Content

A bioreactor typically encompasses three phases: a liquid phase, a solid phase (cells can be regarded as “solid”), and a gas phase (bubbles for aeration). Often, cell mass represents a very small fraction of the culture volume; thus, the content of a cell culture bioreactor is often treated as homogeneous, as if having a single liquid phase, and cells, like nutrients, are treated as part of the liquid phase; no special consideration of the volume taken up by cells in establishing material balance equations .

However, in microbial fermentation and in plant cell culture, cells may make up a large fraction (up to 50%) of total reactor volume. The reactor volume, thus, is partitioned into cell volume and culture medium volume. The cell and nutrient concentrations calculated from medium volume or total reactor volume have different values.

Some so called heterogeneous cell culture bioreactors contain a significant volume fractionof solid phase such as microcarrier beads. In a microcarrier bioreactor for example, microcarrier beads often constitute 10-30% of the culture volume. In this circumstance, even cell concentration needs to bewell defined. For example, itmust bespecified whether 107 cells per milliliter is withrespect to total culture volume or to liquid volume.

Batch and Continuous Processes

Operating Mode of Bioreactors

Theoperationofabioreactorisgenerallyclassifiedasbatch or continuous mode. In a continuous process, the feed is continuously being introduced to the reactor, and product stream is continuously being withdrawn from the reactor. In a batch process, the medium, including all nutrients (except oxygen), is added at the beginning, and no other nutrient addition occurs until the end of cultivation. Continuous culture is not commonly practiced in the cultivation

reactor is rather limited. In a CSTR model, all cells in the reactor encounter the same environment. The nutrients that feed into the reactor are distributed uniformly, albeit at abundant or suboptimal levels.

Fig. 9.7: Schematic of a heterogeneous reactor

microcarrier

Heterogenous Stirred Tank Bioreactor

Fig. 9.8: A reactor supporting cell growth on microcarriers is a heterogeneous reactor


of mammalian cells unless in conjunction with cell recycle. This topic will be discussed in a later chapter.

Batch cultures are often limited by the fact that in many cases the product concentration is not sufficiently high. To achieve a high productconcentration it is necessary to have a high cell concentration; for that the nutrient concentration in the medium must also be high. In many cases high concentrations of nutrients are not attainable in batch culture because the initial concentration at the start of culture is limited by the solubility of some nutrients, or by the concentration at which cells can be grown without growth inhibition. It may be necessary to add additional substrate or product precursor to sustained continued growth to reach higher cell and product concentrations. In other cases, such as intermittent fed-batch cultures, instead of terminating a batch process at the end of culture, a portion of the culture is kept, and fresh medium is added to start the process again.

Batch Cultures Batch processes are simple and are widely used. In fact, batch process culture is used much more frequently than the fed-batch process. Before reaching production scale, cell expansion is carried out in a series of batch cultures with increasing reactor volumes. This series of batch cultures for cell expansion is often referred to as seed train. During expansion in batch cultivations, the culture is discontinued in the reactor and cells are transferred to the next larger reactor while still rapidly growing. If a culture is allowed to run its course, cell growth will be inhibited by the accumulation of metabolites, and the resulting product concentration will be low. In general, batch processes do not result in high productivity. Batch cultures are used in viral vaccine production, or, if used in recombinant protein manufacturing, are largely limited to the cell expansion stage.

Operating Mode of Bioreactors

Fig. 9.9: Kinetics of growth and product formation in a batch culture


Fedbatch For production of recombinant proteins and antibodies, a more traditional fed-batch process is typically used. A fed-batch culture is started in a volume much lower than the full capacity of the bioreactor (approximately 40% to 50% of the maximum volume, or at a level sufficient toallow the impeller to be submerged). Nutrients, usually in a more concentrated form than in basal medium, are added during the cultivation to allow cell and product concentrations to reach much higher levels than can be attained in batch culture.

Fedbatch Cultures

Intermittent Harvest

Fed-batch processes do not differ significantlyfrom batch cultures. The simplest form of fed-batch culture involves intermittent harvest. At a late exponential growth stage of the culture, a portion of the cells and product are harvested, and the culture is replenished with fresh medium. This avoids metabolite inhibition of cell growth and replenishes nutrients for continued cell growth.

This process may be repeated several times. This simple strategy is commonplace for the production of viral vaccines produced by persistent infection, as it allows for an extended production period. It is also used in roller bottle processes with adherent cells. By intermittent harvesting, turn-around time involved in cleaning up and restarting the process is saved. Furthermore, after replenishing the medium, the culture reaches peak cell concentration faster than if the culture is started de novo with fresh inoculum, which usuallyisatasignificantlylowercellconcentration.

Fig. 9.10: Kinetics of growth and product formation in a fed-batch culture with intermittent harvest

Fig. 9.11: Kinetics of growth and product formation in a fed-batch culture with increasing culture volume


Tissue Culture and Disposable Cell Culture SystemsIn this section we will describe reactors used for large-scale cultivation of mammalian cells. Arguably, the oldest bioreactors are animals themselves or their tissues. The application of virus vaccines dates back two hundred years, when Edward Jenner used cow pox to inoculate humans for protection against small pox. Many viral vaccines developed in the first half of the 20th century employed animal oranimal tissues as production vehicles. Use of animal tissues (e.g., chicken eggs, rodent brains) for virus production is still practiced today for some viruses, although cell culture is considered the norm.

Disposable Culture Systems Many different types of disposable, single-use plastic bioreactors including T-flasks, rollerbottles, and multiple flat panels are commonlyused for production of recombinant proteins and viral vaccines, using both attachment-dependent and suspension cells. The plastic surface may be treated to facilitate attachment of adherent cells. Suspension cells can be readily grown in a stirred vessel. However, there is an increasing trend toward the use of disposable (single-use) bioreactors at small and moderate scales. Factors such as ease of operation, shorter time to scale up, lower capital investment, and reduction of process validation burden have contributed to their wide adoption.

Roller Bottles Roller bottles are cylindrical screw-capped bottles with a total volume ranging from 1 to 1.5 liters, and are suitable for a culture volume of 0.1 to 0.3 liters. Stacks of bottles are placed on a rack and rotated at 1 to 4 rpm. For small-scale operations, roller bottles provide many advantages for the cultivation of adherent cells. The system is relatively inexpensive to set up, and allows for rapid adjustment of production throughput in response to changing needs. Furthermore, complete replacement of medium, e.g., from growth medium to product medium, is rather straightforward. Roller bottles

Single Use Bioreactor

• Simple, low-capital investment, fast to implement, ease of equipment validation

• A variety of systems with vastly different mixing mechanisms

• Less well-characterized in fluid dynamics, mass transfer

• Suitable for seed culture, scales up to hundreds of liters

• Possible faster turn around time

• Lacking the strength of steel; some operations routinely done on steel vessels, such as pressurized liquid transfer, cannot be performed on single use vessels


are particularly useful in the case that the serum-containing medium needs to be replaced by a serum-free or protein-free medium for the production phase. The transparent glass or plastic wall allows visual or microscopic examination of the culture status. Microbial contaminated bottles can be readily identifiedanddiscardedbeforemixingwithothers.

For large-scale production of biologics, however, there are numerous drawbacks to roller bottles. On-line environmental monitoring and control is virtually impossible or at least impractical. Aseptic bottle handling for inoculation, protease-treatment for cell detachment and expansion, medium exchange, and product harvest are labor intensive and must be performed by a skilled technicians to ensure a low failure rate. A batch of a size suitable for manufacturing purposes may require hundreds or even thousands of bottles, and the large number of manual steps involved dramatically increases the risk of microbial contamination.

Despite these significant drawbacks, roller bottlesare widely used in the production of recombinant proteins and viral vaccines. Often, they are used primarily because the product involved has already been approved by regulatory agencies and a process changewouldincursignificantcostsforregulatoryapproval and could even result in product comparability issues. In some cases, roller bottles were selected because the process was easy to implement and the production capacity needed was manageable. However, when faced with the need to increase production, e.g., due to market expansion, scale-up of a roller bottle process is challenging.

Notable examples of industrial roller bottle processes include the production of erythropoietin (EPO) using recombinant Chinese Hamster Ovary cells, the production of live attenuated chickenpox (varicella) vaccine, and herpes zoster (shingles) vaccine using theadherentsecondaryhumanlungfibroblastcellstrain, MRC-5. In some manufacturing facilities a fully automatic, robotic roller bottle handling system is available. However, a robotic system cannot handle a large number of bottles in parallel. Handling

Fig. 9.12: A roller bottle and roller bottles on racks for cell culture


a large number of roller bottles is a prolonged process. For example, in a process entailing virus inoculation or a culture condition switch to induce product formation, inoculation and induction time can vary very widely from the processing of the firstbottle to theprocessingof the lastbottle.

Multiple Plate System Nunc Cell Factories® (NCFs) are widely used in laboratories and in industrial production of viral vectors, vaccines, and recombinant proteins. Although designed to culture adherent cells, NCFs can also be used for suspension cells. A 40-tray NCF has a capacity of 25,280 cm2 and ~8 L liquid volume. For large-scale manufacturing, a mechanical handling system can be used. One notable example is the production of multivalent Rotavirus vaccine using Vero cells, a continuous African green monkey kidney cell line. The vaccine is a live, attenuated virus vaccine that contains five different human-bovine virus reassortants,each produced from a different process.

Another multiple plate system (CellCube Module) entails nine-inch square polystyrene plates stacked vertically with 1 mm spacing in a resin case. The space between stacks is completely filled withmedium. Continuous medium circulation is used for oxygen and nutrient supply. CellCube has been used in the cultivation of MRC-5 cells for the production of attenuated hepatitis A virus (HAV) and for the production of VAQTA, an inactivated HAV vaccine. These disposable bioreactors have also been somewhat popular for intermediate levels of production of cells and gene therapy vectors.

Bags, Cylinders on Shakers, Movers Blood bags have long been used for the cultivation of cells for cell therapy. Small bags are placed in incubators for temperature control. For larger single use bag systems, mechanical mechanisms provide mixing and oxygen transfer. Bags are especially suitable for cell expansion before reaching finalproduction scale up. WaveTM consists of pre-sterilized, disposable Cellbags and a special rocking platform. The rocking motion of the platform

Fig. 9.13: A commercial multiple plate system for cell culture

Many cell lines used in the production of vaccines and biologics grow in suspension cells. Disposable systems are invariably limited in their scale of operation. For processes whose production scale is relatively small, a disposable system provides some advantages. For protein therapeutics needed in large quantities, the use of disposable systems may be limited to inoculum preparation. Large scale operations, thus, continue to employ fermentors or other bioreactors.

The majority of cells used for protein production via rDNA are suspension cells, which, as suggested by the name, have no need for attachment to a surface and are simply suspended in the medium in a bioreactor. For growth of adherent cells, cell supports are needed to provide adherent surface. The cell support is placed in a bioreactor which provides a mechanism to supply nutrients and oxygen to cells. In some cases, cell support is also used for suspension cells, allowing cells to be kept in the reactor while the medium is being perfused or exchanged.


induceswavesintheculturefluidtoprovidemixingand oxygen/gas transfer.

In manufacturing settings, validating the equipment for restart of a process contributes a significantpart of the overall operating costs. A more extreme case of single use alternatives to a conventional fermentorisaretrofitproducttomimicanexistingstainless steel bioreactor vessel. It consists of a reusable stainless steel outer support container and a single use cell chamber with a working volume of up to 1000 liters, which can be integrated into the existing bioreactor control system. These modest scale, single use reactors are not intended to replace a conventional stirred tank made of stainless steel withfixedpiping,auxiliaryequipment,etc.Becauseof its single use construction, its operation is limited. For example, a single use fermentor cannot be pressurized to quickly transfer its contents to other process units, or even while receiving inoculum.

Suspension Culture vs. Adherent Cultures

Cell Support Systems

Fig. 9.14: A single-use bag cell cultivation system

Most normal diploid cell strains or primary cells are anchorage-dependent. For large scale operations in a stirred tank bioreactor, microcarriers are used to provide adherent surfaces. Conventional solid or microporous microcarriers are suitable for normal diploid cells which require attachment and spreading. Macroporous microcarriers also allow cells to grow within the interior, and are used for a wide variety of continuous cell lines.

The use ofmicrocarriers for cell culturewas firstdemonstrated by Van Wezel (1967). The basic concept is to allow cells to attach to the surface of small suspended beads so that conventional stirred tank bioreactors can be used for cell cultivation. To facilitate suspension of these cell-laden microcarriers, their diameter and density are usuallyintherangeof100–300μmand1.02–1.05g/cm3, respectively. This diameter range provides good growth surface area per reactor volume. Even at a moderate microcarrier concentration occupying only 8–15% of the culture volume, a significantlylarger surface area per reactor volume can be achieved than that possible with roller bottles.

Most anchorage-dependent cells do not develop their normal morphology, and some do not multiply well on surfaces with an excessively high curvature. These cells do not grow well on small microcarriers. On the other hand, some cells, especially transformed cells, multiply well even on small microcarriers. When these cells are grown on very small microcarriers, they agglomerate to form aggregates and continue to grow to high density. The small microcarriers, usuallywith a diameter of about 50 μm, serve asnuclei for the initiation of aggregate formation.

Microcarriers can be made of many different materials including dextran, gelatin, polystyrene, glass, and cellulose, not all of which are commercially available. In general, microcarriers have a wettable surface, and are sometimes coated with collagen or other adhesion molecules to enhance cell adhesion and spreading. The most widely used microcarriers, which are based on dextran, are derivatized

Human fibroplast FS4 on cytodex 1 microcarriers

CHO on cytodex 3 microcarriers

Fig. 9.15: Cell cultivated on microcarriers


Microcarriers

Solid and Microporous Microcarriers

Cell Support Systems

with charged molecules or denatured collagen.

An advantage of microcarrier culture is the ease of separating cells from culture medium. Many microcarrier cultures require medium exchanges during cultivation to remove lactate, ammonia, and other metabolites and to replenish nutrients. This can be accomplished by simply allowing cell-laden microcarriers to settle so that the spent medium can be withdrawn and replenished. In large-scale operations, continuous or semi-continuous perfusion is more frequently used. This can be accomplished by withdrawing medium through a coarse screen that retains microcarriers in the reactor but allows medium to pass.

A wide variety of cell types have been grown on microcarriers including fibroblasts,epithelialcells,hepatocytes, neuroblastoma cells, and endothelial cells from various species. Overall, microcarrier culture is a convenient laboratory and research tool, and has the advantage of being amenable to large-scale production if a large quantity of product is needed.

Table 1. Desired Characteristics of Microcarriers

Density ~1 .02 g / cm3Only slightly higher than water for easy suspension and setting

Diameter 150 - 200 μmShould be the smallest possible and yet allow for cell spreading

Porosity From solid to nearly 90%

Prefer solid, for use as inoculum bead to bead transfer

Surface Properties

ECM coating, slightly positively charged

Positive charge enhances initial attachment

Macroporous Microcarriers


Macroporous microcarriers contain large internal pores. The void space inside allows cells to be cultivated on internal and external surfaces. Cells in the interior are less susceptible to mechanical damage caused by agitation and gas sparging. On the downside, cells in the interior of microcarriers are more likely to be subject to oxygen limitation due to the long diffusional distance, especially since most macroporous microcarriers have a larger diameter (500μm–2mm)thanstandardmicrocarriers.

Macroporous microcarriers are made of various materials including gelatin, collagen, and plastic. Many cell lines have been successfully grown on macroporous microcarriers including Vero, HepG2, CHO, and 293 cells. The final cell concentrationachieved tends to be higher than that obtained with an equivalent volumetric concentration of conventional microcarriers. In some cases, however, the growth kinetics are slower because the penetration of cells into the interior may be slowed or even retarded by the restrictive opening of the pores.

Cofocal microscopic optical section of HepG2 cells in collagen macroporous

microcarriers

Fig. 9.16: Scanning electron micrograph of a collagen based macroporous microcarrier bead

Fig. 9.17: Cofocal optical section of cells grown in a macroporous microcarrier

Different Types of MicrocarriersDextran (GE Healthcare)

Cross-linked dextran matrix with various surface modifications

Cytodex 1 Positively charged DEAE group Cytodex 3 Denatured type I collagen coatingPolystyrene (SoloHill Engineering)

Cross-linked polystyrene core material with a wide variety of surface modifications

Plastic No surface modification, or positively charged

ProNectin F Recombinant fibronectin coating FACT III Type I porcine collagen coating Glass High silica glass coating Hillex Surface modified with cationic trimethyl ammoniumCellulose Microgranular DEAE-cellulose microcarrier

DE-52 Anion exchange capacity of 1 meq / g dry materials DE-53 Anion exchange capacity of 2 meq / g dry materialsAlginate (Hamilton)

Global Eukaryotic Microcarrier (GEM), composed of alginate matrix with modification of surface

Glass (Biosilon) Hollow glass

Macroporous MicrocarriersGelatin (Percell Biolytica)

CultiSpher-G and CultiSpher-S, cross-linked gelatin matrix with pore size 10-20 µm, CultiSpher-S has a higher thermal and mechanical stability due to a different cross-linking procedure

Cellulose (GE Healthcare)

Cytopore, cross-linked cellulose matrix with a pore size averaging 30 µm. Its surface is modified by the introduction of DEAE group

Collagen (MP Biomedicals)

Cellagen, prepared from bovine corium insoluble collagen by pepsin treatment

Polyethylene(GE Healthcare)

Cytoline, composed of high-density polyethylene weighted with silica

Polypropylene(New Brunswick)

Fibra-Cel disk, composed of polyester mesh with polypropylene support


Table 2. Listing of Microcarriers

Fig. 9.18: Confocal section of HEK293 cell aggregates. Green: viable cells, red: dead cell nuclei


Cell Aggregates

Simple Stirred Tank Bioreactor


Some transformed cells can grow as aggregates when cultivated in shaker flasks or in stirredvessels. Different methods have been used to induce aggregate formation. Aggregation can be promoted by manipulating the calcium concentration in conjunction with the agitation rate. Aggregate cultures have advantages similar to microcarrier cultures. They can be cultivated in conventional stirred tank reactors with environmental control. They can be allowed to settle relatively rapidly by stopping agitation, permitting easy medium replenishment or perfusion.

Stirred tanks, or conventional fermentors, have been widely used for culturing suspension cells since the 1960s. By using microcarriers, adherent cells can also beculturedinastirredtank.Thebasicconfigurationof stirred tank bioreactors for mammalian cell culture is similar to that of microbial fermentors. A major difference is that the aspect ratio (ratio of height to diameter) is usually smaller in mammalian cell culture bioreactors. The power input per unit volume of bioreactor is also substantially lower in mammalian cell culture bioreactors.

While the Rushton type impeller is the norm in microbial fermentors, mammalian cell culture fermentorsmostlyemployaxialflowtypeimpellers.The difference reflects the different purposes ofagitation in microbial fermentation and in cell culture. In microbial fermentation, agitation is needed at a higher power input to disperse air bubblesandto increaseoxygentransferefficiency,whereas in mammalian cell culture, the primary purpose of agitation is to maintain relatively uniform suspension of cells or microcarriers.

In general, the mixing time in a mammalian cell culture bioreactor is substantially longer than that in a microbial fermentor of similar scale. The oxygen transfer capacity in a cell culture bioreactor is also substantially lower than that in a microbial


fermentor. However, the typical oxygen demand in a mammalian cell culture is 10 to 50 times lower than that in microbial fermentation.

Airlift Bioreactor A variant of the bubble column reactor with internal circulation loops is used for the cultivation of mammalian, insect, and plant cells. In these airlift reactors, internal liquid circulation is achieved by sparging through the internal draft tube. The fluidinthedrafttubehasalowereffectivedensitythan the bubble-free section, which, along with the upward momentum generated by air flow,induces liquid circulation. Medium flows upwardthrough the sparged section (riser) and downward in the bubble-free section (downcomer). This method of generating circulation has a low energy requirement compared with stirred-tank reactors.

Airlift bioreactors for cell cultivation are considered to be low-shear devices because there is no mechanical agitation. They have been used successfully with suspension cultures of BHK 21, human lymphoblastoid, CHO, hybridomas, and insect cells.

Fluidized Bed Bioreactor Fluidized bed reactors have long been used in chemical catalysis. The fluid stream (often gasin catalysis) enters through a flow distributorat the bottom at high velocity to suspend the solid catalyst particles. The reactants enter the catalyst and products diffuse out into the fluidto be carried out through the top of the reactor where a separator prevents any particles from beingblownout.Themainadvantageofafluidizedbed is the high heat transfer efficiency betweenthe high velocity fluid and the catalyst surface.

When applied to cells or microcarriers directly, the density difference between solid phase and liquid phase is small, making particle retention difficult.In the 1980’s, collagen macroporous beads were usedincommercialfluidizedbedsofferedbyVerax.The carriers were weighted by inclusion of iron particles to increase the density difference between fluid and carrier so that the particle could beretainedinthereactorattheflowratesrequiredforsupplyingsufficientoxygentosupportcellgrowth.


AirFig. 9.19: Schematic of an air-lift bioreactor


The use of hollow fiber reactors for cultivation ofmammalian cells dates back to the early 1970s. A hollowfibersystemcanbeusedforbothanchorage-dependent and suspension cells. It consists of a bundleofcapillaryfiberssealedinsideacylindricaltube. The basic configuration is rather similar tothe hollow fiber cartridge used in kidney dialysis.

The hollow-fiber entails a porous polymericlayer that provides mechanical support covered by a thin membrane which provides selectivity based on the size of molecules. In most cases, an ultrafiltration membrane is used. The molecularweight cut-off (MWCO) of the membrane differs according to the specific application, ranging froma few thousand to a hundred thousand daltons.

The culture media is pumped through the fiberlumen, and cells grow either in the extracapillary space, or on the shell side. Supply of low-molecular weight nutrients to the cells and the removal of waste products occur by diffusive transport across the membrane between the lumen and the shell spaces. Theultrafiltrationmembranepreventsfreediffusionof secreted product molecules from passing through the membrane and allows them to accumulate in the extracapillary space to a high concentration.

Although the use of microfiltration hollow fibermembranes for cell culture is infrequent, it does appear in various research applications for studying metabolism and for producing small quantities of materials for research or diagnostic applications.

Membrane BioreactorHollow Fiber Bioreactor

Multiple Membrane Plate BioreactorScaling up of a hollow-fiber system is limited bythe ability to extend the axial length of the fiberdue to oxygen transfer limitation. Use of a very high flow rate to supply more oxygen is limitedby the capacity of the pump and the mechanical strength of the membrane. Expanding the cartridge diameter to increase the capacity eventually faces theproblemofunevenflowdistributionamongthefibers.Inprinciple,onecanmixtwodifferentfibersto supply oxygen and medium separately. However, mixingdifferenthollow fibers inacartridgeposes

Fig. 9.20: Schematic of a hollow-fiber bioreactor


a major challenge in manufacturing. An alternative configuration using multiple flat membranes hasbeen attempted. However, this seemingly versatile reactor also suffers from practical manufacturing complexity, and is not used in large scale operations.

Microsphere Induced Cell Aggregates

Other Bioreactor Systems

Some cells do not form aggregates readily. When suspended in culture the rate of aggregate formation is slow and cells lose viability over time. One way to induce aggregate formation is to add microspheres to cell a suspension to promote agglomeration to the microspheres. These agglomerated microspheres become aggregates as cells grow. If the aggregate diameters become too large, necrotic centers can formed as a result of transport limitations. Many cell lines including BHK, CHO, 293, and swine testicular cells have been cultivated as aggregates with sizes ranging between 90 and 400μmwithout the formationofnecroticcenters.

(a) CHO cells attaching to micro-spheres and agglomeration of beads .

(b) Sections of HEK 293 cells forming microspheres induced aggregates .

(c) SEM micrograph of HEK 293 cells on microspheres

Agarose Cell Immobilization Agarose entrapment of cells is usually accomplished by passing a cell/agarose suspension through a small orifice into an air parallel jet stream. Thedroplets of agarose containing entrapped cells are collected in a chilled oil phase to allow the agarose to gel. Alternatively, the cell-agarose suspension may be allowed to drop onto the center of a fast rotating disk. The centrifugal spinning force causes droplets to form and be dispensed outside the disk. Agarose beads tend to be rather large, commonly, hundreds of micrometers in diameter. The large size of agarose beads limits oxygen transfer at high cell concentrations. In addition, agarose beads lack sufficientmechanicalstrength to sustain mechanical optimization even in a moderately small scale (tens of liters) bioreactor.

Figure 9.21: Microspheres induced cell aggregates


The centerpiece of a rotating wire cage bioreactor is the wire cage, which is often mounted on the agitation shaft. The bottom of the cage is solid, while the side is made up of wire mesh with openings ranging from 25 to 60 μm. The averagediameter of cells is approximately 10 to 15 μm.Typically, fresh medium is added continuously outside the draft tube and the culture fluid is

Spin Filter Stirred Tank

Microencapsulation Another method of cell entrapment entails entrapping cells in a polymeric matrix to form spheres followed bycoatingwithapolymericfilmtocontroldiffusivityof molecules. The idea is to allow fast nutrient diffusion while retaining the product molecules.

One of the polymers most often used for cell entrapment is calcium alginate. Cells are suspended in sodium alginate and added dropwise into a calcium chloride solution, which allows for gelation. The alginate beads may be coated with polylysine for increased mechanical and chemical stability. The alginate gel inside the polylysine coated bead can be liquefied through treatmentwith a calcium chelator such as EDTA or citrate.

Large-scale application using this microencapsulation technique is not easy. However, the microcapsule provides a means of immunoisolation of transplanted cells or tissues, and could be suitable for some tissue engineering applications.

The membrane stirred tank was developed by Professor Jürgen Lehmann in the 1980s. This bioreactor uses long microporous polypropylene tubing wrapped around rotating rods. By adjusting the air pressure in the polypropylene tubing, the micropore expands to allow gas to be in direct contact with medium, thereby providing bubble-free aeration. The rotation of the tubing also provides gentle agitation to microcarriers or suspended cells.

Membrane Stirred Tank

Fig. 9.22: Formation of microencapsulated cell beads

Fig. 9.23: A membrane stirred tank bioreactor


withdrawn from inside the cage at the same rate.

Under certain operating conditions, the cell concentration inside the wire cage is lower than that outside the cage, thus achieving cell retention in the bioreactor. Under optimal conditions, the device doesnotactasa filter,andnocakeis formed.Theretention of the cells does not appear to be due to centrifugal force exerted by the rotating motion of the cage, because the terminal velocity of the cells due to centrifugal force is two to three orders of magnitude lowerthanthatofthefluidvelocityacrossthescreendue to perfusion. The electrostatic effect is also unlikely to be responsible, since the ionic strength of theculturefluidisrelativelyhigh,andthethicknessof the Debye layer is only in the order of 1 nm.

The rotating wire cage bioreactor has also been used in aggregate and microcarrier cultures. In these cases, the mechanism of cell retention is relatively straight forward as the particle is much larger than the openings on the cage.

The mechanism of retaining cells in suspension by this device is still not clear. It is plausible that the retention is caused by a fluid mechanical effect,maybe one similar to the behavior of particles of low Reynolds number near the wall in a Poiseuille flow or laminar boundary layer flow along a flatplate. Lacking a mechanistic understanding of cell retentionhasmadescaleupofthisdevicedifficult.

Many recently installed spin filters are operatedat very high rotation rates. At such a high sped of rotation rate centrifugal force plays a key role in cell separation by dispelling cells away from the surface of the rotating cage. Cell separation in these reactors is certainly different from the low speed wire cage reactors described in this section.

Fig. 9.24: A spinner filter stirred tank bioreactor


Concluding RemarksMammalian cells are widely used in the production of viral vaccines and therapeutic proteins. Most of those processes employ cells growing in suspension; however, some applications, particularly in vaccine production, use adherent cells. Stirred tank bioreactors are most widely used for mammalian cell culture processes. When a stirred tank bioreactor is used for growing adherent cells, a supporting surface for cell attachment is provided through the use of microcarriers or macroporous microcarriers. Alternatively, adherent cells may be grown as aggregates in suspension. Fed-batch culture is commonly practiced since it facilitates reaching high cell density and high product concentrations. When a stirred tank bioreactor is operated in a continuous mode, it is a common practice to employ a cell retention device for perfusion in order to increase cell and product concentrations.

As mammalian cell culture processes are increasingly being used in manufacturing, one also witnesses an increasing adoption of disposable (or single-use) cell culture devices. Traditional roller bottles are still oftenseen.Additionally,multipleflatplatesorparalleltrays, along with bag type cell growth chambers and even plastic stirred tanks, are used in moderate scale production and in the seed culture propagation for large scale bioreactors. These disposable devices offer simplicity in operations, and ease process validation in the production of biopharmaceuticals. Some innovative bioreactors developed nearly two decades ago are now finding new applications intissue engineering and cell therapy. As these new technologies advance and the demand of those specialized cells grows we will continue to see innovative developments in bioreactor technology.

A vibromixer uses a perforated disk as the mixing mechanism rather than a conventional impeller. The disk vibrates in a vertical direction (perpendicular to the plane of the disc) at high frequency causing liquid to circulate through the perforated holes and provide mixing. The vibromixer was used in the 1960’s for the cultivation of suspension cells and virus production. Its use in cell cultivation has diminished in recent decades. However, it is still used in some cases to keep concentrated microcarriers in suspension for cell detachment during the trypsinizationstep.Thefluidmixingprovidesgentleshear to detach trypsinized cells from microcarriers.

high frequency vibration

liquid moved by the vibrating plate as going through the holes

Vibromixer

Fig. 9.25: A vibro-mixer based bioreactor

Fig. 9.26: Movement of culture liquid around the vibromix-ing plate

OXYGEN TRANSFER IN CELL CULTURE BIOREACTORS | 213

Oxygen Transfer Through Gas-Liquid Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Oxygen and Carbon Dioxide Concentration in Medium . . . . . . . . . . . . . . . . . . 217Oxygen Consumption and CO2 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219Method of Supplying Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Gas Sparging in Cell Culture Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .221Bubble Size and Sparger Orifice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222Experimental Measurement of KLa and OUR . . . . . . . . . . . . . . . . . . . . . . . . . . . 224Effect of Hydrostatic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Damage to Cells by Gas Sparging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .226Mass Transfer Resistance in Cell Immobilization Reactor . . . . . . . . . . . . . . . . . . . . . . .229Oxygen Transfer in Plug-Flow Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .229Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .231

Oxygen Transfer Through Gas-Liquid InterfaceIn cell cultivation, oxygen is transferred from air through the gas phase (via either gas bubbles or the gas space in the flask) and the liquid phaseinterface into the medium where it is consumed by cells. Simultaneously, CO2 produced by the cells is excreted into the medium and then diffuses into the gas phase. Transfer of oxygen or CO2 molecules from one phase to the other occurs only when oxygen or CO2 in the two phases is out of equilibrium. If oxygen in the two phases are in equilibrium (i.e., the medium is already saturated with oxygen), there will not be any net transfer in either direction.

If a liquid deprived of any gas molecule is in contact with air composed of 79% N2 and 21% O2, both N2 and O2 will diffuse into the liquid and eventually reach equilibrium between the two phases. (Of note, because N2 has a very low solubility and is hardly

Fig. 10.1: Concentrations and partial pressure of oxygen across an air bubble in culture fluid

Oxygen Transfer in Cell Culture Bioreactors


consumed by cells, it will not be further considered in this chapter.) At equilibrium, if the oxygen content in the gas phase is increased to 40%, then oxygen in the liquid phase will no longer be saturated and, gradually, oxygen will diffuse into the liquid phase until the dissolved oxygen concentration becomes equilibrated in the gas phase. Conversely if the oxygen level in the gas phase is decreased to 10%, then oxygen will escape from the liquid into the gas phase until a new equilibrium is reached.

Thus, in order for mass to transfer between the two phases, a deviation from equilibrium is essential. Such a deviation is often referred to as the “driving force for mass transfer”. Quantifying the driving force, or thedegreeofdeviationfromequilibriumisthefirststep toward calculating the rate of mass transfer.

Oxygen level in the gas phase is usually described as mole percentage or mole fraction (yO2). It can also be expressed as partial pressure (PO2), or the mole fraction of oxygen multiplied by total pressure. If the ambient pressure is 1 atm (or 760 mm Hg) and the oxygen mole fraction is 0.21 (yO2 = 0.21), then the partial pressure of oxygen in the air is 0.21 atm (or 159.6 mm Hg).

The oxygen concentration in a liquid phase (or dissolved oxygen concentration) is often expressed as mmole/L. When pure water is saturated with oxygen under a pressure of 1 atm and 21% O2 at 37°C, its oxygen concentration is 2.2 mmole/L. Because this concentration is the equilibrium value with an oxygen partial pressure of 159.6 mm Hg, sometimes it is expressed as 159.6 mm Hg. Expressing dissolved oxygen concentration in terms of its gas phase partial pressure is common in the medical profession. Since oxygen is a critical nutrient in the living world, one needs to be familiar with the multiple descriptors used to describe its concentration.

For example, water which is at 50% of saturation with air at 1 atm and at 37°C has a dissolved oxygen concentration of 0.11 mmole / L, or 79.8 mm Hg. If water containing 0.11 mmole / L oxygen is in equilibrium with a N2/O2 mixture, then that mixture would have 79.8 mm Hg [or 10.5% (mol/mol)] of oxygen.

Description of Dissolved Oxygen Concentration• Solubility in water at 37°C with air of 1 atm, 21% O2

• 0 .2 mmole / L

• 0 .64 mg / L

• Since air in equilibrium contains 159.6 mm Hg or 0.21 atm, sometimes solubility is expressed also in mm Hg, especially for blood oxygen level

To transfer oxygen or CO2 between gas and liquid phases through interface

• The two phases must not be in equilibrium

Fig. 10.2: Driving force for oxygen transfer across air-liquid interface


If water containing 0.11 mmol/L of oxygen is put in contact with air (with 21% O2 at 1 atm at 37°C), it is undersaturated and is 0.11 mmol/L away from saturation. In other words, the water could hold 0.11 mmol more oxygen per liter. One could also say the water is 79.8 (159.6 - 79.8) mm Hg below oxygen saturation. Such concentration difference from saturation is the driving force for oxygen transfer. One can then quantify the driving force using different units; describing it in terms of liquid phase concentration (mM), or gas phase concentration (mm Hg).

Aconcentrationprofileofoxygenacrossaninterfaceof gas phase and liquid phase is depicted in the figure. In the bulk liquid (i.e., the liquid at somedistance away from the interface), the concentration of oxygen is C. In the bulk gas phase, the oxygen fraction is yO2, and its partial pressure is PO2.

To dissect the mechanism of oxygen transfer, we model the interface as having a boundary layer or animaginaryfilmseparatingthetwosides.Letusdenote the oxygen concentration in the liquid as C* if the system is at an equilibrium with gas phase (whose oxygen partial pressure is PO2), and denote the gas phase concentration as P*O2 when it is in equilibrium with liquid phase (which is at a concentration of C). The liquid phase system is therefore C*-C away from equilibrium. In other words, the driving force for the liquid phase to reach equilibrium is C*-C. Recall that we can also use gas phase units to describe the liquid phase concentration, so the driving force can also be described as PO2-P*O2. The two different descriptions of the same driving force are related by a constant as will be seen again later.

Again the driving force across the boundary of the two phases can be expressed in either gas phase concentration or liquid phase concentration. Thus, when the driving force is not zero, oxygen will diffuse across the boundary layer to move the system toward equilibrium. The higher the concentration gradient across the interface is (i.e., a stronger driving force), the faster the oxygen transfer rate will be.

The rate of oxygen transfer is dependent on three


factors: 1) the magnitude of the difference between C* and CL, 2) the area that is available for oxygen transfer, and 3) the mass transfer coefficient.

The second factor, available surface area, is typically expressed as interfacial area per volume of liquid. It, thus, has a unit of inverse of length (L-1, like cm-1). Think of water evaporation, or mass transfer of water vapor, from a liquid water surface into dry air. Therefore, if a given amount of water were contained in a cup, it would take longer for it to evaporate than if that same amount of water was spread out in a flat dish because the surface areais much larger in a dish. Another example of using a large surface to increase oxygen transfer is our lungs, which, when fully expanded, can yield an area of nearly 150 m2. This is why using a lung to transfer oxygen is far more effective than using our skin.

The third factor affecting the rate of oxygen transference is the mass transfer coefficient.This is a measurement of the resistance for mass transfer at the interface. It is affected by the specific molecules that are being transferredand the physical and chemical properties of the liquid. The mass transfer coefficient (KL) has the same units as velocity (L/t, like cm/sec).

One can imagine the boundary layers in the interface as a stagnant film surrounded by a bulk liquid,which is under some mechanism of localized mixing. The oxygen molecules have to diffuse across the boundary layer to reach other phases. If the system is under vigorous mixing, the boundary layer would be thinner and the transfer would be faster. Again considering water evaporation in a dish, if that dish were being stirred and the air is being blown across the dish, the mass transfer coefficient atthe interface would be higher and the evaporation would be faster than when the dish is unperturbed.

Oxygen Transfer Rate (OTR) = KLa • (C*-C) =KLa ΔC [mmole / l • hr] = [cm / hr] • [1 / cm] • [mmole / L] C*: solubility of oxygen, ΔC: oxygen concentration gradient (i.e., the driving force)

Three Factors Affecting OTR • Mass transfer coefficient (KL) • Specific transfer area (i.e., the interfacial area) • Driving force (i.e., the gradient across interface)

To Improve Oxygen Transfer • Increase KL (make the interface more “turbulent”) • Increase a (use smaller bubbles for sparging, or use

silicone tubing) • Increase ΔC (use oxygen enriched air, or don’t maintain

dissolved oxygen (C) at an unnecessarily high level .)


Because the rate of oxygen transference between a gas phase and a liquid phase is dependent on how far it is from equilibrium, it is important to firstdetermine its saturation level, i.e., concentration at equilibrium, to accurately estimate the oxygen transfer rate. Oxygen is sparingly soluble in water. For such gas-liquid pairs, the solubility of a gas solute in the liquid phase is described by Henry’s Law. Henry’s Law states that, at equilibrium, the concentration of a gas in the liquid phase (i.e., its solubility) is proportional to the pressure of the gas.

The concentration in each phase is described in different ways. The most commonly-used variations are partial pressure for gas phase (which is the mole fraction of the gas solute multiplied by the total pressure) and the mole fraction for the liquid phase. For process calculations, one may find itconvenient to express liquid phase concentration in mmole/L. In the medical profession, the gas phase composition is often expressed in mm Hg, with 760 mm Hg = 1 atm. The value of Henry’s law constant for a gas-liquid pair is, thus, dependent on how the concentration is expressed.

Henry’s law applies well to an ideal solution. Despite all of its components, cell culture medium is still close to an ideal solution so the saturation concentration calculated using an aqueous solution applies to cell culture media. However, cell culture media is frequently used under a gas phase containing 1 - 10% CO2. Also, in a bioreactor, the air pressure is usually greater than 1 atm. Thus, oxygen solubility needs to be corrected for the differences in pressure and CO2.

Calculation From Henry’s Law

Oxygen and Carbon Dioxide Concentration in Medium

• Oxygen solubility is not affected by other dissolved species in medium . Its solubility is virtually identical to PBS and water.

Henry’s Law

xA: mole solute A / mole solutionPA: partial pressure of ACA: mole A / LH: Henry’s law constant

xH

PA

A=

Example: at 37° C, in 1 atm air (PO2 = 0.21 atm), O2 concentration in H2O is:

• In ambient air, (oxygen partial pressure = 0.21 atm), C* ~ 0 .22 mmole / L in H20 at 37 °C = 160 mmHg = 5.8 mg / L (5.8 ppm)

(Eq . 1)C* = (0.21atm) / (5.18x104atm / moleO2 / moleH2O) x (55 .5 mole H2O / L H2O) = 0.22 mmole O2 / L H2O

T, ˚C 5 20 25 30 35 3710-4 x H 2 .91 4 .01 4 .38 4 .75 5 .07 5 .18

Table 1 . Henry’s Law Constants for O2 (atm/ mole O2/mole H2O)


The solubility of CO2 can similarly be calculated using Henry’s law. However, in an aqueous solution, CO2 associates with water molecules and then dissociates into HCO3- and H+. The value calculated using Henry’s law is the sum of CO2(aq) and H2CO3 in pure water. Thus, the solubility of CO2 will be affected by the pH of the solution.

( ) ( q)CO g H CO a

CH

P

2 2

CO

CO

2

2

$=

=

6 6@ @

Example: Using 10% CO2 in air, CO2 concentration at 37°C is

Table 2 . Henry’s Law Constants for CO2 (atm / mole CO2 / mole H2O)

T, °C 0 5 10 15 20 25 30 35 40

10-3 x H 0 .728 0 .876 1 .04 1 .22 1 .42 1 .64 1 .80 2 .09 2 .33

(Eq . 2)

( ) ( )

( ) ( )

CO g CO aq

CO aq H O H CO aq

H CO HCO H

2

2 2 2 3

2 3 3

2?

?

?

+

+- +

(Eq . 4)

Solubility of CO2 affected by pH

• Solubility in medium should be adjusted by CO2 used in gas phase (i.e., the fraction of O2 in gas phase is usually not 0 .21) .

(Eq . 3)


Oxygen Consumption and CO2 Production

The objective in oxygen transfer is to supply oxygen at a rate that is enough to maintain the dissolved oxygen at a set level (or optimal level) for cell growth and production. For most mammalian cells, a30%saturationwithambientair issufficient foroptimal growth. Given that the saturation level at 1 atm of ambient air is about 0.2 mmole/L, the optimal value for cell growth is about 0.06 mmole/L. The amount of oxygen cells consume per culture volume per unit of time is called the oxygen uptake rate (OUR). As you may recall from the kinetics chapter, OUR is dependent on the specific oxygenconsumption rate (qO2) and the cell concentration.

Most oxygen taken up by cells is used to oxidize carbon sources. The most important carbon sources for mammalian cells in culture are glucose and glutamine. The oxidation reaction of both compounds yields CO2 at a stoichiometric ratio of 1.0 and 0.9, respectively. This CO2 to O2 ratio is called the respiratory quotient (RQ). The CO2 produced by cells will need to be removed from the culture to avoid excessive accumulation, which would cause a drop in pH or an inhibition of growth.

In cell culture operation, aeration accomplishes two goals: supply of oxygen and stripping carbon dioxide out of culture medium. CO2, inhibitory at high concentrations, is also an essential component of the medium. A certain level of gaseous CO2 is necessary to maintain the pH in a neutral range. CO2 is also used in a number of biosynthetic reactions, notably fatty acid synthesis. If CO2 is completely stripped from the medium, cell growth may be inhibited.

Optimal Oxygen Concentration• Most cells grow well at a dissolved oxygen level of 30%

of saturation with air

Oxygen Demand by Cells• On the average q02 ≈ 1.0 x 10-10 mmole / cell-hr

CO2 Production by Cells• Respiratory quotient of cells is about 1.0, i.e., 1 mole

CO2 is produced for every mole of O2 consumed, or qCO2 ≈ 1.0 x 10-10 mmole / cell-hr

• A high CO2 concentration is inhibitory• Inhibitory level usually starts at 15% to 20% CO2

Stripping of CO2 by Aeration• CO2 is required for cell growth (e.g. in fatty acid

synthesis), in addition to providing pH buffer in vivo.• Cells can grow well even in sodium-bicarbonate-free

medium, especially in a stationary culture, because CO2 produced by cells accumulates in medium to condition their growth.

• When cell concentration is low in a sodium-bicarbonate-free medium, excessive aeration may strip off CO2 to inhibit cell growth, unless some CO2 (~0 .5 – 1%) is in the gas .


Method of Supplying Oxygen To supply oxygen, one can introduce it through a gas phase in contact with culture medium. In a small-scale reactor or flask, a gas head space isoftensufficientinsupplyingoxygen.Inalarge-scalereactor, air or oxygen-enriched air is introduced as gas bubbles from the bottom. In some cases, direct bubbling of gas phase into culture medium is to be avoided to minimize foaming or other adverse effects. Under such conditions, the gas phase may be introduced through a membrane with high oxygen permeability, such as silicone or Teflonmembranes. In other cases, the gas phaseis not introduced into the reactor but rather the medium is pumped out and passed through an oxygenator. The oxygen-enriched medium is then recirculated back to the reactor. A hollow fiberbioreactor is typically operated in this fashion.

To control oxygen transfer, one can manipulate any of the three factors affecting oxygen transfer. One way is to increase the driving force by enriching the air with more oxygen. If surface aeration is used, one can install a surface aerator to increase the mass transfercoefficientontheliquidsurface.Iftubingora membrane is used, one can use a larger membrane or more tubing to increase the surface area.

In small-scale reactor operations, direct sparging (i.e., bubbling air) is more prone to problems. The agitation rate in a cell culture reactor is comparatively low compared to a microbial reactor. A microbial fermenter employs turbine impellers at a very high agitation rate to make gas bubbles entrenched in the liquid. The bubbles take a torturous path to rise and eventually burst from the liquid surface. The holding time of a bubble in the liquid is relatively long. In comparison, in a cell culture bioreactor, the bubbles rise in a relatively straight, vertical direction and emerge from the liquid phase quickly. Therefore, theefficiencyofoxygentransferforagivenairflowrate in a bioreactor is low. But, as the scale increases in cell culture, the holding time also increases with the length of the traveling path of the bubbles, thus increasing the efficiency of oxygen transfer.

Method of Supplying Oxygen• Surface aeration

• Silicon tubing/membrane

• Sparging

KL is about 3 cm / hr – 6 cm / hr for surface aeration in cell culture vessel

Example: What cell concentration can be reached in a 400 ml spinner flask (diameter 8 cm)?Estimate: KL = 5 cm / hrSurface area A= (4 cm)2 • π = 50.2 cm2

a = 50.2 cm2 / 500 cm3 = 0.1 cm-1 (a is surface area per culture volume)Assume D .O . is almost zero at maximum cell concentration (maximum driving force)ΔC = 0.18 mmole / lqO2 = 1.0 x 10-10 mmole / cell • hrqO2 • x = OTR = 5 cm / hr • 0.1 / cm • 0.18 mmole / l = 0.09 mmole / ℓ-hr x = 0.09 mmole / l-hr ÷ 1 x 10-10 mmole / cell • hr= 9 x 108 / l = 9 x 105 / ml

*2 2( )L O

dCV OTR V OUR Vdt

K a V C C q x V

= ⋅ − ⋅

= ⋅ ⋅ − − ⋅ ⋅

Balance of Oxygen in a Reactor

OTR: Oxygen Transfer RateOUR: Oxygen Uptake RateV: Culture volume


In a cell culture reactor, the most practical means of supplying oxygen is by blowing air into the reactor through a sparger. If one were to supply oxygen throughthemediumflow,themediumwouldhaveto be recirculated many times per hour because it can only carry a small amount of oxygen. Thus, air sparging is commonly used in bioreactors.

Direct sparging in microcarrier culture poses a number of challenges. Cells attached to microcarriers render the microcarrier prone to stick to air bubbles. As the bubbles rise to the liquid surface, carriers may also rise as well. The associated fluid shearcaused by bubble rising may also cause cell damage. If a foam layer is allowed to accumulate, one may see microcarriers accumulate on top of the foam. The use of antifoam may alleviate the problem, or one may use a screen/sieve separator to allow bubbles to be sparged in a microcarrier free zone.

To enhance the oxygen transfer rate in a bioreactor, one may make changes to manipulate each of the three factors affecting oxygen transfer rate (OTR): KL, a, and ΔC. Increasing the airflow ratewill increase the interfacial area, a. Increasing oxygen concentration increases the driving force, ΔC.IncreasingtheagitationpowerinputincreasesKL and enhances bubble breakup to increase the interfacial area, a. Smaller bubbles give a larger interfacialareaatagivenairflowrate.Inmicrobialfermentation, a high agitation power input is used to break up air bubbles. The agitation power input for mammalian cell culture is relatively low. The range of airflow rate used in cell culture is alsolow, compared to microbial culture. A high air sparge rate causes excessive foam formation, but an extensive use of antifoam agent causes cell damage. Therefore, enriching the oxygen content in the air supply and using smaller bubbles are commonly practiced, especially in small-scale cultures.

Gas Sparging in Cell Culture Bioreactor

assume (saturatedby air)need to recirculatemedium5 times every hour

.

5

q x mmole cell hr cell l

mmole l hr

Cin mmole l

F V hr

5 10 2 10

1

0 2

O10 9

1

2$ # $ $ #

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`

=

=

=

=

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Example:

Medium flow rate required to supply oxygen without gas phase aeration:-

Sparging• Short bubble residence time in small bioreactors

• Increasing the agitation rate increases oxygen transfer through better surface aeration and longer bubble residence time.

• Direct sparging in microcarrier culture is possible but more difficult

• Can sparge in the "microcarrier free" zone (use a wire cage)

( )vdtdc F Cin C q xvo2= - -

Fig. 10.3: Supply of oxygen through liquid circulation


Bubble Size and Sparger Orifice Dispersions of gases in liquids are widely employed in fermentation processes. The interfacial area can be very large: 3-mm spherical bubbles with a gas holdup of 26% (i.e., a liter culture containing 26% volume of gas bubbles and 74% medium) provide 52cm2/cm3 — a very large value.

Bubbles behave very much like oil drops, but their buoyancy and rising velocity are greater. Mass transfer within the bubble is rapid, because the molecular diffusivity in gases is large. Normally the resistance to mass transfer on the liquid side of the interface is the dominant resistance.

In the case that a single bubble is slowly released from a submerged orifice or small capillary, if thegas flow isveryslow, thebubble is releasedwhenthe buoyant force just overcomes the surface tension, at which time the bubble diameter is governed by the balance between buoyancy and surface tension exerted on the bubble.

The force balance between buoyancy and surface tension indicates a modest decrease of diameter, dp, with an increase in the orifice diameter,do. Inertial effects become dominant at larger flow rates employed in industrial multi-orificespargers, thus rendering the initial bubble size almost independent of the orifice size.

Usingorificesizetocontrolbubblesisthusnotveryeffective. In small-scale bioreactors, one may employ spargerswithafineopeningtogenerateverysmallbubbles.Thefinebubblesagglomerateastheyriseto the liquid surface and coalesce into large bubbles. Thus, in large-scale reactors (on the order of hundredsofliters),theeffectivenessoffinebubblesin an increasing interfacial area begins to diminish.

Bubble size may also affect bubble rising velocity and the mass transfer coefficient. In principle,larger bubbles have a higher terminal rising velocity and a higher KL if bubbles are considered to be rigid spheres. However, as bubble diameter increases, the bubble tends to deform and become flatter on top, thus decreasing the rise velocity.

Effect of Orifice Diameter on Bubble Size

d0= orifice diameters = surface tension Δ ρ = difference in density between liquid and gas

3106

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=ρs


Experimentally, it has been observed that both rising velocity and KL only increase with bubble diameter when the bubble size is small (<3 mm). Beyond that, they are relatively invariant.

Overall, manipulating bubble size to be very fineis beneficial in small reactors as it increases thegas transfer area for both oxygen and carbon dioxide. This is important because the impeller and agitation rate used is not effective for breaking up gas bubbles. On a larger scale, though, controlling bubble size becomes difficult becauseof the high gas flow rate and bubble coalescence.


Experimental Measurement of KLa and OUR

Mass transfer coefficient can be determinedexperimentally. Overall, KL is relatively insensitive to bubble size, except for very small bubbles and undersimpleflowpatterns.Theinterfacialarea,a,isdifficulttoestimateortomeasureinbioreactors.As a result, most experimental measurements of mass transfer characteristics lump KL and a together and report the value as “KLa”. KLa is sometimes called the volumetric mass transfer coefficient.

To measure KLa, one can aerate a reactor that initially has a very low oxygen concentration. The dissolved oxygen concentration will steadily increase until reaching saturation. A semilog plot of C*-C vs. time will give the slope as KLa. One can also perform the experiment by stripping oxygen from medium that was, initially, at a higher dissolved oxygen level. In this case, the slope in the semilog plot will be -KLa. In most operations, the aeration rate will change over time. One can develop a correlation between KLa and airflow rate for use during cultivation.

Plotln(C*— C) vs . t

Initial Condition

( )ln C C K a t*L $- =

After Integration

t=0, C=CO

( )dtdC K a C C*

L= -

Fig. 10.4: Measurement of KLa by degassing, or stripping of oxygen from the liquid phase


Effect of Hydrostatic Pressure In very large bioreactors, circulating cells will occasionally reach a region where the hydrostatic pressure is high. This is particularly a concern for air-lift bioreactors, in which the aspect ratio (height over width) is much larger than stirred-tank reactors. Experimentally, it has been shown that cells grow normally at up to nine atm and exhibit little discernible metabolic differences, compared to normal conditions.

Effect of Hydrostatic Pressure

• 10 m liquid height gives a 1 atm hydrostatic pressure at the bottom of tank

• Up to almost 9 atm has no adverse effect on hybridoma cell growth and specific glucose consumption.

Measure OUR in laboratory

OUR or the specific oxygen consumption ratecan be measured by filling up a container witha cell suspension without leaving room for a gas phase. Since there is no oxygen supply, the change in dissolved oxygen level will be only from cell consumption. The slope of the linearly decreasing dissolved oxygen level will yield the oxygen uptake rate. Then, the specific oxygenconsumption rate can be obtained by dividing OUR by the cell concentration in the cell suspension.

Figure 10.5: Measurement of oxygen uptake rate


Damage to Cells by Gas SpargingIn an aerated mixing vessel, forces exerted on cells may cause cellular deformation in several ways. Forces normal to the cell surface may squeeze the cell, cells may impinge on moving mechanical objects, and/or cells may be subjected toa fluidshear field,suchthatonesideof thecellis subjected to a fast flower than the other side.

Despite the stress, cells have a substantial capability to change their shape. The shape change involves reorganizing the cellular cytoskeleton and redistributing the lipid bilayer membrane (both the cytoplasmic and organelles). If the rate of deformation is fast and the extent is extensive, the cytoskeletal network and the membrane integrity will be compromised; therefore leading to stressed conditions, or even cell death.

Vigorous sparging of air bubbles through the culture can also cause cell damage. The mechanism likely to cause most severe damage is the fluid shear field caused by bubbles.

Damage to cells by gas sparging

• Cells and cell-laden microcarriers adhere to gas bubbles

• Energy dissipation in gas sparging

• It is generally thought that bubble breakup at liquid surface dissipates most energy and is most damaging to cells

• Protective agents are added to medium in sparged (see media section)

Several possible events may dissipate the high intensity of mechanical energy when bubbles traverse through cell suspension:

1. Release of the bubble from the orifice of thesparger. As the bubble escapes from orifice, avolume suddenly becomes void, causing the cellsuspensiontorushintofillthevoidspace.Avelocitygradient, and thusa shear field,willinevitably occur under such flow conditions.

2. A rising bubble will cause velocity gradient around itself and thereby change the liquid velocity in proximity to the surface of the bubble. Conversely, far from the bubble, the liquid velocity is the same as bulk velocity.

3. Similarly, bubbles coalesce or break-up, also causing liquid velocity gradient.

4. Bubbles bursting from the liquid surface cause the volume originally occupied by gas to be rapidly replacedbyliquid,therebycausingashearfield.

Fig. 10.6: Deformation of a cell by compressing force


It is generally recognized that the last event generates the greatest shear stress. As the bubbles burst off the liquid surface, the high liquid velocity at the interface with the air decreases to the bulk liquid velocity over a very small distance (of about a couple of hundred micron). The momentum of the liquid movement caused by the bursting bubbles causes the liquid to rise above the liquid level surface. As a result, the liquid moving downward to fill theempty space leftby thebubble reversesits direction to rise upward. In the upward moving liquid cone, the velocity is highest in the center and lowest on exterior. Also in the process of bubble bursting, there is a rapid change of the gradient direction when the liquid overshoots the surface and then drops down after reaching a maximum height. The damaging effect on cells entrapped in the bubble-bursting zone can be tremendous.

Cell

fast moving object

Liquid Velocity di�erence on two sides of a cell causes shear stress

Fig. 10.8: Possible ways of damaging cells by sparging in a bioreactor

Fig. 10.7: Liquid velocity difference on two sides of a cell causes sheer stress


As visualized through high-speed video photography, a bubble is often surrounded by a layer of cells, possibly due to hydrophobic interactions between the cell and bubble surfaces. Once bubbles begin to rise, the liquid stream forces cells to the tail, or the wake, of the moving bubble. As the bubble bursts from the liquid surface, cells can be severely damaged, if not outright killed.

Block copolymer pluronic F68 reduces the number of cells adhering to the rising bubble through its surface tension modulation effect. Pluronic F68 thus “protects” cells from the damaging effect of sparging.

Fig. 10.9: Conceptual depiction of fluid movement surrounding the point at which a bubble emerges from the liquid surface.

Fig. 10.10: Accumulation of cells to a bubble surface due to hydrophobic interactions and accumulation to the tail end while the bubble rises.


Mass Transfer Resistance in Cell Immobilization ReactorCells are sometimes cultivated inside particles or in a stagnant liquid phase. Notable examples are macroporous microcarrier and hollow fibersystems. In these systems, oxygen not only needs to be supplied continuously to the culture medium, but it also needs to diffuse to the position where oxygen is supplied to the interior, where cells reside. As oxygen diffuses, it is also consumed by cells. An oxygen concentration gradient is, thus, created between the position of oxygen delivery and the deepest point of cells where oxygen must reach.

The rate of concentration decrease is dependent on how fast the cells consume oxygen and the geometry of the particle. Farther into the interior, oxygen may become depleted or become too low to support a high rate of growth or productivity.

To avoid such oxygen limitations, one might resort to using smaller particles or to maintain a higher oxygen concentration in the culture medium. The extent of oxygen transfer limitation can be estimated by comparing the mass diffusion and consumption rates. The theoretical calculation is presented as plots of two variables, often referred to as effectiveness factor and Thiele’s modulus, which are discussed in standard chemical reactor textbooks.

Oxygen Transfer in Plug-Flow BioreactorsSome bioreactors used in cell cultivation employ a cell compartment that lacks direct contact with the gas phase. Hollow fiber bioreactors and somerecirculatory flat bed bioreactors, especially intissue engineering applications, fall into this category. In such a system, oxygen is supplied by recirculating the medium. Often, an external oxygenator,suchasahollowfibermembranedeviceor a mixing vessel, is used to replenish oxygen in the medium before it is recirculated to the cell chamber.

In such cases, the oxygen concentration gradient exists in two dimensions. As the medium flowsdownstream in the cell chamber, oxygen is diffused

• Oxygen transfer limitation occurs in both axial and radial directions

• Periodically reversing the direction of flow should help

Figure 10.11: Concentration gradient of oxygen in a cell aggregate or an immobilized cell matrix particle


through the membrane into the cell compartment. Oxygen concentration, thus, decreases along the axial direction. In the radical direction, oxygen diffuses toward the cell chamber; along the path the oxygen concentration decreases.

In a hollow fiber bioreactor, oxygen in therecirculating medium diffuses from the bulk liquid to the wall of the fiber, then through the fiberand into the cell chamber. Typically, the diffusion resistance is highest across the wall. Outside, the wall diffusion occurs simultaneously with oxygen consumption by cells. At a high cell concentration, the drop in oxygen in the cell chamber is steep so, often, a very high concentration of oxygen is maintained in the medium to ensure the center of the cell chambers receive enough oxygen. To improveoxygentransfer,onemayincreasetheflowrate of medium recirculation, introduce a gas phase compartment(andhavehollowfibersforairflow),orreversetheflowdirectionperiodically.Inanycasethe longitudinal distance that can be used before oxygen transfer becomes limiting is restricted.

Fig. 10.13: Photograph of a hollow fiber bioreactor

Fig. 10.14: Oxygen gradient in the radial direction of a fiber in a hollow fiber bioreactor

Fig. 10.12: Oxygen concentration gradient in the axial direction of fluid flow in a plug flow reactor


Concluding Remarks

The solubility of oxygen is very low in cell culture medium. It must be supplied continuously to sustain cellular demand from a gas phase. The transfer rate of oxygen through the liquid-gas interface is affected by mass transfer coefficient, interfacial area anddriving force, or the deviation from equilibrium. All three factors can be manipulated to enhance oxygen transfer. The most direct and effective method of

supplying oxygen is by bubbling air through the bioreactor. Although air bubbles cause damage to cells, the problem can be overcome. However, the air supply rate per reactor volume tends to decrease as scale increases. Therefore oxygen transfer and carbon dioxide removal are among the key issues that should be addressed in scaling-up or scaling-down. This will be further discussed in the scale-up chapter.


FED-BATCH | 233

Fedbatch Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .233Types of Fedbatch Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .234

Intermittent Harvest and Feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234Extended Fedbatch Culture: Fortified Feed and Addition . . . . . . . . . . . . . . . . . . 235Fedbatch With Metabolic State Manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

Designing Feed Medium for Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .237Feed Medium Design for Consumed Nutrients: Stoichiometric Principle . . . . 238Feed Medium Design for Unconsumed Components . . . . . . . . . . . . . . . . . . . . . 240

Control Strategies for Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .241Control Objective and Criteria: Productivity and Product Quality . . . . . . . . . . 241

Feeding Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .243Feeding by Direct Measurement of Nutrient Consumption . . . . . . . . . . . . . . . . 243Proportional Feeding With Base Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244Proportional Feeding With Turbidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244Proportional Feeding With Oxygen Uptake Rate (OUR) . . . . . . . . . . . . . . . . . . 245

Delivery of Feed Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245Online Estimation of Stoichiometric Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246


Fedbatch CultureInthefirstdecadeofthe21stcentury,wewitnesseda rapid expansion of therapeutic proteins in clinical applications. We also saw productivity increase rapidly through intense process development. During this time, fedbatch culture processes have emerged as the predominant mode for producing recombinant proteins.

In a batch process, the constraint of osmolarity limits the amount of nutrients that can be added initially. This low-nutrient level prevents the culture from attaining high cell and product concentrations. In fedbatch cultures, medium is added during cultivation to prevent nutrient depletion, thus prolonging the growth phase and ultimately increasing cell and product concentrations.

Batch Culture• Limited nutrient concentration in medium

• Solubility of amino acids

• High osmolarity at high nutrient levels

• Growth inhibition by high nutrient levels

• Feed nutrients throughout the cultivation extending the growth and production period

• Higher cell and product concentration

Fedbatch Culture and Dynamic Nutrient Feeding

With contributions from Weichang Zhou

FED BATCH | 235FED BATCH | 234

Types of Fedbatch CultureFedbatch processes are widely used in multi-purpose, multi-product facilities because of their simplicity, scalability, and flexibility. A variety offedbatch operations, ranging from very simple to highly complex and automated, are seen in current production facilities. Compared to a typical batch operation, the operation time of all types of fedbatch cultures are extended, resulting in higher total cell and product concentrations.

Fedbatch Culture• Predominant mode of production rDNA proteins

• Intermittent harvest used in inoculum preparation, also in virus production

The “intermittent harvest and feed” fedbatch processdoesnotdeviatesignificantlyfromabatchculture. The culture is initiated using a medium and culture conditions, very similar to that used in batch culture. Cells are then allowed to grow exponentially, with no additional manipulation, until cells reach the late growth phase. A portion of the cells and product are then harvested and fresh medium is added to replenish the culture volume. This process is repeated several times. This simple strategy is commonly used for the production of viruses which cause persistent infection in cells, as it allows for an extended production period. It is also used in roller bottle processes with adherent cells. In some cases this type of fedbatch culture is also used to supply inoculum for imitating a reactor production chain.

Intermittent Harvest and Feed

Fig. 11.1: Time profile of a fedbatch culture with intermittent harvest

FED BATCH | 235

In “fortified feed and addition” type of fedbatchculture, the period of rapid cell growth is prolonged by adding concentrated feed media (usually a 10-15 times more concentrated than basal medium, often without salts or with 1x concentrations of salts). The concentrated medium is added either continuously (as shown) or intermittently to supply additional nutrients, thereby further increasing cell concentrations and lengthening the production phase. To accommodate the extra addition of media, a fedbatch culture is initiated at a volume less than the full capacity of the bioreactor. This initial volume, typically enough to allow the impeller to be submerged, is kept at a low level to allow for the addition of extra media throughout the expansion phase.

The addition of extra nutrients results in a higher cell concentration, prolongs the production period, and, ultimately, results in a substantially higher product titer. In recent years, it has become common practice to decrease the operating temperature after a period of rapid growth. The temperature shift reduces the growth rate and nutrient consumption rates, further prolonging the production period and increasing the product titer.

Extended Fedbatch Culture: Fortified Feed and Addition

initial �nal

feeding concentratedfeed

nutrients

Extended Fedbatch Culture

Typical Fedbatch Culture for rDNA Protein Production

• 7 - 15 days

• 8x106-2x107 cells / mL

Fig. 11.3: Time profile of a extended fedbatch culture with increasing culture volume

Fig. 11.2: Depiction of a fedbatch culture with incremental feeding


In batch cultures and most fedbatch processes, lactate, ammonium, and other metabolites eventually accumulate in the culture supernatant over time. These metabolites, along with high osmolarity and reactive oxygen species, are growth inhibitory and contribute to the eventual loss of viability and productivity of the culture. The effects of lactate and ammonia on cultured cells are complex. Detectable changes in growth, productivity, and metabolism have all been documented. Additionally, metabolite accumulation has been found to affect product quality.

By limiting the availability of glucose and glutamine using controlled feeding strategies, cell metabolism canbedirectedtoamoreefficientstate,characterizedby the reduced production of lactate. Such a change in cell metabolism is often referred to as metabolic shift. By extending the methodology to limit the consumption of both glucose and glutamine, both lactate and ammonium accumulation can be reduced.

In the example of fedbatch culture shown, glucose concentration was kept at a low level by continuous feeding of a concentrated glucose solution. In this scenario, lactate was still produced and, after a period, lactate production ceased, thus indicating a metabolic shift. The shift can also be seen from the decrease in the stoichiometric ratios of lactate production to glucose consumption during the cultivation.

A metabolic shift alters the stoichiometric ratio of lactate to glucose throughout the entire culture period. Even after averaging the initial culture period (which includes no metabolic shift), less lactate and ammonium are produced for a given nutrient consumed.

Cells can also consume lactate under slow growth and high lactate and low pH conditions. Recently, it has been shown that after the rapid-growth stage in fedbatch cultures, cells can switch to lactate consumption from lactate production. The consumption of lactate alleviates growth inhibition and gives rise

Fedbatch With Metabolic State Manipulation

Table 1 Characteristic Stoichiometric Ratios of Key Nutrients for Cell Growing in Different Metabolic Statesstoichiometric ratio (mmole/mmole)

Without Metabolic

Shift

Metabolic Shift

Lactate consuming

cellslactate/glucose 1 .4 - 2 .2 0 .05 - 0 .5 0 .4 - 1 .0

ammonia/glutamine

0 .5 - 1 .3 0 .1 - 0 .3 -

alanine/glutamine

0 .2 - 1 .3 0 .01 - 1 .3 -

oxygen/glucose 1 .0 1 .0 - 2 .0 -

Figure 11.4: Growth profile of a fedbatch culture with controlled glucose level to induce metabolic shift

Fig. 11.5: Stoichiometric ratio change during a fedbatch culture with metabolic shift

FED BATCH | 237

to a higher product concentration, compared to the cultures that remain at a lactate production state.

The vast majority of fedbatch culture employs fortified nutrientmixtures to prolong growth andproduction phases. Critical to a successful fedbatch process is the design of feed media. A well-designed feed medium should enable cell growth and product formation, without depleting any medium component or allowing the build-up of excessive nutrients or metabolites. Furthermore, adding feed media to the culture should not introduce factors that adversely affect cell growth or product formation.

The chemical species in media fall into two general categories: 1) those whose consumption rates are significant andmeasurable and2) thosewhose concentrations greatly exceed the amount consumed by cells during the cultivation period. Chemical species that are consumed should be replenished by the feeding medium to maintain their concentration in an optimal range. Conversely, nutrients that are hardly consumed may not be added; however, because the culture volume increases substantially in a fedbatch culture, even a nutrient that is not consumed by cells may be diluted due to culture volume expansion. Consequently, feeding of nutrients not consumed by cells is necessary to sustain growth and production.

Medium Components• Consumed by cell (almost all organics, growth factors)

• Concentration decreases unless being replenished

• Not consumed

• Or consumption rate too small to cause concentration change in medium

Fedbatch Culture• Volume can be expaniding due to feeding

• Volume expansion may cause dilution

Designing Feed Medium for Fedbatch Cultures


During the growth phase, the goals of feeding are largely to replenish the nutrients which have been consumed and to ensure that nutrient levels stay within an optimal range for optimal growth or for optimal product formation.

Under balanced growth conditions, the specificconsumption rates of various nutrients are relatively constant and, correspondingly, the consumption of nutrients or production of metabolites relative to one another are proportional. These rate proportionalities are called stoichiometric ratios. A well-formulated feed media is designed to add nutrients at appropriate stoichiometric ratios to match their consumption rates. This strategy should allow all nutrients to be replenished at each particular cell line’s rate of consumption.

Stoichiometric ratios can be calculated using historical culture data obtained from the cell line of interest, as discussed in the stoichiometry and kinetics chapter. Ideally, the data should be obtained from cultures under relevant cultivation conditions. Typically, one medium component is chosen as a reference nutrient. The consumption rates of other nutrients are related to the reference nutrient, using stoichiometric ratios. Common choices for reference nutrients are glucose, glutamine, oxygen, and lactate, as they are consumed or produced in larger quantities among all nutrients and metabolites and are relatively easy to measure.

The idea of stoichiometric feeding is that if all nutrients are consumed in relative proportions, once the amount of the reference nutrient consumed is known, one can calculate the amount consumed of all the other nutrients using the known stoichiometric ratios. The stoichiometric ratio can thus be used as a basis for designing feed media. Using this strategy, one would determine the consumption of the reference nutrient and then feed the culture with the feed medium to appropriately replenish lost nutrients, according to their calculated stoichiometric consumption.

Stoichiometric ratios are not necessarily constant.

• Glucose, amino acids can be added proportional to their stoichiometric ratio during fedbatch culture

• Determine the stoichiometric ratio of medium components

• Choose one reference component (e .g . glucose, lactate, glutamine, oxygen), feed all other nutrients by stoichiometric ratio

• Stoichiometric ratio may change over time of cultivation

Feed Medium Design for Consumed Nutrients: Stoichiometric Principle

FED BATCH | 239

Cells may not necessarily consume all nutrients in the same proportion in different culture conditions or stages. The feed media composition may need to be adjusted, according to known changes of stoichiometric ratios under different culture conditions or in different growth stages.

Feed medium design, using this method, is best approached as an iterative process, where a design is tested, analyzed, and refined until an optimalmedium emerges. Most basal media are designed with an excess supply of amino acids, especially glutamine. In addition to being used for protein synthesis, glutamine supplies precursors for nucleotide synthesis and replenishes intermediates for the tricarboxylic acid cycle. The consumption of glutamine and other amino acids can often be reduced without affecting growth and product formation. The reduced consumption of these nutrients also decreases ammonium production. In the feed medium optimization process, the concentration of some amino acids may also be reduced if excess ammonium accumulation becomes growth inhibitory.

The first round of iteration usually employs datafrom batch culture growth, in which amino acids are supplied in excess. The stoichiometric ratios determined from batch culture are then used to prepare the initial version of feed medium. Often, the concentration of nutrients in the feed medium is about 10- to 15-times that in basal medium, in order to reduce the required feed volume.

During the optimization runs, the concentration of the reference nutrient (usually glucose, glutamine, or oxygen), which can be measured on-line or off-line rapidly, is measured periodically to determine the consumption rate. The amount of feed medium needed to replenish the reference nutrient to the original level or a set point is then calculated and added. If the stoichiometric ratios are estimated correctly, the level of nutrients should remain within the set range. If the stoichiometric ratio for a nutrient is not accurate, its concentration will then increase or decrease. After the optimization run, a new set of stoichiometric ratios is then determined and is used

Normalized Specific Rate (relative to lysine at low ΔL/ΔG state)

Normalized Composition

High ΔL/ΔG state

Intermediate State

Low ΔL/ΔG state

Cell IgG

ALA -11 .50 -0 .93 0 .08 1 .32 0 .76ARG 1 .25 1 .25 1 .25 0 .69 0 .35ASN 0 .23 0 .25 0 .03 0 .00 0 .50ASP 0 .05 0 .23 0 .10 1 .47 0 .57CYS 0 .00 0 .00 0 .00 0 .04 0 .35GLN 16 .25 9 .00 4 .50 0 .00 0 .72GLU 0 .50 0 .25 0 .13 1 .84 0 .74GLY 0 .35 1 .28 0 .10 1 .33 1 .00HIS -0 .75 0 .75 0 .50 0 .32 0 .24ILE -0 .75 1 .25 1 .00 0 .84 0 .35LEU 0 .75 1 .75 1 .25 1 .31 0 .98LYS 1 .50 2 .25 1 .00 1 .00 1 .00MET 0 .78 0 .58 0 .10 0 .33 0 .20PHE -0 .40 0 .38 0 .45 0 .54 0 .50PRO 0 .00 0 .00 0 .00 0 .80 1 .02SER 1 .08 0 .90 0 .45 0 .90 1 .85THR 2 .00 1 .50 0 .75 0 .79 1 .11TYR -0 .50 0 .25 0 .50 0 .40 0 .59VAL -1 .25 0 .75 1 .00 0 .95 1 .30

Table 2. Amino Acid Specific Consumption Rate and Composition in Cell Biomass and IgG

ΔL/ΔG: stoichiometric ratio of lactate to glucose

Fig. 10.6: Use of cumulative nutrient consumption data to calculate stoichiometric ratio


for the design of the next feed medium. Using these methods, a very effective medium is typically achieved with fewer than three rounds of optimization.

Some medium components are consumed in very small quantities and their concentrations remain virtually unchanged in culture. This includes many inorganic ions, such as sodium, calcium, and sulfate. From a stoichiometric point of view, the feed medium does not need to include these nutrients. As feed is added, however, the volume of the culture increases and causes the medium components to be diluted. Thus, these components are included at low levels in feed medium (typically 1X concentration or less). In some cases, inorganic salts, such as NaCl, are completely eliminated from the feed medium to reduce any changes in culture osmolarity.

Some ions, such as magnesium (complexed with ATP), phosphate (as free phosphate or in nucleotides), and potassium, are present at a much higher concentrations inside the cell than in the medium. At high cell concentrations, the amount of these ions takenupby thecellsmaybecomesignificant.Therefore, it is often necessary to compensate their consumption by supplying them in the feed medium.

In addition to basal medium components, protein hydrolysates, serum, insulin, transferrin, vitamins, and lipid additives are also used in culture. These additives supply minute nutrients, which are consumed or become inactive with time. However, the concentrations of these medium components are not usually measured and their consumption rate is mostly unknown. In the absence of a reliable measurement consumption rate for those additives, one has to rely on an order-of-magnitude estimate of the upper and lower limits of their consumption rate. The feeding rate of those additives is then chosen to maintain their concentrations above a minimum threshold, while below a maximum tolerable limit which is experimentally determined.

• For Mg+2 and PO4-3, intracellular concentration >>

medium concentration

• At high cell concentration, cell consumption can cause depletion in medium

• Need to supplement about 1x in feed medium

Feed Medium Design for Unconsumed Components

FED BATCH | 241

Control Strategies for Fedbatch CulturesHow the feed medium is delivered to a culture may affect the performance of a fedbatch process. On-line measurements may be employed to determine the consumption of the reference nutrient in real time, then used to drive a fully-automated system to feed nutrients continuously. At the other ex-treme, one can use off-line monitoring of nutrient level and manually feed the nutrient periodically.

Three elements should be considered in devel-oping a feeding scheme: 1) the control objective and the control criterion (level of different nu-trients to be maintained), 2) the mode of feed-ing (continuous or intermittent feeding), and 3) a control strategy for determining the tim-ing and amount of feed medium to be delivered.

The simplest strategy is to allow nutrient levels to vary, within a wide range, by adding large quantities of feed media at widely-separated time intervals (e.g., once or twice per day) based on off-line measurements or historical data. Such a periodic feeding scheme is very simple and is usuallysufficienttoavoiddepletionoroverfeedingof nutrients. For more specialized cases, especially those aiming to manipulate cell metabolism, a more frequent measurement of parameters, along with well-controlled feeding schemes, are necessary.

Developing Fedbatch StrategyDefine objective of feeding. Possibilities include:

• Sustain nutrient level

• Manipulate growth rate in a range

Define what criteria to manipulate

• e.g., what level of nutrient to control?

Mode of feeding

• Continuous vs. intermittent

How to deliver the feed medium

• By direct measurement of nutrient or infer from indirect measurement

A common objective of nutrient feeding is to increase product titer by increasing cell concentration. In recent years, the objective has been gradually extended to assuring product quality and consistency. Culture conditions may affect glycoform or protein folding. The depletion of a particular amino acid may cause an amino acid to be mis-incorporated into the protein product. Culture conditions may also affect the frequency of deamination or glycation of the protein product after it is secreted into the medium. Furthermore, extensive cell death may cause product degradation or desialylation.

The control objective of feeding may be to reduce glycation by setting a control criterion to keep

• Control objective extends beyond acheiving high productivity to high product quality and consistency

• Require an understanding of relationship between manipulated variables and control objective

Control Objective and Criteria: Productivity and Product Quality


glucose concentrations below a certain level, or to minimize amino acid mis-incorporation by maintaining amino acids above a certain level. Another method of control is to minimize protein quality deterioration by maintaining a long, slow growth phase and avoiding a death phase.

Afterdefiningthecontrolobjective,thenextstepistoidentify the process variables that cause the process to deviate from the control objective. Furthermore, a relationship between the key process variable and the control objective, such as inducing lactate consumption, should be available so that the process variable can be controlled within an optimal range. For mammalian cell culture, although the control objectives can be defined, the factors that affectthe path to the objective are not easily identified.Lacking knowledge of the relationships between manipulated variables and the control objective makestheoptimizationofcontrolcriteriadifficult.

In the past few years, it has become empirically evident that maintaining a moderately high osmolarity in a late stage of culture can increase the productivity of recombinant proteins. A high osmolality increases productivity, perhaps, by enhancing the expression of stress responsive proteins, which facilitate protein folding, although the exact mechanisms are not known. By keeping the osmolality only at a moderately high level to avoid causing a rapid decline of cell viability the culture can be sustained over a longerperiodofhighspecificproductivity, leadingto high final product concentration. This practiceis commonly used in industrial manufacturing.

An increasingly common practice is to employ atypical culture conditions, which may not be optimal for long-term maintenance or expansion of cells in the final production reactor. Theseconditions may include very high glucose concentrations (15 g / L or 83 mM), low pH (6.9 vs. 7.2 for optimal growth), and/or low temperature (33/34 oC vs. 37 oC). These conditions have been found to affect the duration of culture and the overall cell metabolism, resulting in an increased product titer. A high concentration of glucose

Possible Control Objective and Control CriteriaControl Objective

• Reducing desialylation by reducing cell death and sialydase release

• Reducing glycation

Control Criteria

• Control osmolarity below set point

• Control glucose level below set point

FED BATCH | 243

also increases osmolality. This is compensated by reducing the salt concentrations to keep osmolality around 300 mOsm during the cell growth stage.

Feeding StrategiesDirect measurement of nutrient concentrations is the most straightforward way to determine the amount, rate, and timing of feed to be added. Based on current concentrations of nutrients, one can determine how much medium should be added to sustain the nutrient level for a given period of time.

The concentrations of some nutrients can be determined on-line, although off-line nutrient measurement is more common. Glucose and glutamine are the two nutrients most commonly measured and used as controls because their concentrations can be determined rather rapidly in laboratories.

Direct measurement of these compounds on-line can be implemented using an auto-sampling device, in series with commercially-available immobilized enzyme/membrane-based measurement devices.

HPLC can also be implemented as an on-line approach for the measurement of glucose and amino acids. This technique requires a series of processing steps for sample preparation before injection into theHPLC.Asignificantlagtimebetweensamplingand delivering control action is unavoidable. However, since the doubling time of mammalian cellsincultureingenerallyexceedingfifteenhours,even an hour lag time in HPLC measurement of amino acids is acceptable. Industrially HPLC on-line analysis has been implemented at pilot plant scales.

In-Time Measurement for Control• On-line or off-lineExample• On-line sampling coupled

• HPLC for amino acids

• Enzyme assays for glucose, glutamine, lactate

Different Way of Coupling Feeding to On-Line Measurement

• Single stream with fixed stoichiometric ratio

• Multiple streams ratios among different nutrients can be adjusted

Feeding by Direct Measurement of Nutrient Consumption


Proportional Feeding With Base Addition

A widely-used control strategy in bioprocessing is to proportionally add medium according to the amount of base added to the culture for controlling pH. This provides the option of continuous on-line feeding without an on-line nutrient measurement device.

In most cultures, a large fraction of the glucose consumed is converted to lactic acid. To maintain culture pH, one mole of base is added to neutralize each mole of lactic acid produced.

If the stoichiometric ratios between lactic acid production, glucose consumption, and other nutrients are relatively constant, the rate of lactic acid production can be used to estimate the consumption rates of glucose and other nutrients.

Barring the effects of pH buffers (such as sodium bicarbonate or HEPES), the base addition rate is indicative of lactic acid production. This method is simple and easy to implement. However, it is highly sensitive to CO2 level in the gas phase and sodium bicarbonate concentration in the medium.

Proportional feeding according to base addition is not well-suited for processes requiring a highly accurate control of feed rate. Medium buffer capacity can cause delays in base addition and decrease the overall sensitivity of the method.

Rationale• In lactate production state L / G is relatively constant• Base added to neutralize pH at 1 mole base / mole

lactate• Use stoichiometric ratios to lactate to feed

• Easy to implement, but its sensitivity is limited by buffer in medium

The use of an online laser turbidity probe can provide accurate estimations of cell concentration in culture. Simple proportional feeding with turbidity works well during the exponential growth phase, when viability is high and the growth rate is relatively constant. However, near the end of the exponential growth phase, when viability drops, an assessment of viability or metabolic activity must be used to adjust feed rates to avoid over-feeding. An alternative measurement to turbidity is measurement of capacitance,which reflects viable cell volume, i.e.,viable cell concentrations and cell sizes. This may be used for better control of nutrient feeding rate.

• Turbidity is a good indicator of total cell density (but not viability)

• Feed nutrients proportional to cell density• Reliable during growth stage• Capacitance probe can measure viability

Proportional Feeding With Turbidity

FED BATCH | 245

Among different measurements of nutrient consumption, the oxygen uptake rate (OUR) is most accurate for assessing cellular metabolic activities. OUR measurements, unlike pH, are not masked by buffers in the medium. A small amount of oxygen consumed in culture, even in the range of 10 µM, can be accurately determined using a dissolved oxygen sensor. For other nutrients which are often present at millimolal or hundreds of micromollal levels, such sensitivity of measurement cannot be easily achieved especially for on-line measurement.

With its high sensitivity, small changes of OUR can be confidently detected on-line and in realtime, thereby providing an immediate indication of changes in metabolic rate. On-line oxygen consumption data can then be used to determine nutrient demand, using established stoichiometric ratios and control continuous feeding.

• Most sensitive among all on-line measurement of metabolic activity

• Require developing a computer algorithm• Need to develop stoichiometric ratios to oxygen

Proportional Feeding With Oxygen Uptake Rate (OUR)

After determining the amount of feed media to be added, one needs to decide on the mode of medium delivery (e.g., the frequency and amount of feed to be delivered at each feeding). The method of feed media delivery is constrained by equipment and also by the composition of the feed medium. The feed medium usually consists of a solution of concentrated amino acids and other organic nutrients, which, when kept over a long time, tend to precipitate.

The main consideration in determining the proper feeding frequency is the acceptable range of nutrient concentration. The concentration of nutrients will fluctuatebetweenahighlevelatthetimeoffeedingand the low point immediately before the next feeding. More frequent feeding reduces the deviation from the set point. On-line feeding, by coupling to base addition, turbidity, or to OUR measurement, is easily implemented by computer control and is almost continuous. When feeding is coupled to less frequent off-line measurements, medium is typically added manually, a few times a day.

Parameters in Feeding• Initial culture volume vs. feeding volume• Feed concentration

• High concentration desired, but stability/ precipatation concern

• High salt content from solubilization• Single feed vs. multiple feed solution

• Need adjustable stoichiometric ratio

• Continuous vs. step-wise feeding

• Step-wise feeding• Frequency• Equal volume each time or proportional to

cell density

• Operational simplicity vs. controllability

Delivery of Feed Medium


A challenging issue of stoichiometric feeding is the adjustment of the feeding rate, or feed composition, in response to metabolic changes throughout a culture [12]. This is particularly critical when fedbatch cultures are used to elicit a metabolic shift.

Changes in metabolism over the course of a culture are commonly seen, as evidenced by the nonlinear relationships between specific nutrientconsumption rates. Many such changes bear little consequence on cell growth or productivity, but in some cases the effect is profound.

For simplicity, major changes in feed composition or feed rate are only made when profound changes in metabolism are observed. For example, in a late stage of growth, cells may cease to produce lactate and consume glucose at a slower rate. Excessive glucose feeding may, then, reverse the metabolism in the favor of lactate production.

Major changes in the metabolic rates of glucose, lactate, and glutamine can be detected by monitoring their stoichiometric ratios throughout the course of a culture. On-line measurement of nutrient levels would allow for timely detection of changes in stoichiometric ratios; however, its widespread implementation in industrial processes will require further development of reliable, automated sampling methods.

Without on-line, direct measurements of glucose and lactate to detect changes in stoichiometric

Detecting Stoichiometric Ratio Change During the Culture• Monitor glucose, lactate, glutamine, OUR• Monitor ∆L, ∆G, ∆G in, ∆OUR and check their ratios

over time

Online Estimation of Stoichiometric Feeding

Processes employing continuous feeding to control nutrient levels in a small range will require substantially more effort than intermittent feeding strategies. While allowing more control over environmental conditions, the superiority of continuous feeding in terms of extending culture lifespan and increasing productivity has not been clearly documented, except in the case where a metabolic shift is the desired outcome. In fact, with simple off-line monitoring, robust, intermittent feeding strategies is standard industrial practice.

FED BATCH | 247

ratios, one may resort to indirect estimation. For example, changes in OUR and the base addition rate may be an indication of a changing metabolic state, as the ratio of OUR to lactate consumption changes significantly when cells switch from alactate production state to a consumption state.

Concluding RemarksFedbatch culture is the prevailing mode of cell culture in the final production of manufacturingrecombinant proteins. It is also commonly used for extending the period of cell expansion for pre-production culture. In the latter, the culture conditions and feeding strategy are designed for optimal cell growth, whereas in the former, the conditions are often designed to elicit a high

productivity, high cell concentration, and high product accumulation. The design of feed medium and the selection of the feeding control strategy are critical to the successful implementation of fedbatch culture. By applying stoichiometric principles to feed medium design and by using a well-designed feeding strategy, an optimal fedbatch culture process can be implemented with relative ease.


CELL RETENTION AND PERFUSION | 249

Many sectors of the chemical process industry underwent major transformation during the second half of the 20th century. One of the most significant changes is the switch from batchprocesses to continuous processes. By minimizing equipment turnover time and process start-up time, a continuous process can be sustained at a high rate of productivity over a long time and achieve a higher throughput than a batch process.

Increasing adoption of the continuous process method also sparked much research into bioprocessing. Many enzymatic biocatalyst processes have long been operated continuously, similar to most waste treatment or biodegradation processes. Contrary to the practices of the chemical process industry, however, continuous process only became more common in the later years of microbial and cell culture bioprocessing.

A vast majority of biochemical processes involving microbial or animal cell cultivation are batch

Marketed cell culture products using perfusion bioreactors

Product Company

• RecombinantTM, Antihemophilic Factor (recombinant), (Factor VIII) Baxter

• Kogenate-FS (Factor VIII) Bayer• Aldurazyme• Naglazyme BioMarin

• ReoPro (IgG Fab Fragment)• Remicade (IgG1• Simponi (IgG1)

Centocor (J&J)

• Xigris (Protein C) Eli Lilly• Cerezyme• Fabrazyme• Myozyme/Lumizyme

Genzyme (Sanofi)

• Gonal-F (follicle stimulating hormone) Serono (EMD)

• Vpriv (velaglucerase alfa)• Replagal (agalsidase alfa) Shire

• ReFacto (Factor VIII) Wyeth (Pfizer)

Practice of Perfusion Culture

Cell Retention and PerfusionWith contributions from

Sadettin Ozturk, Chun Zhang, and Weichang Zhou

Practice of Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .249Analysis of Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .251

Material Balance on Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251Effect of Recycling Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

Methods of Cell Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .254Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254Incline Settling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256Acoustic Resonance Enhanced Settling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257Spin Filter Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258Microfiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259Alternating Tangential Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260



processes for many reasons. First, unlike catalysts and reactants do not change their behavior over time in chemical reactors (although catalysts may gradually lose their activity), microbes and cells may mutate, evolve, and change the make-up of their population and production capacity. Second, the risks of microbial contamination and equipment failure make long operations undesirable, especially in manufacturing, for which process robustness is of paramount concern. This is especially true when producing high value pharmaceuticals. Finally, the current product capture and purificationoperations are all designed for batch mode. Even if the production is operated in a continuous mode, the process is not designed to realize all of the advantages of a continuous process.

A continuous culture is constrained by the maximal flowrateatwhichitcanoperate.Onecannotoperateata flowratethat is fasterthancells’growthrate;otherwise cells are washed out and incapable of replenishing the culture content. This is particularly daunting for processes that must be performed at a flowratehigherthanthecellgrowthrate.Insomecases, cells produce growth inhibitors that must be continuously removed by media replenishment. In other cases, cells must be grown at a low growth rate to achieve a high productivity, resulting in the media being removed at a faster rate than the growth rate.

To overcome this shortcoming, a cell recycling system can be added to continuous culture. By recoveringcellsfromtheeffluentflowandreturningthem to the reactor, one can operate a continuous culture beyond its natural limitation of the dilution rate (flow rate divided by the bioreactor volume).With a higher cell concentration in the reactor, the overall throughput of the reactor is then also higher.

Whencellculturewasfirstadoptedastheproductionvehicle for biopharmaceuticals, continuous operation was explored as an industrial process. Researchers realized the growth of mammalian cells in batch culture is impeded by lactate and ammonium accumulation. A continuous process, thus, alleviates growth inhibition by removing metabolites through

AdvantagesBetter Product Quality

• Better controlled culture environment (nutrients & byproducts)

• Pseudo steady state operation (ease of control)

• Shorter residence time

• Higher cell viabilities & lower concentration of impurities

• Critical for unstable molecules

More Economical

• Higher cell concentrations & higher productivities

• Smaller bioreactor size

• More flexible

• Faster start up in process development

Disadvantages

Longer Cycle Time

• Longer process development & validation time

• Higher contamination risk

• Higher equipment failure risk

• Potential regulatory/licensing issues


Analysis of Perfusion CultureMaterial Balance on Perfusion Culture

Fig. 12.1: Schematic of a continuous culture with cell recycle

acontinuousflowofmedium.However,thehighcostof medium, especially serum, and the low product concentration made continuous culture impractical.

To enable continuous operation, cells are retained in the reactor while the media flow flushesout metabolites. This can be accomplished by immobilizing cells on some solid particles to prevent them from being flushed out by mediaflow, or by separating cells out from the effluentstream and recycling them back to the reactor. Thus, the general idea is continuous culture with cell retention. Theese processes are often called perfusion, which is reminiscent of the procedure of flowing fluid through an organ or tissue.

A number of biotherapeutic proteins are produced by perfusion processes. Some recombinant antibodies could easily be produced with a fedbatch process instead. It should be noted that the selection of process mode is often based on the availability of in-house expertise and many other factors. However, in the cases of labile products that may be degraded or otherwise inactivated over time, a perfusion culture alleviates the loss of productivity that cannot be easily overcome in a batch culture. For products that accumulate only at very low concentrations, a perfusion process may also present a competitive advantage over a batch or fedbatch

One can analyze a perfusion culture system by performing material balances on the reactor system. The flow rates (F) of fresh medium entering andexiting the system are the same, to keep the volume in the reactor constant. The fresh medium stream is free of cells. We also assume that the reactor is well mixed, so that the nutrient (substrate) concentration in the reactor is the same that it is in the effluentstream. A cell separator is used to process the reactor effluentstreamtoreturnaconcentratedcellstreamwith a given cell concentration, cx, back to the reactor. Notethattheeffluentstreamfromthereactorhasahigherflowratethanthefreshfeed,withaflowrateof(1+α)FinsteadofF,tobalancetherecyclestream


attheαFflowrate.Thesymbolsusedarethesameas those in the stoichiometry and kinetics chapter.

Material balance can be performed on both the reactor and the cell recovery/recycling device for both cells and substrate. An important parameter affecting the performance of the system is the ratio of cell concentration in the purge stream, x2, to that in the reactor, x. This ratio is affected by the recycling factor, α, and the cell concentrationfactor, c by x2/x = 1+α-αc. Note the ratio shouldbe always smaller than 1, so that there is a concentration effect by the cell recovery device.

An important conclusion from the steady state analysis is that with cell recycling, the dilution rate, which is defined as the flow rate divided by thereactor volume, is larger than the specific growthrate;whereaswithoutcell recycling (i.e.,α=0), thedilutionrateisalwaysthesameasthespecificgrowthrate.Asseeninthefigureaboutcellrecycling,thecellconcentration in the reactor is higher than without cell recycling and the reactor can be operated at a dilution rate higher than the growth rate. Therefore, for cells with a doubling time of one day, the maximum flow rate that can be used without cell recyclingwould be one volume a day. With a perfusion culture, even a few volumes a day may be operated depending on the cell retention factor (c). A higher retention factor permits a higher cell concentration. Depending on the retention device used, the efficiency of retention may vary; for instance, itmay decrease rapidly at the high dilution rate, causing the cell concentration to decrease rapidly.

The analysis described above is for a bioreactor with an external cell recovery device. The same principle applies to a system with an internal device. The consequence of employing an internal device is the same: the dilution rate can be higher thanotherwise. It allowsa fastnutrient flowrateto be used to reduce metabolite concentrations, while keeping cells in the reactor and allowing cell concentrations to become maximal.

For the balance equation on biomass for the bioreactor is

Balance on the cell recycle system gives:

2 1x cx

α α= + −

The balance on the substrate on the bioreactor

Defining F/V=D. Applying steady state conditions:

practically c>1, so D>μ

(1 )dxV Fcx Fx xVdt

α α μ= − + +

2)1( FxFcxFx +=+ αα

2(1 )c Fx Fxα α+ − =

0 (1 )ds xV Fs V F s Fsdt Y

μ α α= − − + +

0 (1 )Dcx Dx xα α μ= − + +

(1 )D cμ α α= + −

00x s

xFs V FsYμ

= − −

00 ( )x s

xD s sYμ

= − −

D = Dilution rate

Cell recycle (or retention) can accomplish:

• A higher cell density in reactors

• A higher dilution rate than maximum specific growth rate

Fig. 12.2: Comparison of cell concentration profile at different dilution rates with and without cell recycle


Effect of Recycling Factor In a perfusion system operated at steady state, the amount of cells purged (Fx2) from the cell recovery device equals the amount of cells produced through reproduction (µxV). To keep a high viability and steady state, a small amount of cells are purged,. The system is operated at a dilution rate, D.

For a given dilution rate, one may employ a different recovery device with a different cell retention efficiency. Using the equation as an illustration,giventhesamedilutionrateandspecificgrowthrate,onemaychoosedifferentcombinationsofαandc.Ifonechoosesahighlyefficientcellconcentratorwithalargec,thenαthatisusedwouldbesmaller,andvice versa.

This concept illustrates that at a given dilution rate, when using a highly efficient cell separator (suchas a centrifuge that creates a concentrated recycle stream),alowrecyclingrate(αF)canbeemployed.Conversely,whenusinganinefficientcellseparatorthat gives a low degree of cell concentration, then a large recycling rate needs to be used; in other words, cells will need to be pumped out of the reactor and pass through the cell separator to recycle more frequently.

In large-scale operations, the cell stream could potentially stay outside the reactor for a long time, so oxygen starvation is a concern. Consequently, the fluidstreamoutofthereactorisoftenchilled,first,to reduce oxygen consumption. With a device that gives a low c value, cells will need to be subjected to the extra environmental perturbation of being chilled and pumped more often. This factor should be considered in selecting a cell recovery method.

Fig. 12.3: A high cell concentration factor allows for a low recycle ratio while achieving the same enhancing effect of cell cycle


Selecting the method of cell separation very much depends upon the way cells grow. The larger the particles are, the easier they are separated. Many processes employ microcarriers with particle diameters ranging from 0.2 mm to 2 mm. These cell-laden microcarriers can be easily separated from media stream by sedimentation. In some cases, cells are grown as large clumps, or aggregates, of 1 – 2 mm in size, also making sedimentation readily applicable. Other considerations in selecting a cell separator are the required throughput purge rate, the concentration factor (c), and the scale of operation.

Because of the limit of the concentration factor (c) of a cell separator, even though the purge rate (F) may not be large, a large recycling factor (α) may be necessary. Note that the capacity ofthe separator required is not only based on the dilutionrate (D,orF/V),butalsoon the flowrateoutofthereactor((1+α)F).Ifadevicewithasmallconcentration factor, c, is used, the capacity of the separator will have to be substantially larger than what is needed to process the purge stream alone.

Methods of Cell Retention

Sedimentation The simplest cell separator is perhaps a settling cyclone. The fed stream from the bioreactor enters into the settler in its midsection, where the stream splits into flows in two directions. The upwardstream, drawn by a pump for a purge stream, encounters a large cross sectional area and, thus, the vertical velocity is much smaller than the cell’s setting velocity, so the cells move downward. The downward stream, on the other hand, faces a decreasing cross sectional area and, thus, increases its vertical velocity as it carries cells downward. A transient zone separates the two well-developed upward and downwardflowregions.Inthistransitionzone,cellsare separated and carried downward into the reactor.

Such a settler is a convenient device for laboratory operations.Asthereactorscaleincreases,theflowrate also needs to increase proportionally, but the cross sectional area of the settler increases only

Conical Settler

• Selective removal of dead cells

• Low separation efficiency

• Cell settling velocity ~ 2-10 cm / h

• Good for larger cells

• Long residence time outside of the bioreactor

Fig. 12.4: A settling cone for cell retention


with2/3powerofthesettlervolume.Theefficiencyof cell separation decreases rapidly as the scale increases, making it ill suited for larger operations.

Incline Settling Inasimplesettlingtank,thedirectionoffluidflowand cell settling are along the same axis (both vertical). A sufficiently long transient zone isnecessary to separate the cell and cell-free streams.

To enhance the separation efficiency, thesettler is often inclined so that an angle exists between the fluid flow direction and the particlesettling direction. A particle is considered “collected” once it settles on a settler’s surface, since the fluid velocity on the surface is zero.

In an inclined settler, the feed cell stream enters at the bottom and moves upward. Inside the settler, cells begin to “settle” down vertically due to gravity. If a cell particle hits the surface at the lower plate, it is “collected” and does not get carried out by the effluent stream.

Eventually, the cells settled on the bottom plate form alayeroffluidwithahigherdensitythanthestreamabove. This heavy stream then moves downward, carrying the cells along. At the steady state, there are three streams in the system: the feed stream, the effluent stream (carrying unsettled cells),and the concentrated cell stream at the bottom.

In industrial design, multiple inclined plates are used in a single settler. In such designs, the feed stream and the returning cell stream do not cross each other by partitioning their flow pathin different zones. In some cases, mechanical vibration is applied to the plates to prevent settled cells from sticking to the surface and being lysed.

The residence time in the settler has to be at least as long as the particle settling time. With a high cell concentration in the stream, oxygen starvation is a major concern, as it may induce apoptosis and cell lysis. Therefore, the stream passing through the settler is often chilled to reduce the cell’s metabolic rate.

Operating parameters

• Cell size and concentration

• Perfusion rate

• Settling area

• Length of the plates

Inclined Settling

• While a particle is moving upward with flow, it also settles toward the bottom plate

• It is “collected” upon hitting the bottom

• Eventually, the particle rich zone has a higher fluid density and begins to move downward

• The particle-rich stream is recycled to the bioreactor

ParticleRecovery

ParticleSetting

feed from reactor recycle to reactor

conce

trate

d cell s

tream

purge stream

Methods of Cell Retention

Fig. 12.5: Cell separation in an inclined settler for cell retention


Centrifugation Centrifugation is a standard unit operation in many downstream recovery processes. In the early days of perfusionprocessdevelopment,itwasamongthefirstto be exploited for cell retention. As early as the mid 1980s, the Japanese company Teijin had employed centrifuges for perfusion culture of hybridoma cells used to produce antibodies for cancer imaging.

However, most centrifuges are not designed for long term and aseptic operations. Early use of the centrifuge for perfusion was only intermittent and was for batch harvest and cell biomass recycling, as well as for the replenishment of fresh medium. A number of autoclavable or stem-sterilizable centrifuges are now available. These and the disposable bag-based centrifuge are all capable of processing up to hundreds of liters of medium a day, and are suitable for continuous use in perfusion culture.

The disc-type centrifuge is analogous to a multiple parallel plate settler, except that the parallel plates are rotating and generate a centrifugal field forcell settling. The disposable bag system employs three tubes: a feed tube, an outflow tube for theheavy (cell-rich) stream, and an outflow tube forthe light stream. The unique design of an inverted question mark allows the three tubes to rotate, along with the centrifuge, without being twisted.

Centrifugation Technology

• Excellent separation efficiency

• High perfusion capacity

• Little clogging

• Easy scale-up

• Vulnerable to mechanical failure in long term continuous operation

Three different designs:

• Westfalia Disc type

• Continuous cell recycle back to fermenter

• Centritech Lab

• Disposable separation unit

Fig. 12.6: The cell loading and cell ejecting regions of a disc centrifuge for cell recycle


Acoustic Resonance Enhanced Settling This device uses acoustic energy to enhance cell agglomeration. As the cell stream from the reactor passes through the acoustic chamber, cells are induced to agglomerate. This gives rise to a faster settling velocity. With increased settling velocity, sedimentation is easily accomplished without resorting to multiple plates.

As cell concentration or operating conditions (i.e., flow rate and temperature) change, the energyand residence needed for agglomeration may also

cell

cell movement

feed

dilute stream

concentrated cell stream

Fig. 12.7: A disposable bag based centrifuge (Centritech) for cell recycle

Mechanical/Acoustic Trapping

• Enhanced sedimentation (Cell aggregation)

• Slightly favorable for viable cell retention

• Heat generation may create temperature gradient

• Low separation efficiency

• Moderate capacity (200L/day per unit)

Operating parameters

• Cell density, perfusion rate, power input


change. Sensors for detecting cell agglomeration will help stabilize the operation of the device.

Spin Filter Separation Theterm“spinfilter”isusedtorefertotwodifferentdesigns that may have rather different mechanisms ofoperation.Bothemployhighporosityfilterwithrelatively large openings (~20 - 100 µm) installed on the wall of a rotating cage. Both are submerged in culturefluid.Apumpdrawsmediumfrominsidethecageasthepurgestream.Theculturefluidpassesthroughthefiltertoenterthecage,theniswithdrawnbythepumptobecometheeffluentstreamexitingthe reactor. The flow into the cage has a lowercell concentration than thebulkculture fluid, thusachieving the overall retention of cells in the reactor.

Rotational Cage A rotating cage rotates along the center shaft of the impeller agitator at a low speed. The centrifugal fieldistypicallyinsufficienttopushawaycellsalongthe outside wall of the cage. Yet, a boundary layer of liquid around the cage probably exists, in which the cell concentration is lower than in the bulk. As a result, the fluid drawn across the filter is lowerthan in the bulk. Furthermore, there is little filtercake formation. The screen on the cage is, thus, not exactly a filter. Nevertheless, the system hasbeen employed in scales of up to hundreds of liters.

Internal and External• Remove dead cells/debris

Operating Parameters Affecting Performance• Screen pore size (1-120 μm)• Perfusion rate• Rotation speed• Screen surface area• Draft tube• Screen materials:

• Stainless steel, DNA & RNA deposit, • Teflon, Polyamide 66, polyethylene, better

“light” single cell stream moves upward

“heavy” aggregate stream moves downward

recycle stream cell aggregates

feed

re�ector transducer

agglomeration zone

Fig. 12.8: An acoustic cell agglomeration device for cell retention

Fig. 12.9. Picture of an acoustic cell retention apparatus

Fig. 12.10: A spinning filter (or rotating cage) for cell retention


Centrifugal Cage Therotatingcageisnotoriouslydifficulttoscaleup,as its operating mechanism is not well understood. Later modifications of spin filters increased itsrotation rate up to hundreds of rpm, allowing it to operate like a centrifugal filter. The centrifugalforce is sufficient to push the cells away from thesurface of the filter, thus drawing liquid throughwith a lower cell concentration than in the bulk.

Using such a device, the operation is less prone to variation, due to changes in fluid dynamics.It gives the freedom of operating in different regions in the reactor. In some variations, it is installed outside of the reactor and used as an external cell retention device. However, the device

differs from the traditional centrifugal filter usedin the recovery process in chemical industry in an important way in that no filter cake isformed. In fact, if cell cake is formed on the screen wall of the cage, cell death is likely to occur in the cake and leads to process failure

Microfiltration Microfiltration uses membranes of differentconfigurations,includingparallelplatesandhollowfiberdevices, andhasapore sizeofaround4µm.Microfiltration was among the first techniquesused for cell retention. Its widespread use has been impeded by membrane fouling, which is especially severe when a high concentration of proteins or complex medium is used in the culture. With the use of low protein medium in the past decade, the problem of protein fouling has lessened, but clogging by dead cells remains problematic.

External Loop for Cell/Harvest Separation• High shear, tangential flow

• At high transmembrane pressure, cell deformation occurs

• Fouling caused by high molecular weight DNA, protein, lipids, anti-foam occurs after days of operation

• Difficult to scale-down• The degree of concentration in a single pass is

relatively small

Cross Filtration Model

stationary

spinning

recycle stream

dilute purge stream

cell-free permeate

return to reactor

feed

cell

membrane

very high �ux may cause cell damage

Fig. 12.11: A Centrifugal filter for cell retention

Fig. 12.12: Microfiltration membrane for cell retention


Alternating Tangential Filtration Different configurations of microfiltrationdevices all use tangential flow, meaning thefeed stream flows in a direction parallel to themembrane, while the filtrate flows across themembrane.Themorerecentuseofapulsatileflowsystem, in which a diaphragm is used to rapidly reverse the flow direction, has reduced fouling.

product harvest stream

air

diaphragm in closed position

return to reactor

product harvest stream

from reactor

air

Rapid pulsatile �ow in reverse directions minimizes fouling.

diaphragm in open position

Fig. 12.13: An alternating tangential flow hollow fiber device for cell retention


volumes of inoculum that are used to scale up. The carrying over of a large amount of spent medium from seed culture is undesirable; evidence seems to suggest that a high metabolite concentration at inoculation can negatively affect a cell’s metabolic characteristics in the main culture. Thus, using the cell recovery devices for preparing inoculum, especially to increase the initial cell density, could potentially enhance process performance.

Another area that may change in the near future is the increaseduseof fortifiedmediuminperfusionculture, as we have seen in fedbatch cultures. Using fortified medium (instead of medium witha standard composition) can reduce the flow rateand increase cell and product concentrations.

Given the intrinsic advantages of continuous operations and the advances in cell retention technology, we may begin to see more widespread application of perfusion culture in the coming years. It has considerable potential to increase the capacity of high throughput processes, reduce reactor sizes, and possibly minimize product quality fluctuations through steady state operations.

Perfusion culture operation was explored very early on in cell culture development for obvious reasons: (1) the low throughput from batch operation can be enhanced by switching to continuous operation, (2) a simple continuous culture would have too low of a cell concentration to make the process economical, and (3) a dilution rate faster than the cell growth rate is needed to purge the metabolites accumulated in culture. Its widespread adoption was inhibited, however, by concerns about regulatory requirements and the lack of a clear definition of BATCH for product manufacturing.Researchers were also concerned about the lack of a scalable cell recovery device for long operations. These hindering factors have largely been overcome. Although cell line stability for sustained production of a product is still a concern, evidences have shown that, with proper control of cell age and cell stock, stability may not be an overriding concern.

As cell retention technologies become mature, one may also see this application go beyond perfusion culture. The same devices can be used to concentrate cells and remove metabolites, as well as to quickly replacethefluidphaseforcellfreezingoperations.Current inoculation operations are limited by the

Concluding Remarks


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Translation of the process scale is one of the most difficultissuesinbioprocessing,anditisprobablyoneoftheleastsystematicallystudiedsubjectsinthefield.Few engineers are involved in designing large-scale equipment using small-scale experimental data, but many will be developing processes in laboratories and at pilot plant scales for eventual implementation in a production scale. Others may be involved in troubleshooting investigations for production plants using laboratory equipment. Therefore, an understanding of the factors affected by scale-translation is important in carrying out those studies.

Scaling Up and Scaling Down for Cell Culture Bioreactors

Introduction

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .263Mechanical Agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .265

Mechanism of Agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265Power Consumption and Mixing Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .266

Power Consumption of Agitated Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266Other Dimensionless Numbers for Stirred Tank Reactors . . . . . . . . . . . . . . . . . 268Effect of Scale on Physical Behavior of Bioreactors . . . . . . . . . . . . . . . . . . . . . . . 269

Mixing Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .271Nutrient Starvation Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271Mixing Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272Nutrient Enrichment Zone, Mixing Time vs . Starvation Time . . . . . . . . . . . . . 272Mixing Time Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Scaling Up and Mechanical Forces on Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .274Scaling Up and Oxygen Transfer and Carbon Dioxide Removal . . . . . . . . . . . . . . . . . .275

Material Balance on Oxygen in Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276Aeration Rate and Superficial Gas Velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278Gas Flow Rate in Scaling Up: A Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280Effect of Scaling Up on CO2 Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281


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A reactor may be scaled up geometrically similarly or non-similarly. Geometrical similarity refers to maintaining the ratio of the main geometrical lengths, such as height over diameter, as well as the relative size of the internal parts (e.g., impeller, flow diverter, etc). In this chapter, ourdiscussion will focus on geometrically similar cases, which are conceptually easier to grasp, although this is not always the best approach in scaling.

In scaling up geometrically similarly, all length dimensions of the rector are scaled proportionally. Consequently, the surface area will increase with the length dimension to the second power, while the volume will increase to the third power of the length. As a result of scaling up, the scale-related surface area per unit volume of equipment will decrease. In microbial fermentation, the decrease in surface area to volume ratio causes the impediment for removal of heat generated from metabolism and mechanical agitation. In mammalian cell processing, the metabolic heat generated is less a concern. However, the process may still be sensitive to other variables related to scale change.

As the scale of an equipment changes, the physical and mechanical parameters may not be maintained constant. Frequently, it will not be possible to keep all key operating parameters constant between different scales. This may lead to changes in the chemical environment and, ultimately, cell physiology and productivity. The objective of scaling-up and scaling-down is, therefore, not to strive to keep scale-related parameters at constant, buttodefinetheoperatingrangeofscale-sensitivephysical and mechanical parameters so that the cellular physiological state and productivity can be maintained within an acceptable range. At times, this may require an adjustment of the chemical environment at different scales.

Translation of Scale Objective• Prediction of process performance

• Specify operating conditions from one scale to another

Major Effect of Scale

• Oxygen (Gas) transfer

• Heat transfer

• Shear force

• Compression force

• CO2 removal

Fig. 13.1: Schematic of a large scale cell culture fermenter

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Mechanical agitation in a stirred bioreactor keeps cells in suspension, provides mixing to create a more homogeneous chemical environment, and creates a flow pattern that increases theretention time of gas bubbles in the culture fluidto enhances oxygen transfer. In the cases that cells are grown as aggregates, agitation also helps reduce the formation of oversized particles.

In microbial fermentation, oxygen demand is rather high (often exceeding 150 mmole / L-hr). To increase the efficiency of the oxygen supply, extensiveagitation is used to break up air bubbles. In many fermentations of mycelial mold or actinomycete, extensive agitation is used to overcome the high viscosity of culture fluid and to reduce themycelial pellet size to enhance oxygen transfer.

In cell culture processes, the oxygen demand is nearly two orders of magnitude lower than that in microbial fermentation. Adequate oxygen supply can usually be accomplished by much less intensive agitation, which is also sufficient forprovidingsufficientmixingandsuspensionofcells.

Purpose of Agitation• Gas-liquid mass transfer

• The higher shear field near the impeller tip produces small bubbles, thereby increasing gas-liquid interfacial area (provided that bubble coalescence is not correspondingly increased)

• Suspension of solid (e .g . microcarriers, soymeal) or dispersion of liquid

• Liquid-liquid, liquid-solid mass transfer (e .g . hydrocarbon culture, quick mixing of pH neutralizing base)

• Minimization of pellets or aggregates

• Pellets are cell aggregates or mycelial microorganisms (streptomyces, molds)

• Mixing, especially for viscous fluid (e.g. xanthan gum)

• Fermentation, broth of mold culture

Theflowpatternsgeneratedbydifferentimpellersinastirredtankaregenerallyclassifiedasoneoftwotypes:axialfloworradialflow.Anaxialflowpatternreferstoprimarilyupwardordownwardflowduetothepumpingactionoftheimpeller.Inaradialflowpattern, the liquid moves primarily outward toward the wall of the vessel. In cell culture processing, impellers generating axial flow are used becausetheshearfieldsgeneratedbyaxialflowpatternsarelowerthanthosegeneratedwithradialflowpatterns.

The Rushton disk turbines, as are often used with multiple installations in large reactors, are the predominant type used in microbial fermentation. In this design, the sparger is placed directly underneath the disk turbine. Gas bubbles from the sparger rise to hit the disk and are directed outward.

Table 1. Characteristics of Impellers

Characteristics Propeller Disk Turbine

Flow direction Axial Radial

Gassing Less suitable Highly suitable

Dispersing Less suitable Highly suitable

Suspending Highly suitable Less suitable

Blending Highly suitable Suitable

Mechanism of Agitation

Mechanical Agitation

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The blades, rotating at a fast speed, then break the bubbles up. In the immediate surrounding region of the blade, a very high-energy dissipation is predicted by computer simulations, which would contribute to bubble break up, but also create a high shear zone which can potentially cause damage to cells.

The propeller or pitch blade type impellers, on the other hand, focus on moving the liquid to create the lift for mixing and suspending solids. The impeller diameter to tank diameter ratio should be higher for microcarrier culture. While the “propeller three blades” is used extensively in microbial fermentation to enhance oxygen transfer, the “axial flow three blades” provides less shearstress and a more uniform velocity in the entire discharged area than the “propeller three blades.”

Flat Blade (Rushton Turbin)

Axial Flow Blade

Propeller Three Blade

N PO

H

D

V

Di

T

Fig. 13.2: Fluid flow patterns in a stirred tank reactor: axial flow type vs. radial flow type

Fig. 13.3: Three types of impellers commonly seen in stirred tank reactors

Fig. 13.4: Notation of an impeller based mixing reactor. H: liquid height, V: liquid volume, N: impel-ler rotation rate, Po: agitation power, Di: impeller diameter, DT: tank diameter.

Power Consumption and Mixing CharacteristicsPower Consumption of Agitated Bioreactors

In designing equipment and analyzing physical systems, of which the scale spans over a wide range, one often needs to develop a correlation between different design variables. When data collected from different scales and under different conditions are plotted together, they inevitably give rise to different correlations. Each of those correlations is good for that particular scale or a range of scales. They are, thus, of limited value.


Fig. 13.5: Relationship between impeller Power number and impeller Reynolds number for different types of impellers

In order to find a correlation that is applicable todifferent scales over a wide range of operating variables, the experimental data are often plotted in “dimensionless variables” to develop correlations among the experimental data. These dimensionless variables are a combination of experimental variables. In such combinations, the units of dimensions from individual variables cancel each other out with the idea being that a correlation between dimensionless variables is not sensitive to scale. The dimensionless correlations obtained from experiments performed on different scales should hold on any scale, including those that have not been investigated.

Various relationships expressed in dimensionless numbersarefundamentaltofluidmechanics,masstransfer, heat transfer, etc. The correlation between friction factor (f) and Reynolds number (Re) is used universallyinthedesignoffluidflowinpipes.Theplot of friction factor and Re show two regions: at low Re, f decreases linearly until Re = ~2000, where there is a short break, then it continues at a relatively constantvalueatahighReregion.Thefirstregionisrecognizedasthelaminar(orviscous)flowregionand the constant tail is the turbulent flow region.

A similar plot has been generated for power consumption in a stirred tank reactor. The Reynolds number is now denoted as ReI (Impeller Reynolds number) to indicate that it is based on the length (diameter) of the impeller. The dimensionless number for power consumption by impeller is the power number, Np. Correlations between Np and ReI have been generated for various types of impellers. They all exhibit a similar behavior to the f vs. Re plot for fluid flow inapipe. In all theseNp vs. ReI plots, a rapid decrease with increasing impeller Reynolds number is followed by a constant value region; the decreasing region and the constant region represent the two correlations for viscous flow and turbulent flow regimes respectively. Inthe turbulent regime, the power number is constant over a wide range of impeller Renolds number, but the value changes with different types of impellers.

Impeller Reynolds Number (ReI): ND2

i

nt

Power Number (Np): N DP

3 5i

o

t

Po = Impeller power (ungassed power, Po indicated indicated ungassed)

N = Impeller speed

Di = Impeller diameter

ρ = Fluid specific gravity

μ = Fluid viscosity

• For all practical purposes a bioreactor is always operated in the turbulent region

In turbulent regions: (Np) is constant, independent of (ReI)

N DP K3 5

i

o

t=

(Eq . 1)

Power Consumption and Mixing Characteristics


v: tip velocityQp: liquid pumpingθ : mixing time

Other Dimensionless Numbers for Stirred Tank Reactors

Three other dimensionless numbers are frequently used to predict the performance of a stirred tank when the scale changes. They deal with three important aspects of bioreactor operations: velocity, volumetric flow rate, and mixing time.

The maximum velocity in a mixing tank occurs at the tip of the impeller. This velocity can be represented by the multiplicative product of the rotation speed of the impeller times its diameter, NDi. (Note: in this chapter, wewill ignore π in the discussion ofperimeter, area of circle, etc. The constant valueπis cancelled out when comparing different scales.)

The amount of fluid that the impeller can move(called “pumping”) is directly dependent on its rotating velocity and the area of the impeller blades. Because we are considering scale translation under geometrically similar conditions, we can use the length of impeller, instead of the impeller blade, to represent the length scale. The pumping, then, is the projected area (D2) of the impeller multiplied by the velocity of its rotation (ND), which gives ND3.

For mixing time, the representative time scale (called “characteristic time”) in a mixing tank is the inverse of rotation speed (1/N).

The dimensionless numbers for the three properties can by obtained by taking the representative velocity (v),liquidvolumetricflowrate(Qp), and mixing time (Ө), and divide by their respective characteristiccounterpart (e.g., ND, ND3, and 1/N). The plots of dimensionless velocity, pumping, and mixing time against ReI all show profiles similar to the powernumberplot,withtwodistinctflowregimes:laminar(viscous) flow and turbulent flow. The values athigh ReI turbulent regimes are relatively constant.

Three other dimensionless numbers, in addition to power number, are used:• Dimensionless velocity, v / ND

• Dimensionless pumping number, Qp / ND3

• Dimensionless blending (mixing time), ϴN

In bioreactor operations the flow is always in theturbulent regime. Viscous flow is encountered ina stirred tank onlywhen a very viscous fluid, likeglycerol, is used. Therefore the power number for impellers is constant for the same type of impeller. In other words, the impeller power divided by N3Di

5 (N is agitation rate, Di is impeller diameter) is constant.

• At turbulent regions, all those numbers are relatively constant.


Using the correlations based on the dimensionless numbers, one can explore the effect of a changing scale on different variables. We assume that the equipment in different scales will remain geometrically similar. In that case, the effect of different reactor sizes can be compared using characteristic length D (the tank diameter). If the tank diameter increases by 10 fold, all the other reactor parts (tank height, impeller diameter, etc.) will increase by the same proportion of 10 fold.

In scaling up different processes, one needs to keep the most important variable(s) constant or within an acceptable range. The commonly used criteria for scaling up are (1) a constant KLa, so that mass transfer can be maintained, (2) a constant impeller tip speed, to sustain a critical value of high shear velocity to break up agglomerating particles or pellets of mycelial cells, (3) a constant power input per volume (usually for less power intensive processes such as crystallization, blending), and (4) a constant mixing time.

Consider the case of scaling up by maintaining power input per reactor volume constant. Recall that the power number is constant in a turbulent region and the power input (PO) is proportional to N3D5. The reactor volume is described by πHD2. Because of geometrical similarity, we can represent H by D and ignore the constant π that does not contribute toscale comparison. The reactor volume (V) is thus represented by D3. In keeping PO/V constant, N3D2 is also constant in different scales. In scaling up as D increases, the rotation speed must decrease by 1/D2/3. It is inevitable that larger reactors will need to be operated at lower rotation speeds.

By similar algebraic manipulation one can also see that scaling up by a constant power per volume (PO/N3D2) constant will lead to increasing the amount f total pumping (ND3) with the scale. However, pumping per volume will decrease as the scale increases. For mixing time, the trend is an increase with scale.

The table compares the effects of scaling up

Effect of Scale on Physical Behavior of Bioreactors

Table 2 . Examples of Scaling Up by KeepingDifferent Parameters Constant. The reactor is scaled up 15.6 times by volume while keeping geometric similarity .

Property Pilot Scale 160 l Plant scale, 2500 l

P 1.0 15 .6 98 .0 6 .2

P/volume 1 .0 1.0 6 .2 0 .4

N 1 .0 0 .54 1.0 0 .4

D 1 .0 2 .5 2 .5 2 .5

Qp (pumping) 1 .0 8 .5 15 .6 6 .2

Qp/volume 1 .0 0 .54 1.0 0 .4

NDi(tip speed) 1 .0 1 .35 2 .5 1.0

Scale Translation Approaches• Constant KLa

• Constant impeller tip speed (NDi)

• Constant power per unit volume (Po/V)

• Constant mixing time


by setting different parameters at a constant value. Cases considered include constant power per volume, constant agitation rate, constant pumping rate, and constant tip speed. Often, one chooses not to keep a single value constant, and not to scale entirely geometrically similarly.

Scaling Up Geometrically Similarly By Keeping Power Per Unit Volume Constant

PO/N3DI5ρ is constant in turbulent region. Density of water, ρ, is constant. Thus,

PO = KN3DI5 (Eq . 1)

The volume of reactor can be expressed as characteristic length raised to the third power, V = πHDt

2 = cDi3 (Eq . 2)

The power per unit volume is described as Po/V = K N3DI

5/ cDI3 = K’N3DI

2 = constant This leads to the conclusion that when power per unit volume is kept constant, N3DI2 is also constant . N3DI

2 = constant (Eq . 3)

Effect on Agitation Rate Comparing scale 1 and scale 2 N1

3DI12 = N2

3DI22

N1/N2 = (DI2/ DI1 )2/3 (Eq . 4)

The agitation rate N decreases with increasing scale. When the diameter increases eight times, the agitation rate is ¼ in the larger scale .

Effect on Impeller Tip SpeedTip speed is described by N multiplied by Di . from Eq . 3

N13DI1

3/ DI1 = N23DI2

3/ DI2

N13DI1

3/ N23DI2

3 = DI1/ DI2

N1DI1 / N2DI2 = (DI1/ DI2)1/3 (Eq . 5)

Tip speed increases with increasing scale, but only at 1/3 power of the length of scale .

Effect on Liquid Pumping

The capacity of liquid pumping can be described by the impeller tip speed, NDi, by the area that it moves against the liquid, Di2 .

Under the condition of constant power per volume, N1

3Di12 = N2

3Di22. Multiply both sides by diameter to

the seventh power .

N13Di1

9 / Di17 = N2

3Di29 / Di2

7 N1Di1

3 / N2Di23 = (Di1 / Di2)

7/3 Liquid pumping capacity increases with scale . By dividing both sides by characteristic length raised to the third power, we can obtain the pumping capacity on a per volume basisNDi

3 / Di3 = pumping per volume = Qp/V

(Qp1/V1) / (Qp2/V2) = (Di1 / Di2)-2/3 = (Di2 / Di1)

2/3

The pumping capacity per volume actually decreases with increasing scale .

Effect on Mixing Time

The decreased pumping per volume also causes an increasing in mixing time when scale increases.The dimensionless mixing time is ΘN. Its value is relatively constant in the high Re number turbulent region .

Θ1N1 = Θ2N2

From Eq . 4

Θ1 / Θ2 = N2 / N1 = (Di1 / Di2)2/3

(Eq . 7)

(Eq . 8)

(Eq . 6)


In cell culture processes, oxygen is almost always the firstnutrienttobedepleted.Duetoitslowsolubilityin medium, it must be continuously supplied. In comparison, the concentration of glucose maintained in the medium is usually a couple orders of magnitude higher than oxygen. Their ratio of molarspecificconsumptionratesrangesfromabout1.0 (when most glucose is converted to lactate) to close to 6.0 (when most glucose is converted to CO2). For oxygen, at a high cell concentration, the depletion time can be as short as a few minutes, whereas the depletion time for glucose is orders of magnitudes longer. Therefore, in reactor scaling, special attention is always paid to oxygen.

Nutrient Starvation TimeBecause of its solubility, oxygen is the first nutrient species to be completely consumed

Table 3. Comparison of Oxygen and Glucose Saturation Time in a Typical Culture (For 1010)

Oxygen Glucose

C in Culture0 .1 mM(50% saturation with air space) 1 g / L (55 mM)

Specific consumption rate

1 x 10-10 mmole / cell-hr

0 .15 – 1 .0 X 10-10 mmole / cell-hour

Volumetric consumption 1 mmole / L-hr 0 .15 – 1 mmole / L-hr

Time to depletion 0 .1 hr (6 min) 12 hr

The nutrients that are added at the beginning of the culture will eventually become well mixed in the culture fluid. (Note: This may not be truefor some microbial fermentation in which some nutrients are supplied in a solid form and dissolve gradually). Mixing problems may arise for those components that are added continuously or intermittently during the cultivation. If the nutrient isaddedinafixedposition(s),itmaynotgetcarriedto other locations in the reactor fast enough to meet the cells’ metabolic needs. In some cases, the additive needs to be supplemented at a high enough concentration that has an adverse effect to the culture, and must be dispersed quickly. In these cases, adequate mixing must be provided.

Mixing Time


To measure the mixing time, one can inject a dye into the reactor under operating conditions and then use a sensor placed in a fixed locationto record the dye concentration over time. The concentration will fluctuate over a large rangeinitially, but will eventually reach a steady value. The time needed for the concentration to reach a range of steady value (such as 90% of its finalhomogenous concentration) is considered the “mixing time.” If one plots the concentration deviation from its final steady value, ΔC, the timeprofilecanbeapproximatedbyfirstorderkinetics.

Mixing TimeMixing Time Measurement• Measurement

• At t = 0 add tracer• Measure terminal mixing time, defined as the

point when an arbitrary chosen uniformity, 90%, is reached

Inastirredtankreactor,anutrientisaddedatafixedposition.Considerafluidelementcarryingcells.Whenit passes by this position, it acquires the nutrient and then moves away to circulate around the reactor. On average,thesamefluidwouldreturntothisfeedingzone after a duration of one characteristic mixing time. It is important that the amount of nutrient that the fluid acquires at the nutrient enrichmentzoneissufficienttosustainthemetabolicneedsofthe cells before it returns to the zone. Therefore, the mixing time needs to be shorter than the starvation time. The starvation time is dependent on cell concentration and the consumption rate.

If the mixing time is longer than the starvation time, a discernible concentration difference of the nutrient would appear in different locations in the reactor. Pockets of low concentrations would emerge and their locations may change over time, asthefluidflowpatternandcellconcentrationsarenot constant. A sensor that is at a fixed positionmay, thus, not reveal the presence of such pockets.

Nutrient Enrichment Zone, Mixing Time vs. Starvation Time

Fig. 13.6: Measurement of averaged mixing time in a stirred tank


Mixing Time Distribution If we follow a particular fluid element in areactor, it will come to the nutrient enrichment zone, go away, and come back repetitively. The time interval between its return to the nutrient enrichment zone, however, will not be uniform.

The mixing time described above is an average mixing time. But the mixing time that is physiologically important (e.g., the return time to the nutrient enrichment zone) is not a single value of the average mixing time, but is distributed over a range.

Imagine that we use a ball that emits a radio signal. The ball has the same density as the fluid and iscarriedaroundby the fluidmotion. A sensorat afixed position in the reactor would pick up thesignal when the ball is close and record the time interval between consecutive returns. This time interval of return will distribute over a range as the ball sometimes returns shortly after it moves away, while at other times it roams around the reactor for a while before returning to the sensor.

The histogram of the time-interval distribution can be converted to a mixing time distribution function. In general, the distribution follows a logarithmic normal distribution. The mean or median of mixing time is a descriptor of mixing characteristics, but it does not present the entire picture of mixing. Given the same median or mean mixing time, two reactors may still have a very different mixing time distribution.

Imagine that the location of the sensor is also the position of nutrient feeding. Those cells circulating with the particular fluid elementreceive nutrient only when the fluid returns tothat position. If the circulation time is longer that a critical time, then the nutrient level seen by those cells may fall below the critical value. A wide distribution of mixing time can be a concern. Even though the frequency of exceedingly long circulation time is low, nutrient starvation may occur in those rare occasions and trigger apoptosis or cause other irreparable damage to cells.

In reactor operation, the fraction of mixing

Mixing Time Distribution Measurement• Add radio emitter to the reactor i, a sensor picks up

the signal when the emitter circulates around and passes by

• Measure circulation time for each encounter and plot the frequency distribution of the circulation time

• Determine the mean and median circulation time and the standard deviation σ

• One can plot distribution of circulation time as a population density function. The portion of circulation whose time of circulation lies between t and t + Δt is the area under the curbe between t and t + Δt .

Fig. 13.7: Measurement of circulation time distribution in a stirred tank


time that is longer than the critical time should be minimized by either improving mixing or by setting a limit on the cell concentration.

Scaling Up and Mechanical Forces on CellsIn a turbulent flow, the fluid’s kinetic energy istransferredbyswirlingpocketsoffluid,called“eddies”.Turbulent regimes are comprised of eddies of differentsizes,characterizedbyvelocityfluctuations.

The hydrodynamic forces experienced by the cells may arise from fluid-cell and cell-mechanicalparts interactions. Cells, being neutrally buoyant particles, follow the motion of the relatively larger eddies. In general, the direct impact of cells on the impeller is minimal and cells generally flow by impeller blades without suffering muchdirect mechanical impact. However, these large eddies cause the formation of cascades of smaller eddies, which may impart damage to cells by dissipating all of their energy on the cell surface.

The size of the eddy relative to the size of the cell is thought to be an important factor that damages cells. Smaller eddies in the size range of cell surface motif, i.e., much smaller than cell diameter, are considered to be more damaging than eddies that are larger than cells. Studies examining cell death

Mixing Time Distribution• The mixing time in a tank is not uniform. If the aver-

age mixing time is 6 minutes, many fluid elements will have a mixing time shorter than 6 minutes, and others will have longer times.

• If the critical mixing time is 6 minutes, the average mixing time should be shorter than that.

Circulation Time

Popu

latio

n D

ensi

ty D

istr

ibut

ion

f(t)

Probability of Starvation

Critical Circulation Time0 1 2

τ

Fig. 13.8: Mixing time distribution and critical mixing time for nutrient depletion


caused by turbulent flow often assume that thecell death rate is proportional to the Kolmogorov eddy concentration, and cell damage occurs when the eddy size is smaller than a critical eddy size.

As the scale increases, the eddy length increases. Thus, strictly from the viewpoint of the average eddy size, the effect of turbulence on cell damage will not become more severe in scaling up. One should be cautioned that the eddy size is not uniform, but distributed over a range. As the scale increases, the distribution function of the eddy size also varies. Further, the discussion here does not take into account the effect of gas mixing. The phenomenon of mechanical stress caused by combined agitation and aeration is rather complex in scaling up.

Scaling Up and Oxygen Transfer and Carbon Dioxide RemovalWhen scaling up a process, we aim to produce cells and product in quantities proportional to the scale. To meet that goal, we normally provide all nutrients in a proportional amount, to meet the increased metabolic needs of cells. For liquid nutrients, increasing the nutrient provision in proportional to cellular needs on a large scale is easily met. However, for oxygen and CO2, which are supplied and removed through gas aeration, scaling up presents a challenge because of the constraints of physical factors.

• Microeddies cause cell damage

• Eddy size increases with scale if the power per unit volume decreases with scale

Scaling Up and Mechanical Forces on Cells

Fig. 13.9: Eddies surrounding a cell suspending in a stirred tank


Supplying oxygen to the growing cells in the bioreactor involves blowing air through the culture fluid,andtransferringoxygenfromthegasbubblesintotheculturefluid.Theamountofoxygencarriedout from the reactor is less than that supplied into the reactor. This difference is the amount transferred into the liquid phase. Material balances on oxygen can be performed on the gas entering and leaving the reactor (the gas phase balance), as well as on oxygen being transferred from gas bubbles to liquid (the liquid phase balance). The oxygen transfer rates calculated from the gas phase balance and from the liquid phase balance are equal.

Material Balance on Oxygen in Bioreactor

Gas phase balance is performed by taking the difference between the oxygen input rate at the inlet and the oxygen output rate at the outlet. That difference is the amount of oxygen that has been transferred into the liquid. While oxygen is transferred into the liquid phase, CO2 (produced by cells in the medium) and water vapor are stripped outoftheculturebroth,thusthevolumeflowratein the outlet may differ from the inlet. Assuming idealgasbehavior(PV=nRT),themolarflowrateofthe oxygen at the inlet and the outlet is the total air flowrate(PQ/RT)multipliedbythemolarfractionsof oxygen at the inlet and the outlet YO2,in and YO2,out respectively. The oxygen transferred into the liquid is simply the difference between the molar flow rates of oxygen in the inlet and the outlet.

• On the gas side, the oxygen transferred from the gas side to liquid side is reflected in the difference of oxygen concentration between gas inlet and gas outlet .

Oxygen Balance on Gas Phase

(Eq . 9)

(Eq . 10)

(Eq . 11)

• The dynamics of the dissolved oxygen concentration are described by the balance between OTR and OUR at a quasi-steady state .

At steady state, OTR=OUR


Oxygen Balance on Liquid Phase On the liquid side, the oxygen transfer rate (OTR) isdescribedbytheoverallmasstransfercoefficient(KLa) and the driving force (C*-C). For small reactors, we assume that liquid is well mixed. This assumes that the concentration measured at the outlet is the same as the concentration in the reactor. C* at the air outlet should be used for the driving force calculation. For a large reactor, one assumes that the gas phase behaves like a tubular reactor (plug flow), and a logarithmic mean ofthe driving force is used. We assume that a quasi-steady state, i.e., the change in dissolved oxygen (dC/dt), is very slow compared to the oxygen consumption and rate of transfer. Thus, the oxygen uptake rate (OUR) can be approximated by the OTR.

On the liquid side OTR and OUR is described by Eq . 11 since the rate of change of dissolved oxygen is small . In cell culture process, the air flow rate can be considered to be the same at the inlet and outlet. Overall, the relationship is described as:

C* is the dissolved oxygen concentratoin in equilibrium with the gas, which may differ in different parts of the bioreactor .

For small scale bioreactors, one can assume both liquid phase and gas phase are well mixed . The gas phase in the reactor is thus the same as that in the exit gas stream . Thus, C* is related to the oxygen concentration at the ex-haust gas by Henry’s law constant:

For large scale bioreactors, the inlet and outlet oxygen con-centrations may be very different. One uses the logrithmic mean driving force described below:

(Eq . 12)

(Eq . 13)

(Eq . 14)

(Note: P/RT converts volume flow rate Q to molar flow rate using ideal gas law . P is the pressure of gas phase)


Aeration Rate and Superficial Gas Velocity

Now we examine the gas phase balance. In scaling up, if we keep Q/V constant, we will be able to maintain the same oxygen level at the inlet and outlet (Yin, Yout) and sustain the same oxygen transfer rate (OTR). However, when scaling up, Q/V is likely to decrease. If OUR is to be sustained, then Yout has to be smaller to maintain the material balance.

Then, consider the liquid phase balance. OTR is KLa multiplied by (C*-C). Because oxygen level at outlet, Yout, is lower, C* is also lower. How can OTR be kept at the same level as in the small scale? One possibility is to increase KLa while keeping C at the same level as in the small scale (thus allowing (C*-C) to be smaller). The will require an increase in agitation power. The other possibility is to increase (C*-C) to the same level as in the small scale by allowing C to be maintained at a lower level, if the reduced C has no adverse effect on culture performance. Alternatively one can use oxygen enriched air to increase C* to maintain (C*-C) at the same level as in the small scale. However, this will lead to increased level of CO2

accumulation as will be discussed in the next section.

Therefore, overall oxygen transfer becomes a challenge in scaling up because the aeration rate cannot be increased proportionally with the scale.

D = tank diameterQ = aeration ratevl = culture volume in reactorVs = gas superficial velocityA = cross-sectional area of the tankH = heights of culture volume

Superficial gas velocity = gas flow rate/reactor cross sectional area vs = Q/A = Q/(πDt

2) (Eq . 15)

The reactor volume increases with length scale raised to the third power, while the cross sectional area increases only with the second power . V1 / V2 = D1

3/ D23 A1 / A2 = D1

2/ D22

When scaling up one may choose to increase the air flow rate proportional to the increasing reactor volume, Q1 /Q2 = V1 / V2

One can see that vs1 / vs2 = D1/ D2 (Eq . 16)

Superficial gas velocity will increase linearly with in-creasing scale .


Now we examine the gas phase balance. In scaling up, if we keep Q/V constant and keep KLa constant in the liquid phase, we will be able to maintain the same oxygen level at the inlet and outlet (Yin, Yout) and sustain the same oxygen transfer rate (OTR). However, when scaling up, Q/V will actually decrease so if OUR is to be sustained, then Yout has to be smaller to maintain the material balance.

Then, consider the liquid phase balance. We have kept KLa constant in this analysis. To keep OTR also constant, the driving force must be sustained. However, the driving force is the logarithmic mean of the oxygen concentration at the inlet and the outlet. With a lower level of Yout, the driving force for oxygen transfer will actually be lower.

Therefore, overall oxygen transfer becomes a challenge in scaling up because the aeration rate cannot be increased proportionally with the scale.

Aeration Rate and Oxygen Transfer Driving Force

When scaling up, we aim to maintain OUR and OTR at the same level .

Considering mass balance in the gas phase:

From Eq. 12:

Given that Q2/V2 is smaller, and Yin (oxygen concentration in the inlet air) is the same, Yout,2 in the large scale will be smaller

From Eq . 13 and Eq . 14, since Yout,2 is smaller, so is C* .

Considering the liquid phase, OUR and OTR will be maintain at the same level in the two scales:

(KLa)1(C*1 – C1) = (KLa)2(C

*2 – C2)

This can be accomplished by

• Allowing C*2 to be lower while maintain C2 = C1 . KLa2 in the large scale must increase .

Keep KLa2 at the same level as in small scale, then the concentration driving force of oxygen must be increased to the level of the small scale by

• Allowing C2 to decrease

• Increasing C*2 by using enriched oxygen


Table 4. Effect of Scale on Oxygen Transfer

Reference Scale

Constant Air Flow

Constant Superficial

velocityScale (volume) 1 1,000 1,000

Cross Sectional Area 1 100 100

Air Flow Rate 1 1,000 100

Superficial Air Velocity 1 10 1

O2 Consumption/CO2 Production 1 1,000 1,000

Q(yin –yout) 1 1

(yin –yout) has to be very

large, i .e . yout is small

Comments May reach flooding

Need to increase KLa or

power input

If air supply increases proportionally with scale, foaming can become serious and flooding may occur.

The physical constraint of a reduced cross-sectional area to a reactor volume ratio with increasing scale poses a challenge in oxygen transfer. In scaling up, the airflow rate may not be increasedproportionally to the culture volume because an overly high superficial air velocity is problematic.

In microbial fermentation, a high airflow rateeventually causes “flooding” (i.e., the impeller isswamped by gas bubbles) and loses its capacity for pumping liquid. For cell culture, the aeration rate usedissubstantiallylowerthanthefloodingaerationrate. However, potentially a different problem may arise. Antifoam agents are not used as extensively in cell culture as in microbial fermentation because of potential damage to cells. At a high superficialvelocity control of foaming may become problematic.

When scaling up, aeration rate is not increased proportionally to the culture volume. Because less air is given to the same volume of culture, more oxygen has to be taken out from the gas phase to meet the oxygen demand. This causes the oxygen level in the gas phase to be lower. As a result the driving force for oxygen transfer is also lower in the large scale.

In scaling up it is a common practice to take a middle ground. The airflow rate per reactorvolumeisdecreasedsomewhat,butthesuperficialvelocity is allowed to increase (albeit less than proportionally to the culture volume) to minimize the loss of the oxygen transfer driving force. In some cases, the air is enriched with oxygen, while in other cases, the agitation rate is increased when oxygen falls below a set point during the cultivation.

Gas Flow Rate in Scaling Up: A Summary


The respiratory quotient for most cells, using glucose and glutamine as the main source of energy, is very close to 1.0. Thus, every mole of oxygen consumed by the cells generates about one mole of CO2. A very active culture with a high cell concentration can produce more than 100 mmole/L of CO2 per day. In comparison, a cell culture medium has about 20 - 40 mM of sodium bicarbonate. The amount of CO2 produced by cells, thus, far exceeds that which is added as buffer to the media.

Many cells, such as hepatocytes, are rather tolerant to CO2 but others are more sensitive. The growth of most cells may begin to be affected at a CO2

concentration of 15% (114 mm Hg) so continuous removal of CO2 from the culture is important.

When scaling up, the airflow rate per reactorvolume may not increase proportionally. As a consequence, less air is used to strip CO2 from an equal volume of culture media. If the metabolic activity of the culture remains the same, the same amount of CO2 produced by the cells is now being removed using less air. The CO2 level will then be higher in the air exiting from the large-scale reactor.

The rate of CO2 stripping is dependent on the concentration difference of CO2 in the liquid and the gas phase. A higher concentration in the gas phase diminishesthestrippingefficiency. TheincreasedCO2 concentration in the exit air, thus, causes further accumulation of CO2 in the liquid phase.

To compensate for the reduced O2 driving force from scaling up, one can use O2-enriched air. However, such a measure cannot compensate for the diminished stripping efficiency of CO2. Therefore CO2 accumulation patterns in large scale and small scale bioreactors can be rather different.

Effect of Scaling Up on CO2 Removal

• The mass transfer coefficients for oxygen and carbon dioxide are about the same .

• R.Q. (moles of CO2 produced/ moles O2 consumed) for mammalian cells is very close to 1 .0 . So, oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) are about equal .

• The toxic level of CO2 is around 15–20% (110 mm Hg – 150 mm Hg) .

• The carbon dioxide removed from the reactor (at steady state) is the difference between its concentrations in the inlet and outlet gas:

• CER and CCO2, in are the same in reactors of different scales

• If cell concentration and metabolic activity remain constant .

• Q / V is smaller in large scale.

• Inevitably CCO2, out will have to be higher on a large scale .

• The driving force for CO2 removal decreases with increasing scale .

(Eq . 17)


TheoverallmasstransfercoefficientforCO2 is slightly lower than that of oxygen because of its larger molecular weight. However, the difference, approximately the square root of their molar weight ratio, is very small. One can consider the KLa to be about the same.

Unlike O2, the solubility of CO2 in aqueous solution is very high. At the gas bubble interface, O2 in the liquid phase can be assumed to be in equilibrium with the gas phase, so (C*-C) is a good estimate of the driving force. The same assumption is not always valid for CO2.

CO2 in the medium exists as CO2, HCO3, and CO3-2. At

the interface, CO2crossesthefilmandistransferredout of solution, but HCO3

- does not. So, HCO3-must

dissociate to CO2 before being transferred to the gas phase. The kinetics of HCO3

-, the dominant form of CO2 in aqueous solution, to dissociate to CO2 is slow. Because of the slow kinetics, the actual driving force is smaller than what can be estimated from the total CO2 (g) concentration.

• To achieve the same molar transfer rates for oxygen and carbon dioxide (although in opposite directions), the driving force for carbon dioxide has to be around 100 mm Hg, a level similar to that for oxygen transfer .

• The upper bound of CL for CO2 should be below the inhibitory level of 110-150 mm Hg . If the driving force is 100 mm Hg, then C* will have to be 10-50 mm Hg . That is 1 .5-6 .5% CO2 in air .

• To strip off carbon dioxide a sufficiently fast air flow rate must be used to ensure the CO2 concentration in the gas phase is low enough to provide a large driving force .

(Eq . 18)


Chemical Environment in Scale Translation

In scaling up or scaling down, the chemical environment may also be affected. For cell culture processes, many of those changes are caused by different CO2 accumulations at different scales. CO2is a major contributor of the pH buffer in a cellular environment. CO2 produced by the cells is excreted to maintain a physiological range of intracellular pH.

Carbon dioxide is transported through the plasma membranes as CO2 and HCO3

-. CO2 can diffuse through cellular membranes, while HCO3 transport is mediated by transporters. A symporter co-transports HCO3

- and H+, while another antiporter co-transports Cl- and HCO3

- in the opposite direction.

As CO2 builds up in the culture fluid, a higher Cl-

gradient is needed to “drive” HCO3- out, otherwise

the intracellular HCO3- level will be higher. Because

the airflow rate per reaction volume is not keptconstant in scaling up, the CO2 level in the culture will be higher on a larger scale. This will affect the intracellular CO2/HCO3

- levels. However, experimental evaluation of the effect of scale on cellular level of CO2 and intracellular pH is still lacking.

• CO2 produced by cells can diffuse through the cell membrane

• Most CO2 becomes HCO3- and is excreted through

transporters

• Accumulation of CO2 in medium may affect intracellular pH

Fig. 13.12: Schematic of the removal of carbon dioxide produced by cells


In scale translation, the relationship of geometry-related physical parameters are not kept constant, because the volume and the surface area of the reactor change in different proportions relative to length. Therefore,onehas todefinethecriticalrange of various scale-sensitive variables and choose scaling up criteria to ensure the operation is within the optimal region. In most cases, one chooses not to scale up completely geometrically similarly. Most large-scale reactors have a larger height to diameter ratio than the smaller scale ones. Nevertheless, the physical constraints on scaling up are the same regardless of whether one scales up geometrical similarly or not. In scaling up, the gas flowrateisalsolikelytochangeinitsproportiontothe reactor volume. This causes the mass transfer characteristics to be different for different scales. While the dissolved oxygen can be controlled at the same level, the CO2concentrationsprofilesarelikelydifferent for reactors of different scales. Differences in CO2,concentration in the reactor will cause pH control actions, including base and CO2,addition and

CO2 stripping, to vary at different scales. Difference in pH control actions may further change the chemical environment of the culture. Given that the physical and chemical parameters related to scaling up cannot easily be manipulated or controlled, one may resort to selecting cells which are less sensitive to those parameters. Understanding scale-sensitive parameters and a sound knowledge of estimating the range of those critical parameters will greatly facilitate the scale translation of cell culture processes.

In process development involving scale translation, one should aim to reproduce or to predict the conditions of physical constraints, as well as the resulting chemical environment. It may not be possible to replicate all physical and chemical parameters on drastically different scales. Ultimately, one should identify and aim to control critical physical parameters, to minimize variations in the chemical environment.

Concluding Remarks

CELL CULTURE GENOMICS | 285

Gene and Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .285What is a Gene? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285Organization of Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287Packaging DNA into Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

Epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .291Molecular Mechanisms that Mediate Epigenetic Regulation . . . . . . . . . . . . . . . 291

Genome Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .293Transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293Proteome Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

Sequencing Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .302Sanger Method and Sequencing Technology Evolution . . . . . . . . . . . . . . . . . . . 302High Throughput NextGen Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

Gene Expression Exploration in Cell Culture Processing . . . . . . . . . . . . . . . . . . . . . . . .306Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .308

Cell Culture Genomics

What is a Gene?

Gene and Genome

A gene is a sequence of DNA that encodes for an RNA and protein product. Up until a quarter century ago the prevailing notion was that one linear sequence of DNA directly encodes for one gene. After the discovery of alternative splicing, however, our understanding quickly changed. Recent findingshave highlighted a relatively large number of alternatively splicing genes in the mouse genome. A large number of these genes are translated into different protein sequences. Our knowledge of the relationship between a gene and its expression product is evolving. For instance, we now know that two genes may reside on opposite strands of the same segment of DNA (and will be transcribed in opposite directions), or they may reside in the same strand of DNA and are overlapping..

A gene may encode for two types of end products: 1)

Mouse Genome Encodes• ~30,000 genes coding for proteins

• ~1,600 genes coding for RNA

• 800 tRNA genes

• 350 tRNA genes

• ~450 other ncRNA (noncoding RNA genes)


a protein, or 2) a non-protein-coding RNA (ncRNA). In its entirety, the mouse genome encodes for a total of about 37,000 genes. Among them, just over 30,000 genes code for proteins, 4,000 code for so-called pseudogenes (genes whose identity can be traced to an ancestor but are no longer translated into a functional protein), and the remaining 1,600 code for RNAs (including 800 rRNAs, 350 tRNAs, and other non-coding RNA, or ncRNA).

The average mammalian gene spans a region encompassing approximately 23 to 28 thousand base pairs (kbp) in length. Typically, the promoter region is located upstream of the transcribed region (about 1 kbp or longer, sometimes as long as 10 kbp) and the transcribed region is used to generate a primary transcript, or pre-mRNA. The primary transcript starts with a 5’ end untranslated region (5’ UTR) and ends with a 3’ UTR. It also contains a number of protein coding exons and introns. After the introns are spliced out, the mRNA, or mature transcript, remains and includes: 1) the 5’UTR, 2) the exons, 3) the 3’UTR, and 4) a newly-added polyA tail. The mRNA is ultimately exported out of the nucleus for translation in the cytoplasm.

Many primary transcripts undergo alternative splicing. In these cases, portions of exons are stitched together under different conditions (e.g., in different tissues, at different times, or occurring at different frequencies) to give different mature mRNA species. The stitching point of two consecutive exons may not be the same under alternative splicing. In this case, splicing may result in a shift of the reading frame, which would affect the exons downstream of the splice junction and perhaps give rise to completely different protein sequences.

The Functional Annotation of the Mammalian genome (FANTOM) consortium has generated the most complete mouse gene sequence database to date. It has uncovered a large number of protein coding genes with alternative splicing. These events have been found to produce very different protein sequences, a large number of polymerase II transcribed ncRNAs (with polyA tail), and

Protein coding genes

~30,000

Pseudogenes ~4,000

RNA coding genes ~1,000

rRNA ~800

tRNA ~350

snRNA ~150

miRNA ~300

rcRNA ~450

Table 1. Distribution of Mouse Genes

Gene Structure in Eukaryotes• Exons make up the mRNA; intervening sequences

called introns are also present

• Splicing of pre-mRNA entails removal of intronic regions, addition of polyA tail and 5’ cap

• Alternative splicing is common


Exon1 Exon2Intron1 Intron2 ExonsPromotor

Organization of Eukaryotic Genes

Transcription

protein 1 protein 2

Translation

Export to cytosol

Splicing polyadenylation

Alternative splicing

UGA

5’ UTR 3’ UTR

primary transcript

5’ UTR 3’ UTR

AAAAAAAAAAAA

AAAAAAAAAAAAAUG

UGAAUG

AUG UGA

AUG UGA

AUG UGA

23-28kbp

intergenic region intergenic region

~1 kbp

UGAAUG

start stop

start stopAAAAAAAAA

Fig. 14.1: Expression of an eukaryotic gene. Alternative splicing into two different proteins are shown.

Organization of Genome The number of genes in each organism varies greatly, from 700 genes in a simple parasitic mycoplasma to over 30,000 in mammals. Those studying the “minimum set of genes” that are required for life have derived a gene set ranging from 270 to 350 genes.

As the number of genes increases with increasing complexity of organism, the additional genes and gene products acquired tend to affect a cell’s interaction with its environment (e.g., transporters, signaling molecules, and their receptors). In other words, data suggest that the increased complexity of higher organisms requires the formation of a more sophisticated communication, both at the cellular and organism level.

With an increasing number of genes, one also sees an increasing size of the genome. By convention, the sizeofagenomeisquantifiedusingthenumberofbases in a haploid genome. A bacterium’s genome

Composition of a Typical Mammalian Genome • Coding Sequences ~ 1-2%• Intronic Sequences ~ 20-25%• Repetitive Sequences ~45-55%• Other intergenic sequences

antisense RNAs that may have regulatory roles. The prevalence of so many classes of variants poses ambiguityinthedefinitionofa“gene”.Forinstance,if one were to count each independent gene product as a different “gene”, the total number of genes could be estimated to be upwards of 50,000.


Eukaryotic Genome is Organized into Chromosomes• Human: 22 pairs; x,y

• Mouse: 19 pairs; x,y

• Rat: 20; x,y

• Chinese hamster: 10 pairs; x, y

• Mouse chromosome size: 61 Mbp (Chr 19)to 197 Mbp (Chr . 1)

Repetitive Sequence• Short RNA-derived interspersed elements (SINES,

90-300 bp)

• Long interspersed nucleotide element (LINES,>500 bp)

• Retrovirus like elements with long terminal repeats (LTR)

• DNA transposons

• Widely distributed simple sequence repeats:

• Direct repetitions of short k-mers such as (A)n, (CA)n or (CGG)n

• Segmental duplications, consisting of blocks of 10–300bp of the genome

• Blocks of tandem repeated sequences, such as at centromeres, telomeres, the short arms of acro-centric chromosomes and ribosomal gene clusters, these also include satellites and microsatellites .

is about 3 Mbp to 8 Mbp, while a fungus’s genome is a bit less than one order of magnitude larger than that of bacteria, from 12Mbp to 30Mbp.

In contrast, mammals have about a genome size of 2-3 Gbp, nearly 1,000 fold higher than bacteria. While the mammalian genome is nearly 1,000-fold larger, it contains only 10 times more genes. A typical protein-coding gene in bacteria is about 1 kbp, or 330 amino acids, while an equivalent sequence in mammals is about 1.3 kbp. Including introns and UTRs, a mammalian gene in total is 23-28 kbp, which is substantially larger than a bacterial gene.

The gene-encoding regions, including introns, account for only about 25% of the mammalian genome. The rest of the genome consists of other intergenic sequences, including promoters, regulatory elements, and regions not yet explored by scientific inquiries. Additionally, nearly 50%of a mammalian genome consists of repetitive sequences. That number is even higher in some plants, giving them a genome size even larger than mammals. The repetitive sequences reside not only in intergenic regions, but also in introns, UTRs and upstream or regions adjacent to genes. Repetitive sequences fall into different categories; some are short, while others are long, up to 500 bp. Some are the result of transposons or the remnants of retrovirus infection throughout evolution.

The presence of repetitive sequences presents a barrier to the quick and accurate assembly of DNA sequencing reads. In DNA sequencing, the DNA moleculeisfirstfragmentedandthentheindependentfragments are sequenced. The assembly algorithm searches for the overlapping regions and attempts to stitch them together into longer contiguous sequences (or contigs). Therefore, a fragment that has a repetitive end can often be assigned to multiple loci as it is not a unique sequence. One solution to this is to sequence very long fragments of DNA, over the stretches of repetitive sequences.


Common Name Taxonomy Species Genome Size Estimated Number of Genes

Mycoplasma TENERICUTES Mycoplasma pneumoniae FH 8 .11E+05 670

Bacteria PROTEOBACTERIA Escherichia coli DH10B 4 .69E+06 4,271

Bacteria FIRMICUTES Bacillus subtillis subtillis 168 4 .21E+06 4,354

Yeast FUNGI-ASCOMYCOTA Saccharomyces cerevisiae S288C

1 .21E+07 6,273

Slime Mold PROTISTS-MYCETOZOA Dictyostelium discoideum AX4 3 .40E+07 13,362

Roundworm NEMATODES Caenorhabditis elegans 1 .00E+08 20,935

Fruit Fly ARTHROPODA Drosophila melanogaster 1 .37E+08 21,116

Chicken CHORDATA-BIRDS Gallus gallus 1 .00E+09 17,935

Frog CHORDATA-AMPHIBIA Xenopus tropicalis 1 .70E+09 20,500

Human CHORDATA-PRIMATES Homo sapiens 3 .17E+09 53,894

Mouse CHORDATA-MAMMALS Mus musculus C57BL/6J 2 .72E+09 37,261

Rat CHORDATA-MAMMALS Rattus norvegicus BN/SsNHs-dMCW

2 .70E+09 35,427

Dog CHORDATA-MAMMALS Canis lupus familiaris 2 .40E+09 24,661

Chinese Hamster CHORDATA-MAMMALS Cricetulus griseus 2 .70E+09 32,476

Table 2. Genome Size of Representative Organisms


DNA molecule

packed with histone proteins to form chromatin

chromatin forms fiber-like structure

may condense tightly or packed loosely in different regions of chromosomes

Packaging DNA into Chromatin In bacteria, the genome is typically arranged into a single, large chromosome. Most chromosomes are circular, although linear chromosomes are also seen. Conversely, in eukaryotes, the genome is segmented into different chromosomes. Chromosomes are not merely DNA molecules wrapped loosely together. A typical E. coli cell (2 µm x 1 µm in size) has a genome of about 1.5 mm in length. If all 46 diploid chromosomes of the human genome were strung together, they would form a ~2 m x 2 nM string. The chromosome is, thus, not merely a string of DNA packed haphazardly into the nucleus. It requires extensive manipulation and the work of specialized machinery to allow it to be packed into a dense volume, while still remaining accessible for transcription.

Each chromosome is a molecule of double-stranded DNA. Packaging of DNA occurs at multiple levels. At the local level, small regions of a DNA molecule form a complex with DNA binding proteins, chiefly histones, to form a “beads-on-a-string”-like structure. That form is further condensed into packed beads, called nucleosomes. In further condensation of the structure, some regions are more open and accessible to transcription (this is called euchromatin), while other regions are more densely packed, and are called heterochromatin.

• Chromatin: complex of DNA and protein in which genetic material is packaged within the cell

• Histones: principal protein components of chromatin

• Nucleosome: fundamental sub-unit of chromosome which consists of 165 bp of DNA wrapped around an octamer of core histones

Fig. 14.2: Packing of a segment of DNA into highly condensed region and relatively open region


Epigenome

Although the genome of each cell of a higher mammal may encode more than 30,000 genes, at a given time it may actively transcribe only a fraction of these. A typical CHO cell in culture, for instance, expresses only about 16,000 genes. Some genes are ubiquitously expressed in all tissues; others are tissue- or time-dependent. At the transcriptional level, a large array of transcription factors are responsible for regulating the expression of genes according to the tissue type, timing by developmental stage, or by event (such as stress or exposure to some signaling molecule). At a higher level, transcription is also regulated by the accessibility of the gene loci, which is further controlled through epigenetic events.

The term ‘epigenetics’ was introduced in the 1940’s to describe “the interactions of genes with their environment, which bring the phenotype into being.”

• ‘Heritable changes in gene expression not encoded in the DNA’

• Essential for genome packaging and fundamental to development

• Epigenetic alterations influenced by the environment; for example, identical twins can be susceptible to different diseases

• Epigenomics: Representing the totality of epigenetic marks in given cell type

Molecular Mechanisms that Mediate Epigenetic Regulation

Epigenetic regulation does not result in a mutation, as no change occurs to the underlying DNA sequence. It does, however, cause a heritable change in the cellular phenotype. Unlike a mutation, which originates in a single genomic locus of a single cell, epigenetic changes can occur and affect gene expression on a global level. For instance, when stem cells differentiate, or when fibroblasts are transformedinto iPS (induced pluripotent stem) cells, global epigenetic changes occur on the chromosomes to affect the reprogramming of genetic circuits.

In cell culture, cells are often “adapted” to new culture conditions, such as differing growth factors or adjusting to growth in suspension. In such processes, the entire population of cells shift their phenotype. Such processes are less likely to be mutation events and are more likely to involve epigenetic regulation.

In the generation of producing cells, the host cell transforms from a non-secretor to a professional secretor in a short time, accompanied by a vast change of cellular properties. Although it is possible that mutations may be responsible for some of the changes, it is very likely that epigenetic events are the major


drivers to in the dramatic shift to high productivity.

Themajormechanisms of epigeneticmodificationinvolve DNA modifications and histonemodifications. Of the DNA modifications, themethylation of Carbon 5 of cytosine is one of the most common. Once methylation occurs, the mark is highly stable and can be passed on to daughter cells. Note that this change does not constitute a change or mutation in the DNA sequence. Much of the DNA methylation occurs on cytosines that reside in CpG dinucleotides (its complementary strand, 3’- GpC -5’, is also methylated). Regions of the genome with a high GC content, where the CpG sequence is very frequent, are often called “CpG islands”. Such regions, when upstream of a promoter, can play key roles in the regulation of gene expression.

Methylation of a CpG islands primarily leads to gene silencing, as has been shown in the cholesterol dependence of NS0 cells. For instance, Hsd17b7, a key gene in cholesterol synthesis, is silenced by CpG methylation in NS0 cells. Accordingly, demethylation treatment of NS0 cells led to the rapid emergence of cholesterol-independent cells. Methylation is also likely to be involved in the glutamine dependence of CHO cells, due to methylation of a CpG island upstream of the glutamine synthetase gene.

Of note, methylation does not only occur in CpG islands; nearly a quarter of methylation seen in embryonic stem cells is not in a CG context. This non-CG methylation, however, is more transient and mostly disappears after differentiation.

At the histone level, acetylation, methylation, phosphorylation, and ubiquitination may occur on different amino acid residues of histone proteins. Each histone protein has multiple sites that may be modified, resulting in a largecombination of possible histone modifications,each affecting the packing of DNA and the accessibility of genes in the region. Both histone and DNA chemical modifications require specificenzyme-mediated reactions. Their maintenance and removal also requires specific enzymes.

Chromatin Modifications

Residues Modified

Functions Regulated

Acetylation Lysine Transcription, Repair, Replication, Condensation

Methylation Lysine Transcription, RepairMethylation Arginine Transcription

Phosphorylation Serine, Threonine

Transcription, Repair, Condensation

Ubiquitination Lysine Transcription

• Chromatin modification

• Covalent modifications of histones

• Histone variants

• Nucleosome remodeling

• DNA Methylation

• Non-coding RNAs

Table 3. Types of Histone Modifications


At a given time a typical mammalian cell transcribes about 15,000 genes into RNA. The vast majority of those transcripts are present at only very low levels, with some even as low as a few copies in each cell, with another small fraction expressed at intermediate levels. Only a very small number of genes are expressed at extremely high levels. Genes in the last class, the so-called “abundant genes”, encode proteins such as ribosomes, GAPDH (3-phospho-glyceraldehyde dehydrogenase), and actin.

In some recombinant cells, the product gene, which is highly amplified, also falls into this category.Because the sheer number of transcripts for each abundant gene is very high, the total mass of those RNAs can constitute up to ~10% of all mRNA. Such genes usually do not undergo a very large degree of change in their expression level. For instance, one rarely sees even two-fold changes in the transcript level of most abundant genes under different culture conditions. However, keep in mind that, because they are so abundant, even a 10% change in the level of a gene in this category is much greater than even a 10-fold change in a rare gene.

The genes that most frequently undergo very large changes in expression are the rare genes. These rare genes often encode for products that are gene regulators or other products that are powerful even at minute levels. For this reason, they are kept at very low expression levels and are not expressed when not needed.

Mouse Genome Encodes• ~15,000 genes are expressed in a given cell

• Highly abundant genes generally don’t change transcript level over a wide range

• Rare genes can be very dynamic

• 1,000 fold change in transcript level in 30 min is common in bacteria . A similar change in differentiating stem cells usually takes days.

Table 4. mRNA in a Typical Somatic Human CellNumber of Species % of mRNA by

mass

Superprevalent (Abundant)

10 - 15 10 - 20

Intermediate 1,000 - 2,000 40 - 50

Complex (rare) 15,000 - 20,000 40 - 45

Transcriptome

Genome Scale Analysis

Knowing the mechanisms of epigenetic regulation, it is possible to intervene using chemical inhibitors of the necessary enzymatic reactions. For example, 5’-Azacytidine is used to facilitate demethylation. TrichostabinA and sodium butyrate are also used to interferewithhistonemodifications.Thealternationof the epigenetic status can also be induced by the introduction of exogenous genes, as in the process of reprogramming induced in the derivation of iPS cells.

reprogramming induced in the derivation of iPS cells.


Expressed Sequence Tags (ESTs)

• Transcripts from cells of tissues are isolated, reverse transcribed to cDNA and cloned into E . Coli to construct an EST library

• Clones of E . Coli are sequenced to give rise to fragment or the entire length of transcript

• Sequences are assembled and annotated

• The data gives transcriptome profiles of (abundance level) of transcripts of different genes under different conditions

• The sequence data are used to design microarray and assemble the genome

The transcript levels of many genes are relatively stable at different times and under different conditions, while some are relatively dynamic. Overall, the rate of change of gene expression in mammalian cells is rather slow compared to bacteria. We see over three orders of magnitude decrease in transcript levels within half an hour in bacteria; however, even under stem cell differentiation conditions, a similar level of change in mammalian cells usually occurs over days.

To explore the dynamics of gene expression in different tissues and in different diseases or differentiation stages, transcripts were isolated from those tissues and directly sequences. Those transcripts are typically called expressed sequence tags (ESTs). The collection of those ESTs form the core of the database of various genes in different species.

Capturing the dynamics of transcripts at a global level, i.e., on a genome scale, has become readily available in the past decade through the use of DNA microarrays and, more recently, through deep sequencing. Transcriptome profiling through arrays andsequencing remains the cellular analytical tools that are truly global and capable of genome-wide survey.


DNA Microarrays Two prevailing forms of microarrays are currently utilized: cDNA microarrays (commonly used for complex mammalian genomes) and oligonucleotide-based microarrays. The difference between these methods lies in their type of probes. In the case of DNA microarrays for microbial species the primers aredesignedtospecificallyamplifygenefragmentsfrom genomic DNA that will then serve as probes. These probes are designed specifically for uniquesegments of gene sequences. The probes spotted on a cDNA microarray for mammals, on the other hand, are amplifiedfromclonedcDNAsusinguniversalvectorprimers.Usuallydesigningprobesbasedonspecificgene sequences is too costly with the large number ofgenesinvolved.Theyusuallylackthespecificitytodifferentiate many isoforms or alternatively-spliced variants. Oligonucleotide-based microarrays, in contrast, utilize much shorter (20 - 80 bp), specifically designed, then chemically-synthesizedprobes. Multiple probes covering different regions ofeachgeneareoftenusedtoincreasethespecificity.

With the decreasing cost of oligoDNA microarrays and direct sequencing, cDNA microarrays are being phased out. cDNA arrays rely on using two fluorescence channels for relativemeasurement renders them inconvenient for comparison of a large number of samples.

Long oligo microarrays are typically comprised of 50 - 70 bp probes synthesized onto a glass slide. Affymetrix arrays are made by the direct synthesis of eleven sets of short 25-mer probes onto the chip through photolithography-based technology. Typically, multiple probes are employed for a given gene or contig over a region of a few hundred base pairs of each target transcript.

The photolithographic in situ synthesis technique requires the construction of masks for each layer of nucleotides added to the probes. The process is extremely costly. In contrast, using a digital micromirror, NimbleGen technology directs light to tiny spots to allow chemical reactions to occur only in the lighted spots without using masks, thus drastically

Table 5 . Available Microarray TechnologiesAffymetrix Agilent Nimblegen

Manufacturing technology

Photolithographic manufacturing

Non-contact inkjet printing

Maskless synthesis using digital micromirro device

Probe LengthFeature sizeMultiplexing

25 - mer5 - 18 μmNo

60 - mer65 μm2-, 4-, and 8-plex

60 - mer16 μm2- and 4 - plex

Fig. 14.3: Classical DNA microarray prepared from cDNA clones of an EST library and the use of it as a two-dye microarray


With the ability to generate tens of gigabases in a single run, high throughput sequencing technologies are becoming an affordable and powerful tool for transcriptome profiling.

AfirststepintranscriptprofilingistheremovalofrRNA by oligo(dT) capture of mRNAs, which targets their polyA. Subsequent reverse transcription for cDNA synthesis is performed either by polyA-based priming or by using random primers.

RNA-seq methods take mRNA samples, shears them

RNA-seq

• Direct counting of abundance level of reads for each gene, normalized to gene length

• Very wide dynamic range of detection

• Quantification not affected by low sensitivity or saturation as in fluorescence detection

• Does not require sequence information for

light source

mask (each layer four masks for A, T, G, C)

array “layer by layer” synthesis (total ~25 layer for 25-mers of DNA)

digital mirrorlight source

no mask needed

“layer by layer” synthesis ~60-mers of DNA

reducing the manufacturing cost of making the array.

Both Agilent and NimbleGen allow multiplexing (i.e., multiple independent samples can be hybridized to separate arrays on a single slide). Both of these array formats also support a dual mode system that provides the option of using the routine two-color experimental design (Cy3 / Cy5 based) or one-color (Cy3 only) on a single platform.

When the DNA array is used for two-channel comparison, usually a common reference is used. Such common references are usually acquired by mixing mRNA samples from different tissues under different culture conditions to ensure that the vast majority of transcripts are present.

When the DNA array is used for two-channel comparison, usually a common reference is used. Such common references are usually acquired by mixing mRNA samples from different tissues under different culture conditions to ensure that the vast majority of transcripts are present.

Fig. 14.4: Mask-based layer-by-layer in-situ photosynthesis of oligonucleotide on array surface (Affymetrix system).

Fig. 14.5: Digital mirror-based photosynthesis of oligo-nucleotides on array surface (NimbleGen system).


into fragments, and reverse transcribes them into cDNAs, which are then sequenced using one of the high throughput sequencing technologies. The resulting output is a large number of sequences on the order of 50 - 150 bp. Each string of sequence is called a “read”. Depending on the depth of sequencing and the abundance level of the nucleic acid fragment, a segment of nucleic acid may be sequenced only a couple times or up to million times.

RNA-seq is not 3’ end biased; the abundance level of a transcript is represented by the number of times that segments of the transcript have been sequenced. The more abundant and the longer the transcript is, the more frequently its sequence reads will appear.

Using this method, one can sequence as deeply as is necessary to detect almost all transcripts in the cells, even those that are rare. In microarray analysis rare transcripts usually do not yield enough signal to give confidence to results. Furthermore,very high abundant genes are usually detected in the non-linear, near-saturation range of signal, thus lacking good sensitivity for quantification.Such problems are not present when RNA-seq is used, as it gives a much wider dynamic range.

Another major advantage of RNA-seq is that it requires no prior EST database or genome sequences of the species to be probed, whereas for DNA array probe design, at least the sequences of the genes to be probed must be available. For the species whose genome sequence or EST database is available, the reads from sequencing are mapped to the exon sequences for enumeration of hit reads and for normalization to sequence length. Even if no genome sequence or EST database is available, the reads can be still assembled into contigs. In most such cases, the contig annotation can be obtained from a related species, and the reads can be mapped back to each contig and then counted to give an estimate of transcript abundance.


Table 6 . Commonly Used DNA Microarray FormatsMicroarray

TypeProbe sequence

generationCommon Uses Sample Numbers Hybridization

methodQuantification

Method

cDNA Universal primer amplified EST probes

Commonly used for mammalian EST library based array

Normally two channel comparisons (can go up to four)

Typically mRNAs are isolated and reverse transcribed to cDNA which is the labeled with a fluorescent dye

Measure the ratio of intensity of the same transcript from different samples. Comparison of multiple specimens from different samples must use ratio of ratios unless a common reference for all samples is used . Can use a pooled RNA as a common reference to facilitate the comparison of multiple samples. Direct comparison of levels of different transcript in the same specimen is difficult.

Sequence specific primer amplified probes

Generally used for microbial species whose genome has been sequenced

oligoDNA Synthetic specific 50-60mer, can have multiple probes per gene

Cross hybridization among different closely related sequences can be minimized

Two Channel

Single Channel

Photolithographically synthesized 25mers, multiple probe set per sequence

Interrogate multiple segments of about 500 bp region using both “perfect match” and “mismatch” probes to compute the signal

Single Channel mRNA is reverse transcribed, then transcribed into cRNA, which is then fragmented and biotin labeled before hybridizing to the probes on the array .

The intensity gives an estimate of the abundance of transcript for each gene . Data can be used for both intra-array comparison (different genes in the same specimen) as well as inter-array comparison (ratio of expression level as in cDNA array) .

RNA-Seq Direct sequencing . Coverage depends on sequencing depth . For CHO, 20GB gives good coverage of most genes

Use for reaching depths sufficient to detect rare genes . Discerning heterogeneity in transcript or genome . Also for transcriptome profiling of unsequenced genome

Single sample per channel, or multiplexing barcoded sample

Short reads are sufficient for sequenced genome . If assembly is required, long reads are preferred .

Direct counting of sequence reads per gene, normalized to gene length .


Proteome Profiling Proteome profiling is anothermeans of surveyingglobal changes in gene expression. It is a powerful complementtomicroarraytranscriptomeprofiling,serving to examine gene function directly at the protein level. Proteomic analysis allows for the investigation of the primary amino acid sequence, protein-protein interactions, and post-translational modificationson a large scale. In proteomics, mass spectrometry has established itself as an indispensable tool.

2D-gel electrophoresis allows for the simultaneous analysis of a many protein molecules. In this method, a complex protein sample is resolved in two dimensions according to charge (isoelectric point) and mass on a polyacrylamide gel, followed by staining. The stained protein spots are then characterized using sophisticated image analysis tools (such as PDQuest). The difference in staining intensity of observed spots allows for relative quantification between protein samples.

This method, however, is not suitable for some proteins. For instance, proteins present at lower levels are not easily detected. Also, some proteins co-migrate and cannot be resolved. Finally, proteins with charges outside of the isoelectric range of the gel or highly hydrophobic proteins are also unable to be resolved in the gel.

Once a protein spot of interest is identified, thespot is excised and purified for further analysis,such as direct sequencing or mass spectrometry for protein identification. Electrospray ionization(ESI) and matrix-assisted laser desorption/ionization (MALDI) are two techniques commonly used to volatize and ionize the proteins or peptides for mass spectrometric analysis.

2D Gel Electrophoresis

• 2D gel electrophoresis

• First Dimension: Isoelectric focusing (IEF), separation by charge. IPG (immobilized pH gradient) strips; usually pH 4 to 7

• Second Dimension: SDS-PAGE, separation by molecular weight

• Staining types: Coomassie Blue (sensitivity in the ug range); silver staining (sensitivity in the ng range); SYPRO RUBY(sensitivity in the pg range; fluorescent dye; most suitable for quantification)

• Image Analysis: identification of differentially expressed spots by eye or with the aid of specific software packages (PDQuest)

• MALDI-TOF and ESI

• Differentially expressed spots are extracted from 2D gel, and subjected to proteolytic digest, and peptide finger print analysis using MALDI-TOF

Figure 14.6: Typical flow of 2-D electrophoresis-based protemics.


In applying two-dimensional liquid chromatography (LC) for analysis of protein mixtures, a proteolysis of theprotein(typicallyusingtrypsin)isfirstperformed.This process reduces proteins to oligopeptides of mostly 15 - 30 amino acids long, which facilitates separation by liquid chromatography. In the firstdimension, the peptides are separated into fractions. Each of the selected fractions is then injected into the second LC for further separation before injection into the mass spectrometer (MS) for detection.

LC-LC/MS methodologies have the advantage of being capable of analyzing complex protein mixtures; however there was previously no way to quantify different expression levels among samples until recently, due to the application of stable-isotope labeling to LC-LC/MS. This method makes use of the fact that pairs of chemically-identical analytes with different isotope compositions can be differentiated in a mass spectrometer by their difference in mass-to-charge ratio. The ratio of signal intensities for the pair accurately indicates an abundance ratio for the two analytes. Two commonly used labeling techniques are SILAC (Stable Isotope Labeling by Amino Acid in Cell Culture), and iTRAQ (Isobaric Tagging for Relative and Absolute Protein Quantitation).

Although all proteins in a sample are, technically, included in the analysis using non-gel-based techniques, these methods are still not a true global surveying tool. In actuality, only a few hundred to a fewthousandproteinsareidentifiedintheanalysisof a sample due to limitations in the resolving power of liquid chromatography. The capture of a peak from a sample is stochastic, and highly abundant proteins are detected over those present at lower levels. The isolation and identificationof low abundance proteins can be enhanced by repetitively analyzing the same fractions in the mass spectrometer and by excluding peptides that have alreadybeenidentifiedinthepreviousanalysis.Thisis very expensive and tedious so exhaustive surveys of the proteome space are not commonly practiced.

Shotgun LC Based Methods

SILAC

• Use non-radioactive isotope for labeling cultured samples

• Mix samples for isolation or enrichment of some cellular fractions

• Sample can be analyzed by PAGE or 2D LC

• The fractions can be analyzed in mass-spec for identification

iTRAQ

• Use isobaric tag for different samples

2D LC

• Protein mixture is subjected to proteolytic digestion peptides are amenable to LC separation to reduce complexity

• Column types: First dimension often exchange, second dimension reverse phase

• Electrospray injection into mass spectrometer

• For identification of molecules, not quantification

2D LC

Fig. 14.7: iTRAQ labeling of proteolytic peptides for 2-D liquid chromatography.


Chemical isotope labeling of proteins in vitro (after protein isolation) using isobaric Tags for Relative and Absolute Quantification (iTRAQ) is ahighly versatile and widely-used method. iTRAQ tags have a reporter group, a balancer group, and a peptide reactive group that tags the N-terminus of every peptide. One of the unique features of iTRAQ is that up to eight samples can be labeled with different tags and analyzed simultaneously, while other methods can only label two samples.

The combined balancer and reporter groups have an identical mass of 145 for all four labeling pairs of balancers (mass 28 to 31) and reporters (mass 114-117). The MS spectra for each of the four labeled samples look identical. The reporter-balancer fragment stays intact, giving rise to the same m/z ratio. This allows for protein identificationusing the combined signal of the four samples. Upon MS/MS fragmentation, the bond between the reporter and balancer group is broken. The reporter groups then appear as peaks in the low massregion,andquantificationofthepeakareaforeach reporter group gives the relative abundance for a peptide between the labeled samples.

ITRAQ

SILAC In vivo labeling methods, such as Stable Isotope La-beling with Amino acids in Cell culture (SILAC), use deuterated leucine, or other isotope labeled amino acids, to differentially label one of the protein sam-ples by replacing an amino acid in the cell culture me-dium, thereby allowing the isotope to be incorporat-ed into the cellular proteins. The combined labeled and unlabeled samples are then analyzed by LC-LC/MS, generating two spectra for each peptide frag-ment, one shifted precisely by the mass of the deu-terated amino acid. Differences in peak height pro-videthemeansofquantificationbetweensamples.This method allows two samples to be mixed prior to protein isolation, thereby eliminating systematic errors due to protein isolation efficiencies. It iswell suited for use when subcellular enrichment protocols are used (as in the case of organelle fractionation) prior to LC-LC/MS analysis. One

Fig. 14.8: Isotopic labeling of peptides.

Fig. 14.9: Stable isotope labeling of amino acids in cell culture (SILAC) for proteomics.


notable limitation of this method is that the labeling timerequiredisrelativelylong,toallowforsufficientincorporation of the isotope into cellular proteins.

Sequencing TechnologiesDNA sequencing technology has undergone revolutionary changes in the past few years. Traditional Sanger sequencing has dominated the field for nearly three decades, and it is stillthe prevailing method used for sequencing small regions of DNA. For large-scale, or genome-wide sequencing, a number of high throughput methods have rapidly changed the scope of sequencing. They are now used for genome sequencing, transcriptome profiling, transcription initiationsite surveys, transcription factor binding site profiling, and epigenetic alteration studies.

Sanger sequencing gives relatively long reads, while newer methods give shorter reads. Some of the reads are too short for efficient assemblyand are used primarily for “resequencing”, i.e., for sequencing the genome of individuals of a species whose genome sequence is already available. Other newer methods, such as 454 and Illumina, produce reads that are at least long enough for assembly.

All of the new methods and the Sanger method share the same “reading” scheme. Each time a nucleotide is incorporated into an elongating DNA molecule a signal is emitted. Two approaches are adopted to detect the emitted signal, (a) amplifying the signal by having many DNA molecules emitting the same signal, (b) using very sensitive detection methods to detect even the signal emitted from a sigle molecule.

In Sanger sequencing, each target DNA molecule is firstcloned intoE. coli so thatasufficientamountof pure DNA molecules can be obtained by growing the E. coli clone. Those DNA molecules are then used as templates for DNA synthesis. By using altered nucleotide analogues that cannot be used in DNA synthesis, the elongation stops randomly as soon as an analogue (instead of the genuine nucleotide) is incorporated. Given a proper titration of the ratio

• Target DNA template is used for DNA synthesis

• Fluorescently labeled nucleotide analogues (dideoxynucleotides) is added to the synthesis reaction; wherever an analogue is incoporated into the elongating DNA, the reaction terminates

• Each of the four dideoxynucleotide chain terminators is labelled with a different florescent dye.

• The newly synthesized and labeled DNA fragments are separated by size (with a resolution of just one nucleotide)

Table 7 . Summary of Sequencing MethodologiesTechnology Read Characteristics Applications

Sanger Sequencing (ABI)

500-1000 bp 384 reads/run

Gold standard all purpose sequencing

Roche Sequencer FLX/454

400 bp/read1-7 Gbp/run

Re-sequencing, expression profiling, SNP/methylation

Illumina/Solexa 36-150 bp/read20 Gbp/run


SOLiD (ABI) 30-50 bp/read20 Gbp/run


Helicos 35 bp/read20 Gbp/run


Sanger Method and Sequencing Technology Evolution


of analogues to normal nucleotides, there will be a DNA molecule terminated at every base of the DNA template. After synthesis, the mixture is elongated and the terminated molecules are separated by chromatographic separation at a single base resolution. Since each analogue is labeled with a fluorescent color, the colorimetric chromatogramis finally used to read off the sequence.

This clone handling is very expensive and time consuming. Furthermore, single-base resolution can be accomplished only up to at most 1.2 kbp, even with capillary electrophoresis.

Sanger Sequencing

• Enrich target DNA fragments by cloning into a E . coli plasmid

- Specific primers at two ends of target DNA Collect plasmids, use specific primer to start DNA synthesis from one end

• Add nucleotide analog, ddNTPs in addition to dNTPs at low frequency in the reaction mixture

• Incorporation of a ddNTP terminates DNA elongation.

• Probabilistically a small fraction of elongating DNA is terminated at every base, resulting a collection of synthesized DNA fragments of different length

• Separate those DNA molecules using capillary electrophoresis

• Each of the four ddNTPs fluoresce at a different wavelength . As each DNA fragments comes out of electrophoresis, the fluorescence is “read” to give the identity of the base

Fig. 14.10: Conventional Sanger sequencing of DNA.


The most fundamental change from the Sanger method to newer methods is the omission of E. coli clones. Two approaches were taken to bypass cloning. Inthefirstapproach,localizedPCRamplificationisperformed: a single DNA fragment is amplified ina droplet or in a locus on a surface. This allows a large number of identical DNA fragments to be used to generate signals for detection as they incorporate nucleotides when used as templates for synthesis. In the second approach, single molecule detection is achieved. As long as each molecule is separated from the others, the signal from nucleotide incorporation into an elongating DNA can be detected. The increased sensitivity eliminates the need for cloning.

The current prevailing methods, sometimes referred to as “next generation sequencing technologies”, fragment the DNA molecules randomly without resorting to cloning. A key feature of these methods is the massive and parallel amplification of each DNA fragment individually,thus creating up to millions of clusters, or colonies, of DNA molecules of the same sequence.

With the 454 technology, each fragment is immobilized on a bead contained in an oil / water emulsion droplet, thus forcing all of the PCR products of that particular fragment to also be immobilized on the same bead.

On the other hand, the Illumina Solexa sequencing technology immobilizes the fragment on a solid surface and confines the PCR products ofthe fragment in the locale, thereby forming a cluster of identical sequences on the surface.

These ingenious approaches allow a large number of identical sequences to be isolated so that the light emitted from reactions in these clusters can be detected using a single sensor, such as a CCD camera. This contrasts with the traditional Sanger method, which requires a sensor at the end of each electrophoretic capillary, thus drastically reducing the cost of sequencing.

Both methods then employ base-by-base synthesis. The 454 technology works by sequential addition of

High Throughput NextGen Sequencing

Next Generation Sequencing Technologies

• Massively Parallel Local Amplification

• ‘Sequencing-by-Synthesis’

Fig. 14.11: Two strategies of localized PCR amplification of DNA molecules for enhanced signal detection.


one of the four nucleotides and photons are emitted upon the incorporation of a base. The Illumina Solexamethodology employs fluorescently-labelednucleotides that are also reversible terminators. One base is incorporated and interrogated at a time, since further elongation of the chain is prevented. When all clusters are scanned at the end of a cycle and the base has been determined for each colony, the fluorophores are cleavedoff and terminating bases are activated, thereby allowing another round of nucleotide incorporation.

The drawback of the current generation of these new sequencing technologies is their reliance on the complete synchronization of serial reactions. The reads obtained are relatively short, compared to those from Sanger sequencing. However, this drawback is more than compensated by their improved capability of massively paralleled “reading” of millions of sequences at high speeds and at relatively low costs. Their tremendous parallel sequencing abilities allow for up to a million fragments of a very abundant mRNA species (such as the transcript for the recombinant protein product) to be “read” in a single run, while only single reads can be obtained for thousands of rare genes. Such a dynamic range in sequencing outputs has made them great tools for assessing transcriptome-wide abundance levels.

454 Sequencing Technology

• Immobilize one DNA fragment per bead

• Suspend each bead in an emulsion droplet

• PCR amplify DNA in each bead

• Place each bead in one well

• Perform synchronized synthesis by adding one nucleotide at a time

• Each nucleotide incorporation emits a photon

• Homo-oligomer emits stronger light intensity (works only for short homo-oligomer sequences)

• Can sequence up to 1000 bp in length

Illumina Sequencing Technology

• Immobilize DNA fragments on surface with adaptor

• Localize PCR reaction to create clusters of DNA fragments with complementary strands

• Perform synchronized DNA synthesis from one strand, then on the other strand

• Upon the extension of one base, the reaction is terminated because the functional group for further extension of the added nucleotide is protected; this prevents incorporation of more than one nucleotide when homo-oligomer sequences are encountered

• Can sequence up to over 100 bp; very high throughput


Transcriptome analysis is a powerful tool for studying cell culture processes, as it is extremely revealing for both subtle and not-so-subtle changes that can occur in many situations (e.g., cell cultivation, the variation of cell behavior over time, and cell line development). It has been used to compare cell performance in different reactor scales, cells of varying productivities, and cells growing under different media composition, and to probe cell changes after long-perfusion culture.

In a striking example, a cell line was subjected to studies under differing culture conditions. The variables included temperature, pH, and reactor size. There was also an uncontrolled variable. Two different lots of the same hydrolysate were used in each run, as is customary (when one lot of material is exhausted, another lot is used). The time-course transcriptome data from all runs were collected and subjected to clustering. All the runs with the same lot of hydrolysate were found to be clustered together, thus overriding the other variables of the experiment.

Such revelation is possible because of the large number of genes probed. Many subtle changes which, individually,may not be used to draw a confidentconclusion, can collectively point to a trend or correlation that is not otherwise easily discernible.

There has been some effort in comparing cells of varying levels of productivity. It has now been realized that the “hyperproductivity trait” is a complex phenomenon that is not simply a result of turning on a small number of master genes. Rather, the generation of high producers is likely to involve colossal changes in gene expression that occur in a wide range of genes, but each only to a modest extent.

A most important feature of either sequencing- or microarray-based transcriptome analysis is its global coverage of gene expression. This type of analysis sometimes reveals unnoticed cellular processes that may play a key role in some physiological events. For example, it was found that the endocytosis and secretory vesicle retrograde transport pathways

Gene Expression Exploration in Cell Culture Processing

Fig. 14.12: Comparative transcriptome dynamics between (a) mouse hybridoma cells treated with butyrate and (b) differentiating mouse stem cells (MAPC). MAPC cells were grown on liver lineage differentiation medium for 6 days. Mouse hybridoma cells were treated with 1mM butyrate for 27 h. Both MAPC and hybridoma cell samples were referenced to a corresponding, untreated time sample. The transcript levels were probed with the Affymetrix MOE430A array.For each dataset, average intensities are plotted along the y-axis, and the log2 of the expression ratios are plotted on the x-axis. Each marker represents a gene on the array. The vertical lines mark the bounds of a two-fold expression change. Markers lying outside of these lines are more than two-fold up or down regulated between the two samples compared. In comparing Figures 2a and b, the number of genes that are more than two fold differentially expressed is substantially higher for the stem cell differentiation study than for the butyrate treated hybridoma cells.


are upregulated in high recombinant protein-producing cells. This led to the realization that high producers have an increased capability to recycle components of membrane vesicles that carry proteins destined for secretion from the Golgi apparatus to the cytosolic membrane.

One of the important features of transcript profiling in cell culture processes is the smallnumber of genes differentially expressed and the lower magnitude in the observed fold-changes, as compared to those observed in other biological processes. For microbial cells, it is not unusual to observe more than 20% of the genes as differentially expressed by more than two-fold within ten minutes of changing culture conditions. For cultured mammalian cells, it is rare to see over two-fold expression changes occurring in more than a small percentage of the genes surveyed.

It is important to realize that the cells we deal with are cultivated in artificial conditions, not in theirnative niche. For an organism the development of a fertilized egg to an adult body incurs many major events and gene expression changes to guide those events. Once the cell is guided to its destined developmental status, the perturbations of other environmental factors are relatively minor in magnitude by comparison. The various events that cells in culture may encounter are only relatively small perturbations compared to developmental or differentiation events that their genome has been evolved to accommodate. It is not surprising that colossal big changes in gene expression are rarely seen in cell culture bioprocess but are frequently seen in stem cell differentiation.

The small change in gene expression, as compared to other organisms and to the in vivo processes, requires one to be more versatile and more skillful in data analysis. However, the power of global transcriptome analysis will fundamentally change our practice in cell culture processing.

nor perturbations in comparison to development.

The small degree of gene expression changes, as

Fig. 14.13: Intracellular processes differentially expressed between high and low producing NS0 cell lines. (a) Each node depicts an intracellular process with a large number of differ-entially expressed genes. (b) Schematic of the steps involved in vesicle-mediated transport (nodes 4, 5, and 6).


Concluding RemarksIn the past few years we have seen dramatic advances in genome science and technology. The availability and affordability of sequencing technology has changed sequencing effort from species sequencing (i.e., focusing on obtaining a “representative genome sequence”), to individual sequencing (i.e., aimed to acquire genome sequence of an individual human, organism or cell line). The depth of information we are acquiring from genome, transcriptome, proteome and epigenome, is transforming the

compared to other organisms and to the in vivo pro-cesses, requires one to be more versatile and more skillful in data analysis. However, the power of glob-al transcriptome analysis will fundamentally change our practice in cell culture processing.

way we deal with bioprocess challenges. The application of “-omics” technology in cell culture processing is still largely limited to process analysis. One can foresee that, in a not too distant future, “-omics” technology will be increasingly used for the generation of producing organisms, as well as in the design of biological processes. In some ways, genomics science may also bring about transformative changes in cell culture bioprocessing.

INDEX | 309

IndexNote: Page references followed by f or t indicate material in

figures or tables, respectively.

AABC transporter, 43, 44f, 84Abraham, Edward Penley, 2Abundant genes, 293, 293tAcetylation of histones, 292–293, 292tAcetyl CoA

in cholesterol synthesis, 87in lipid metabolism, 86, 86fmetabolism of, 81in tricarboxylic acid cycle, 61, 73

Acetyl CoA shuttle, 86, 86fAcoustic resonance enhanced settling, 257–258, 258fActin fibers, 37, 38–39, 38f, 46Active transport, 40–42, 41fActivity parameters, 156, 160fAdaptation, in stable cell lines, 131, 142, 142f, 143fADCC. See Antibody dependent cellular cytotoxicityAdenosine, in medium, 109, 109tAdenosine triphosphate (ATP)

in active transport, 40consumption in glucose metabolism, 60–61mitochondrial production of, 29production in glucose metabolism, 60–64, 66, 70

Adenosine triphosphate synthase (ATP synthase), 61, 63Adherent cultures, vs . suspension cultures, 202Adhesion, cellular, 45–46, 53, 142

microcarriers for, 203–204, 204t, 205tvs . suspension, 202

Aeration. See also Oxygen transfer, in bioreactorsstripping of carbon dioxide by, 219surface, for oxygen supply, 220

Aerobic glycolysis, 65–66Affym etrix microarrays, 295–296, 295tAgarose cell immobilization, 209Aggregation, 206, 206f, 209, 209f, 212Agilent microarrays, 295t, 296Agitation, in bioreactors, 265–266

impellers for, 206, 265–268, 265t, 266fmechanism of, 265–266purpose of, 265

Airlift bioreactor, 207, 207f, 225Akt, in regulation of glucose metabolism, 77, 77fAlanine

in medium, 108metabolism of, 81–82

Albuminproduction of, 13recombinant, 122secretion of, 31serum, in medium, 118, 122

Alginate microcarriers, 205tAllosteric regulation, differential, of glucose metabolism, 75–76,

75fAlternating tangential filtration, 260, 260fAlternative splicing, 285–286Amino acid(s)

alterations, in bioprocessing, 16–17, 16tanalysis of, 299–302in cellular growth, 150, 151, 151tdegradation of, 81–82essential, 81, 108, 108t, 151in fedbatch culture, 238–240, 239f, 239t, 246–247in intracellular fluid, 153, 153tin medium, 81, 106–108metabolism of, 58, 81–82, 81f, 82f, 186non-essential, 81, 108, 108t, 151stock solutions of, 108transport of, 40–41, 43, 81

Aminophospholipid translocases, 84Ammonia, in cultured cell metabolism, 67, 236Amphipathic lipids, 22–23Amplification

localized, in DNA sequencing, 304, 304ffor stable cell lines, 131–132, 138–141, 140f

Amplification maker, 135Anaerobic glucose metabolism, 58, 65Aneuploid cells, 54, 133, 143, 150Animal(s)

as production vehicles, 199transgenic, 12, 14, 14tvaccines for, 8t, 9

Animal component-free serum, 102–103Animal serum, in medium, 102, 117–119Anterograde transport, 34, 35, 35fAntibiotic(s)

declining price of, 2–3development of and advances in, 2–4in medium, 117, 117tas selection markers, 137t

Antibody dependent cellular cytotoxicity (ADCC), 90Antibody products, 5–8, 7t

manufacturing of, 11–12quantity for administration, 11, 11tstoichiometric ratio in, 11

Antibody synthesis, analysis of, 186Antioxidants, in medium, 116Antiporters, 41, 42fApf-1, 52Apoptosis, 51–53

cell cycle and, 50fdeath receptor pathway in, 51–52mitochondrial pathway of, 52–53, 53fmorphological changes in, 51vs . necrosis, 51

Apoptosome, 52Ascorbic acid (vitamin C), in medium, 109

INDEX | 310

Asparaginein medium, 108metabolism of, 81

Aspartate, metabolism of, 81–82ATP . See Adenosine triphosphateATP-binding cassette (ABC) transporter, 43, 44f, 84Attenuated vaccines, 4–5Autocatalytic growth, 156Automation, for cell line production, 145–146, 145fAxial flow impellers, 206, 265–266, 265t, 266fAzacytidine, 293

BBaby hamster kidney cells . See BHK cellsBacterial genome, 287–288, 289t, 290Bag-based centrifuge, 256Bag culture systems, 201–202, 202fBak protein, 52Balance equations, 162–165, 175–180. See also Material balanceBasal medium, 101

components of, 106–117optimal concentration of organic nutrients in, 110, 110f

Base addition, proportional feeding with, 244Base-by-base synthesis, 304–305, 304fBatch culture (processes), 233 . See also Fedbatch culture

in bioreactors, 196–198, 197fvs. continuous processes, 249–251

Bax protein, 52B cells, transition to plasma cells, 33, 33f, 130Bcl-2 proteins, 52Bcl-xL protein, 52Beef extract, in medium, 124Beta-carotene, in medium, 116Beta cells, pancreatic, 31BHK cells, 8t, 19t

adhesion of, 142aggregates of, 209as continuous cell line, 49for transient expression, 129veterinarian vaccines from, 8t

Bicarbonate buffer, 113–114, 115f, 115tBiogen, IDEC ISM facility, 12Biological fluids, in medium, 118Biologics, 127. See also specific typesBiomass, 147–148, 148t

growth of . See also Growthmaterial balance on, 149–154, 149fas objective of medium, 97–99, 125overall synthesis equation for, 149–150

intracellular fluid in, 153–154medium to generate, 103–104metabolic flux analysis of, 184–185, 185fas output of reaction, 176on substrate, yield of, 158

Bioreactors, 191–212. See also specific typesagitation in, 265–266

impellers for, 265–268, 265t, 266fmechanism of, 265–266purpose of, 265

basic types of, 193–196batch processes in, 196–198, 197fcell culture, 206–212cell retention in, 249–261. See also Perfusion culturecell support systems in, 202–206continuous processes in, 196–197disposable or single-use systems in, 199–202fedbatch cultures in, 198, 198f, 212mixing characteristics of, 193, 266–270mixing time in, 206, 268–274operating mode of, 196–198oxygen balance in, 220, 276–280oxygen supply for, 220–228

by medium recirculation, 221fby silicon tubing/membrane, 220by sparging, 220–228by surface aeration, 220

oxygen transfer in, 206–207, 213–231driving force for, 214–216, 220, 279–280enhancing or improving, 216, 220, 221experimental measurement of, 224–225, 224f, 225fhydrostatic pressure and, 225in immobilization reactor, 229, 229fmass transfer coefficient in, 216objective of, 219in plug-flow reactors, 229–230, 230frate of, 215–216, 220scaling up and, 275–280, 280f, 280tsurface/interfacial area and, 216through gas-liquid interface, 212–219, 213f, 214f

phases in, 196power consumption in, 266–270, 266freactions and reaction kinetics in, 194–196scale translation for, 263–284

and carbon dioxide removal, 275, 281–283and chemical environment, 283, 283fconstant parameters in, 269–270, 269tdimensionless variables in, 267–270and driving force, 279–280effect of scale on physical behavior, 269–270geometric nonsimilarity in, 264geometric similarity in, 264major effects of scale, 264and mechanical forces on cells, 274–275, 275fmixing time in, 271–274, 271tnutrient starvation time in, 271–274, 271tobjective of, 264and oxygen transfer, 275–280, 280f, 280tphysical and mechanical parameters of, 264Reynolds number in, 267–268, 267fand superficial gas velocity, 278, 278f

tracer concentration response in, 193–195, 194fvelocity of, 268, 278, 278f

INDEX | 311

volumetric flow rate in, 268–270Biosimilars, 3, 9–10

approval in Europe, 10, 10tmarketed in China, 11tmarketed in India, 10tuncertainty about quality of, 10

Biotin, in medium, 109BiP chaperone protein, 33, 35Bispecies-transporters, 41–42Blasticidin S, as selection marker, 137tBlood bags, 201–202Bok protein, 52Bristol Myer Squibb, Devens (Massachusetts) facility, 12Bubble size, in gas sparging, 222–223Buffer systems, in medium, 113–115, 115f, 115tBulk ions, in medium, 111–112, 111t

CCalcium

intracellular and extracellular concentrations of, 100, 100tin medium, 111, 111t

Calcium phosphate precipitation, 133–134, 134tCalculated differentiated data, 169Calculated integrated data, 169Carbohydrates, as cellular component, 21, 148Carbon

in amino acid metabolism, 81–82in cellular growth, 149–150flow or flux of, 67in glucose metabolism, 59, 61–67in glutamine metabolism, 59metabolic flux analysis of, 182f, 183in pentose phosphate pathway, 64–65

Carbon dioxidein buffer system, 113–114cellular tolerance of, 281concentration in medium, 217–218in glucose metabolism, 60, 61, 66production of, 219removal in bioreactors, 219

scaling up and, 275, 281–283transfer (diffusion) of, 212–219

g-Carboxylation, 12, 14, 16Carrier-mediated diffusion, 40–42, 41fCarrier proteins

in medium, 117, 118, 122transport by, 122, 122t

Caspases, in apoptosis, 51–53Catabolism

of glucose, 60of lipids, 36in metabolic flux analysis, 188t

Catalase, in medium, 116Catalytic macromolecular components, of medium, 105–106CDK4/6-cyclin D complex, 48–49CDK inhibitors (CDI), 48–49

cDNA microarrays, 295, 295f, 298tCell(s)

chemical environment of, 21–22, 21t, 97–101composition of, 21–22, 21t, 148, 148tin culture . See Cultured cellsdeath of, 51–53

apoptosis vs . necrosis, 51cell cycle and, 50fconsideration in growth rate, 157death receptor pathway in, 51–52from injury (necrosis), 51mitochondrial pathway of, 52–53, 53fmorphological changes in, 51

diameter of, 21mass of . See Biomassmovement of, 46–47nutritional requirements of, 97–99. See also Mediumsenescence of, 53–55size of, 21, 148, 148tsources of, 19–21, 19tvolume of, variation in, 150–151, 150t, 151f

Cell adhesion, 45–46, 53, 122–123, 142microcarriers for, 203–204, 204t, 205tvs . suspension, 202

Cell adhesion molecules, in medium, 122–123, 123tCell aggregates, 206, 206f, 209, 209f, 212CellCube Module, 201Cell culture engineering. See also specific processes and

materialsadvances and growth in, 1–4process robustness in, 14–17

Cell culture products, 4–12. See also specific productsalternative technologies for, 12–14biosimilar or follow-up biologics, 3, 9–10, 10t, 11tin-process structural alterations to, 15–17, 16tmanufacturing of, 11–12from perfusion bioreactors, 249tquality of, 14–17

Cell cycle, 47–49, 48fand apoptosis, 50fcheckpoints in, 47–49cyclins and CDKs in, 48–49positive and negative cues in, 47–48

Cell expansion . See also Growth; Growth controlmedium for, 97–99, 125

Cell linesadaptation of cells in, 131, 142, 142f, 143famplification for, 131–132, 138–141, 140f, 141fautomation and high throughput technology for, 145–146,

145fbasic steps for generating, 131–133, 132fcontinuous, 49, 143–144development of, 127–146gene expression in, 306–308, 306f, 307fgenomics and, 146, 306–308, 306f, 307fhost cells for, 127–129

INDEX | 312

hyper-producing, 129–133, 130t, 131fimmortalization of, 54industrial, 8t, 9screening for, 131selection of cells for, 131–132single-cell cloning for, 131–133sources of, 19–20, 19tstability of clones selected for, 143–145stability of product quality in, 144–145stable, 127, 129–146transfection for, 131–137veterinary, 8t, 9vs . cell strains, 53f, 54

Cell membrane, 22–27composition of, 22–25dynamic nature of, 23, 26homeostasis of, 26lipid bilayer of, 22–27, 83, 87potentials across, 25–26, 25tprotein content of, 25transport across, 23, 26–27, 39–47turnover rate of, 26

Cell pool, 132–133Cell recycling, 250–251Cell retention

methods of, 249–261in perfusion culture, 249–261

Cell separation methods, 254–261Cell signaling, 45–46Cell strain, 53f, 54Cell support systems, 202–206Cellulose, for microcarriers, 203–204, 205tCentrifugal cage, 259, 259fCentrifugation, 256, 256f, 257fCentritech Lab centrifuge, 256Ceruloplasmin, in medium, 116Channel-mediated diffusion, 40–42Chaperone proteins, 33, 35, 90Checkpoints, in cell cycle, 47–49Chemical environment

of bioreactors, scale translation and, 283, 283fof cells, 21–22, 21t, 97–101

Chemically defined medium, 102, 103Chemical reaction systems

cellular system vs ., 175–176material balance for, 175–180

Chick egg, 4, 9, 19China, biosimilars marketed in, 11tChinese hamster ovary (CHO) cells, 8t, 9, 19t, 20

adhesion of, 142aggregates of, 209, 209fas continuous cell line, 49doubling time of, 154tgenome of, 288, 289ton microcarrier, 203f, 204non-antibody products from, 6t

recombinant proteins from, 8tfor stable expression, 129–130, 130t, 146therapeutic antibody products from, 7tfor transient expression, 129volume of, 150t

Chloride ionsintracellular and extracellular concentrations of, 100, 100tin medium, 104transport of, 43–45

Chlortetracycline, in medium, 117CHO . See Chinese hamster ovary cellsCholesterol

biosynthesis of, 86–88, 88ffunction of, 83, 86–87in lipid bilayer, 24–25, 24f, 83, 87structure of, 85, 85fturnover rate of, 26

Choline, as backbone of phospholipid, 22Chondroitin sulfate, in extracellular matrix, 46Chromatin

DNA packaging into, 290, 290fmodifications of, 292–293, 292t

Chromium, in medium, 112Chromosome(s)

abnormalities, in cell line, 143–144in cultured cells, 54–55in mammalian genome, 288

Cis Golgi, 32, 33Cisternae maturation model, 33Citric acid cycle . See Tricarboxylic acid cycleCity water, contaminants in, 106Clonal growth, 110Clones

in Sanger sequencing, 302–303selected for cell lines, 131–132, 143–145

Cloning, single-cell, 131–133CMV promoter, 135cMyc, in regulation of glucose metabolism, 77, 77fCobalamin, in medium, 109Cobalt

in intracellular fluid, 153in medium, 112

Codon optimiziation, 135Collagen

in extracellular matrix, 45–46in medium, 123tfor microcarriers, 204, 204f, 205t

Combustion, 61–63, 176–178Compartmentalization, 184Complex glycans, 92, 92fComplex medium, 102Complex supplements, in medium, 117–125Concentration factor, in perfusion culture, 254Concentration gradient

across cell membrane, 21–22, 21t, 40–42, 43–45across mitochondria, 29–30, 30f

INDEX | 313

Conditional promoters, 134–135Conical settler, 254–255, 254fConstitutive promoters, 134–135Contact inhibition, 46, 48, 53, 54fContinuous cell lines, 49Continuous processes, 249–251

advantages of, 250in bioreactors, 196–197with cell retention, 251. See also Perfusion culturedisadvantages of, 250economics of, 250flow rate vs. growth rate in, 250

Continuous stirred tank reactor (CSTR), 193–196, 193f, 212reaction and reaction kinetics in, 194–196tracer concentration response in, 193–195, 194f

Copperin intracellular fluid, 153in medium, 112

COS cells, for transient expression, 129Co-transporters, 41–42, 42fCow pox vaccine, 4, 199CpG islands, 292CRFK cells, 8tCrisis, 54Cross filtration model, 259, 259fCSTR. See Continuous stirred tank reactorCultured cells

crisis of, 54–55epigenetic changes in, 291–293growth of . See GrowthHayflick’s phenomenon and, 54–55life span of, 54–55medium for . See Mediummetabolism of . See also Metabolism

glucose, 58–70overview of, 58–59

passages of, 53–55stoichiometry and kinetics of, 147–166

Cumulative data, 171Cyclins, 48–49Cylinders on shakers, 201–202Cytidine, in medium, 109tCytochrome C

in apoptosis, 52in electron transfer chain, 63

Cytokine(s)in medium, 97–98quantity for administration, 11

Cytoplasm, 27–36Cytoskeleton, 27, 37–39Cytosol, 27–29

acetyl CoA shuttle in, 86lipid metabolism in, 83, 85, 86, 87

DData, types of, 169

Data analysis, 167–174Data processing, 167–174

cell culture, 169–171fedbatch culture, 160, 160fmapping data to pathways, 173, 173f. See also Metabolic flux

analysispipeline for, 168–173spreadsheets for, 168, 170–171standardized templates for, 168–169

Data visualization, 172, 172f, 174fDeath, cellular

death of, 51–53apoptosis vs . necrosis, 51cell cycle and, 50fconsideration in growth rate, 157death receptor pathway in, 51–52from injury (necrosis), 51mitochondrial pathway of, 52–53, 53fmorphological changes in, 51

Death phase, of cell growth, 154–155, 154fDeath rate, specific, 157Death receptor pathway, 51–52Decline phase, of cell growth, 154–155, 154fDelivery of feed medium, 245–247Deoxyribonucleic acid . See DNADerived parameters, 160, 160fDextran

in medium, 116tfor microcarriers, 203–204, 205t

DH82 cells, 8tDHFR amplification system, 138–141, 140fDifferential allosteric regulation, of glucose metabolism, 75–76,

75fDifferentiated data, calculated, 169Diffusion

in bioreactors, 212–219, 213f, 214fcarrier-mediated, 40–42, 41fchannel-mediated, 40–42facilitated, 40–42

Dihydroxyacetone-phosphate (DHAP), 61Dilution rate, in perfusion culture, 254, 254fDimensionless variables, in scale translation, 267–270Diploid cells, 53–55, 143, 150Diploid chromosomes, 290Direct DNA sequencing, 295Direct measurement, for fedbatch culture, 243Disc centrifuge, 256, 256fDisposable bag-based centrifuge, 256Disposable cell culture systems, 199–202Dissociation, of cells from surface, 53–54, 54fDisulfide-bond formation, in protein therapeutics, 5, 14DNA

as cellular component, 21location in cell, 28methylation of, 292–293mitochondrial, 29–30

INDEX | 314

modifications of, 291–293packaging into chromatin, 290, 290fsynthesis of, 28, 47

DNA–calcium phosphate Co–precipitation, 133–134, 134tDNA microarrays, 294–297, 298t

cDNA, 295, 295f, 298tlayer-by-layer synthesis in, 295, 295t, 296foligonucleotide-based, 295–296, 298tfor pathway-related data, 173RNA-seq, 296–297, 298tfor two-channel comparison, 296

DNA sequencing, 288–289, 302–305in cell culture processing, 306–308, 306fdirect, 295evolution of technologies for, 302–303next generation technologies for, 304–305Sanger method of, 302–303, 302t, 303f

Dog (MDCK) cells, 8t, 9, 19–20, 19tDominant marker, 136Doubling time, 154–157, 154tDriving force, for oxygen transfer, 214–216, 220

scaling up and, 277, 279–280Dry mass, of cells, 148, 148t

EE . coli . See Escherichia coliE2F transcription factor, 49ECM . See Extracellular matrixEddies, in bioreactors, 274–275, 275fEF-1. See Elongation factor 1Electron transfer chain, 61, 63–64, 66Electroporation, 133–134, 134tElectrospray ionization (ESI), 299Elongation factor 1 (EF-1), 135Endocytosis, 26–27, 36Endoplasmic reticulum, 27, 31

expansion of, 33glycan biosynthesis in, 93glycosylation in, 90lipid metabolism in, 83, 88fprotein secretion through, 31–35, 35f, 83rough, 31smooth, 31

Endosomes, 27Endothelial cells, volume of, 150tEnzyme(s)

in glucose metabolism regulation, 74–76, 75fMichaelis–Menton kinetics of, 41

Epigeneticsin cell culture, 291–293definition of, 291inhibition of, 293mechanisms of, 292–293molecular mechanisms mediating, 291–293

Epigenome, 291–293Epigenomics, definition of, 291

Epithelial cellsmovement of, 46–47use in bioprocessing, 19–20, 19t

EPO. See ErythropoietinER. See Endoplasmic reticulumErythropoietin (EPO)

development of, 5glycan and, 89product quality and process robustness, 15quantity for administration, 11

Escherichia coligenome of, 28, 290as host system, 12in Sanger sequencing, 302–303

ESI. See Electrospray ionizationEssential amino acids, 81, 108, 108tESTs . See Expressed sequence tagsEthanol, transport of, 39Euchromatin, 290Eukaryotes

chromosomes of, 290, 290fgene structure in, 286genome of, 287–288, 289t

European Union, biosimilar approval in, 10, 10tEx-Cyte, 124Exocytosis, 39–40Exons, 286Exponential phase, of cell growth, 151f, 154–155, 154fExpressed sequence tags (ESTs), 294Extended fedbatch culture, 235, 235fExtracellular fluid, 100–101, 100tExtracellular ion concentration, 21–22, 21t, 100–101, 100tExtracellular matrix (ECM), 45–46

composition of, 45–46functions of, 45–46

Extracellular matrix extract, in medium, 123

FFacilitated diffusion, 40–42F actin, 39Factor VIII

in-process structural alterations to, 16recombinant, 5tissue-derived, 5

FADD . See Fas-associated death domainFADH2, from glucose metabolism, 63, 68FANTOM. See Functional Annotation of the Mammalian genomeFas-associated death domain (FADD), 51–52Fatty acids

in lipid bilayer, 23–24in medium, 84metabolism of, 85–86oxidation of, 36saturated, 85transport of, 39, 84, 122

FBS . See Fetal bovine serum

INDEX | 315

Fedbatch culture, 233–247in bioreactors, 198, 198f, 212control objective and criteria in, 241–243control strategies for, 241–243data processing and plotting of, 160, 160fdelivery of feed medium in, 245–247feeding parameters in, 245feeding strategy for, 241–245

direct measurement of nutrient consumption, 243proportional with base addition, 244proportional with oxygen uptake rate, 245proportional with turbidity, 244

fortified feed and addition, 235, 235fhabitation-conducive components of, 104for industrial production, 125intermittent harvest and feed, 198, 198f, 234, 234flactate and production in, 59medium for, 233, 237–240

for consumed nutrients, 238–240, 239f, 239tfor unconsumed components, 240

with metabolic state manipulation, 236–237, 236f, 236tmetabolic stress in, 161, 161ton-line estimation of stoichiometric feeding in, 246–247productivity and product quality of, 241–243for stable cell lines, 130stoichiometric ratio in, 160f, 236–240, 236f, 236t, 239f, 239t,

246–247types of, 234–237

Fermentation technology, 1Fermentors, 206–207

large-scale, 264fFerrous ion, in medium, 111Fetal bovine serum (FBS), 118Fetuin, in medium, 123tFibroblast(s)

doubling time of, 154, 154thuman vs . chicken embryo, 19life span in culture, 54on microcarrier, 203fuse in bioprocessing, 19–20, 19tvs . epithelial cells, 20

Fibroblast growth factor, 48Fibronectin

in extracellular matrix, 45in medium, 123, 123t

Filopodia, 38–39, 46Filtration, 259–260FL72 cells, 8tFleming, Sir Alexander, 2Flippases, 84, 90Flooding, in bioreactors, 280Fluid

extracellular, 100–101, 100tintracellular, 153–154, 153t

Fluidized bed bioreactor, 207Flux vectors, 179–180

Folding, of proteins, 32–33, 89–91Follow-up biologics, 3, 9–10

approval in Europe, 10, 10tuncertainty about quality of, 10

Formalin, viral inactivation with, 4, 9Fortified feed and addition culture, 235, 235f454 sequencing technology, 304–305Friction factor, in scale translation, 267Fructose

in glycan biosynthesis, 93–94in medium, 108transport of, 41, 71

Fructose 1,6-bisphosphate (F16BP), 75, 75fFructose 2,6-bisphosphate (F26BP), 77Fructose-6-phosphate, 67FS-4 cells, 19tFunctional Annotation of the Mammalian genome (FANTOM),

286–287Fungus, genome of, 288Fusion proteins, 8

GG1 phase of cell cycle, 47–48, 48fG2 phase of cell cycle, 47–48, 48fG actin, 39Galactose

in glycan biosynthesis, 93–94in medium, 108transport of, 71

Gangliosides, in lipid bilayer, 22, 24GAPDH . See Glyceraldehyde dehydrogenaseGap phase of cell cycle, 47–48, 48fGas-liquid interface, oxygen transfer through, 212–219, 213f,

214fGas phase, in bioreactor, 196, 276, 278–279Gas sparging, 220–229

damage to cells by, 226–228, 226f, 227f, 228fdirect, 221orifice and bubble size in, 222–223

Gelatin, for microcarriers, 203–204, 205tGene(s), 285–287

abundant, 293, 293talternative splicing of, 285–286coding for non-protein RNA, 285–286coding for proteins, 285–286, 288coding for RNA, 285–286definition of, 285environment and, 291–293minimum set of, 287number of, 287, 289trare, 293–294, 293tstructure in eukaryotes, 286

Gene expression, 285in cell culture processing, 306–308, 306f, 307fepigenetic regulation of, 291–293proteome profiling of, 299–302, 299f

INDEX | 316

transciptome analysis of, 293–297Genentech, Vacaville (California) facility, 12Geneticin, as selection marker, 137tGene transfer

amplification in, 131–132, 138–141, 140f, 141fcell adaptation to, 142, 142f, 143fdirection to transcriptionally active region, 141methods of, 133–134, 134tpromoters for, 134–135selectable marker in, 135–137, 137tfor stable cell line, 131–145for transient expression, 128–129

Genome(s)and complexity of organism, 287environment and, 291–293eukaryotic, 28mammalian, 287–289mitochondrial, 29–30mouse, 285–287, 288, 289t, 293organization of, 287–289, 287fprokaryotic, 28, 287–288repetitive sequences in, 287–288species comparison of, 287–288, 289t

Genome engineering, 3Genome scale analysis, 293–297Genomics, 3, 285–308

cell line, 146, 306–308, 306f, 307fprotein (proteome profiling), 299–302transcriptome, 293–297

Gentamicin, in medium, 117, 117tGeometric-nonsimilar scale translation, 264Geometric-similar scale translation, 264Germanium, in medium, 112Glass, as microcarrier, 203–204, 205tGluconeogenesis, 67Glucosaminoglycans, in extracellular matrix, 45–46Glucose

in cellular growth, 149–150in fedbatch culture, 236–237, 238–240, 239f, 246–247lactate ratio to, 158–159, 236–237, 236f, 236tin medium, 106–107, 149metabolism of . See Glucose metabolismspecific consumption rate of, 156transport of, 39–43, 42f, 71–72, 71f, 72t

Glucose-6-phosphate, 64, 67Glucose metabolism, 58–77, 62f, 70f

aerobic, 65–66amino acid metabolism and, 81–82, 82fanaerobic, 58, 65ATP consumption in, 60–61ATP production in, 60–64, 66, 70carbon flow or flux in, 67carbon production in, 59, 61–67in culture, 149in electron transfer chain, 61, 63–64, 66glutamine and, 80, 80f

insulin in, 120lactate consumption in, 59, 78–80, 79flactate conversion in, 58–59, 60, 65–66, 67lipid metabolism and, 86metabolic flux analysis of, 186NADH balance in, 68–69, 69freaction intermediates in, 67regulation of, 74–78

differential allosteric, 75–76, 75fgrowth control and, 77, 77fisozymes in, 74–76, 75fsignaling pathways and, 77, 77f

transport in, 71–74Warburg effect in, 65–66yield of, 60–61, 70

Glucose oxidation, 58–64, 70fGlutamate, metabolism of, 81–82Glutamine

in cellular growth, 150in medium, 106–108, 149metabolism of, 58, 59, 67, 81–82in regulation of glucose metabolism, 80, 80f

Glutamine oxidation, 58Glutamine synthetase (GS)

for amplification, 138–141, 141ffor glutamine synthesis, 108

Glutathionereduced, in medium, 116reduction of, 64

GLUT transporter(s), 71, 72tGLUT1 transporter, 40–41, 43, 71, 72t, 107GLUT2 transporter, 72tGLUT3 transporter, 72tGLUT4 transporter, 43, 71, 72t, 77GLUT5 transporter, 41, 71, 72t, 107GLUT6 transporter, 72tGLUT7 transporter, 72tGLUT8 transporter, 72tGLUT9 transporter, 72tGLUT10 transporter, 72tGLUT11 transporter, 72tGLUT12 transporter, 72tGlycan(s)

biosynthesis of, 67, 89–96diversity among species, 95–96effect/importance of, 89–90extension in Golgi apparatus, 91–92, 91f, 93–94and immunogenicity, 95–96macroheterogeneity of, 89microheterogeneity of, 89, 92–93, 92fN-linked, 89–92, 90f, 91f, 174, 174fnucleotide sugar precursors of, 93–94, 93f, 94fO-linked, 89types of, 92–93, 92fvisualization of data on, 174, 174f

Glyceraldehyde dehydrogenase (GAPDH), 135

INDEX | 317

Glycerol, as backbone of phospholipid, 22, 22f, 24Glycine

as buffer in medium, 115tin medium, 108

Glycoforms, heterogeneity in, 89Glycolipids, in lipid bilayer, 22, 24Glycolysis, 58–70

aerobic, 65–66carbon flow and reaction intermediates in, 67in culture, 149lactate consumption in, 78–80metabolic flux analysis of, 186pentose phosphate pathway as shunt from, 60, 64–65regulation of, 74–78transport and, 71–74yield of, 60–61, 70

Glycosylation, 5, 12–14, 89–96, 90fdiversity among species, 95–96multiple sites of, 89N-linked, 89–92, 90f, 91fO-linked, 89, 90in secretion process, 33visualization of data on, 174, 174f

Glycosyltransferases, 34Glycylglycine, as buffer in medium, 115tGlyeraldehyde-3-phosphate (G3P), 61Golgi apparatus, 27, 32–34

classical view of, 32compartments of, 32dynamic nature of, 32glycan extension in, 91–92, 91f, 93–94glycosylation in, 90protein secretion through, 32–35, 35f, 83transport across, 33–34

Green monkey kidney cells, 8t, 9, 19t, 129, 201Growth

autocatalytic, 156balance equations for, 162–165cell death consideration in, 157doubling time of, 154–157, 154tkinetic model of, 162–165mammalian cell, 154–155, 154f, 154tmaterial balance on, 149–154, 149f, 162–165medium for . See Mediummultiplicative model of, 165overall synthesis equation for, 149–150phases of, 151f, 154–155, 154fquantitative description of, 156–161

Growth controlapoptosis in, 51–53cell cycle and, 47–49contact inhibition in, 46, 48, 53, 54fHayflick’s phenomenon and, 54loss, in continuous cell lines, 49in regulation of glucose metabolism, 77, 77ftelomeres and, 54–55, 54f

Growth curve, 154–155, 154fGrowth factors

in cell cycle, 48, 49in cell migration, 46–47in medium, 97–98, 117, 118

Growth hormone . See Human growth hormoneGrowth rate, 53–55, 53f, 156–157GS . See Glutamine synthetaseGTP-mannose, 67Guanosine, in medium, 109t

HHabitation-conducive components, of medium, 103–104Hamster cells, 8t, 9, 19t, 20 . See also BHK cells; Chinese hamster

ovary cellsHayflick’s phenomenon, 54Heavy metal ions, in medium, 112HEK 293 cells, 8t, 19t

adhesion of, 142aggregates of, 209, 209ffor transient expression, 129

Helicos sequencing technology, 302tHenry’s law, 217–218Heparin

in extracellular matrix, 46in medium, 123

Hepatocyte(s)cell membrane of, 25, 25tendoplasmic reticulum of, 31size of, 21, 151

Hepatocyte growth factor, 46–47HEPES buffer, 115, 115tHepG2 cells, on microcarrier, 204, 204fHeterochromatin, 290Heterogeneous bioreactor, 196, 196fHexokinase (HK), in regulation of glucose metabolism, 75hGH . See Human growth hormoneHigh-mannose glycans, 92, 92fHigh-molecular-weight supplements, in medium, 117–125High-performance liquid chromatography (HPLC), for fed-batch

culture, 243High throughput technology

for cell line production, 145–146for DNA microarrays, 296–297for DNA sequencing, 302–305for proteome profiling, 299–302, 299f

Histone, 28, 290, 290fHistone modification, 292–293HMG-CoA, 87–88, 88fHMG-CoA reductase (HMGCR), 87–88, 88fHMG-CoA synthase (HMGCS), 87–88, 88fHolding time, in secretion, 32, 34Hollow fiber bioreactor, 208, 208f, 229Homeostasis, of cell membranes, 26Hormone(s)

in interstitial fluid, 100

INDEX | 318

in serum, 118Host systems, 5–6, 12–14, 127–129. See also specific systemsHot spot integration, 141HPLC, for fedbatch culture, 243Human growth hormone (hGH)

biosimilar, 9–10quantity for administration, 11

Humira, expiration of patent, 9Hyaluronic acid, in extracellular matrix, 46Hybrid glycans, 92Hybridoma, 5

adhesion of, 142gene expression in, 306, 306fgrowth of, 149stoichiometric ratio and metabolic stress in, 161, 161ttherapeutic antibody products from, 7tvolume of, 150t

Hydrogenin cellular growth, 149–150transport of, 43–45

Hydrogen peroxide, 116Hydrolysates, in medium, 124, 240Hydrostatic pressure, and oxygen transfer, 225Hydroxylethyl starch (HES), in medium, 116tHygromycin B, as selection marker, 137tHyper-producing cell line, 129–133 . See also Stable cell lines

basic steps for generating, 131–133, 132fcharacteristics of, 129–131, 130t, 131fgene expression in, 306–308

IIEF . See Isoelectric focusing (IEF)IGF . See Insulin-like growth factor(s)IgG . See Immunoglobuin GIllumina sequencing technology, 302t, 304–305Immobilization reactors

agarose, 209oxygen transfer in, 229, 229f

Immortalization, 54Immunogenicity, glycans and, 95–96Immunoglobuin G (IgG)

in cellular growth, 151, 151tFc fragment, in fusion protein, 8secretion time of, 34f, 34t

Impellers, in bioreactors, 206, 265–268, 265t, 266famount of fluid moved by (pumping), 268–270constant tip speed, for scale translation, 269–270, 269tvelocity of, 268

Inactivated vaccines, 4, 9Incline settling, 255, 255fIndia, biosimilars marketed in, 10tInducible promoters, 134Industrial cell lines, 8t, 9Industrial production

bioreactors for, 192–193 . See also Bioreactorsmedium for, 125–126

scale translation for, 263–284. See also Scale translation; Scaling up

In-process calculations, 169Insect cell culture, 12–13, 13tInsulin

in cell cycle, 48, 49in glucose metabolism, 120in glucose transport, 43, 71in medium, 120–121, 240mitogenic response to, 120recombinant, 121as tissue-derived protein therapeutic, 5

Insulin-like growth factor(s), 48Insulin-like growth factor-1, in medium, 120–121Insulin-like growth factor-2, in medium, 120Integral cell concentration, 159, 159fIntegrated data, calculated, 169Interactive data exploration, 172Interferon(s), as tissue-derived protein therapeutic, 5Intermediate(s), of reaction modes, abbreviation of, 189tIntermediate filaments, 37, 38, 38fIntermittent harvest and feed, 198, 198f, 234, 234fInterstitial fluid, 100–101, 100tIn-time measurement, for fedbatch culture, 243Intracellular fluid, 153–154, 153tIntrons, 286, 287, 288Ion(s)

bulk, in medium, 111–112, 111tintracellular vs. extracellular concentration of, 21–22, 21t,

100–101, 100ttransport of, 43–45

Ion channels, as transport mechanism, 40–42, 41fIron

free vs . bound form of, 45in intracellular fluid, 153–154in medium, 111, 112reactivity of, 45transport of, 45, 122t . See also Transferrin

Iron chelators, 121, 121tIsobaric Tagging for Relative and Absolute Protein Quantitation

(iTRAQ), 300–301, 300f, 301fIsoelectric focusing (IEF), 299Isoleucine, metabolism of, 81–82Isozymes, in glucose metabolism regulation, 74–76, 75fiTRAQ, 300–301, 300f, 301f

JJenner, Edward, 199

KKaryotype, variable, in cell lines, 143–144a-Ketoglutarate

in amino acid metabolism, 81in glucose metabolism, 61, 68, 80in medium, 108

Kinetics

INDEX | 319

in bioreactors, 194–196of cell cultivation, 147–166model of growth, 162–165

KL. See Mass transfer coefficientKrebs cycle . See Tricarboxylic acid cycle

LLactate

accumulation in culture, 78, 236in fedbatch culture, 236–237, 246–247metabolism of, 58–59, 65–66, 78–80

consumption in glucose metabolism, 59, 78–80, 79fcorrelation with productivity, 58f, 59production in glucose metabolism, 58–59, 60, 65–66, 67

specific production rate of, 156transport of, 41–42, 42f, 43, 72–73, 72f

Lactate dehydrogenase, 66, 67, 79Lactate-to-glucose ratio, 158–159, 236–237, 236f, 236tLag phase, of cell growth, 154, 154fLamellipodia, 38–39, 46Laminins

in extracellular matrix, 45in medium, 123, 123t

Large scale, translation for. See Scale translation; Scaling upLarge-scale fermenter, 264fLDL . See Low-density lipoproteinLehmann, Jürgen, 210Length, in scale translation, 264Leucine, metabolism of, 81–82Lipid(s)

amphipathic, 22–23catabolism of, 36in cell membrane, 22–27as cellular component, 21functions of, 83in medium, 84, 125, 240metabolism of, 83–88

acetyl CoA shuttle in, 86, 86fsubcellular localization of, 83

transport of, 84Lipid bilayer, 22–27, 83, 87

characteristics of, 23composition of, 22–25dynamic nature of, 23, 26function of, 25permeability of, 23, 39phase transition of, 23, 23f, 24transport across, 23, 26–27, 39–47turnover rate of, 26

Lipofection/lipid-mediated gene transfer, 133–134, 134tLipoprotein(s)

in medium, 105–106transport of, 84

Liquid chromatography, 2D, 300–302Liquid chromatography/mass spectroscopy, 300Liquid phase, in bioreactor, 196, 277–279

Live attenuated vaccines, 4–5Liver, endoplasmic reticulum of, 31Localized amplification, in DNA sequencing, 304, 304fLogging data . See also Data processing

standardized templates for, 168–169Low-density lipoprotein (LDL)

in medium, 105–106transport of, 84

Lymphoid cells, use in bioprocessing, 19–20, 19tLysis, 157Lysosome, 36

MMA104 cells, 8tMacroheterogeneity, of glcyans, 89Macromolecules

catalytic, in medium, 105–106transport of, 39–40

Macroporous microcarriers, 203, 204, 204f, 205t, 229Madin-Darby bovine kidney (MDBK) cells, 8tMadin-Darby canine kidney (MDCK) cells, 8t, 9, 19tMagnesium ions

in fedbatch culture, 240intracellular and extracellular concentrations of, 21, 100,

100t, 153, 153tin medium, 111, 111t

Malate-aspartate shuttle, 68–69, 186MALDI. See Matrix-assisted laser desorption ionizationMammalian cells. See also specific cells

bioreactors for, 192–193 . See also Bioreactorscritical feature of rDNA proteins from, 14fragility of cells in, 22genome of, 28growth of, 154–155, 154f, 154tlimitations of, 14product quality and process robustness in, 14–17products from, 5–6, 6ttransgenic animals in, 12, 14, 14t

Mammalian genome, 287–288, 289tManganese, in medium, 112Mannose, in glycan biosynthesis, 90–94Manufacturing plants, 12Manufacturing processes, 11–12, 12f. See also specific processes

and productsMass . See BiomassMass spectrometry, in proteome profiling, 299–302Mass transfer coefficient (KL)

for carbon dioxide, 282for oxygen, 216

constant, in scale translation, 269–270, 269texperimental measurement of, 224–225, 224fscaling up and, 277sparger orifice and bubble size and, 222–223

Material balanceon cell growth, 149–154, 149f, 162–165in fedbatch culture, 236–237, 236f, 236t, 238–240, 239f, 239t,

INDEX | 320

246–247on oxygen, in bioreactors, 220, 276–280on perfusion culture, 251–253, 252f, 253ffor reaction systems, 175–180. See also Metabolic flux

analysissetting up equations, 178–179systematic way to solve problems, 178

Mathematical modelof growth, 162–165information needed for developing, 163Monod and Monod derivatives, 163–165, 164fpurposes of, 162

Matrigel, in medium, 123Matrix-assisted laser desorption ionization (MALDI), 299Matrix operations, 179–180MDBK cells, 8tMDCK cells, 8t, 9, 19–20, 19tMDR. See Multidrug resistance geneMeasurement data, 169Mechanical/acoustic trapping, 257–258, 258fMechanical damage protective agents, in medium, 116–117,

116tMedial Golgi, 32, 33, 35fMedium

amino acids in, 81, 106–108, 238–240, 246–247animal component-free, 102–103antibiotics in, 117, 117tbasal, 101, 106–117buffer systems in, 113–115, 115f, 115tbulk ions in, 111–112, 111tcarrier proteins in, 117, 118, 122cell adhesion molecules in, 122–123, 123tfor cell expansion, 97–99, 125chemically defined, 102, 103classical, composition of, 58complex vs. chemically defined, 102components of

basic, 103–117classes of, 103–106non-nutritional, 113stochiometric vs. catalytic macromolecular, 105–106stochiometric vs. habitation-conducive, 103–104

design for, 97–126for fedbatch culture, 233, 237–240

for consumed nutrients, 238–240, 239f, 239tfor unconsumed components, 240

fundamental influence of, 97high-molecular-weight and complex supplements in, 117–125for industrial production, 125–126insulin and insulin-like growth factors in, 120–121, 240lipids in, 84, 125, 240nucleosides in, 109objectives of, 98optimal concentration of organic nutrients in, 110, 110foptimization of cell growth environment in, 97–101osmolarity of, 111–112

for production, 97–99, 125–126protective agents in, 116–117, 116tprotein-free, 103protein hydrolysates in, 124, 240serum albumin in, 118, 122serum-free, 102–103, 108, 109serum in, 102, 117–119, 240for stem cells, 97–99, 117sugars and energy source in, 106–107tolerance of deviation from optimum, 100–101, 101ttrace elements in, 111–112, 112ttransferrin in, 105, 116, 118, 121, 240types of, 101–103vitamins in, 109, 240water in, 106

Membrane, cell . See Cell membraneMembrane bioreactor, 208–209Membrane fusion, 39–40Membrane potentials, 25–26, 25t, 29–30, 30f, 43–45Membrane stirred tank, 210, 210fMercaptoethanol, in medium, 116Messenger RNA (mRNA), 21, 286

abundant, intermediate, and rare, 293–294, 293tin RNA-seq, 296–297

Metabolic flux analysis (MFA), 173, 173f, 175–187abbreviation of intermediates in, 189tbiomass equations in, 184–185, 185fcatabolism reactions for, 188ton cellular system, 181–186compartmentalization in, 184example of, 186general approach to, 182f, 183–185selecting reactions for, 183, 183fsolution and analysis in, 185utility and limitations of, 181–182

Metabolic state, of culture, 158, 236–237, 236f, 236tMetabolic stress, stoichiometric ratio as indicator of, 161, 161tMetabolism. See also specific types

amino acid, 58, 81–82, 81f, 82fcentral, in cultured cells, 58–59glucose, 58–70lactate, 58–59, 65–66, 78–80lipid, 83–88transport and transporters in, 71–74

Methane combustion, 176–178Methionine sulphoximine (MSX), in glutathione synthetase

amplification, 138–141Methotrexate (MTX), in DHFR amplification, 138–141, 140fMethylation

DNA, 292–293histone, 292

Methylcelluloses (MC), in medium, 116tMFA. See Metabolic flux analysisMicelles, 22–23Michaelis–Menton enzyme kinetics, 41Microarray analysis . See DNA microarrays

INDEX | 321

Microcarriers, 203–204, 205tcharacteristics of, 204tmacroporous, 203, 204, 204f, 205t, 229microporous, 203–204, 203f, 205tsolid, 203–204, 203f

Microencapsulation, 210, 210fMicrofiltration, 259–260, 260fMicroheterogeneity, of glcyans, 89, 92–93, 92fMicroporous microcarriers, 203–204, 203f, 205tMicrosphere-induced cell aggregates, 209, 209fMicrotubules, 37, 37f, 46Minerals, in medium, 111–112Minimum set of genes, 287Mitochondria, 29–30

acetyl CoA shuttle in, 86as cell’s power plant, 29genome of, 29–30lipid metabolism in, 83, 85, 86, 88fmembrane potential of, 29–30, 30fpH of, 29–30protein content of, 25size of, 29transport across, 73–74

Mitochondrial apoptotic pathway, 52–53, 53fMitochondrial DNA, 29–30Mitogenic factors, 48, 49Mitosis, 47–48, 48fMixing time, in bioreactors, 206, 268–274

distribution of, 273–274, 273f, 274fmeasurement of, 272, 272fin scale translation, 269–274vs. starvation time, 272

Molybdenum, in medium, 112Monkey cells, 4, 8t, 9, 19t, 129, 201Monocarboxylate transporter (MCT), 42, 43, 72–73, 72fMonod-derivative models, 164–165Monod models, 164–165, 164fMonolayer, 53MOPS buffer, 115tMouse cells, 8t, 9, 19t, 49 . See also NSO cells; SP2/0 cells

embryonic stem, doubling time of, 154tgene expression in, 306, 306f

Mouse genome, 285–287, 288, 289t, 293Movement, cellular, 46–47M phase of cell cycle, 47–48, 48fMRC-5 cells, 19tMRCS cells, 8tmRNA . See Messenger RNAMSX, in glutathione synthetase amplification, 138–141MTX, in DHFR amplification, 138–141, 140fMultidimensional data exploration, 172Multidrug resistance (MDR) gene, 137Multiple membrane plate bioreactor, 208–209Multiple plate system, 199, 201, 201f, 212Multiplicative model, of growth, 165Myc, in regulation of glucose metabolism, 77, 77f

Myelin membrane, protein content of, 25Myeloma cells, 20 . See also NSO cells

as continuous cell line, 49for stable expression, 129–130, 130t, 146stoichiometric ratio and metabolic stress in, 161, 161t

NNADH

balance of, 68–69, 69fcarrier system for, 68–69in electron transfer chain, 63–64, 66in glycolysis, 60–64, 66, 68–69, 69fin lactate metabolism, 79–80in lipid metabolism, 86in tricarboxylic acid cycle, 61–63, 68–69, 69f

NADPHin lipid metabolism, 85in pentose phosphate pathway, 64–65

Necrosis, 51Neomycin

in gene transfer, 139–140in medium, 117

Next generation sequencing technologies, 304–305Nickel, in medium, 112NimbleGen microarrays, 295–296, 295tNitrogen

in cellular growth, 150in cultured cell metabolism, 67metabolic flux analysis of, 182f, 183

N-linked glycosylation, 89–92, 90f, 91f, 174, 174fNon-essential amino acids, 81, 108, 108tNon-nutritional components, of medium, 113NSO cells, 8t, 19t, 20

as continuous cell line, 49doubling time of, 154tgene expression in, 307ffor stable expression, 130ttherapeutic antibody products from, 7t

Nuclear envelope, 29Nuclear membrane, 29Nuclear pores, 29Nucleoid, 28Nucleoside(s), in medium, 109Nucleosomes, 290Nucleotides, precursors of glycans, 93–94, 93f, 94fNucleus, 27–29, 28fNunc Cell Factories, 201Nutrient consumption curve, 154–155Nutrient consumption rate, specific, 156–157Nutrients, transport of, 43Nutrient starving time, 271–272, 271tNystatin, in medium, 117t

OOAA . See OxaloacetateOff-line data, 160, 160f

INDEX | 322

Oligonucleotide-based microarrays (oligoDNA), 295–296, 298tOligopeptides, transport of, 43O-linked glycosylation, 89Omnitrope, 9–10On-line data, 160, 160fOn-line measurement, for fedbatch culture, 243Organelle(s), 23–24, 27–36, 28f. See also specific organellesOrganic nutrients, in medium, optimal concentration of, 110,

110fOsmolarity

of interstitial fluid, 100of medium, 111–112, 125

OTR . See Oxygen transfer rateOUR . See Oxygen uptake rateOxaloacetate (OAA), in glucose metabolism, 61, 68Oxidative phosphorylation pathway, 61Oxygen

cellular demand for, 219concentration in medium, 217–218consumption of, 219dissolved concentration of, 214optimal concentration of, 219transport of, 39

Oxygen balance, in reactor, 220, 276–280on gas phase, 276, 278–279on liquid phase, 277–279

Oxygen starvation time, 271–272, 271tOxygen supply, for bioreactors, 220–228

by medium recirculation, 221fby silicon tubing/membrane, 220by sparging, 220–228

damage to cells by, 226–228, 226f, 227f, 228fdirect, 221orifice and bubble size in, 222–223

by surface aeration, 220Oxygen transfer, in bioreactors, 206–207, 213–231

driving force for, 214–216, 220, 277, 279–280enhancing or improving, 216, 220, 221experimental measurement of, 224–225, 224f, 225fhydrostatic pressure and, 225in immobilization reactor, 229, 229fmass transfer coefficient in, 216objective of, 219in plug-flow reactors, 229–230, 230frate of, 215–216, 220scaling up and, 275–280, 280f, 280tsurface/interfacial area and, 216through gas-liquid interface, 212–219, 213f, 214f

Oxygen transfer rate (OTR), 215–216, 220balance with oxygen uptake rate, 220, 276–280scaling up and, 276–280

Oxygen uptake rate (OUR), 160, 160f, 219, 220, 224–225, 225fbalance with oxygen transfer rate, 220, 276–280in fedbatch culture, 245–247scaling up and, 276–280

Pp53 tumor suppressor, in regulation of glucose metabolism, 77Pancreas, beta cells of, 31Passage, 53–55PAT. See Process analytical technologyPatents, expiration of, 9–10Pathway-related data, 173, 173f. See also Metabolic flux analysisPCR. See Polymerase chain reactionPDI. See Protein disulfide isomerasePDQuest, 299Penicillin(s)

declining price of, 2–3development and production of, 2–4, 2fdiscovery of, 2

Penicillin Gin medium, 117tproduction outside U.S., 2–3

Pentose phosphate pathway (PPP), 60, 62f, 64–65carbon flow or flux in, 67in culture, 149molecular transformation in, 64–65oxidative segment of, 64

Peptides, in medium, 108, 123tPeptone, in medium, 124Perfusion culture, 249–261

analysis of, 251–254cell culture products from, 249tcell retention in, 251, 254–261external vs . internal recovery device in, 252material balance on, 251–253, 252f, 253frecycling factor in, 254, 254f

Permeability, of lipid bilayer, 23, 39Permeases, 39Peroxisomes, 27, 36

lipid metabolism in, 83, 85, 87, 88fPFK . See PhosphofructokinasePFKFB . See Phosphofructokinase/fructose biphosphatePFR. See Plug-flow reactorpH

of bioreactors, scale translation and, 283, 283fof fedbatch culture, 244of medium, 113–115of mitochondria, 29–30

Phenotype, epigentic changes in, 291–293Phosphate

in fedbatch culture, 240intracellular and extracellular concentrations of, 100, 100t,

153, 153tin medium, 104, 111, 111ttransport of, 43–45

Phosphatidyl choline, in medium, 125Phosphatidyl ethanolamine, in medium, 125Phosphofructokinase (PFK), in regulation of glucose metabolism,

75, 75fPhosphofructokinase/fructose biphosphate (PFKFB), in

regulation of glucose metabolism, 75–76, 75f

INDEX | 323

Phospholipidsin cell membrane, 22–27turnover rate of, 26types of, 22, 22f

Phosphorylationof histones, 292–293, 292tin protein therapeutics, 14, 16

Photolithographic synthesis, in microarray analysis, 295, 295t, 296f

Physiological state, of culture, 158Pichia (yeast) cell culture, 12–13, 13tPinocytosis, 39–40Piston-flow reactor. See Plug-flow reactorPK . See Pyruvate kinasePK cells, 8tPlants, transgenic, 12Plasma cells, 20, 33, 33f, 130, 151Plasma membrane . See Cell membranePlasmid(s) . See also Gene transfer

basic elements on, 134–137, 134ffree, 135method of delivery, 133–134, 134tselectable marker for, 135–137, 137tfor stable cell line, 131–137for transient expression, 128–129

Plastic, for microcarriers, 204, 205tPlug-flow reactor (PFR), 193–196, 193f

oxygen transfer in, 229–230, 230freaction and reaction kinetics in, 194–195tracer concentration response in, 193–195, 194f

Pluronic F68, in medium, 116–117, 116t, 123Pluronic F77, in medium, 117Pluronic F88, in medium, 116t, 117PolyA tail, 286Poly-d-lysine, in medium, 123tPolyethylene glycol (PEG), in medium, 116tPoly-L-lysine, in medium, 123Polymerase chain reaction (PCR), 304Polymyxin B, in medium, 117Polypropylene microcarriers, 205tPolystyrene, for microcarriers, 203–204, 205tPolyvinyl alcohol (PVA), in medium, 116tPolyvinylpyrrolidone (PVP), in medium, 116tPost-process calculations, 169Post-translational modification

analysis of, 299in endoplasmic reticulum, 31in protein therapeutics, 5, 12–17

Potassium chloride, in medium, 111–112Potassium ions

intracellular and extracellular concentrations of, 21–22, 100–101, 100t, 153, 153t

in medium, 111–112, 111ttransport of, 43–45

PPP . See Pentose phosphate pathwaypRB. See Retinoblastoma protein

Process analytical technology (PAT), 167–168Product accumulation rate, 156, 159Product concentration profile, 154–155Product formation. See also specific products

in fedbatch culture, 241–243kinetic model of, 162–165lactate metabolism and, 58f, 59quantitative description of, 156–161specific rate of, 156–157, 159, 165–166

Programmed cell death . See ApoptosisProline, in medium, 108Promoter

for gene transfer, 134–135in mammalian genes, 286, 288for transient expression, 128–129

Pro-oncogenic genes, in regulation of glucose metabolism, 77, 77f

Proportional feedingwith base addition, 244with oxygen uptake rate, 245with turbidity, 244

Protease inhibitors, in serum, 118Proteases, for dissociation, 53Proteasome, 36Protective agents, in medium, 116–117, 116tProtein(s)

carrierin medium, 117, 118, 122transport by, 122, 122t

in cell membrane, 25in cellular growth, 151in cytoplasm, 27, 27tin cytoskeleton, 37in extracellular matrix, 45–46folding of, 32–33, 89–91gene expression in, 299–302genes coding for, 285–286, 288in interstitial fluid, 100secretion of

and cell membrane, 26in endoplasmic reticulum, 31–35, 35f, 83in Golgi apparatus, 32–35, 35f, 83time of, 32, 34, 34f, 34t, 35, 35f

Protein C, in-process structural alterations to, 16Protein disulfide isomerase (PDI), 33Protein-free medium, 103Protein hydrolysates, in medium, 124, 240Protein molecules, as therapeutics, 7–8. See also Protein

therapeuticsProtein therapeutics, 4–8

alternative technologies for, 12–14biosimilar or follow-up biologics, 9–10g-carboxylation in, 12, 14, 16disulfide-bond formation in, 5, 14fedbatch culture for, 233, 235f . See also Fedbatch culturefrom fusion proteins, 8

INDEX | 324

glycosylation in, 5, 12–16, 89–96, 90fgrowth and advances in, 1host cells for, 127–129immunogenicity of, 95–96industrial cell lines for, 8t, 9in-process structural alterations to, 15–17, 16tinstability in production of, 144from mammalian cells, 6tmanufacturing of, 11–12from non-mammalian host, 6tphosphorylation in, 14, 16post-translational modification of, 5, 12–17product quality and process robustness for, 14–17quantity for administration, 11recombinant technology for, 5stable cell lines for, 127, 129–146stoichiometric ratio in, 11tissue-derived, 5transient expression of, 127–129

Proteoglycans, in extracellular matrix, 45–46Proteome profiling, 299–302, 299f

iTRAQ labeling in, 300–301, 300f, 301fSILAC labeling in, 300–302, 301f2D liquid chromatography in, 300–302

Proton pumps, 36Pseudogenes, 286Pulsatile flow, in microfiltration, 260, 260fPumping, in bioreactor, 268–270Purines, in medium, 109, 109tPuromycin, as selection marker, 137tPyridoxine, in medium, 109Pyruvate

in amino acid metabolism, 81as controlling node, 67in glycolysis, 60, 61, 66, 67, 70lactate conversion to, 79–80in lipid metabolism, 86in medium, 107NADH balance and, 68–69, 69ftransport of, 43, 72–73, 72fin tricarboxylic acid cycle, 60, 61

Pyruvate kinase (PK), in regulation of glucose metabolism, 75–76

QQuality, of cell culture products, 14–17Quality by Design, 162Quantitative description, of growth, 156–161Quasi-steady state, 179

RRadial flow impellers, 206, 265–266, 265t, 266fRare genes, 293–294, 293tRate-limiting enzymes, 74Rate-limiting nutrient, 165Reaction systems

cellular vs . chemical, 175–176

material balance for, 175–180Reactive oxygen species (ROS)

glutathione and, 64, 116in medium, 116

Reactor state parameters, 160fRead, in DNA sequencing, 297Recessive marker, 136Recombinant technology, 5, 192. See also specific applications

and productsRecovery process, 11–12Recycling factor, in perfusion culture, 249–261, 254, 254fRemicade, expiration of patent, 9Repetitive sequences, in genome, 287–288Reporter gene, 136Respiratory quotient (RQ), 219, 281Retention, cell

methods of, 254–261in perfusion culture, 249–261

Retinoblastoma protein (pRB), 48–49Retrograde transport, 34, 35, 35fReynolds number, 267–268, 267fRGD peptide, in medium, 123tRiboflavin, in medium, 109Ribonucleic acid . See RNARibose

in medium, 107synthesis of, 67

Ribosomal RNA (rRNA), 21, 27removal, in microarray analysis, 296–297

Ribosomes, 27, 31Rice hydrolysate, 124RNA

abundant, intermediate, and rare, 293–294, 293tas cellular component, 21genes coding for, 285–286location in cell, 28non-protein coding, 285–286synthesis of, 28

RNA-seq, 296–297, 298tRobustness, of cell culture processes, 14–17Roche sequencer, 302tRoller bottles, 199–201, 200f, 212ROS. See Reactive oxygen speciesRotational cage, 258, 258fRough endoplasmic reticulum, 31RQ. See Respiratory quotientrRNA . See Ribosomal RNARubidium, in medium, 112Rushton impellers, 206, 265–266, 265t, 266f

SSacchromyces cerevisiae systems, 12Saturated fatty acids, 85Scale translation

and carbon dioxide removal, 275, 281–283and chemical environment, 283, 283f

INDEX | 325

constant parameters in, 269–270, 269tdimensionless variables in, 267–270and driving force, 279–280effect of scale on physical behavior, 269–270geometric nonsimilarity in, 264geometric similarity in, 264major effects of scale, 264and mechanical forces on cells, 274–275, 275fmixing time in, 271–274, 271tnutrient starvation time in, 271–274, 271tobjective of, 264and oxygen transfer, 275–280, 280f, 280tphysical and mechanical parameters of, 264Reynolds number in, 267–268, 267fand superficial gas velocity, 278, 278f

Scaling down, 263–264. See also Scale translationScaling up

and carbon dioxide removal, 275, 281–283and driving force, 279–280and mechanical forces on cells, 274–275, 275fand oxygen transfer, 275–280, 280f, 280tand superficial gas velocity, 278, 278f

Secretion time, 32, 34, 34f, 34t, 35, 35fSedimentation, 254–255, 254fSelectable marker, in gene transfer, 135–137, 137tSelenate, in medium, 111Selenium

in intracellular fluid, 153in medium, 112, 116

Senescence, 53–55Separation methods, 254–261Sequencing, DNA . See DNA sequencingSerine

as backbone of phospholipid, 22, 24in medium, 108

Serumdisadvantages of use, 119in medium, 102, 117–119, 240

Serum-free medium, 102–103, 108, 109Settling cyclone, 254–255, 254fSGLT transporters, 71–72Shotgun liquid chromatography, 300Sialic acid, in glycan biosynthesis, 93–94Signaling, cellular, 45–46Signaling pathways, in regulation of glucose metabolism, 77, 77fSignal recognition particles (SRPs), 32–33, 35, 35fSILAC, 300–302, 301fSilicon tubing/membrane, for oxygen supply, 220Simple stirred tank bioreactor, 206–207Single-cell cloning, 131–133Single molecule detection, in DNA sequencing, 304Single-use bioreactor, 199Smooth endoplasmic reticulum, 31Sodium beta-glycero-phosphate buffer, 115Sodium bicarbonate buffer, 113–114, 115f, 115tSodium butyrate, 293

Sodium chloride, in medium, 111–112Sodium/glucose transporter, 42Sodium ions, 21–22, 21t

intracellular and extracellular concentrations of, 21–22, 21t, 100–101, 101t, 153, 153t

in medium, 104, 111–112, 111ttransport of, 43–45

Sodium/potassium ATPase transporter, 44–45, 44fSoftware, for data visualization, 172Solid microcarriers, 203–204, 203fSolid phase, in bioreactor, 196SOLiD sequencing technology, 302tSolutes, cellular transport of, 40Soybean hydrolysate, 124SP2/0 cells, 19t, 49

for stable expression, 130ttherapeutic antibody products from, 7t

Sparging, 220–228damage to cells by, 226–228, 226f, 227f, 228fdirect, 221orifice and bubble size in, 222–223

Specific death rate, 157Specific growth rate, 156–157Specific nutrient consumption rate, 156–157Specific product formation rate, 156–157, 159, 165–166Specific rate, 156–157, 169Spectinomycin, in medium, 117tS phase of cell cycle, 47–48, 48fSphingomyelin, in medium, 125Spin filter separation, 258, 258fSpin filter stirred tank, 210–211, 211fSpreadsheets, 168, 170–171SRPs. See Signal recognition particlesStable cell lines, 127, 129–146

adaptation of cells in, 131, 142, 142f, 143famplification for, 131–132, 138–141, 141fautomation and high throughput technology for, 145–146,

145fbasic steps for generating, 131–133, 132fgene expression in, 306–308genomics and, 146screening for, 131selection of cells for, 131–132single-cell cloning for, 131–132stability of clones selected for, 143–145stability of product quality in, 144–145transfection for, 131

Stable Isotope Labeling by Amino Acid in Cell Culture (SILAC), 300–302, 301f

Standardized templates, for data logging and processing, 168–169

Starvation time, in bioreactors, 271–272, 271tState of cultures, 158Stationary phase, of cell growth, 151f, 154–155, 154fST cells, 8tStem cell(s), 4

INDEX | 326

differentiation in culture, 143medium for, 97–99, 117mouse embryonic, doubling time of, 154tsize of, 21, 151

Stirred tank bioreactor, 193, 193fagitation in, 265–266

mechanism of, 265–266purpose of, 265

continuous, 193–196, 193f, 212reaction and reaction kinetics in, 194–196tracer concentration response in, 193–195, 194f

heterogeneous, 196, 196fimpellers in, 206, 265–268, 265t, 266fmembrane, 210, 210fmixing time in, 206, 268–274power consumption in, 266–270, 266fsimple, 206–207spin filter, 210–211, 211fvelocity of, 268volumetric flow rate in, 268–270well-mixed, 193–196

Stock solutions, amino acid, 108Stoichiometric components, of medium

vs. catalytic macromolecular components, 105–106vs. habitation-conducive components, 103–104

Stoichiometric-limiting nutrient, 165Stoichiometric matrix, 179–180Stoichiometric ratio, 11, 156, 158–159

in data processing, 169, 171, 171fin fedbatch culture, 160f, 236–240, 236f, 236t, 239f, 239t,

246–247as indicator of metabolic stress, 161, 161tof lactate to glucose, 158, 236–237, 236f, 236tof product to substrate, 158

Stoichiometry, of cell cultivation, 147–166Streptomycin, in medium, 117Stress, metabolic, stoichiometric ratio as indicator of, 161, 161tSubstrate

stoichiometric ratio of product to, 158yield of biomass on, 158yield of product on, 158

Sugars, in medium, 106–107Superoxide dismutase, in medium, 116Superoxide radical, 116Support systems, cellular, 202–206Surface aeration, 220Surface area, and scale translation, 264Surfactants, in medium, 116–117Suspension culture, 202, 212SV40 late promoter, 135Symporters, 41–42, 42fSyrian hamster cells (BHK), 8t, 9, 19t

adhesion of, 142aggregates of, 209as continuous cell line, 49for transient expression, 129

veterinarian vaccines from, 8t

TTangential flow, in microfiltration, 260, 260fTaurine, in medium, 116TCA . See Tricarboxylic acid cycleTelomerase, 54Telomeres, 54–55, 54fTemperature, and lipid bilayer, 24T-flasks, 199Thiamine pyrophosphate, in medium, 1093T3 cells, 54Thymidine, in medium, 109, 109tTIGAR, 77Tin, in medium, 112Tissue culture systems, 199–202Tissue plasminogen activator (tPA)

development of, 5in-process structural alterations to, 16product quality and process robustness, 15–16quantity for administration, 11

Titers, increases in, 2–3, 2fTNFa . See Tumor necrosis factor a (TNFa)tPA. See Tissue plasminogen activatorTrace elements, in medium, 111–112, 112tTranscription, 28Transcription factors, 28, 291Transcriptome analysis, 293–297

in cell culture processing, 306–308, 306f, 307fDNA microarrays for, 295–297, 295t, 298t

Transfection. See also Gene transferfor stable cell line, 131–137

Transferriniron chelators as alternative to, 121, 121tiron transport by, 45, 122tin medium, 105, 116, 118, 121, 240recombinant, 121secretion time of, 34, 34t

Transgenic animals, 12, 14, 14tTransgenic plants, 12Trans-Golgi network (TGN), 32, 33, 35, 35fTransient expression, 127–129

host cells for, 129production life of system, 129

Translation of scale. See Scale translation; Scaling down; Scaling up

Transportclasses of processes, 40mechanisms of, 39–47. See also specific mechanismsin metabolism, 71–74nutrient, 43

Transporters, 40–42. See also specific typesTricarboxylic acid cycle (TCA), 60–64, 62f

amino acid metabolism and, 81–82, 82fcarbon flow or flux in, 67in culture, 149

INDEX | 327

glutamine and, 80, 80flipid metabolism and, 86NADH balance in, 68–69, 69fregulation of, 74–78transport and, 71–74

TrichostabinA, 293TRICINE buffer, 115tTrypsin

for dissociation, 53, 212for proteolysis, 300

Tubular bioreactors, 193–196, 193foxygen transfer in, 229–230, 230freaction and reaction kinetics in, 194–196tracer concentration response in, 193–195, 194f

Tumor necrosis factor a (TNFa), in fusion protein, 8Turbidity, proportional feeding with, 2442D liquid chromatography, 300–302

UUbiquitin, 36Ubiquitination of histones, 292–293, 292tUDP-galactose, 67UDP-glucose, 67Unfolded protein response (UPR), 33Uniporters, 41, 42f, 71Untranslated regions (UTRs), 286, 288UPR . See Unfolded protein responseUridine, in medium, 109tUrokinase, as tissue-derived protein therapeutic, 5UTRs . See Untranslated regions

VVaccine(s)

veterinary, 8t, 9viral . See Viral vaccines

Vanadium, in medium, 112Vector . See Gene transferVelocity, in bioreactors, 268, 278, 278fVero cells, 8t, 9, 19t, 201Vesicle diffusion model, 33Veterinary vaccines, 8t, 9Vibromixer, 212, 212fViral vaccines, 4–5

cell sources for, 20inactivated, 4, 9industrial cell lines for, 8t, 9live attenuated, 4–5manufacturing of, 11–12principal, in prevention of human disease, 5tquantity for administration, 11

Viscosity, of medium, 116–117Vitamin(s), in medium, 109, 240Vitamin A, in medium, 109Vitamin B6, in medium, 109Vitamin B12, in medium, 109Vitamin C, in medium, 109

Vitamin D, in medium, 109Vitamin E, in medium, 109, 116Vitamin K, in medium, 109Vitronectin, in medium, 123tVolume of cells, variation in, 150–151, 150t, 151fVolumetric flow rate, in bioreactor, 268–270Volumetric rates, 156–157

WW1-38 cells, 19tWarburg effect, 65–66Water

as cellular component, 21, 148, 153in medium, 106

Water for injection (WFI), 106WaveTM, 201Well-mixed stirred tank reactor, 193–196, 193fWestfalia disc centrifuge, 256WFI. See Water for injection

XXBP-1, 33

YYeast (Pichia) cell culture, 12–13, 13tYield coefficient, 158–159

ZZeocin, as selection marker, 137tZinc

in intracellular fluid, 153in medium, 111, 112

Zirconium, in medium, 112Zwitterionic buffers, 115

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Cell Culture Bioprocess Engineering