Plant and Mammalian Tissue Culture

Introduction to bioprocessing and pharmacutical biotechnology of plant and animal cell culture

Industrial Application of Cell Culture Technology

Large Scale-Up of cell culture Bioprocessing Pharmacutical Biotechnology Industrial Production

Production of cell material, protein, phytochemicals and other molecules from cell culture

Market – 1 billion upstream processing industry with 5,800 employees Follow-on biologic or “biosimilar” market is going to grow

Refer to products marketed after expiration of patents Product can only be made that is similar not identical due to complexity of

biologics Investment and market is driven by a number of successful therapeutic

proteins going off-patent between 2013 and 2017 European and Asian guidelines and competition is an unknown impact

Examples of Bioprocess

Cell Culture and Fermentation Process Therapeutic Antibody Products

• Treat lymphoma, inhibit transplant rejection, anti-metastatic breast cancer, rheumatoid arthritis

Growth Factors (HGH, PDGR, Insulin) Veterinarian Vaccines – Diarrhea, parvovirus, distemper Many metabolites – alcohols, citric acid, amino acids Antibiotics

Blockbuster Proteins Remicade – monoclonal antibody against TNF-.

• Used to treat Rheumatoid arthritis and Chron’s disease• License approved August 1998• Possible mechanism of action is inhibiting cytokine receptor activation• $900 for a 100 mg dose! Responsible for $2.1 billion in sales 2009• Produced in 1,000 liter production reactors

Examples of Manufacturing Plants

Genentech New Vacaville Started construction in 2004, FDA approval 2009 $800 million invested Eight 25,000 liter bioreactors Production of Herceptin, Avastin and Rituxan

Bristol Myer Squibb Started construction 2007 – validation I 2011 $750 million invested Six 20,000 liter bioreactor, one purification strain Productioin of Orencia and other biologics

Non-Mammalian Examples Insect Cell Culture – Baculovirus

25 compounds in clinical trials Possible combitorial proteomic approach could lead to more effective protein

therapeutics

Yeast – Pichia expression systems. Need to humanize the glycoprotein expression

• Immune system keys in on different “sugared” proteins• Glycofi(Merk) is creating a multistep genetic engineering process to eliminate

non-human glycosylation enzymes• Working to batch processing of uniformly glycosylated products

Plant – alfalfa, barley, corn, rice and duckweek have been given field trials “Edible vaccines” and plant-made pharmacuticals No current PMP product on market – first will likely be animal health vaccine

“Concert”

Production Workflow

After discovery comes development, lots and lots of it!

Expression SystemDevelopment

Flasks Clone Evaluation Media Development*

Process Optimization**

• Screen and select the highest producing and most stable clone

• Develop optimal growth and production media for each cell line

• Optimize conditions for biomanufacturing process in a “scale-down” version

Scale Up

• Scale up process for use in large bioreactors for production of therapeutic

• Identify target, isolate gene, and develop expression system

Knowing gene for the protein you want is great, but what cell line to use? What clone form that cell line is best. 100s of possibilities!

60 or more nutritional components in culture media, how many combinations? When to feed them? Inducers, promoters?

What temperature? What oxygen level? CO2? pH any shifts? When to harvest? A strategy of multi-factorial design is the natural way to attack this type of

problem, but is difficult to execute in cell culture because the parameters interact strongly-requiring a lot of experiments. This means models!

Bioprocessing

Use of biological materials to create a material for medical or scientific purposes Upstream and downstream processing

Bioprocessing

Use of biological materials to create a material for medical or scientific purposes Upstream processing – from gene/cell to harvesting off cell culture

media or cell biomass Downstream processing – lysing, isolating and further purification of

bioproduct All sections require validation, quality control and quality assurance

Some High-Throughput Cell Culture System Requirements

Deliver meaningful scalable data Sustain cells, control temperature, O2, CO2, pH,

agitation Maintain sterility Monitor cell density, pH, DO, metabolites, product titer Operate with accuracy and precision and provide control

of process parameters comparable to bench top bioreactor systems

Automatic operation with minimal operator supervision Integration with tools for designing experiments and

handling data

Cell Culture Concerns Mammalian cells

Fragile and shear sensitive – membranes lyse Suspension culture cells are needed for scale

up• Fluidized bed, hollow-fiber and packed-bed do

provide some scale up potential

Slow growing compared to bacteria or yeas (24 hour doubling time)

Low production titer Extended batch times – facilitate potential

contamination Virus removal and or inactivation is required

for further processing Must start with smaller cultures then move up

to large 10,000 and 25,000 liter cultures

Scale up issues

Operating issues that affect reactor designHeat transferFoamingSterilityOxygen transfer

Bioreactor

A bioreactor is a system in which a reaction or biological conversation is effected

Different from fermentor Enzymes – to produce new product (biofuels) Microorganisms (beer fermentor) Animal and Plant Cells

Basic Design of Reactor Control temperature Maintain and analyze pH Measure viability of cells Culture composition

• Sugar, protein, carbon substrate

Oxygen Product and byproduct removal Clean and Sanitize In Place (CIP/SIP)

Types of Bioreactors

Internal Mechanical AgitationMost common and highly

flexibleMechanical agitation – paddles

• Disperses gas bubbles• Increases times of bubbles

(oxygen transfer)

Types of Bioreactors

Internal Mechanical Agitation Bubble-Column Reactor Disperse gas through reactor

with plates to enhance dispersion and mixing

Low-Sheer – but air / liquid interface produces denaturation and cell lysis

Energy efficient – low power required

Types of Bioreactors

Airlift Loop Commonly used Air is fed through sparger ring in

center-bottom of draught tube Air flows up the tube, forming bubbles

and exhausts at top Degassed liquid (now more dense)

flows down creating a circulation flow Larger fermentors and reactors use

this style to meet oxygen and cooling needs

Packed Bed Reactors Used for monolayer (adherent) cell cultures Initially used glass beads to grow cells then flow media through beads

to change media and oxygen Glass is still used but also macroporus glass beads, ceramic, polyester and

polyurethane disks are used as a growth surface

Critical issues include: high surface to volume ration, diffusion through packed bed, bed height vs. shear and pressure effects

Reservoir of media can be external or internal

Packed Bed Reactors

Hollow Fiber Cell Bioreactor

Packed Bed Reactors

Hollow Fiber Cell BioreactorEnhance mass transferProvide 3D space for cells to growUsed with hepatocytes as an artificial

Liver (Bioartificial Liver – BAL)

Packed Bed Reactors

Fluidized Bed BioreactorCells are immobized – cultured,

on small particles which move with the fluid

Large numbers of particles create a large surface area for high rate of heat, nutrient and oxygen transfer

Works best with high viscosity or gaseous substrates or products are used

Bioreactor Operating Modes

Batch – Inoculate culture and allow to cultivate without changing media Simple and allows for reduced risk of contamination Lower capital investment and greater flexibility with media

adjustments Slower – must prepare one batch at a time Small amounts of product are produced

Fed Batch – allows cells to grow to high density. Use concentrated feedstock Add in growth limiting nutrient/substrate – not a change in media Allows for high cell density with higher working time Must know very specific details on cell cultured used

Continuous

Bioreactor Operating Modes

Batch – Inoculate culture and allow to cultivate without changing media

Fed Batch – allows cells to grow to high density. Continuous- perpetual feeding process

Culture medium is fed to cells constantly May be automated and thus less expensive Less non-productive time spent emptying, filling and sterilizing

reactor Higher risk of contamination Greater processing costs – more media Used in high volume production

Regulatory Concerns

Mammalian Production SystemsPotential for Adventitious Virus

• Indicate Breach in cGMP Practices Even if Virus Has No Pathogenic Effect in Humans

• Likely Source is Raw Material• Potentially Costly Impact --- Equipment and Facility

Antibiotics to Prevent Microbial Contamination,• Not Ideal• Has Been Done for Repeated Mycoplasma Problems

Inactivation / Disposal, Environmental Concerns• What Happens if 10,000L Catastrophic Failure• Safeguards Available to Prevent Back-flow?• Method to Inactivate Prior to Release to Environment

Regulatory Concerns

Living Production System Rather than Synthetic Importance of Cell BankVariability of Living Organisms

• Complex Physiology • Balancing Growth vs Production• Spent Culture Medium is Full of Enzymatic Activity• Impurity Profile

Adventitious Agents, a Host for Propagation• Endogenous• Adventitious• Both Theoretical and Demonstrated Concerns

Unique Features of Bioreactor Production

Often Complex MoleculesPost-translational modification may / may not be

important to:• Biological activity --- increase or decrease• Purity Profile• Serum Half Life• Immunogenic Nature of the Molecule(s)• Stability• Subsequent Chemical Modification

“Family” of molecules rather than single entity• Differential Toxicity or Clinically Relevant Activity Differences

How to get the cells?

Cell Isolation/Harvesting

Heat Transfer

Large masses of cells actively respiration will produce heat

Control of heat by transfer is one of the two main limitations on size of bioreactorsMay use internal coils or external water jacket to control

tempCoils can pose problem for contamination but is more

effective with higher surface for potential heat transferCoils can also adversely affect mixing with additional

unwanted turbulence

Foaming

Foam is a natural byproduct – mostly protein bubbles but some lipidFoam will block and wet filters causing pressure

back-up and contaminationFoam must be controlled by chemical dispersing

agents (antifoams)Maintaining 75% volume capacity of reactor

allows for foam to be retained within the vessel

Sterility

Sterilization in place (SIP)– cleaning of reactor and bed without dismantling reactor or feed tubes

Pressurized steam is used for in-place sterilization of probes, valves and seals

All crooks, crevices and surfaces are potential contaminants and must be sterilized

Sterilization must be verified and validated

Cleaning

Cleaning in place (CIP) is performed after each run and before a new run is initiated

Highly alkaline detergents, bases and acids are used with copious amounts of water

Cleaning solutions are often plumbed into system for automation