Burger's Medicinal Chemistry and Drug Discovery || Process Development of Protein Therapeutics

PROCESS DEVELOPMENT OFPROTEIN THERAPEUTICS

JAMES N. THOMAS

S. SAM GUHAN

DEAN K. PETTIT

Amgen, Inc., Seattle, WA

1. BIOTECHNOLOGY: INTRODUCTION

The term “biotechnology” dates from 1919,when Karl Ereky, a Hungarian engineer, de-fined it as “any product produced from rawmaterials with the aid of living organisms” [1].One modern definition proposed by the UnitedNations Convention on Biological Diversity de-fines biotechnology as “any technological appli-cationthatusesbiologicalsystems,livingorgan-isms, or derivatives thereof, to make or modifyproducts or processes for specific use” [2].

The discovery of the structure of DNA byWatson and Crick in 1953 resulted in an ex-plosion of research in molecular biology andgenetics. Molecular biologists devised meth-ods to isolate, identify, and clone genes as wellas to mutate, manipulate, and insert theminto other species.

Today, biotechnology has far-reaching ap-plications in health care, environmentalsciences (bioremediation), agriculture, andsynthesis of products such as biofuels and bio-degradableplastics.Fromitsrelativelyhumblebeginnings in the late seventies and early eigh-ties, the biotech industry is a thriving businesstoday. According to BIO (Biotechnology Indus-tryOrganization) as ofDec 31 2005, therewere1415 biotechnology companies in the UnitedStates of which 329were publicly held [3]. Bio-technology companies, in the context of thepharmaceutical industry, are those companiesthat focus primarily on large-molecule proteintherapeutics, often referred to as “biologics.”

1.1. Process Development of ProteinTherapeutics

Biologics are often large molecules (usuallyproteins) andmuchmore complex compared totraditional small molecular weight pharma-ceuticals. A typical biologic is hundreds oftimes larger than the compounds found in

most pills or tablets.Moreover,most currentlyavailable biologics have to be directly intro-duced into the blood stream, either via sub-cutaneous injections or via intravenous infu-sions to achieve their intended affects.

In contrast to small-molecule therapeuticsthat can be made through organic synthesis,large-molecule proteins must be made usingbiological systems. Proteins, in addition totheir primary structure (amino acid se-quence), have secondary, tertiary, and qua-ternary structures. This three-dimensionalstructure is often critical for the activity ofthe protein and is an important differencebetween small molecular weight drugs andprotein therapeutics. Figure 1 schematicallyillustrates the size difference between small-molecule and protein therapeutics, illustrat-ing the significant difference in complexitybetween these drug classes.

The development of a manufacturing pro-cess for the production of a biological is verycomplexandistypicallycomprisedoffourparts:

(1) Upstream Development. Describes theactivity of integrating a gene of interest(cDNA) into a host and generating arecombinant cell line or strain. It in-cludes subsequent steps to isolate asingle cell clone ormicrobial colony andto preserve such recombinant cells inthe form of frozen cell banks. Upstreamdevelopment also refers to the processthrough which the product is made,typically by fermentation or cell cultureusing live organisms (e.g., bacteria,mammalian cells, etc.).

(2) Downstream Process. In this step, theproduct of interest is isolated from thevarious contaminants and impuritiespresent in the cell culture or fermenta-tion broth and concentrated to the levelrequired.

(3) Formulation Development. This de-scribes the development of the opti-mum product containing solution, andits delivery device.

(4) Analytical Development. Developmentof analytical methods and technologiesthat are used throughout process devel-opment in order to provide critical

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Burger’s Medicinal Chemistry, Drug Discovery, and Development, Seventh Edition,edited by Donald J. Abraham and David P. RotellaCopyright � 2010 John Wiley & Sons, Inc.

information on the quantity and qual-ity of the product.

The integration and overlap of the fourbasic components of protein process develop-ment are shown in Fig. 2.

A challenge to the organization and execu-tion of protein biologics process developmentis the interdependence of each part of theprocess on the other. The upstream processhas a large impact on the downstream pro-cess. It is not just the quantity or type of

Figure 1. Protein process development requires coordinated integration of upstream, downstream, for-mulation, and analytical activities. Primary functional activities are indicatedwithin each circle and areas ofoverlapping circles indicate shared activities. The major inputs required to initiate protein process devel-opment and the key deliverables or outputs from these activities are indicated at the top and bottom of thefigure, respectively.

290 PROCESS DEVELOPMENT OF PROTEIN THERAPEUTICS

contaminants that need to be removed, butalso the challenges created when the up-stream process produces more product thanthe downstream system can handle. Thedownstream/purification process is highlydependent on the final formulation aswell. The last step of the downstream processdelivers the drug substance, preferably,in the final formulation. The use of appropri-ate analytical tools is important to monitorproduct quality during process develop-ment [4]. Any treatment during processingmay subject the protein to stress (pH, con-centration, salt, etc.), which would poten-tially impact chemical and/or structuralcharacteristics.

The following sections discuss each of theseindividual parts of process development indetail.

2. UPSTREAM PROCESSING

Since the first gene cloning experiments weresuccessfully performed by Boyer andCohen atthe University of San Francisco [5,6], variousexpression systems have been used to producerecombinant proteins of potential therapeuticvalue. The variety is staggering in complexity,ranging from simple microbes such as Escher-ichia coli to complex transgenic whole animalsystems. While a complete review of all the

available systems is beyond the scope of thischapter, a few examples will be cited to under-line the numerous expression tools availableto the modern scientist. The majority of mar-keted recombinant proteins, especially glyco-proteins, are expressed usingmammalian cellculture systems.With this inmind, the bulk ofthis section will be devoted to a more in-depthdiscussion of current mammalian cell culturetechnology.

2.1. A Plurality of Expression Systems

Many prokaryotic expression systems havebeen evaluated over the years for their abilityto make recombinant proteins. Some of theseinclude various strains of Bacillus and Strep-tomyces [7,8] and of course the workhorsesystem E. coli [9–11]. The desire has been todevelop host strains with the ability to secreteproperly folded protein, but yields employingthis approach are still relatively low. Prokar-yotes such as E. coli produce enormous quan-tities of recombinant protein, especially if theprotein is protected from host proteolytic de-gradation within inclusion bodies. Protein ininclusion bodies is usually misfolded and in-soluble, so one of the first downstream proces-sing steps involves refolding the protein into acorrectly folded and active form. The yieldsassociated with refolding steps are usuallylow for complex proteins containing several

Figure 2. Size comparison of small-molecule and protein therapeutics, illustrating the significant differencein complexity between these drug classes.

UPSTREAM PROCESSING 291

disulfide bonds. E. coli is also missing cellularenzymes for adding carbohydrate to proteins.Many human proteins are glycoproteins andrequire the proper amount and type of carbo-hydrate for full in vivo activity. All prokaryoticand most eukaryotic systems are incapable ofproper glylcosolation and are best used forexpressing proteins that are not dependenton carbohydrate for potency or optimumphar-macology. Recent attempts have beenmade toaddress some of the drawbacks of usingmicro-bial systems such as E. coli by optimizingsecretion signal sequences, coexpressing cha-perones, foldases, selecting protease deficientmutants [8,12], optimizing expression vectors,codon usage [13], and engineering cellphysiology [14].

Anumber of eukaryotic expression systemshave been used to produce recombinant pro-teins during the last several years. The mostpopular of these have been yeast systems thatinclude Pichia pastoris, Saccharomyces cere-visiae, Kluyveromyces lactis, and Hansenulapolymorpha [8]. Yeast has rapid doublingtimes, which can grow to high cell densitiesin fermentors and can secrete complex pro-teins. S. cerevisiae (baker’s yeast) has been apopular expression host for 25 years, butP. pastoris has recently become the yeast sys-tem of choice due primarily to its ability tosecrete complex proteins [15–20]. Expressionlevels in yeast can be very high for someproteins (>10 g/L), but in general these sys-tems have greater difficulty expressing com-plex glycoproteins thanmammalian-based ex-pression systems. While yeast adds carbohy-drate to proteins, these organisms tend toattach more primitive high mannose speciescausing glycoproteins to be cleared more ra-pidly in vivo. A traditional method for gettingaround this concern is to remove N-linkedglycosylation sites from the protein if this canbe done without negatively impacting activityor immunogenicity. Still another, more so-phisticated approach has been developed byGlycoFi, a wholly owned subsidiary of Merck.By knocking out 4 genes in P. pastoris respon-sible for glycosylation, and inserting 14 het-erologous genes, GlycoFi has successfully ex-pressed human glycoproteins with fully com-plex, terminally sialyatedN-glycans [21]. Thisallows the expression of essentially fully

“human” glycoproteins, with the added ad-vantage of greater homogeneity of glycoforms.

Another eukaryotic expression systemused for expression of a variety of glycopro-teins is based on efficient transduction of in-sect cells with baculoviruses [17,22]. The hostrange of these viruses is restricted to inverte-brates, primarily insect cells from moths andbutterflies [7], making the system safe for usein manufacturing. Because insect cells arehigher eukaryotes, cellular machinery isavailable for performing proper posttransla-tion modifications in most cases. Glycosyla-tionpatterns are still somewhatdifferent thanhuman, although some have addressed thisissue by engineering insect cell lines to ex-press enzymes necessary for proper mamma-lian carbohydrate structure [23]. Anotherchallenge is the transient nature of the sys-tem; cells need to be transduced in everyproduction phase. This requires preparationof both the cell inoculum and the baculovirusused for transduction [7].

Transgenic systems have been exploredover the past several years for use as expres-sion vehicles for recombinant proteins, espe-cially those needed in very large amounts(metric tons) [24]. Awide variety of transgenicsystems exist and include expression into themilk of transgenic mammals, the eggs oftransgenic chickens, the cocoons of silkworms,and the seeds, leaves, or tubers of transgenicplants [24–29]. The core of the technology liesin the molecular approach for introductionand expression of the transgene in the hostorganism.Expression is frequently targeted totissues or organs, such as the mammaryglands of cattle or goats, designed for makingenormous amounts of milk protein, and easilyaccessible for harvesting. Because of the long-er development cycles and perceived regula-tory hurdles of transgenic systems, somecompanies are developing molecules usingtransgenics in parallel to their primary devel-opment route, (i.e., mammalian cell culture).

While improved yields of conventional pro-duction technologies have made many trans-genic systems less attractive than in the past,transgenic plants may still hold some long-termpromise as production systems of proteintherapeutics. Like mammalian transgenicsystems, expressed proteins are targeted into


plant organs, often concentrated, and readilyaccessible for harvest. Depending on the loca-tion of concentration in the plant, this offerssome advantages for downstream proces-sing [30]. If the transgenic protein is expressedin seed crops such as rice, wheat, maize, orpeas; stability of the unprocessed seeds can beextremely favorable (>1 year), providing anexcellent opportunity for optimizing the effi-ciency of downstream processing steps [31].While plants can perform complex posttran-slational processing of glycoproteins, they canalso add immunogenic plant carbohydratespecies at the same N-linked sites used bymammalian systems [30]. The same generalstrategies are usedwith plant systems aswithorganisms such as yeast or insect cells tocontrol glycosylation patterns; either removalof the N-linked sites or expression of mamma-lian glycosyltransferases in the host plant tohumanize plant N-glycans [30,32]. Time willtell whether these approaches are successfuland how well these systems can compete withever improving conventional expressionsystems.

In the 30 years since the beginning of themodern biotechnology industry, several mam-malian expression systems have been usedextensively to produce recombinant glycopro-teins. Some of the major workhorse systemshave been developed from Chinese hamsterovary (CHO), baby hamster kidney (BHK),mousemyeloma (NS0), humanembryonic kid-ney (HEK-293), and human retina (PER.C6)cell lines [33–35]. It is important to note thatthese cell lines, while derived from normaltissues, are all transformed and can theoreti-cally be passaged indefinitely in culture asopposed to normal diploid cells. The genomesof these cell lines are generally stable, butdefinitely aneuploid andmore adaptable thannormal diploid cells. Each cell line can begrown in suspension culture making scale-upin conventional bioreactor systems straight-forward. Important qualities in a host cell lineare several: the ability to grow in suspensionculture to high cell densities with high viabi-lity; relatively fast doubling times (24h orless); easy to transfect with foreign genes;ability to grow in chemically defined media;low protease production; not susceptible totransformation by viruses that are human

pathogens; and ability to perform posttransla-tional modifications, such as glycosylation,similar to humans.

Of the cell line options, CHO cells havebecome the overwhelming choice of many bio-technology companies for producing complexglycoproteins, including recombinant antibo-dies. NS0 myeloma cells have been used ex-tensively for the production of recombinantantibodies, but less frequently for the produc-tion of other complex glycoproteins due to aless desirable carbohydrate profile [36].

There are several CHO cell lines in usetoday in the industry, originally derived fromCHO-K1 cells characterized by Kao and Puckin the 1960s [37]. Their laboratory createdauxotrophic mutants for nutrients such asproline and glycine to study mammalian ge-netics [38–41], and performed the founda-tional work needed for the development of thedhfr- (dihydrofolate reductase-deficient) se-lection and amplification system used sowidely today. Urlaub and Chasin [42] ex-tended this work by deriving variants ofCHO-K1 deficient in expression of dhfr bymutating CHO-K1 cells. The popular DXB11cell line was derived from these mutationalstudies and while this cell line can revert todhfrþ , the frequency is very low. Furtherwork by this laboratory [43] created otherCHO-K1 mutant cell lines such as DG44, adouble deletionmutant for dhfr, containing nofunctional copies of the hamster gene. CHOcells can be further adapted for enhanced ex-pression or improved growth under a varietyof culture conditions [44,45].

The importance of having a good host cellline was foundational for the beginning ofmodern pharmaceutical biotechnology, andparticularly for the production of complex gly-coproteins. CHO cells have remained a popu-lar expression system because of the manypositive qualities mentioned previously. For-eign genes can be efficiently transfected, se-lected and then amplified using dhfr as aselectable and amplifiable marker [46].DXB11 or DG44 cells deficient in dhfr expres-sion cannot be cultured in selective medialacking glycine, hypoxanthine, and thymi-dine [42]. Functionally, dhfr is the enzymeresponsible for the formation of intracellulartetrahydrofolic acid, a cofactor for one-carbon


transfers important in several cellular biosyn-thetic reactions.The role ofdhfr is particularlyimportant in the formation of precursors forDNA synthesis. Cotransfection of dhfr defi-cient CHO cells with both the foreign gene ofinterest and the gene coding for dhfr, followedby culturing these cells in selective medialacking glycine, hypoxanthine, and thymidinewill select for cells expressing both dhfr andthe foreign gene of interest. Only cells coex-pressing dhfr and the foreign gene of interestwill survive. After selection, amplifying thenumber of foreign gene copies can be accom-plished by exposing the cells to methotrexate(MTX), an efficient binder and inhibitor ofdhfr. Increasing the concentration of MTX,usually in a stepwise fashion, forces cells toexpress higher levels of the gene for survival.Since the dhfr gene and the foreign gene willgenerally integrate into the same region of thecell genome, amplifying dhfrwill also result incoamplification of the foreign gene.

Other selectable and amplifiable markershave been used to increase the expression offoreign genes in CHO and other cell lines. Forexample, glutamine synthetase (GS) has beenused to express foreign genes in cell lines suchas CHO-K1 and NS0 myeloma [47,48]. Thisenzyme is essential for the conversion of glu-tamate and ammonia into glutamine, a nutri-ent for most transformed cells. While somecells can be adapted to grow in the absence ofglutamine by increasing the endogenous ex-pression of glutamine synthetase, other cells,such as myeloma, appear to have an absoluterequirement for exogenous glutamine. TheGSsystem is particularly useful in these cell linesas only those transfected with exogenous GSwill survive inmedia lacking glutamine. Geneamplification can be accomplished by expos-ing the transfected cells to methionine sul-phoximine (MSX), a specific inhibitor of GS.A similar selectable and amplifiablemarker isasparagine synthetase (AS), based on the E.coli gene for this enzyme that catalyzes theconversion of aspartic acid to asparagine [49].This system has been used in cell lines thatexpress endogenous AS by using albizziin forinitial drug selection. The exogenous bacterialAS is more resistant to inhibition by albizziinthan the endogenous mammalian AS gene, soselection of transfected cells in albizziin con-

taining medium favors the exogenous markerover the internal. Amplification is accom-plished using a second AS inhibitor moreeffective against the E. coli enzyme. Otherselectable and amplifiable markers have beenused to enhance recombinant gene expressionin animal cells, but most have not found wide-spread practical application in the industry.

2.2. Creating Recombinant Production CellLines

Nowwe will turn to a more specific discussionof the approaches used to create recombinantproduction cell lines using the components ofthe CHO system.

2.2.1. Expression Vectors Transcription ofmRNA is generally not rate limiting for theexpression of recombinant proteins in mostcurrent production cell lines [50]. This is dueto advances in understanding gene regulationat the molecular level, and the design ofpowerful vectors used for driving expression.

cDNA, coding for the gene of interest, ispackaged with other DNA sequences to facil-itate efficient transcription and translation inthe host cell. This DNA package is called theexpression vector or plasmid and also containssequences to facilitate replication in bacteria(generating sufficient plasmid for transfec-tion). Our understanding of the regulation ofgene expression in mammalian cells has ad-vanced over the past 30 years, but there is stillmuch we do not understand. Our knowledgehas come from the study of how viruses repli-cate in host organisms. Some of the mostcritical elements for achievinghighexpressionof foreign proteins in mammalian cells areviral promoter regions that influence tran-scription. They contain components such asthe Goldberg-Hogness box (TATA sequence)approximately 25–35 bp upstream of the RNAinitiation site, as well as an enhancer regionthat stimulates transcription rate. Some pro-moters have host specificity, working well inone cell line to drive expression but ineffectivein others [51]. Promoters such as human cy-tomegalovirus immediate-early gene promo-ter and enhancer (huCMV P/E) are powerfuldrivers of transcription in multiple cell hosts.InCHOcells, the huCMVP/Ehas beenused to


drive expression of both the foreign gene ofinterest and the selectable marker from thesame transcript [52,53]. This promoter can betransactivated by certain adenovirus proteinssuch as E1A, so one strategy for enhancingexpression has been to include the gene codingfor E1A in the expression vector [47]. Earlyadenovirus proteins function as transcriptionenhancers; so, when coexpressed with thegene of interest, they can serve as powerfulstimulators of protein production from just afew integrated gene copies.

Expression vectors usually contain otherelements to enhance either transcription ortranslation of mRNA in the host cell. The capand poly(A) tail are regulatory determinantsthat establish the translational efficiency ofmRNA [54]. The IRES sequence (internal ri-bosomal entry site) from encephalomyocardi-tis virus also enhances efficiency of transla-tion [55]. Other elements, such as the EASE(expression augmenting sequence element)sequence, increase the speed of obtaining highexpressing cell clones after transfection, andincrease the stability of integration in theabsence of selective pressure [53]. EASE is aCHO genomic sequence discovered by map-ping the flanking sequence of a single inte-grated gene copy expressing relatively highlevels of a recombinant protein. There arelikely many such elements in the mammaliangenome that serve as regulatory elements forgene expression [56–61]. Vectors can also bedirected to a known active transcriptionallocus in the cell genome by incorporating atargeting sequence to facilitate integrationinto the active site through homologousrecombination [33,50,62–64].

2.2.2. Transfection and Gene AmplificationEfficient translocation of a plasmid throughthe cell membrane is essential for creatinghigh-yielding cell lines. This process is calledtransfection, and a variety of methods havebeen employed to accomplish this over theyears. The three most widely used methodsfor transfection of plasmid DNA are calciumphosphate precipitation, electroporation andthe use of liposomes (lipofection) [65,66].

Calcium phosphate transfection involvesthe coprecipitation of calcium chloride, phos-phate buffer and plasmid DNA to facilitate

uptake of DNA via endocytosis [67,68]. Thistechnique has been used extensively with anumber of different cell types, and has beenmodified to improve its efficiency.

Electroporation is a simple method thatuses an electrical field to create reversiblepores in the plasma membrane of cells [69].While this method is applicable to a variety ofcell types, it must be carefully optimized forbest results.Efficient transfectionwhilemain-taininghigh cell viability is a challenge for thistechnique.

Lipofection is based on the properties ofcationic lipids (modification of lipids with qua-ternary amines) to package negativelycharged DNA for transport through nonpolarmembranes. Transport is achieved througheither endocytosis or direct membrane fu-sion [70]. Optimization of the ratio of DNA tolipid is probably the most important aspect ofthis method.

Transfection can be accomplished by pla-cing both genes on the same vector or by usingmultiple vectors and cotransfecting. Fortu-nately, even cotransfected genes tend to inte-grate into the same site on a chromosome,creating the opportunity of coamplification ofthe amplifiable marker gene with the foreigngene of interest. This happens because largeregions of the chromosome near the amplifi-able marker are replicated during drugselection.

Due to the complexity of gene regulation inthe mammalian genome and the random nat-ure of integration, the selection process priorto amplification must be stringent. Parachut-ing foreign DNA into a complex mammaliangenome subjects foreign gene expression toa variety of positional influences. Variousmethods have been used to minimize theseinfluences including the use of matrix attach-ment regions and anti-chromatin repressorelements to insulate against positional ef-fects [58–60,63]. As described earlier in thischapter, the dhfr system functions as a highlystringent selection and amplification methoddue to the availability of dhfr-cell lines andthe critical role this enzymeplays in the synth-esis of cellular DNA. After initial selection inmedium lacking glycine, hypoxanthine andthymidine, cells are usually exposed to in-creasing concentrations of MTX in a stepwise


fashion [46]. Copies of the integrated foreignDNA can number in the thousands [46,65]after amplification. Integration is generallyinto one site or locus in the genome where,after exposure to selective pressure, gene am-plification occurs causing elongation of theintegration site chromosome [71]. By FISH(fluorescence in situ hybridizationanalys) isother “satellite sites” can be observed in thegenome, probably arising through homolo-gous recombination.Uponremoval of selectivepressure (MTX), these “satellite sites” usuallydisappear suggesting they are unstable.

One approach to ensuring high levels ofstable expression froma small number of genecopies has been the use of targeted integrationmethods. Since some loci in the genome aremore actively transcribed than others, theability to target integration into a highly tran-scribed locus becomes attractive. Twoprimarysystems have been successfully used in thisapproach, the Cre/loxP system and the Flp/FRT system [62–64,66]. The former system isfrom the bacteriophage PI and the latter isfrom yeast. Identification of the active tran-scriptional locus is accomplished by screeningclones after transfectingwith a vector contain-ing the target site (lox or FRT), amplifiablemarker and reporter gene. Selection of cellswith high expression of the reporter gene pro-vides evidence of transcriptional activity. Co-integration of the recombination target sitefacilitates future targeting of that site usingrecombinase enzymes (either Cre or Flp) thatcatalyze excision/integration of the gene ofinterest.

2.2.3. Cloning and Banking the Production CellLine After amplification, cell pools expres-sing the desired quantity and quality of thetherapeutic protein are cloned. This has beentraditionally accomplished by limiting dilu-tion of the transfected cell pools into 96-wellplates. The objective is to dilute the pool toobtain a single cell per well, which can beconfirmed by microscopically examining eachwell for colony growth. Typically clones arescreened for growth and productivity, but thetrend is to perform increasingly rigorous pro-duct quality analysis at this stage as well.Selecting thebest clonemaybe the singlemostimportant activity in process development for

ensuring high productivity and acceptableproduct quality. Due to the importance of thisstep, other methods have been developed toimprove the selection of the best clone. Manyof these are based on fluorescence-activatedcell sorting (FACS) techniques as a way ofvisualizing and sorting the best expressingcell clones [72,73].

Since the cell line is a controlled reagentand a critical part of the manufacturing pro-cess, it must be banked and rigorously testedto confirmidentity, purity, andsuitability [74].Banks are qualified by testing for the presenceof adventitious agents including bacteria, fun-gi, mycoplasmas and viruses. Since recombi-nant cell lines aremost often employed for theproduction of biotherapeutics, genetic stabi-lity is also an important characteristic and isdetermined at the molecular and cellularlevel.

A two tiered cell banking system isgenerally used for the manufacture of mostbiotherapeutic products [74]. This system iscomposed of a master cell bank (MCB) and aworking cell bank (WCB). The MCB is rigor-ously characterized and serves as the corerepository of the recombinant cell line. ManyWCBs can be made from the MCB, ensuringplenty of well-characterized cell stock for thelifetime of drug manufacturing. Because theWCB is derived from theMCB after only a fewpassages, a much smaller subset of tests arerequired for qualification. An additional cellbank is usually established at the limit of invitro cell age for determining genetic stabilitythat spans the production window of the man-ufacturing process.

2.3. Upstream Process Formats

The primary objective of any manufacturingprocess should be to produce a high-qualityproduct in the most cost effective way to meetmarket demands. As a way to achieve this, awide variety of manufacturing formats havebeen used in the biotechnology industry. Thechoice of production format is influenced by anumber of factors including the cells beingcultured, themolecule being produced and theoverall productivity required of the process. Acomplete review of all process formats used toculture mammalian cells is beyond the scope


of this chapter, but a few of the most popularsystems will be covered for illustration.

Every process format must meet the basicrequirements for culturing mammalian cellsincluding physical demands such as dissolvedoxygen and temperature aswell as nutritionaland hormonal requirements. A cloned recom-binant cell line will consistently produce aknown amount of product/cell/unit time. Toincrease the concentration of secreted productin the cell culture medium therefore requiresan increase in viable cells per volume of med-ium per unit of time. This can be achieved in avariety of ways and has therefore led to multi-ple choices in upstream process formats.

Some cells require attachment to a matrixor surface for growth, while other cells growreadily in suspension. This characteristic willhavea significant influence on the formatusedfor production.

2.3.1. Attachment Dependent Cultures Manycell types can be cultured in suspensionattached to small particles called microcar-riers [75]. This method was originally devel-oped for normal diploid cells requiringattachment to a matrix for growth [76–78],but has also been used to culture transformedcell lines such as BHK, VERO, MDCK, andCHOcells.At one time, an enormous variety ofmicrocarriers were available that varied inmaterial frommacroporous gelatin to ceramicor glass. One of the original and most popularmaterials is diethylaminoethanol (DEAE)—Sephadex A50 ion exchange resin, optimizedfor both charge density and the size to supportefficient cell attachment and growth [79]. Oneadvantage of microcarriers is the ability toculture attachment dependent cells in modi-fied STRs (stirred tank bioreactors). Specialimpellors and sparging systems are requiredto minimize shear in bioreactors used for mi-crocarrier culture. Another potential advan-tage of this method is the increased sedimen-tation velocity achieved when cells are at-tached to microcarriers. This allows easy se-paration of culture supernatant from cells,making this type of cultureamenable to repeatbatch or continuous processing.

Other formats for culturing cells attached toa surface include roller bottles, hollow fiberbioreactors and fluidized-bed systems to name

a few. Roller bottle culture is perhaps the sim-plest system in its basic form; involving theattachment of cells to the surface of a rotatingdisposable plastic bottle [78]. This system canbe scaled-up using an automated format, butdue to the complexity and size of the automa-tion,thissystemshouldonlybeusedifneededtoinsure production of a product with a specificproduct quality profile or a product with verylow production requirements.

Hollow fiber reactors have been used formany years to producemolecules such as anti-bodies from hybridoma cells. This format isparticularly useful when only a few grams ofproduct are needed, but can be especiallychallenging when hundreds of kilograms arerequired. Cells are cultured in the extracapil-lary spacewhilemedium is circulated throughthe intracapillary space or lumen to deliveroxygen and nutrients [80]. This system isrelatively simple and will concentrate theproduct with the cells in the extracapillaryspace. Oxygen and nutrients will diffusethrough the capillary to the cells while CO2

and other metabolites will diffuse away fromthe cells into the perfused medium [81,82].Scale-up can be a challenge due to the forma-tion of significant diffusional gradients alongthe length of the hollow fiber reactor, particu-larly at the distal end.

Fluidized bed systems involve the immobi-lization of cells on some type of matrix andthen “fluidizing” this matrix within the con-fines of a usually cylindrical reactor. The ad-vantages of such system are improved masstransfer and mixing within the reactor con-taining as much as 50% volume as matrix andcells. One such system employedmacroporouscollagen microcarriers weighted to a specificgravity of about 1.6 withmetal particles. Cellsgrew both inside the approximately 500mmmacroporous carriers and on their surface andat the proper flow rate remained fluidizedwithin the reactor [83]. Fluidization was nota result of perfusion, but was due to flowthrough an external loop recirculating med-ium through the reactor. Fresh medium wasadded and spent medium was removedthrough a second pumping system. The recir-culation loop also contained an oxygenatorand inline oxygen probes to control the levelof oxygen in the reactor and help exchange out


excess CO2. This reactor format can be scaled-up more easily than hollow fiber reactors, butalthough eloquent in design, is complex tooperate and may not be suitable for a com-mercial manufacturing environment.

2.3.2. Suspension Cultures Perhaps the sim-plest and most widely used process format isbatch culture of suspended cells. This is aprocess where cells are cultured in bioreactorsof increasing size in order to inoculate the fullscale production reactor, usually a STR (stir-red tank reactor). Originally cell culture STRswere modified versions of bacterial fermen-tors, but over time the design has evolved tobetter meet the environmental needs of mam-malian cells. This includes low shear impel-lors and baffling for improved mixing, specialspargers for delivering dissolved oxygen androbust probes and ports for themeasuring andsampling needs of mammalian cells. The in-tegrity of the sterile envelope is critical for anymammalian cell culture system, so very so-phisticated utilities for CIP (clean in place)and SIP (steam in place) are usually asso-ciated with cell culture STRs. For batch cul-ture, cells are inoculated into the productionSTR and allowed to grow and produce productfor several days before harvesting. Cells willgrow until they reach either limiting concen-trations of an essential nutrient or inhibitingconcentrations of a metabolic byproduct. Atthis point, cells will begin to lose viability andthe volumetric production rate of the culturewill decrease. A modification of this approachis the fed-batch format. In this case cells areinoculated into the production STR and cul-tured in batch format; at periodic intervalsadditional nutrients are added that increasethe total number of cells per volume and thelength of the culture period. Other approaches(such as adjustment of temperature and pH)are also used to limit the production of meta-bolic byproducts in order to increase the totalnumber of viable cell days in the productionreactor.

There are many variations in the use ofSTRs for culturing mammalian cells, but theprinciple of optimizing the total number ofviable cells per unit volume of medium perunit time (cells/volume/time), is the same.Some process formats constantly perfuse

fresh medium into the production STR whileretaining, through various retention meth-ods, very high cell density cultures in thebioreactor [83].

Over the past several years, the most pop-ular system for production of recombinantproteins has been a fed-batch process usingCHO cells cultured in large scale STRs ofseveral thousand liters. The productivity ofthese systems continues to improve, and hasnow surpassed the capacity of many oldercommercial downstream processing facilitieswithout substantial facility modifications.While continuous processes are more elo-quent in design and better mimic an in vivoenvironment for cultured mammalian cells,the relative simplicity and high productivityof current fed-batch systems will be difficultto supplant in commercial manufacturingoperations.

2.4. Upstream Process Development

Cell culture process development of large mo-lecules attempts to integrate the properties ofthe production cell line, the process formatand the physical and chemical environmentof the cell culture into a robust manufacturingprocess. Some fundamental goals of upstreamprocess development, particularly as appliedto batch and fed-batch formats are as follows:

. To create an optimum environment forculturing cells to high viable cell densi-ties for an extended period of time in theproduction bioreactor.

. To optimize conditions for expressinghigh levels of a therapeutic protein withthe desired critical quality attributes(CQAs).

. To facilitate efficient harvesting anddownstream processing.

Specific productivity has increased sev-eral-fold over the past 25 years due to ad-vances in molecular biology and ourunderstanding of gene expression. The im-portance of creating a high expressing cellline with robust growth suitable for commer-cial manufacturing cannot be overempha-sized. A survey of the essential elements fordoing this have already been covered in this


chapter, so the next section will be devoted toa short discussion of considerations for opti-mizing culture conditions for increasingviable cells per unit volume per unit time.Figure 3 denotes the two dominant ap-proaches for improving productivity.

2.4.1. The Physical Environment As alreadymentioned, the bioreactor must create theoptimum physical environment for culturingmammalian cells. It should be recognizedthat although cell lines used to producebiotherapeutics today are transformed froma genotypic and phenotypic perspective, theystill require a physical environment that issimilar to that found in vivo in the animal oforigin. In other words, the pH, temperature,dissolved oxygen, dissolved carbon dioxide,osmolality, and other physical parametersmust be carefully optimized and controlledfor the best performance of the culture. Insome cases, there are advantages to control-ling some of these parameters outside thenormal range to improve viability, productquality or specific productivity; but this mustbe carefully defined during the course of pro-cess development.

2.4.2. Medium Design Complex cell culturemedia, the nutrient source for cells, are com-posed of over 50 different components includ-

ing amino acids, vitamins, trace elements,salts, lipids, carbohydrates, growth factors,buffering components and shear protectingagents [84]. These factors provide the buildingblocks and stimulus for driving cell growth andthe protein product being made. In addition,medium components are interlinked with thephysical environment of the culture influen-cing pH, osmolality, the tolerance to bubbleandmechanical shear. Requirements are simi-lar, although not quite the same as those foundin the host animal. For example, 13 aminoacids are generally required in cell culturewhile only about 8 or so are required for thehost animal [85]. The past 50 years have wit-nessed a progression of cell culture mediumfrom an ill-defined mixture to one that is che-mically defined (i.e., all chemicals are known).There are many commercial media availablethat can serve as starting points for optimiza-tion [84], but the ultimate medium used in aprocess will depend on the desired cell densityand process format. In the case of fed-batchcultures, thegoal is toprovideanenrichedbasemedium and feeds to support the highest pos-sible cell density for the desired culture period.This is a complex optimization exercise com-posed of understanding the utilization rate fornutrients, their concentration change relativeto other nutrients, the conversion rate intometabolic byproducts and the solubility limits

Figure 3. Greater productivity requires improved fundament scientific understanding leading tomore cells/volume/time and/or more product/cell/time.


for nutrients in both the base medium and thefeed medium.

Amino acids that serve as the buildingblocks for proteins must be carefully opti-mized to maximize culture performance.Amino acid transporters in cell membranestransport multiple amino acids, providing acompetitive situation whenever differentialutilization rates cause significant imbalanceover time [86].Metabolic byproducts of aminoacids may also be toxic for some cells, and inbatch or fed-batch cultures the concentrationof these toxic compounds will build over time.Due to the complexity of optimizing the nu-tritional and physical environment of cellculture processes, the industry is beginningto rely more heavily on DoE (design of experi-ments) and other statistical approaches andtools. While the work continues to be challen-ging, if done well, it provides an opportunityto push cell culture densities to much higherlevels once nutrient utilization, metabolicproduction kinetics, and interactions arewellunderstood.

2.4.3. Understanding Metabolism Under-standing metabolism is closely linked to med-ium optimization as metabolic byproducts aresimply a conversion of available nutrients andoxygen into other chemical forms. Metabolicflux through various biochemical pathwayswithin the cell can, to some degree, be regu-lated or influenced by a variety of controllablefactors within the culture environ-ment [86,87]. The complexity of optimizingbatch or fed-batch cultures stems from thecell’s expectation that the system contains adetoxifying organ, such as a liver, to convert orrepackage metabolic byproducts into either ausable form or a detoxified form for elimina-tion. Controlling cell culture inputs that arenutritional, physical and sometimes hormo-nal provides a mechanism for preventing me-tabolic byproducts from reaching toxic orinhibitory levels. The basis for successfullyperforming this type of work resides in under-standing physiology and intermediary meta-bolism in thewhole animal. It is only from thiscontext, and from an understanding of thetweaks to normal physiology brought aboutby cell transformation, that one can produc-tively approach this area.

2.5. Future Trends

While there are many potential areas for im-proving technology associated with produc-tion of biotherapeutics, one that promises sig-nificant benefit is continued improvement ofthe expression host. The complexity of biolo-gical systems, even at the cellular level, can bedaunting. This poses a significant barrier tounderstanding andmanipulating the host cellline for improved performance. Even so, thereis growing interest and work in this area asacademic and industrial laboratories usegenomic and proteomic tools to improve hostcell lines. The fundamental premise is thatunderstanding the limitations or metabolicbottlenecks in cells will provide an opportu-nity for removal or improvement through cel-lular engineering [88].

In some cases experimental observationshave led to engineering of host cells to improvecell viability by preventing or decreasingapoptosis [89–91]. The level of lactic acid incultures can be controlled by transfecting andover expressing the GLUT5 transporter inCHO cells [92]. CHO cells are normally cul-tured with glucose as the source of carbohy-drate, but when engineered with GLUT5 theycan be cultured with fructose, and the slowerrate of transport prevents the buildup of lac-tate in the culture. Culturing CHO cells at lowtemperatures (30–33�C) induces many “coldstress” genes and can induce recombinantprotein expression. Over expressing a specificcold stress protein (CIRP) in a recombinantcell line and culturing at 37�C led to a 40%increase in recombinant protein production insuspension culture [93].

Another approach for this work is to com-pare gene expression under different cell per-formance conditions by genomic analysis, pro-teomic analysis or by using a combination ofboth approaches [94–97]. Analysis of this datais often complex as one finds multiple genesupregulated or downregulated in a given cul-ture condition. Interpretation of expressionpatterns must rely on a fundamental under-standing ofmolecular and cellular biology, cellphysiology, and the biochemical pathways ofmetabolism. While this can be a challengingexercise, the power of understanding howcellsbehave at the molecular level under a variety


of conditions will provide critical informationfor designing better cell hosts in the future.

3. DOWNSTREAM PROCESSING

3.1. Overview of Downstream Processing

The production of protein therapeutics withliving cells, such as bacteria or mammaliancells, poses different challenges for purifica-tion than small molecule NCEs (new chemicalentities) made using organic synthesis. Impu-rities such as solvents or isomers found in themanufacture of an NCE are “inherent” to theprocess. Impurities found during the produc-tion of biopharmaceuticals are more varied incomposition and come from many sources,including the host itself. An added complica-tion is that the protein product is susceptibleto environmental conditions such as heat andpH, and canbecomeunstable either physically(e.g., denaturation and aggregation) or chemi-cally (e.g., deamidation).

The use of bioseparation for isolation of thetarget protein from a fermentation broth orcell culture supernatant [98] is called down-stream processing. Thematerial produced up-stream is generally dilute and contains theprotein in low concentrations (e.g., in a mam-malian system, the product may only be pre-sent at a concentration of 1 g/L of cell culturefluid). The majority of cell culture fluid is anaqueous solution containing undesirable dis-solved components such as soluble proteinsfrom the host cell, imperfect forms of the pro-duct (e.g., aggregates), lipids, nucleotides, cellculture reagents, bacterial endotoxins, andother components. Downstream processingmust successfully remove these impuritiesand contaminants, including a significant por-tion of the water, to render a concentratedfinal product at the desired purity. Typicallythe final product is formulated at about10–100 g/L, corresponding to a 10–100-foldincrease in concentration. Viral inactivation/removal stepsarealso required to ensurea lowrisk of viral infectivity formammalian derivedproducts [4]. Additional key attributes of asuccessful downstream process include scal-ability, regulatory compliance, and favorableeconomics. Most importantly, the processshould be robust and be insensitive to small

perturbations [99]. A variety of separationtechniques are used for purification. Chiefamong these are centrifugation and filtrationfor primary (crude) separations, chromatogra-phy, and membrane filtration for polishingoperations (fine separations). Together, chro-matography and filtration membranes enablethe downstream scientist to provide purifiedproduct at the target concentration and for-mulation conditions, and with the necessaryproduct quality [100].

Some separation techniques play a key rolein both analytical and purification processdevelopments. In its analytical role, a separa-tion technique is used to quantify and char-acterize theprotein of interest. In purification,the chief aim is to isolate and purify the pro-duct. This difference in the goal often leads tovery different operating conditions for thesame separation technique. As an example,the chromatographic process used to charac-terize a protein employs a smaller column,typically 2–5mm in diameter, with an empha-sis on high resolution and high throughput. Inpreparative or process chromatography, thecolumn sizes are much larger, ranging from30 cm to 2m in diameter, and the focus is onmaximizing product recovery at a desired pur-ity level while minimizing cost.

A good purification process must producethe final product at the desired purity andconcentration, and be cost efficient. Studiesindicate that downstream costs can be as highas 80% of the total production costs [101–103].An increase in cell culture titer will shift thiscost even further to downstream proces-sing [4,104]. An efficient downstream processbecomes crucial at this stage, underscoringthe importance of continuous processimprovements [105].

3.1.1. Typical Impurities and Contaminants Amore complete description of the classes ofimpurities that must be separated from therecombinant therapeutic protein is summar-ized in Table 1. Impurities can be classified asthose that are process related versus thosethat are product related. Process related im-purities typically require clearance to pre-scribed levels that are basedonproduct safety.These include large cell debris and fragmentsthat are removed during primary separation

DOWNSTREAM PROCESSING 301

processes; DNA derived from host cells; pro-teins that are encoded and coexpressed by thehost cell; material leached from chromatogra-phy resins; reagents that are added asprocessing aids such as antifoam or mediacomponents; viruses that are potential con-taminants in animal cell cultures, and can beinadvertently introduced via raw materialsources [106]; endotoxins introduced either byE. coli derived fermentation systems or byinadvertent bacterial contamination duringprocessing; and salts and buffers.

Product related impurities are those thatoccur as a result of physical or chemical mod-ification of the primary species in the product.Whether these species are considered as im-purities or simply product variants dependson their impact on product safety and efficacy.For example, a product isoform that impactsrelative potency and pharmacokinetic profile

would be considered an impurity whereas achemically modified species that does notmeasurably impact product performance maynot. Product impurities require removal,whereas product variants or isoforms requirethorough characterization. Some potentialproduct related impurities or variants includeaggregates and protein particles that are un-desirable; glycosylation variants such as al-pha-galactose (a glycan linkage found in ani-mal cells that is immunogenic in humans) andothers that may impact product circulationhalf-life; misfolded forms of the protein thathave the same primary sequence as the pro-duct of interest, but do not have the correctsecondary and tertiary structure; structuralisoforms that may occur naturally or as aresult of improper disulfide bridging; andother posttranslational modifications includ-ing deamidation, oxidation, cyclization, and

Table 1. Common Impurities Encountered During Protein Process Development That mayRequire Removal and/or Analytical Assay

Process RelatedImpurities

Purification Modalities Analytical Assaya

Cell debris and fragments Depth filtration and centrifugation VariedDNA Ion exchange (IEX) chromatography,

flocculationQPCR, others

Host cell proteins IEX chromatography, affinity,hydroxyapatite (HA)

Immunoassay

Protein A IEX chromatography ImmunoassayProcess reagents

(e.g., antifoam, mediacomponents, etc.)

Varied Varied

Virus Low pH inactivation, nanometerfiltration, chromatography

Varied, transmissionelectron microscopy (TEM)

Endotoxin IEX chromatography Limulus amebocyte lysate (LAL)Salts, buffers Ultrafiltration/diafiltration (UF/DF) Liquid and gas chromatography

Potential product related impurities

Aggregates and proteinparticles

IEX chromatography, hydrophobicinteraction (HIC), HA,precipitation

Chromatography and otherbiophysical methods

Glycosylation variants IEX chromatography ChromatographyMisfolded forms

of the proteinHIC, IEX chromatography Varied

Structural isoforms Refolding, chromatography (various) Varied biophysical methodsPosttranslational

modifications (e.g.,deamidation, oxidation,clips etc.)

Chromatography (various) Mass spectrometry and otherbiochemical methods

aSee Section 5 for discussion of analytical methodologies.


others. The topic of product characterizationwill be discussed in greater detail in latersections of this chapter.

3.1.2. Design of Downstream Processing Agood purification process must take into ac-count properties of the target protein, proper-ties of impurities, and contaminants (bothproduct related and nonproduct related), scal-ability, and process fit into a manufacturingfacility. An immense number of possible com-binations [107] exist, but heuristics can beused to provide a good starting point suchas [98]

. perform the crude separations first,

. understand the properties of the mole-cule (e.g., the protein’s pI will decidewhich pHwill be best for separation (pro-pensity to aggregate, etc.),

. lower the volume of the feed stream asearly as possible,

. use high-resolution separation steps aslate in the process as possible, and

. use orthogonal steps to maximize theseparation ofmoleculeswith diverse phy-sical and chemical characteristics (e.g.,affinity, ion exchange, and hydrophobicinteractions).

3.1.3. Key Classifications in DownstreamProcessing Downstream processing is gener-ally classified as primary recovery (alsoknown as harvest operations) and secondaryrecovery (also known as purification or polish-ing steps). The objective of primary recovery isto bring the feedstock into a state suitable forthe application of more refined purificationmethods by removal of cells and cell deb-ris [108]. The distinction between primaryrecovery and purification is at times unclear(e.g., when highly discriminating bioaffinitymethods such as protein A chromatographyare used to selectively extract the productfrom a crude feedstock). However, even inthese applications primary recovery is re-quired to prevent unrefined feedstock fromfouling costly bioaffinity materials. Polishingsteps take the product stream from the pri-mary recovery operation and isolate the pro-tein of interest using orthogonal techniques.

These operations determine the quality attri-butes of the final product (e.g., aggregate le-vels, product related impurities). The two keytechnologies used in this area are chromato-graphy and filtration membrane processes.

An important concern of primary recoveryis the removal of particulate materials thatare incompatible with downstream opera-tions. A most common method for removingparticulates of a defined size is filtrationthrough membranes of an absolute pore size.However, the soft and easily deformable solidsfrombiological production can create virtuallyimpermeable filter cakes leading to very lowcapacity on absolute filters. Open three-dimensional structure depth filters are wellsuited for clarification of biological feedstocks.However, they can become overwhelmed bythe debris from high-density cultures typicalof today’s processes. Centrifugation and mi-crofiltration have therefore been adopted inbiotechnology to perform an initial removal ofcellular solids [109]. In these operations, thefragile nature of cells poses a challenge as themechanical shear generated tends to micro-nize a portion of the cells. In microfiltration,mechanical stress caused by repeated recircu-lation of the retentate can lead to rapid foulingof the microfiltration membranes. Most mam-malian cell-based processes therefore employcentrifugation, where shear forces causedby rotational velocity of the unit will causegeneration of fine particles, butwithin a rangethat allows subsequent removal by depthfiltration.

The most common approaches used forpurification and polishing are chromatogra-phy and membranes. Process chromatogra-phy, in particular, continues to be the work-horse and forms the basis ofmost downstreamoperations in biopharmaceutical produc-tion [110]. Chromatography (termed in 1906by Mikhail Tswett) [100] literally means“color drawing” [111] since it was originallyused for separation of natural pigments onfilter paper. From this relatively humble be-ginning, chromatography has become the cen-tral technology for both protein characteriza-tion and downstream processing. The wideuse of chromatography is due to the largevariety of stationary phases or chromato-graphic supports available. For themost part,


these can be broadly categorized as eitheraffinity or nonaffinity approaches [112].

Affinity chromatography refers to the spe-cific interaction of a ligand to a well-definedsite on the protein [112]. This is usually anexcellentmethod to effect separation since theinteractions are specific and strong. However,there are trade-offs in terms of high cost of theligand and, in some cases, availability of thespecific ligand at commercial scale. The mostcommon use of affinity chromatography intherapeutic protein production is the use ofprotein A for the purification of monoclonalantibodies and Fc fusion proteins.

Nonaffinity techniques include a host ofchromatographic modes such as ion exchange,hydrophobic interaction, hydoxyapatite,metalchelate, and size exclusion chromatographythat use awide range of stationaryphases. Thethreemost commonof thesearedetailedbelow.

Ion exchangers (IEX) are themost commonadsorbents used inprocess chromatography inbiotechnology. They exploit the net charges ofthe protein at a given pH. If the operating pHis below the pI of the protein, the protein has anet positive charge and vice versa. The mostcommon ion exchangers are anion (positivelycharged support) and cation (negativelycharged support). Within these categories area variety of subcategories (e.g., weak cationexchangers, strong cation exchangers, etc.). Ingeneral, in a bind and elute mode, the proteinis loaded under low salt (conductivity) condi-tions resulting in its binding to the stationaryphase, and is then eluted using a higher saltconcentration. The choice of salt, conductivityand pH of the operation are critical para-meters when optimizing these separations. Ina “flow through”mode, the protein of interestflows through the columnwhile impurities areheld behind. In general, the throughput(amount of protein purified per time per literof resin) is higher for the flow through modebut resolution may not be as good as the bindand elute mode.

Hydrophobic interaction chromatography(HIC) exploits the interactions of hydrophobicpatches on the surface of the molecule withhydrophobic adsorbents. In analytical chro-matography and in small molecule separa-tions, reverse phase chromatography is fre-quently used, where the interaction of the

product with the stationary phase is so strongthat it requires organic solvent to elute theproduct from the column. These are typicallyharsh conditions for protein isolation andmaycause irreversible loss of activity. In proteinpurification, the use of HIC is performed atmore gentle conditions. The protein is typi-cally adsorbed on the stationary phase usinghigh salt conditions and is eluted off the col-umn in a decreasing salt gradient. Just as inIEX, HIC can be performed in either bind andelute mode or flow through mode.

Hydroxyapatite chromatography (HA) orceramic hydroxyapatite (CHT) uses calciumphosphate as both the ligand and the basematrix. CHT is a mixed-mode support withfunctional groups consisting of calcium, phos-phate and hydroxyl groups (Ca5(PO4)3OH)2.In a bind and elutemode, the protein is loadedat or near neutral pH in a low ionic strengthphosphate buffer and eluted with a higherphosphate buffer or salt (depending on theseparation mechanism). CHT is generallyused to decrease the level of aggregate, DNA,host proteins and endotoxin.

The chromatography system may be oper-ated in a number of ways. The most commonare step gradients and linear gradients, wherethe material is bound to the column at condi-tions where it is held by the chromatographicstationary phase, and then eluted using anappropriate buffer (e.g., higher salt, change inpH, etc.), removing the protein of interest fromthe column. There are other modes such asdisplacement chromatography [113], butthese are less commonly used in commercialprotein production.

3.2. Protein Purification in E. coli Systems

Heterologous protein accumulation in E. colisystems often appears as inclusion bodies(IBs) due to the high expression levels andinability of bacterial cells to properly fold re-combinant human proteins [114–116]. If pro-tein renaturation is simple, IBs offer a sourceof highly concentrated product and can pro-vide an excellent starting point for a verystraightforward downstream process. On theother hand, problematic protein renaturationcan make downstream process developmentdifficult [117].


There are excellent examples of proteinfolding techniques and advances in thisarea [114,115,117–121]. De Bernardez Clarket al. [122] suggest the low yields across therefold step may be due to formation of inac-tive misfolded species, particularly aggre-gates, and the authors detail various techni-ques to “inhibit aggregation side reactions toensure efficient in vitro protein folding.”Cowgill et al. [123] present a practical intro-duction to protein refolding anddiscuss scale-up effects. They also discuss the emergingand promising technology of high pressurerefolding. Another good review on this topic isthe publication by Sahdev et al. [124], wherethe authors discuss soluble protein expres-sion. It must be noted, however, that findingthe optimum conditions for correct proteinrefolding is still relatively empirical and con-sidered somewhat of “an art.”

The quality of the IBs is critical in overallprocess yield and product quality. Wong et al.[125] have shown that maximizing the IBrecovery during centrifugation does not guar-antee high overall yield. The contaminantspresent in the IB “paste” can lead to a loweryield, depending on the nature of thecontaminants.

Once the protein is refolded, it undergoeschromatographic purification to remove bothproduct-related impurities (misfolds andothers) and contaminants (e.g., host cell pro-teins). A typical flow chart describing purifi-cation of a protein from an E. coli host isdetailed in Table 2.

Table 2 is only one example of a processpath that can be used for purification of aprotein from IBs. There are many exampleswhere a different strategy has been employedto obtain the final product produced in an

Table 2. Typical Flow Chart Describing Purification of Protein from E. Coli

Purification Process Description

Cell productintermediate (CPI)

Washed Inclusion Bodies

#Solubilization Dissolution ofwashed inclusion body cell product intermediate (CPI) to release the

desired protein into solution. Denaturation of the desired protein to eliminateelements of secondary, tertiary, and quaternary structure. Reduction of proteindisulfide bonds to ensure desired protein is fully denatured and present inmonomeric form.

#Refold Renaturation of the desired protein to achieve the correct secondary, tertiary, and

quaternary structure. Typically the most chemically complex operation in thepurification process but necessary to eventually produce biologically activeprotein from inclusion bodies.

#UF/DF Concentration of the relatively dilute clarified refold solution. Reduction of low

molecular weight solutes derived from CPI. Reduction of low molecular weightchemical components that would inhibit desired precipitation of impurities.

#Precipitation Species separation based, predominantly, on solubility. Condensation of impu-

rities into relatively large precipitates that readily sediment during centrifu-gation or filtration enabling their removal.

#Capture column

(chromatography -1)Volume reduction and gross removal of DNA, host protein, and product related

impurities#

Polishing columns(generally 2)

Removes DNA, host proteins, and other product related impurities to deliverproduct at desired purity.

#Formulation UF/DF Concentrate and buffer exchange the protein to formulation buffer.


E.colisystem.Asanexample,Khalizadehetal.[126] discuss an approach for purification ofrecombinant human interferon-g expressed inE.colithatinvolvespurificationoftheproteinina denatured state over two columns and a finalcolumn purification after the refold step.

3.3. Protein Purification in MammalianSystems

Monoclonal antibodies are the most commonclass of therapeutic proteins expressed inmammalian systems. These molecules sharecommon features such as framework of theFab region and the Fc region. Due to structur-al similarity, many companies are adoptingplatform approaches to process developmentand manufacturing, applying a predefined se-quence of unit operationsandanalyticalmeth-odologies to multiple molecules. For example,monoclonal antibodies bind with high specifi-city via the Fc region to proteinA derived fromStaphylococcus aureus. This property isexploited byemployingproteinA impregnatedresins resulting in one the most powerful pur-ification steps in monoclonal antibody proces-sing. This step provides high specificity andhighyieldand forms thebackboneof thedown-stream platform process approach.

While the isoelectric points (pI) of antibo-dies generally vary from slightly acidicthrough the basic region, polishing steps cangenerally be developed using ion exchangechromatography. Other polishing options thatlend themselves to a platform approach in-clude hydrophobic interaction chromatogra-phy and hydroxylapatite.

A platform approach to process develop-ment does not mean that the purification pro-cesses will be identical for all antibodies. Theproperties of the molecule are varied enough(different pI, hydrophobic nature, etc.), thatthe same purification process may not workacross multiple molecules, andmay not be theoptimum process. The platform is used todefine the overall scheme, provides ranges ofoperating conditions for each unit operationand limits the experimentation required todevelop the final process.

3.3.1. Harvest Operations Themost commontechnique used in the harvest step of mam-

malian cell culture processes is the use ofcentrifugation for cell removal [108]. Centri-fuges exploit the density differences betweenthe solid particles, the surrounding liquidmedium, and the centrifugal force to achieveseparation. Althoughmany large-scale centri-fuges are available, the most common aredisk-stack separators, which have proven sui-table for clean-room operation and have suffi-cient clarification performance to removesmaller sized cell fragments. An added benefitis the ability of disk-stack centrifuges to re-move solids from the centrifuge bowl eithercontinuously or intermittently, allowing un-interrupted clarification of large volumes ofcell culture feed in relatively short timeperiods.

The performance of the centrifuge is deter-mined by the interaction of multiple factors.The most common operational control para-meters are rotational velocity of the bowl,residence time, and feed interval. In addition,the centrifuge design itself (e.g., pump type,back pressure control, mechanism of dis-charge, etc.) can affect the separation perfor-mance. Perhaps, the most important factor ishow the feedstock fluid is accelerated as itenters the bowl. This is a complex hydrody-namic problem that is influenced by feed zonedesign, bowl speed and the feed flow rate. Allof these factors affect the degree of cell dis-ruption that can result in the generation ofparticles too small to settle in the centrifuge.

Centrifugation is a good primary separa-tion step due to its rapid, crude separationability, but is not the complete harvest solu-tion. Typically, small particles that escape thecentrifuge must be removed by a subsequentdepth filtration step. In fact, the depth filter iswhere purification scientists often experiencedifficulty (e.g., high filter areas required), evenif the cause lies upstream in the process.

It is difficult to develop good scale-upand scale-down models for centrifuges. Sheareffects can cause large deviations from theo-retical centrifuge capacity and the changes insuch effects are little understood during scal-ing.Furthermore, the role of the cell culture oncentrifuge performance is not well under-stood, but is likely significant.Anongoingareaof research is in understanding scale-downperformance of centrifuges [127,128].


One way to address harvest capacity is bychanging the character of the debris (i.e., par-ticle size). The use of flocculants has a longhistory in diverse industries such as waste-water, chemicals and food. This technology isnow being applied to the cell culture harvest.Use of flocculants can result in a range ofperformance changes to the process; thesevary from being a “patch” on an existing pro-cess to significant purification of impuritiesnot previously affected during recovery (e.g.,virus,HCP, and aggregates). Chitosan (a poly-cationic linear polymer) has been shown toimprove clarification throughput of a large-scale cell culture harvest without negativelyimpacting mAb recovery or purity [129].

Tangential flow microfiltration (MF) is an-other technique used for clarification of mam-malian cell culture [108]. An advantage overcentrifugation is a particle-free harveststream that requires minimal additional fil-tration prior to further processing [130]. Fineparticle generation during MF is no worsethan during centrifugation, but leads to fail-ure of the operation by fouling the MF mem-branes. The situation is exacerbated in thatthe usualmode of overcomingmembrane foul-ing, increasing the cross-flow velocity to sweep

the membrane free of debris, only amplifiesthe problem by increasing the intensity ofshear and hence particle generation.

A depth filter is a porous medium that canretain particles throughout its matrix [108].While the primary mode of action of depthfiltration is to trap particles, many modernfilters have been shown to have an additionaladsorptive capability, useful for host cell pro-tein and DNA reduction. Therefore, depthfiltration could potentially combine filtrationfor particulate removal with adsorptive bind-ing for the removal of contaminants [109].Some understanding exists in the use of ad-sorptive properties of these depth filters, butthat knowledge remains largely empirical atpresent and further development is required.

3.3.2. Polishing Steps Table 3 describes acommon process used for purification ofmonoclonal antibodies. The development, op-eration, and validation of chromatographicprocesses used in monoclonal antibody puri-fication at industrial scale has been recentlyreviewed byFahrner et al. [131]. In this paper,the focus is on a commonly used three-columnpurification (protein A, followed by cation ex-change and flow through anion exchange).

Table 3. Typical Process Used for Purification of Monoclonal Antibodies

Purification Process Description

Cell productintermediate

Harvested cell culture supernatant. The supernatant has undergone primarypurification through centrifugation and depth filtration. Removal of cells andcell debris. Some impurity removal on the depth filter.

#Protein A Affinity chromatography.Able to obtain highpurity (>95%) and lower volumes (up

to three to ten times concentration) across this step. Gross removal of DNA andhost cell protein.

#Viral inactivation Typically done at low pH; inactivates enveloped viruses such as XMuLV.

#Depth filter For clarification of feed stream in preparation for downstream polishing chro-

matography steps.#

Polishingchromatographystep 1 and step 2

An example is CEX chromatography (bind AND elute) and AEX chromatographyflow through [131]. Reduction of DNA, host cell proteins, leached protein A,aggregates, and process reagents as well as viral clearance.

#Viral filtration Typically a parvovirus filter (�20nm). Removes viruses, both large (XMuLV) and

small (MMV).#

Formulation UF/DF Concentrate and buffer exchange the protein to formulation buffer.


ProteinAaffinity chromatography is a verypowerful first step for thepurification ofmono-clonal antibodies. The use of protein A as theligand is common for this class of proteins dueto its affinity for the Fc portion of antibodies,with an affinity constant KD reported to be70nM [4,132]. Product purities of >95% (bySEC-HPLC) are possible after just one passacross this column.

Affinity of protein A for antibodies func-tions across a broad pH range, and conductiv-ity is not a critical parameter; therefore, load-ing the column directly from the cell culturefluid is possible [4,133]. A benefit of the high-affinity interaction is that one can employ abroad range of washing conditions to ensurehigh purity of the eluted product. These washsteps can have the dual advantage of increas-ing product purity aswell as extending the lifetime of these expensive resins [134]. In gen-eral, the product is eluted off the columnusingan acidic pH (typically 3–4). The column isthen stripped (acidic solution around pH� 2)and regenerated.

A variety of protein A resins are availablefor commercial use. Hober et al. [135] detail, asummary of the most commonly used proteinA affinity media in antibody production. Re-sins differ in their backbone chemistry, thetwo commonly used base matrixes are poly-mer (agarose based) and silica glass (con-trolled pore glass).

Due to the nature of the protein-basedligand, the protein A resin cannot be regener-ated using harsh cleaning agents, potentiallyimpacting the lifetime of the resin. Recent ad-vances have provided more base-stable resins,which allows for cleaning using caustic solu-tions, such as 0.1–0.5M NaOH [134,136,137].

Although the protein A step is extremelypowerful, it is not without development chal-lenges. The need to elute the columnat lowpHmay induce aggregation in some mAbs. Shuk-la et al. [132] provide data from 14 differentmolecules that indicate problems of aggrega-tion and precipitation occur frequently duringprotein A chromatography. It is also impor-tant to ensure that leached protein A from thecolumn is captured and removed in the sub-sequent purification steps [138]:

Although the product from the protein Astep has a purity of>95%, additional steps are

required to achieve target purity. Additionalchromatographic steps are generally requiredfocused on reducing host cell protein impuri-ties, aggregates, clipped species, DNA, lea-ched protein A, and other contaminants.

These column steps are generally chosenfrom unit operations such as cation exchange(CEX) chromatography, anion exchange(AEX) chromatography, HIC, and less com-monly, HA. A common process used in mAbproduction is the use of a cation exchangecolumn followed by an anion exchanger [139].Pete Gagnon [140] has detailed the variouspolishing methods available for mAb purifica-tion.Typically, one of these steps are run inflow through mode (where the product doesnot bind to the resin but flows through, whilecertain impurities are retained behind).

While similar, monoclonal antibody pro-ducts are, of course, not identical. Specificchromatography resins and operating condi-tions are chosen based on intrinsic moleculeproperties and impurities to be separated.Differences in subclass and variable regionsequences contribute to variations in molecu-lar properties (e.g., charge, hydrophobicity,and other heterogeneity) that will impact pur-ification process design [141]. A cation ex-change step, for instance, can be used fordepletion of HCP and leached protein A, butwill not be as efficient as HIC for aggregateremoval.

Viruses are potential contaminants ofconcern in animal cell cultures, and can beinadvertently introduced via sources such asserum-derived raw materials, contaminatedproteins added to the nutrient broth, infectedproduction cell lines, or accidental contamina-tion during bioprocessing. The downstreamprocess must ensure that these potential con-taminants are effectively cleared by removaland inactivation [106]. Even in caseswhere novirus is introduced, manufacturers still needto demonstrate through validation that theprocess has significant capability to removeboth enveloped and nonenveloped viruses.

The FDA Q5A guidance document [142]requires “demonstration of the capacity of theproduction process to inactivate or removeviruses” in order to assure safety of productsproduced by mammalian cell culture. Theguidance document mentions that effective


clearancemaybe obtained froma combinationof inactivation and separation steps.

Chromatographic steps used in the down-stream process each provide some degree ofviral clearance. For example, greater than 4logs of retroviral and parvoviral clearancehave been achieved for monoclonal antibodiesby use of AEX chromatography flow throughsteps [132,143]. In addition, the process alsotypically includes specific viral clearance bylow pH viral inactivation and nanometer fil-tration. The depth filter step in the processalso results in some viral clearance. Althoughthis is currently not recognized by regulatoryagencies as a robust orthogonal method forviral clearance, it does provide an additionalsafety margin [144].

Viral inactivation steps must ensure a per-manent reduction of viral infectivity [4].Manymethods are possible to effect viral inactiva-tion—chemical (lowpH), heat,UV irradiation,etc. A comparison of different techniques usedis detailed in other reviews on this to-pic [4,145]. Due to its operational simplicity,low pH (acid) inactivation is the most com-monly used technique in antibody purifica-tion, the mechanism of inactivation is mosteffective against large enveloped viruses.

The most robust unit operation currentlyused to remove viruses is size-based nan-ometer filtration. It has been shown to bescalable and robust [146]. Typically, 50 and20nm pore sizes are used in the process—although the smaller pore size is being in-creasingly used due to its ability to removeboth large viruses (e.g., x-MuLV) and smallvirus particles (e.g., MMV) [144].

The capability of each unit operation isvalidated by viral clearance studies using ap-propriate scale-down models. In general, pro-cess validation is performed in the commercialmanufacturing facility. However, for safety,financial and technical reasons, viral clear-ance validation is performed in small-scalemodels (�1/1000 scale) taking care to ensurethat relevant parameters are appropriatelyscaled to those used at full-scale produc-tion [147]. Model viruses that can be detectedand quantified are used to characterize thecapacity of the downstream process to clearadventitious viral agents. Xenotropic murineleukemia virus (x-MuLV) and murine minute

virus (MMV) are common model viruses usedto test the viral clearance capacity of each unitoperation [144]. Zhou and Tressel [148] re-viewed the operations for viral clearance inmAb downstream processing operations, in-cludingviral clearance strategiesused in earlystage (or Phase I processes) to commercial(biological license application) filings.

After purification, the product is deliveredin the final formulation buffer by buffer ex-change [132]. This is typically done by usingUF/DFmembranes [149]. Several parametersare important in the development of this stepincluding the membrane type, the transmem-brane pressure (TMP), cross-flow rate, finalproduct concentration, etc. In the past, finalproduct concentrations were lower (typicallyless than 30mg/mL), but concentrations areincreasing to 70–150mg/mL. This has impli-cations for solubility and viscosity of proteinsand needs to be dealt with on a product-by-product basis. As an example, salts may beused to decrease the viscosity in some cases,but may not be an optimum solution in allinstances.

3.4. Future Trends

Typically, downstream scientists develop theprocess at bench-scale by using either batchbinding experiments or small chromato-graphic columns of �1–2 cm in diameter (or�5–15mL column volume). This is, by neces-sity, a batch operation and limits the totalnumber of data points that can be obtainedduring development. Advances in instrumen-tation technology as well as use of platformconditions allow for high-throughput purifica-tion development. Industry uses high-throughput 96-well plate formats for rapidpurification development using a minimum ofresources [150–153].

Tomake therapeutic protein at commercialscale the downstream process should be easilyscalable. The process should also be capable ofutilizing the facility in the most optimumwayto maximize asset utilization and lower costs.While this is a strategic business objective, thesolution will come from science and engineer-ing. As an example, processes that utilize aminimal number of buffer tanks at lower vo-lumeswith faster cycle timeswill contribute to


lower costs and smaller capital investment.The term “process intensification” is definedas the ability of the process to utilize theproduction facility in the most optimumman-ner. A specific example would be the develop-ment and use of a resin that supports highercapacity and can operate at higher flow rates.

Another example of process intensificationwould be the use of a two-column processversus the standard three-column process.The two-column process reduces rawmaterialcosts, supports a smaller plant footprint, andwill utilize less pieces of capital equipmentsuch as buffer tanks [154]. In addition, havingfewer unit operations allows for fewer errorsand improved success rates. Finally, the use ofa two-column purification process may enablean easier semi-continuous (tandem process)approach. There are several examples of suc-cessful application of the two-column ap-proach [141,155,156] “Connected processing”or tandem operation is one possible futuredirection. This is the connection of two con-secutive unit operationswithout an in-processpool and/or tank between the two. This hasbeen demonstrated in bothmicrobial andmAbpurifications [157].

In microbial systems, a key area of focus isthe protein refold or renaturation step. Thisunit operation is not only a critical part of thepurification process but also one of the leastunderstood and modeled areas. Research onincreasing refold concentrations and yields[158,159] is necessary to make microbial sys-tems more productive and to decrease cost ofgoods manufactured (COGM).

Protein A has been well established as theaffinity resin of choice in mAb purification.However, the technology development aroundthis resin isnot stagnant.Many companiesareengaged in research to improveproteinAresinas well as develop alternatives to this affinityadsorbent. There is on-going work to develop“protein A mimetics” that are synthetic li-gands that mimic the interactions of the pro-tein productwith thenatural ligand [112,160].The use of more conventional resins, such asion exchangers and mixed mode resins, hasalso been utilized as the first step in mAbpurifications [154,160].

There have long been arguments that thecost of purchasing and operating protein A

resin is high [161]. While costs may be moresignificant for early stage programs the selec-tivity and platform capability that protein Aoffers is essentially unmatched. Base stableresins and development of effective columnregeneration processes commonly allow thesecolumns to be used for>100 cycles in commer-cial applications, significantly reducing theper batch cost. For the time being, protein Aremains the resin of choice as the capture stepfor most monoclonal antibody purifications.

Techniques such as simulatedmoving beds(SMBs) [162] and sequential multicolumnchromatography (SMCC) have the potentialto utilize lower solvent (buffer) consumptionand deliver higher productivities. There isresearch ongoing for the extension of thistechnology in bioprocessing, especially in thecapture step of mAb purification. This tech-nology evaluation is in its early days andneeds to be evaluated both at lab-scale andduring scale-up.

Disposable technologies have long been ofinterest in the industry as they can providecost and operational efficiency without clean-ing, lifetimeand storage validations. Themostimpactful benefit is the use of disposables toincrease the rate of equipment turnaround inhighly scheduled plants. While single-use dis-posable systems are familiar in filtration, theyare not common in packed-bed chromatogra-phy. A recent area of interest is membraneabsorbers that may be used in a single-passapproach [163].

A popular alternative to ion exchange chro-matography is membrane chromatography inflow through mode. The use of this technologyhas been demonstrated in mAb purifications.It isa rapid, cost-effectiveunit operation that iseasy to scale up [104,164]. In contrast to beadchromatography, the binding capacity of thesesystems is independent of flow rate sincemem-branes arenot diffusion limited [130,165]. Thistechnology is still under evaluation in terms ofits overall benefits versus costs compared tothe packed bed approach. An advantagewith single-use membrane chromatographyis reduced development and validation costssince there is no column packing or cleaningvalidation involved [166]. A more detailedevaluation of this technology is providedelsewhere [163,166,167].


While chromatography has been the work-horse inpreparative separations, it does sufferfrom certain limitations (e.g., resin availabil-ity, column packing, batch operations, etc.).There has been significant research in alter-nate bioseparation operations [164,168].How-ever, at least for recombinant antibodies, thecurrent platform is capable of production of upto 10 tons/year [155]. A paradigm shift towardroutine use of nonconventional approaches formonoclonal antibody purification in the nearfuture is unlikely.

There is an increasing need to deliver high-concentration formulations. As an example,there are monoclonal antibody products beingformulated at >150 g/L. This represents agreater than fivefold increase in concentrationfrom just a few years ago. This trend towardhigh concentration causes significant chal-lenges to downstream processing with respectto solubility, filter capability, particle forma-tion, and yield optimization. Collaborationwith formulation and manufacturing isneeded to understand and solve these issues.

4. FORMULATION AND DELIVERY

4.1. Introduction

Protein drug formulation is a challenging andtime consuming process. The formulationscientist is faced with the requirement of sta-bilizing a protein pharmaceutical in an accep-table liquid or lyophilized formulation for sto-rage periods of up to 2 years. This long termstabilitymust be achieved despite the stressesthat are exerted on the protein during manu-facturing (e.g., copurification with enzymesand other destabilizing compounds, and shearstresses exerted on the protein by vial fillingoperations), shipping and handling (e.g., agi-tation and temperature excursions), storage(e.g., temperature, light, excipient/buffer andpH exposure), and delivery (e.g., dilution andshort term exposure within an IV bag). Theformulated protein must also be compatiblewith a primary container over this storageperiod. These contact materials (typicallyglass vials or syringes) introduce concerns forprotein adsorption and denaturation, and canalso potentially release extractables andleachables such as rubber stopper monomers

and initiators, ions from glass materials, andsilicone coatings. The formulation scientist isalso challenged by the fact that while candi-date formulations may be decided based ondata collected from accelerated storage condi-tions, such as incubation across a range of pHsolutions or exposure to temperature ex-tremes over a relatively short period of time,only real-time processing and storage can con-firm the stability of a final protein formula-tion. These facts can lead to extended time-frames for formulation development relativeto other process development activities. Re-view articles and book chapters have dis-cussed general considerations for protein for-mulation development [169], and more recentliterature has focused on the formulation ofmonoclonal antibodies [170–172].

4.2. Protein Degradation Pathways

The biological activity of proteins is defined bytheir primary, secondary, tertiary, and qua-ternary structure. Changes in any of theseparameters may lead to diminished productpotency, altered pharmacokinetic behavior, orpotential increases in immunogenicity. Dur-ing manufacture and long term storage, pro-teins can degrade by chemical and/or physicalpathways. Early reviews in the literature de-scribe a range of physical and chemical degra-dationmechanisms that need to be consideredwhen formulating proteins [173–175]. Themost likely pathways for degradation of aparticular protein cannot be generalized orpredicted in advance. Rather, degradation de-pends on the specific amino acid sequence andstructural characteristics of the protein phar-maceutical that requires probing for potentialdegradation “hot spots”.

Chemical degradation of proteins can beclassified as either covalent or noncovalent.Covalentdegradationpathwaysthathavebeendescribed in the protein formulation literatureinclude isomerization and deamidation,succinimide formation, oxidation, cyclization,photodegradation, cysteinylation, glycation,disulfide scrambling, hydrolysis, beta-elimina-tion, carbamylation, and enzymatic degrada-tion from proteases copurified in the drug sub-stance. The primary noncovalent degradationpathways for proteins include denaturation,

FORMULATION AND DELIVERY 311

aggregation, and precipitation. Several of themore common degradation pathways will bediscussed below and summarized along withpotential formulation remedies in Table 4.

Deamidation and isomerization resultfrom the hydrolysis of side-chain aminegroups from asparagine or glutamine aminoacid residues. The chemical reaction proceedsthrough a succinimide intermediate to thedeamidated or isomerized forms as dictatedby pH conditions and local amino sequence.The stability of reactive succinimide inter-mediates have been described in the formula-tion literature [176,177]. Deamidation andisomerization have been characterized ingreat detail, in part because they can havesignificant impact on protein activity, and inpart because they can be controlled by theselection of appropriate pH in the bufferingsolution (lower pH disfavors deamidation,whereas higher pH disfavors isomerization),or through the development of a lyophilizedversus a liquid formulation (low levels of re-sidual moisture disfavors deamidation) [178].

Early work from Patel and colleagues withmodel peptides showed the effect of aminoacid sequence on the rates of deamida-tion [179]. More recent work describes theimpact that local conformational flexibility,imparted by amino acid sequence, can haveon the extent of deamidation [180]. The factthat primary sequence can play a large role indeamidation/isomerization may impact deci-sions on selecting candidate molecules to de-velop as protein pharmaceuticals.

Oxidation of therapeutic proteins is alsoknown to occur during protein processing andunder long term storage conditions. Proteinoxidation can occur through several aminoacids, including methionine, tryptophan, tyro-sine, phenylalanine, and cysteine residues.There are several known catalysts for proteinoxidation including heat, photochemical en-ergy, and metal ions [181,182]. Researchershave found that the extent of exposure of labileamino acids can impact rates of oxidation [183]and that oxidation of amino acid residues canleadtoconformationalchangesinproteins[184]

Table 4. Some Common Protein Degradation Pathways and Potential Formulation Remedies

Degradation Pathway Primary Cause Potential Formulation Remedies

Deamidation Solution pH; localpeptide sequence

Lower formulation pH [178]; lyophilization [218].

Isomerization Solution pH; localpeptide sequence

Raise formulation pH [178]; lyophilization [218].

Succinimide formation Solution pH; localpeptide sequence

Neutral formulation pH [176,177];lyophilization [218].

Oxidation Exposure to oxygen,metal ions, solvents

Limit exposure to oxidants and catalysts duringprocessing and long-term storage [181,182];add antioxidants to formulation (e.g.,methionine) [185].

N-Terminal cyclization Solution pH Control upstream processing conditions; raiseformulation pH [186]; lyophilization [218].

Photodegradation Exposure to light Limit exposure to light; add antioxidants [187].Glycation Upstream processing

conditionsEliminate reducing sugars from upstream process

and formulation [190–192].Cysteinylation/adducts Upstream processing

conditionsControl upstream and downstream processing

conditions (e.g., nutrients, and redoxreagents) [188].

Aggregation Surface hydrophobicity Control processing conditions; addition ofsurfactants to formulation [197,206].

High concentration Addition of salt or other exdipients (histidine)to formulation [205]; Protein crystalformulation [247].

Denaturation Appropriate excipient choice [202,203].


thatmaylimitbioactivity.Knowledgeaboutthepropensity of a protein to undergo oxidationmay influence selection of a container closuresystemor lead to filling containerswith oxygendepleted headspace (both approaches are in-tended to limit exposure to oxygen), ormay callfor the addition of an excipient such as meth-oinine that is intended to serve as an oxygenscavenger [185].

One common instability known for antibo-dies (since most recombinant antibodies con-tain glutamic acid or glutamine at their N-terminus) is the cyclization of N-terminal glu-tamate residues forming pyroglutamate(pGlu) [186]. This reaction may occur duringcell culture production as a post-translationalmodification, or may occur during long termstorage in formulation buffer. Pyroglutamateformation is favored at pH 4 and 8, but is lesscommon at neutral pH. The impact of pGluformation on the biological activity of proteinsis likely limited due to the fact that this in-stability is localized on the N-terminus of theprotein in the antibody framework.

Photodegradation of proteins can also oc-cur as a result of light exposure during man-ufacturing or long term storage. Photoinst-ability of proteins may lead to degradationincluding photooxidation that can result inprotein backbone cleavage and other bypro-ducts. The major protein photodegradationpathways were recently reviewed by Kerwinand Remmele [187]. Knowledge of photoinst-ability of aprotein productunder developmentmay lead to storage recommendations thatlimit exposure to light.

Two other covalent degradation pathwaysfor proteins that can be induced by the man-ufacturing process include cysteinylation, andglycation. Cysteinylation occurs as a result offree cysteine binding to unpaired cysteineresidues on proteins (other adducts may alsobe formed through unpaired cysteine) [188].Structural changes induced by cysteinylationhave been shown to impact the rate of proteinaggregation and negatively impact bioactiv-ity [189]. These results suggest that cell cul-ture processes where free cysteine is added asa nutrient may need to be monitored andoptimized when producing proteins with freecysteines tominimize the potential for proteincysteinylation. Glycation is a condensation

reaction between the aldehyde groups of re-ducing sugars and the primary or the second-ary amines of proteins, primarily lysines andthe N-terminal amines of proteins. This mayoccur during cell culture processes that are fedwith glucose, or during long term storage ofproteins in solutions that contain reducingsugars such as sucrose [190–192].

Protein denaturation describes the pro-cess of unfolding of proteins, or perturbingthem from their native conformation. Dena-turation can occur as a result of stresses suchas shear forces applied during a vial or syr-inge filling process, exposure to a liquid–airinterface, or exposure to a polymer IV bag orglass vial surface [193]. Often protein dena-turation is a reversible process, however,under some threshold conditions the associa-tion of multiple denatured molecules mayultimately lead to protein aggregation, a pro-cess known as nucleation. Whether dena-tured proteins nucleate to form aggregatesor reverse back into properly folded mono-mers depends on solution conditions such astemperature, pH, protein concentration, salttypes and concentrations, excipients, surfac-tants, and other factors [194].

Protein aggregation is arguably the mostcommon and troubling degradation pathwayfor the process development scientist, encoun-tered in almost all stages of protein drug devel-opment. Numerous review articles have beenpublished describing fundamental under-standing of themechanisms by which proteinsaggregate [195,196]. Protein aggregation,along with other physical and/or chemical in-stabilities of proteins, remains one of themajorbarriers to rapid development and commercia-lization of potential protein biopharmaceuticalcandidates. One recent report describes theimportance of understanding where aggrega-tion occurs during protein manufacturing andprocessing and how these processes can becontrolled to reduce protein aggregation [197].Reports from the formulation development lit-erature have also detailed approaches to theprevention of aggregate formation during longterm storage. These approaches include thecontrol of solution pH, and the use of excipi-ents, salts, and surfactants [196,198].

Larger aggregates of proteins may evenform precipitates that may be visible to the


eye and sediment upon centrifugation. Visibleand subvisible precipitates and particles havebecome a topic of increasing interest. In arecent publication from Carpenter et al., theauthors suggest that particles may pose im-munogenicity concerns and that these parti-cles should be measured and characterized bybiopharmaceutical manufacturers [199]. Theconcern for monitoring particles in proteinformulations on the basis of immunogenicityis supported by some reports in the litera-ture [200,201]. Current particle counting in-strumentation in the subvisible size range(0.1–10mm) is limited and characterization ofthese particles will be challenged by the ex-ceedingly small proportion of protein masscontained within these particles at typicalbiopharmaceutical concentrations.

4.3. Stability Considerations

Typically, the formulation scientist works todevelop a stable protein formulation in stages.In the early stages of development, a periodoften called “preformulation,” some basicproperties of the protein are evaluated suchas primary sequence (looking for chemicalfeatures that may lead to degradation “hotspots”), isoelectric point, glycosylation pat-terns, size distributionpatterns, andpotentialstructural features that may present chal-lenges to protein stability. As a result of thisearly analytical evaluation, in later stages ofdevelopment accelerated and real time stabi-lity testing is conducted to further determinethe primary pathways of degradation. Theseaccelerated studies may involve temperatureor pH stressing, agitation, or other means toevaluate pathways of degradation.Knowledgeof these accelerated degradation pathwaysallows for testing of candidate formulations,which have been designed to reduce degrada-tion under real-time storage conditions. Final-ly, these candidate formulations must betested for functionality in manufacturing pro-cesses such as syringe or vial filling, and in thepatient delivery setting.

4.3.1. Liquid Formulations The formulationadditives or excipients available for stabiliza-tionofproteins includebuffers, bulkingagents,salts, surfactants, andpreservatives [202,203].

The formulation scientistwill payconsiderableattention to selecting appropriate pH levelsand buffer types during formulation develop-ment. Optimization of pH and buffer strengthhas been shown to significantly impact struc-tural and chemical stability of proteins. Inves-tigatorshavealso realizedthatwhileprovidingstabilizing effects, buffers can cause deleter-ious effects such as stinging upon injec-tion [204], and other stability issues duringfreezing (e.g., sodium phosphate can crystal-lize out of the protein amorphous phase duringfreezing resulting in large shifts in pH). Saltscan affect the physical stability of proteins byshielding charged species on the surface of theprotein, thereby impacting protein–protein as-sociations. Salts have also been shown to beuseful to reduce solution viscosity [205] andensure osmotic balance. Surfactants are oftenadded to protein formulations in order tomini-mize protein denaturation and aggregation atinterfaces [206]. Preservatives such as benzylalcohol have also been added to protein for-mulations to allow for multidose parenteraladministration, however, preserved formula-tions can be challenging to develop since pre-servatives may also destabilize proteins [207].

The stability of additives themselves mustalso be considered when selecting these addi-tives for formulation development. For in-stance, surfactants such as polysorbate areknown to degrade during storage [206,208].Histidine, when used as a buffer or additive isknown to oxidize and discolor on exposure tolight. Additives such as sucrose (used as astabilizer against freeze/thaw induced degra-dation) are known to undergo hydrolysis un-der acidic conditions.

Another challenge faced by the protein for-mulation scientist is the development of high-concentration formulations. This is especiallythe case for antibody therapeutics that mayrequire drug product concentrations ap-proaching or exceeding 100mg/mL. At suchhigh concentrations, unique formulation chal-lenges emerge that result in several manufac-turing, stability, analytical, and deliverychallenges [209–211]. High-concentrationchallenges include solubility limitations, buf-fering capacity limitations, concentrationdependent aggregation, andhigh solution visc-osity that may complicate manufacturing and


delivery by injection. Some solutions to theseproblems have been proposed including self-buffered formulations with antibodies [212],and histidine [213] or high salt containingformulations [214] to reduce solution viscosity.

4.3.2. Lyophilized Formulations Lyophilized(“freeze-dried”) formulations have also beendeveloped for protein biopharmaceuticals thatmay not be stable in liquid formulations forextended periods of time. Several recent re-views have summarized the challenges of de-veloping lyophilized formulations for proteinbiopharmaceuticals [215–218]. The develop-ment of a lyophilized formulation can be com-plex and requires sophisticated processingequipment; however, once developed, theseformulations canbeparticularly robust to longterm storage stability stresses. The lyophili-zation process consists of two phases: freezingof a protein solution and drying under va-cuum.The drying phase is further divided intoprimary and secondary drying. Primary dry-ing removes the frozen water and secondarydrying removes the nonfrozen “bound” water.The levels of residual moisture are monitoredfollowing the lyophilization process to assureappropriate dehydration. Excess residualmoisture allows excessive molecular mobilityand chemical degradation processes to occur,and inadequate residual moisture can disfa-vornativeprotein conformation [219].Rates offreezing, drying and the level of residualmoisture must be carefully controlled suchthat the protein retains its native conforma-tion throughout the lyophilization process.Lyophilized formulations typically contain abuffer, to optimize conformational stability ofthe protein, a cryo-protectant designed to pro-tect the protein during freezing (e.g., sucroseor trehalose), and a bulking agent designed tocreate a pharmaceutically acceptable cake (e.g., mannitol or glycine).

4.4. Interactions with Container ClosureMaterials

The selection of appropriate container closuresystems for biopharmaceutical products is im-portant, and depends to a large degree onpatient delivery issues as well as productcompatibility. Contactmaterials such as glass

and plastics may delaminate or leach follow-ing long term storage of formulations. Signifi-cant differences in glass characteristics andperformance are known among suppliers ofglass vial and syringe materials. Detailedforensic investigations of vial surfaces frommultiple suppliers have demonstrated dela-mination (flaking) and pitting in the presenceof a parenteral solution on some of thesesurfaces [220].

Proteins may also adsorb or denature oncontact with container closure materials, re-quiring the addition of surfactants to reducesurface interactions. For example, insulin hasbeen shown to be destabilized by adsorption athydrophobic interfaces [221]. Protein destabi-lization at hydrophobic surfaces is thought toresult from nucleation, that is, formation ofsmall intermediate aggregates that serve asprecursors to larger precipitates. Specialtyglass coatings have been developed to reducesurface hydrophobicity and limit product in-teractions [222]. Alternative materials havealso been developed to minimize protein bind-ing, (e.g., plastic CZ resin vialmaterialmay bea suitable candidate for packaging parenteralprotein formulations since it offers signifi-cantly less protein binding compared withglass vials) [223].

Finally, silicone oil used to lubricate vialstoppers, syringe walls and plungers has longbeenknowntointeractwithproteinsresultingindenaturation [224].Earlyworkwith insulinhashighlighted the potential hazard of plastic insu-lin syringes coated with silicone oil [225]. Morerecent reports suggest that silicone oil may cat-alyze protein aggregation. Jones and coworkerssuggested that silicone oil could induce confor-mational changes as measured by circular di-chrosim and derivative UV spectroscopy thatmay lead to protein aggregation [226].

4.5. Alternate Modes of Delivery

Protein biopharmaceuticals are typically de-livered via parenteral injection. In order toimprove patient comfort and compliance, ef-forts are underway to deliver proteins throughalternate routes, including oral, mucosal,transdermal, and inhalation. However, eachof these routes of administration presentstremendous challenges. Oral delivery of


proteins has been considered by many to bethe “holy grail” of biopharmaceutical drugdelivery. Drug delivery scientists have at-tempted to reduce the impact of digestive en-zymes in the gastrointestinal tract throughcoadministration of enzyme inhibitors, ab-sorption enhancers, encapsulation in lipo-somes, microemulsions, biodegradable parti-cles, and the modification of proteins throughtheattachmentof chemicalmoieties [227,228].Mucosal delivery of proteins has achievedsome modest success. In particular, low mo-lecular weight proteins and peptides havebeen delivered through the nasal mucosa andacross the oral cavity with the aid of bioadhe-sive polymers [229,230]. Transdermal deliv-ery of proteins has been equally challenging.Peptides have been driven across the skinwith the aid of skin perturbation devices; how-ever, bioavailability for this route remainslow [231]. Pulmonary delivery of proteins hasalso met with mixed success. Appropriatelysized particles (1–5mm) may be inhaled anddeposited in thealveolar space, and ithasbeendemonstrated that proteins such as insulinhave measurable bioavailablity and efficacythrough this route of administration [232].

For parenteral injection, many advances indrugdeliverymodalitieshave been consideredand implemented including, degradable de-pots, PEGylation (polyethylene glycol conju-gation), needle free injectors, andmicroneedlesystems [233–237]. Depot systems fashionedfrom degradable polylactic coglycolic acid(PLGA)-based polymers have been introducedinto the market, including gonadotropin-releasing hormone (GnRH) agonist (LupronDepot�) and human growth hormone (Nutro-pin Depot�) [238,239]. Protein release ki-netics from these systems can be controlledthrough the appropriate selection of polymersand fabrication techniques [240]. ProteinPEGylation has been utilized to enhance thecirculation half-life of peptides and proteinsand reduce dosing frequency and improve pa-tient compliance. Commercially successfulPEGylated products include PEGylated G-CSF (Neulasta�) and PEGylated interferonalpha-2a (Pegasys�) [241]. Needle free injec-tors have been developed to improve patientcompliance, and safety. These devices operatethrough pressurization of the drug container

in order to force the aqueous formulation toexit an orifice at high velocity [242]. Finally,microneedles have been developed to piercethe skin with blunt or hollow needles fabri-cated with a range of sizes, shapes and mate-rials. Microneedle delivery systems take ad-vantage of the highly vascularized nature ofskin for drug absorption while minimizing thepain and safety issues for patients associatedwith traditional needles [243].

4.6. Future Trends

The task of protein formulation developmentrequires the evaluation of a large number offormulation conditions, sample testing andconsiderable analytical characterization tosupport the selection of a final formulation.Since this process also requires the collectionof real-time stability data that can require 2years after the manufacturing process hasbeen developed, there is considerable pres-sure to develop formulations rapidly. Someefforts to increase protein formulation devel-opment throughput have been consideredand reported in the literature [244,245]. Ahigh-throughput formulation (HTF) platformis based on the use of microplates and ro-botics systems. Microplates allow for theincubation and analysis of multiple formula-tions simultaneously under stressing condi-tions and robotics systems automate theprocess of sample preparation and analysis.For a high-throughput system to be effectivethe formulation scientist will need to consid-er what should be the appropriate stressconditions of the protein that will accuratelypredict stability (agitation, temperature, pH,others) and what assays are stability indicat-ing for the protein being tested under theseconditions (size exclusion chromatography tomeasure aggregation, mass spectrometry tomeasure biochemical degradation, others).While HTF appears challenging given thecurrent state of technology, the integrationof high-throughput tools coupled with in-sightful testing strategies will undoubtedlyyield robust formulations in a more rapidtime frame in the near future.

Another area of increasing interest in pro-tein formulation is in the production of proteincrystals designed to be delivered as suspen-


sions. In recent years, investigators have pro-duced protein crystals that are suitable forlong-term storage and delivery [246,247]. Pro-tein crystals provide one solution to the pro-blem of storage of high concentrations of bio-pharmaceuticals for prolonged periods oftime.When delivered as a suspension, proteincrystals assume lower viscosity than solubi-lized protein of the same concentration, alle-viating high-concentration delivery issues.Also, investigators have recently begun to en-gineer protein crystals, controlling their dis-solution characteristics such that their deliv-ery rates may be controlled. Govardhan andcoworkers demonstrated that crystals of hu-man growth hormone coated in poly(arginine)could be injected once per week and achievethe same results in rat growth studies as dailyinjections of soluble growth hormone [248].Despite the challenges in producing proteincrystals at a reasonable commercial manufac-turing scale and unknown immunogenicityissues, this technology may provide signifi-cant advances in protein formulation, storage,and delivery in the future.

5. ANALYTICAL

5.1. Introduction

The utilization of appropriate analyticalmethods is crucial in order to evaluate proteinproducts and in-process intermediates thatare produced throughout the process develop-ment cycle. Analytical methods are used tomeasure protein concentration, product qual-ity attributes, process and product relatedimpurities, product identity, and product po-tency. These product quality attributes arecontinuously monitored during the develop-ment of the upstream and downstream pro-cesses in order to assess the impact of indivi-dual processing steps on the physical andchemical characteristics of the protein. Overthe course of the development process, analy-tical methods are refined and improved toprovide relevant information regarding theconsistency of the process and key attributesof the protein product. Finally, throughout theentire development process, the AnalyticalScientist must consider that many of themethods developed to support process and

formulation development will need to be uti-lized in a quality control environment in orderto release the product.

In-process assays used in the developmentof upstream and downstream processes typi-cally provide critical information to ProcessDevelopment Scientists such as protein con-centration, levels of aggregate and chargeheterogeneity, levels of impurities such asin-process contaminants and host cell pro-teins, and bioactivity [249]. Early develop-ment stage assays are often suitable for devel-opment purposes but require further“qualification” to demonstrate they are fit forpurpose and “validation” to ensure that theyare useful as product release methods [250].Product release specifications are set based onknowledge of key product attributes and keyprocess attributes and an understanding ofhow controlling the process will produce aproduct of desired characteristics [251]. Com-mon in-process and protein product releasetest methods include size exclusion chromato-graphy (product purity) and ion exchangechromatography (product purity and iden-tity), SDS-capillary electrophoresis or SDS-PAGE (product purity), immunoassay (iden-tity and host cell protein impurities), DNAtesting (impurities), and bio- or binding assay(potency). Table 1 summarizes the utility ofmany of these methods for the detection ofprocess impurities.

Analytical methods are also used for pro-duct characterization and product or processcomparability. Throughout the developmentprocess greater refinement of a product’s ana-lytical characteristics are determined. Duringearly stages of development, features such asprotein primary sequence, size characteristics,charge isoforms, biophysical structure, andchemical degradation products are deter-mined. Later stages of development allow forgreater refinement inproduct characterizationas the analytical characteristics that impactproduct attributes such as bioactivity are bet-ter understood. When processes are modified,for example, when changing a commercial pro-cess in order to take advantage of potentialtiter, purity, or yield improvements, analyticalcharacterization methods are essential indetermining product comparability. Typically,appropriate characterization methods are

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developed in order to ensure that the newprocess generates material that is comparableto the original process. Analytical methodsthat are commonly used for product character-ization and comparability testing include chro-matographicanalysis, peptidemapswithmassspec analysis (to monitor post translationalmodifications, instabilities, impurities, pro-duct heterogeneities, disulfide bonding pat-terns, etc.), oligosaccharide maps (glycanheterogeneity), isoelectric focusing (chargeheterogeneity), bioactivity, and biophysicalmethods (structural characterization). Fig-ure 4 illustrates the application of productcharacterization methods for highly complexglycoproteins that are used to support processdevelopment.

Appropriate analytical methods and char-acterization techniques are also essential forthe development of protein formulations andthe evaluation of protein product stability. Asupstream and downstream processing stepsare developed analytical methods are as-sessed for “stability indication” (i.e., analyti-cal methods that can quantitatively demon-

strate changes in critical product parametersunder normal and accelerated storage condi-tions). The development of stable protein for-mulations supports the overall developmentof release methods and characterization as-says by providing key input on determiningwhich analytical parameters are useful tomonitor.

5.2. In-Process and Product Release Testing

5.2.1. Chromatography Chromatographicseparations are the workhorse techniques ofprotein isolation and characterization. Chro-matography is particularly well suited to ana-lysis of proteins because of the ability to se-parate based on physical heterogenities thatare common to biopharmaceutical productssuch as size, charge, and hydrophobicity andbiochemical specificities such as ligand bind-ing affinity. Chromatographic techniques canalso be developed into rapid, high-resolution,high-throughput, and robustmethodsmakingthem well suited in a quality control environ-ment. Finally, chromatographic techniques

Figure 4. Depicted in this figure is a glycoprotein. The protein, defined by the amino acid sequence, folds toform secondary structures such as alpha-helices (red cylinders) and beta-strands (yellow ribbons) which areconnected by ordered loops (green threads). The glycosylation attached to the protein is shown by blue andgreen spheres. Some common physical and chemical attributes of recombinant glycoproteins and an array ofanalytical tools that are utilized to characterize these proteins are listed.


are desirable because of the ability to couplethese separation methodologies with otherhigh-resolution techniques such asmass spec-trometry for in depth characterization.Size Exclusion Chromatography Chromato-graphic separation based on size and shape,called size exclusion chromatography (SEC),is the most commonly utilized chromato-graphic technique for the characterization ofaggregates in protein biopharmaceuticals. InSEC, separation is achieved in an aqueousmobile phase by molecular sieving (ideally),whereby larger species are excluded from pe-netration into porous resins and elute morerapidly relative to smaller species that dopenetrate the pores of resins [252].

Frequently, protein products are producedwith monomeric, dimeric, and potentially lar-ger aggregated species that can be separatedand quantified by SEC. Several examplesfrom the literature demonstrate how SECmethods can be developed to provide a simple,direct measure of protein aggregates in a for-mat that is suitable for evaluation of processdevelopment intermediates, and product sta-bility [253,254]. The ability to quantitate ag-gregate levels in a protein product is thoughtto be critically important because of the po-tential for these species to induce immunereactions [255]. In order to further character-ize sizes of protein species orthogonal techni-ques have been utilized to estimate levels ofsoluble aggregate including dynamic lightscattering (DLS), analytical ultracentrifuga-tion (AUC), and asymmetrical flow field flowfractionation (AF4) (these techniques are de-scribed later).Ion Exchange Chromatography Analyticalscale ion exchange chromatography (IEC) isused to separate and characterize proteins onthe basis of surface charge [256]. Anion ex-changers interact with negatively chargedprotein ions and cation exchangers interactwith positively charged protein ions. In eithercase, the strength of the ionic interactionsbetween the protein surface and the ion ex-changer dictates protein retention times andleads to charge based separations. Typically,ion exchange assays are developed by testingstrong or weak ion exchangers with aqueousmobile phases containingappropriate levels ofsalts or buffer pH conditions that selectively

bind and elute or separate charged species ofinterest.

Some exampleswhere IEChas proven to beuseful to separate proteins on the basis of sur-facechargeheterogeneityincludedeamidation,charge associated with glycosylation, phos-phorylation, and truncated or oxidizedforms [257–261]. Charge-based separationscanbehighlyselectivewithevenasingleaminoacid substitution, if the change causes signifi-cant modification of the charge distribution onthesurfaceof theprotein, resulting incompleteseparation by IEC [261,262]. Also, since IECdepends on surface charge interactions,changes in conformation that result in buryingof charged regions of proteins may be detectedby IEC[263].Because the ionexchangeprofilesof proteins can be highly specific for proteinsequence, this assay has been utilized by somebiopharmaceutical manufacturers as an iden-tity test. Characterization of the individualpeaksthatcontributetoanionexchangeprofilehas also proven useful in understanding im-portant product heterogeneities [261].Reverse Phase HPLC Reverse phase chroma-tography operates on the principle of hydro-phobic interactions, i.e. hydrophobic regions ofproteins interact with the hydrophobic resinsto a degree that can be impacted by resin sidechain length, and aqueous phase conditions,resulting in separations. ReversephaseHPLC(RP-HPLC) is highly resolving and thereforehas been utilized to characterize peptide frag-ments, and specific chemical modifications.RP-HPLC of proteins is carried out underdenaturing conditions and therefore the en-tire unfolded protein sequence is capable ofinteraction with the stationary phase. Be-cause of the high resolving power of RP-HPLCit is also commonly coupled to other detectionsystems such as mass spectrometers.

RP-HPLC has been shown to be a usefultechnique to separate proteins with heteroge-nities in carbohydrates, oxidation, and deami-dation. Reverse phase is also very useful forseparation of smaller peptides, such as pep-tide maps for characterization [264] and forimplementation as a release assay [265,266].Dillon and coworkers showed that reversephase methods could distinguish between in-tact and fragmented monoclonal antibodies,and structural heterogeneity of IgG2 [267].

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Quantitativemethods suitable for quality con-trol have also been developed from RP-HPLCmethods [268,269].Hydrophobic Interaction ChromatographyHIC is complementary to RP-HPLC in thesense thatHIC separates proteins on the basisof surface hydrophobicity whereas RP-HPLCseparates proteins based on surface and corehydrophobic residues that are exposed duringdenaturation of the protein. The relative hy-drophobicity of columnmaterials used forHICwill influence the ability to separate hydro-phobic species. Mobile phase buffer selectionalso plays an important role in the develop-ment of analyticalHICmethods. Inparticular,salts impact the relative hydrophobicity ofexposed protein surfaces.

Because of the selectivity ofHIC for surfacehydrophobicity of proteins this technique hasbeen used to distinguish properly and impro-perly folded isoforms. Two examples are de-scribed by Scheich and Aizawa who developedHIC methods to assay for proper protein re-folding from inclusion bodies produced in E.coli. [270,271].Affinity Chromatography Affinity chromato-graphy takes advantage of the binding of pro-teins to specific ligands suchas occurswith thebinding of protein A or protein G to the Fcdomain of IgG. Affinity resins are commer-cially availablewith preconjugated ligands formany applications (e.g., MabSelect� proteinA resin from Amersham Biosciences). Affinitychromatography is carried out by passing ana-lytes of interest such as heterogeneous popu-lations of proteins over affinity columns. Ana-lytes bound to the affinity resins are theneluted under high salt or low pH conditions.Affinity chromatography has been commonlypracticed to quantitate levels of immunoglo-bulin, immunoglobulin fragments, or Fc fu-sion proteins bound to protein A [272]. Thistechnique forms the basis for Fc protein quan-titation from a complex mixture as occursduring production of monoclonal antibodies.

5.2.2. Electrophoretic Techniques Electro-phoretic techniques such as polyacrylamidegel electrophoresis (PAGE) and capillary elec-trophoresis (CE) are commonly used to sepa-rate proteins on the basis of size or charge. Insize-based applications sodium dodecyl sul-

fate (SDS) is added to the native or denaturedprotein solution prior to separation. The SDS,which carries negative charge, becomes asso-ciated with the protein and draws the proteinthrough the gel under the force of the appliedelectrical gradient. For many years, SDS-PAGE has been the technique of choice forsize-based protein separations. This is due tothe relatively small mass of material that canbe detected (1mg following silver staining), theconvenience of commercially available PAGEgels, and the relative simplicity and transfer-ability of the technique.

In recent years, capillary electrophoresis(CE-SDS) has become more widely utilized inthe analysis of bioharmaceuticals because ofthe advances in the robustness of instrumen-tation, the ability to quantitate peaks directlythrough absorbance measurements, and therelative speed and reproducibility of the tech-nique [273–275]. Biotechnology manufac-turers have successfully tested and releasedtheir products based on CE-SDS methodolo-gies [276]. The superior resolving power ofCE-SDS also lends itself to product characteriza-tion applications [277,278], and the ability tocoupleCEwithmass spectrometers is increas-ingly finding application in glycoproteinanalysis [279].

For charge-based separations electro-phoretic methods can be utilized to focus pro-teins according to isoelectric point in a slab gelor capillary subjected to a voltage gradient.Focusing is achieved by preequilibrating thegel or capillary with polyampholytes thatestablish a pH gradient in response to theapplied voltage between the anode and thecathode. As a technique isoelectric focusing iscapable of extremely high resolution with pro-teins differing by a single charge being focusedinto distinct bands.Recentwork fromHanandcoworkers demonstrated the utility of capil-lary isoelectric focusing (C-IEF) to monitorupstream and downstream processes, theanalysis of in-process samples, and the char-acterization of soluble interleukin-1 receptor(IL-1R) [280].

5.2.3. Nucleic Acid Testing The ability toanalyze residual levels of host cell DNA isimportant in order to minimize the potentialof aberrant gene expression by insertion of


DNA into the genes of patients receiving re-combinant proteins. Regulatory guidelines re-commend that the final product contains nomore than 100pg cellular DNA per dose [281].Traditional assays for the measurement ofDNA include threshold, slot blot, and pico-green dye assays, however, these methods arerelatively time consuming and error prone.More recently, the quantitative polymerasechain reaction (QPCR) has been developed forthe detection of host cell DNA [282,283]. Withthis method host cell DNA is quantified usingprimers targeting repeat regions of the hostcell genome. The sensitivity of QPCR is in thefg/ul range that is about a million-fold moresensitive than traditional DNA assays. Recentpublications fromLovatt andKubista and cow-orkers reviewed the QPCRmethod for quanti-tation of residualDNA inprotein therapeutics,and biosafety applications such as the detec-tion of the level of virus removal in processvalidation viral clearance studies [284,285].

5.2.4. Immunoassays Immunoassays arehighly specific and highly sensitive biochem-ical techniques, with limits of quantitation inthe range of 1–10part per million (ppm). Theimmunoassay system typically consists of a“capture” antibody to bind the antigen of in-terest and a secondary antibody for the pur-pose of detection. Immunoassays are mosttypically carried out in a 96 or 384 well-plateformat and are suitable for use in a qualitycontrol environment. In protein process devel-opment, immunoassays are often used as atest for product identity. Product identity im-munoassays require the production of mono-clonal, and/or polyclonal antibodies that spe-cifically detect the product of interest withoutcross-reacting with other products in develop-ment. Immunoassays are also commonly usedfor the detection of process-related impurities.These impurities include host cell protein con-taminants and often protein A, which may beleached from purification columns in pro-cesses developed for products that containimmunoglobulin Fc domains. Kits for the de-tection of protein A by immunoassay are com-mercially available from several companies (e.g., Repligen and Assay Designs, Inc.).

The development of immunoassays formeasurement of host cell protein contami-

nants has gained considerable attention in theliterature [286]. Host cell protein contamina-tion consists of a complex mixture of proteinsand peptides, and measuring all them with asingle assay poses a challenge [287]. Given thefact that any single host cell protein antigenmay be weakly immunogenic or even nonim-munogenic, an immunoassay may be insensi-tive to elevated levels of these particularantigens. Therefore, host cell protein immu-noassays can only provide relative data,rather than absolute quantiation of host cellcontaminants. Nonetheless, recent studies byKrawitz et al. [288] suggest that a genericassay, developed to detect host cell proteinsfrom a single cell line, may be useful in detect-ing impurities when this same cell line is usedto produce other products.

Other recent advances in immunoassaytechniques include the Gyrolab from Gyros,AlphaLISA (Amplified Luminescent Proxi-mity Homogeneous Assay) from Perkin El-mer, and Octet from ForteBio [289–291]. Inthe Gyrolab application the immunoassayprocess has been automated and simplifiedwith theuse of a compact disc that is preloadedwith capture and secondary antibodies anddetection reagents. Spinning the compact discat predetermined speeds allows sequentialmixing of analyte, capture antibodies, andsecondary antibodies with incubation steps atappropriate intervals. In the bead-based Al-phaLISA application, donor and acceptorbeads conjugated with ligand and detectionantibodies are utilized to amplify antigen–antibody interactions through chemilumines-cence. The Octet application relies on the useof a 96-well-biosensor system that utilizesinterferometry to detect analytes bound to thebiosensor tip for rapid and label free proteinquantitation.

5.2.5. Bioactivity Assays Potency assays arecritically important for the assessment of thebioactivity of protein pharmaceuticals, both toensure that product potency is maintainedduring processing and storage and for thepurpose of product characterization. Typicallypotency assays fall into two categories: (1)bioassays, which utilize cells to directly assessthe ability of protein products to “activate” orblock target pathways, and (2) binding assays,

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which assess the ability of protein products tobind to receptors or ligands in amanner that isknown to represent themechanismof action ofthat particular protein product. For agonisticproducts, it is typical to use cell-based bioas-says since both binding to a target receptor orligand and signaling into the cell for the de-sired activity are required. The activity ofantagonistic products may be assayed withbinding assays if the desired mechanism ofaction can be demonstrated. A recent reviewarticle from Meager described the measure-ment of cytokine potency by bioassays [292].Binding assays for product characterizationhave included ligand binding-based systemsas well as surface plasmon resonance technol-ogy (Biacore�, from GE Healthcare) that issensitive to changes in binding affinity be-tween an analyte of interest and a surfaceimmobilized ligand [293].

Potency assay scientists must develop re-lease assays that are quantitative and able tobe qualified for product release testing. Inorder to be useful for release testing bio- andbinding assays should be sensitive to confor-mational and chemical characteristics of theprotein product. Since the impact of degradedproteins on potency may not necessarily bedemonstrated by other means (e.g., chromato-graphy or immunoassay) it is essential to in-clude a bioassay or binding assay in productrelease testing [294]. Also because of the in-herent variability of bio- and binding assays, athoroughdevelopment processwill be requiredto set useful specifications [295,296]. For cer-tain products, bioactivity must include multi-functional assessment, for instance, a mono-clonal antibody that relies on its ability to binda specific target ligand as well as binding to Fcreceptors for effector function [297].

Potency assays are also critical for thecharacterization of protein products. Caseshave been shownwhere chemicalmodificationof a single amino acid, such as deamidation ofan Asp residue in the CDR of an antibody tohuman IgE, entirely inactivated these pro-teins [298] and other cases where degradedspecies such as aggregates became inactive oreven hyperactive [299]. New technologies forbioassays include the use of “platform” sys-tems such as the introduction of reporter geneelements to take advantage of common cell

signaling pathways (including STAT and NF-kB transcription factors) for biopharmaceuti-cal compounds with different mechanisms ofaction [300].

5.3. Mass Spectrometry and BiophysicalCharacterization

High-resolution and specialty analyticalmethods are powerful techniques that areoften utilized during product characteriza-tion, determination of product comparability,or in support of product investigations. Thesetechniques include methods for biochemicaland biophysical analysis. Often, the results ofthese analyses provide insight into proteinprocessing steps that cannot be measured bytypical release methods or lower resolutiontechniques described in the sections above.

5.3.1. Mass Spectrometry Mass spectrome-try (MS) is perhaps the single most powerfultechnique used in the biopharmaceutical in-dustry for detailed biochemical characteriza-tion of proteins. MS instrumentation includesan ion source that transforms molecules froma sample into ionized fragments, a mass ana-lyzer that separates the ions based on theirmass-to-charge ratio (m/z), and a detector thatmeasures the abundance of each of the ionspresent. MS is typically coupled with onlinechromatographic separations (LC-MS), whichis particularlyuseful for analysis of separationproducts. Recent review articles provide ex-haustive descriptions of common MS techni-ques and applications for the analysis of pro-tein pharmaceuticals [301,302].

As a techniqueMS is well-suited to proteinpharmaceutical characterization because ofthe wide range of problems that can be solved.So-called “top–down” analysis describes MSfor intact proteins. Applications for top downanalysis include whole mass analysis, assess-ment of overall product heterogeneity, identi-fication of labile posttranslational modifica-tions, and confirmation of N- and C-terminalsequences. Bottom–up analysis describes MSfor protein digests, or peptidemaps, which areproduced by incubation of samples in enzymessuch as trypsin, Lys-C, Asp-N, and Glu-C.Applications for bottom–up analysis includecharacterization of glycosylation patterns,


determination of disulfide linkage patterns,and identification of posttranslational modi-fications such as Met and Trp oxidation, Aspisomerization, Asn deamidation, glutamatecyclization, glycation, fragmentation, andothers.

As examples of upstream applications MShas provided useful information on the stabi-lity of master cell banks as measured by gly-cosylation profiles in product expressed fromBHK 21A cells at 0 and 62 generations [303].MS has been utilized to monitor the degrada-tion of oligosaccharides during proces-sing [304] and to demonstrate that duringprotein expression fromCHO cells the cultureconditions can have a significant impact onoligosaccharide profiles [305]. MS has alsoproven useful to map disulfide pair heteroge-neity in IgG2 produced by CHO cell cultureprocesses [263,306,307].

During the purification process MS hasproven to be a useful aid in the identificationof product impurities and instabilities fromintermediate hold steps. Senderoff and col-leagues utilized laser desorption MS to iden-tify racemization of glucagon-like peptide-1(rGLP-1) and to influence the purificationscheme and the in-process storage and hand-ling conditions [308]. More recently, Wein-berger and colleagues described the use ofprotein biochip arrays carrying functionalgroups typical of those employed for chroma-tographic materials [309]. Application of pro-teins to these chips followed by typical testwash conditions resulted in a “retentate”bound to the chip that was then analyzed bysurface-enhanced laser desorption-ionizia-tion (SELDI) MS.

During the development of protein formu-lations and storage of proteins over prolongedperiodsMS is frequently utilized to character-ize degradation products. Among these, de-gradation products characterized by MS tosupport formulation development includecovalent or disulfide linked aggregates, Metoxidation, Asn deamidation, and Asp isomer-ization following storage of formulated bulksolutions or products under moderate andaccelerated conditions [310–312].

5.3.2. Biophysical Techniques Numerous bio-physical techniques are utilized by the analy-

tical scientist during protein process develop-ment and protein characterization in order toassess the structural characteristics of pro-teins. This panel of techniques includes thefollowingmethods to assess: (1) molecular sizeand size distribution of proteins such as lightscattering, assymetric flow field flow fractiona-tion (A4F), and AUC; (2) secondary proteinstructural assessment such as Fourier trans-form infrared spectroscopy (FTIR) and far-UVcircular dichroism (CD); and (3) tertiary pro-tein structure and conformational assessmentsuch as near-UV CD, intrinsic and extrinsicfluorescence spectroscopy, and a calorimetrictechnique known as differential scanning ca-lorimetry (DSC). As a biophysical “tool box,”these techniques form a powerful set of analy-tical capabilities that complement chromato-graphic, biochemical, immunochemical, andother methodologies for product characteriza-tion and investigative purposes. Biophysicaltechniques have also been widely applied dur-ing protein formulation development wherethe ability to assess structural stability is con-sidered to be a key factor in predicting andlimiting aggregation behavior. Although sev-eral other structural/biophysical techniquessuch as nuclear magnetic resonance (NMR),andX-ray crystallography are highly resolvingand are useful in some circumstances, thesetechniques have not traditionally been aswidely utilized during protein processdevelopment.Biophysical Assessment of Size and Size Distri-bution Static light scattering (SLS) mea-sures the average light scattering intensity asa function of sample concentration. For a largesize molecule, multiangle laser light scatter-ing spectroscopy (MALLS) is commonly used.The combination of MALLS with a chromato-graphic method such as SEC has been used toobtain the accurate molecular masses of pro-tein species such as monomers, dimers, etc.,independent of chromatographic retentiontimes [313]. Often, SEC leads to complicatedprofiles that result from the existence of di-mers,multimers, and other species in solutionthat may have affinities for the size exclusionresin. In these examples, MALLS can providea relatively rapid and orthogonal assessmentof size [314]. For example, MALLS has beenutilized in combination with SEC to estimate

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the size of polyethylene glycol conjugated (PE-Gylated) proteins and the size of protein–ligand complexes [315].

DLS is based on the principle of detectingfluctuations of scattered light due to theBrow-nianmotion of molecules in solution, indepen-dent of the protein concentration in solution.Dynamic light scattering is used to measurethe hydrodynamic radius, or size distributionof molecules in solution [316]. DLS measure-ments have greater utility for protein mix-tures with a wide range of masses (chromato-graphic separations not required).

A4F is a separation technique that relies onthe application of a perpendicular physicalflow field to a solution mixture. Unlike chro-matography, A4Fdoes not rely on interactionswith a stationary phase, rather, separation isachieved by differing diffusion coefficients ofthe various components in the field [317,318].For A4F it is possible to inject samples con-taining precipitatedmaterialwithout anypre-treatment (SEC requires filtration to avoidcolumn clogging). A4F can cover a broaderspan of molecular weights than SEC, whichis limited by the exclusion limit of the columnand this technique is capable of separatingaggregates and particles from 0.001 to 50mmin size. Despite the advantages of A4F, wide-spread use of this technique has been limitedby the difficulty in the use of the instrumenta-tion and the complexity of data analysis [319].

AUC allows real-time UV absorption mea-surement of the sedimentation of a macromo-lecular sample as a result of an applied cen-trifugal field. Mathematical analysis of thesedimentation equilibrium (SE) or sedimenta-tion velocity (SV) data through specializedsoftware (SEDFIT) [320] can provide veryhigh-resolution size information about themolecules in a given sample [321]. The kindsof information that can be obtained from ananalytical ultracentrifuge include the grossshape, conformational changes, and size dis-tributions of macromolecular samples. Formacromolecules such as proteins that exist inchemical equilibrium with different noncova-lent complexes, the number and subunit stoi-chiometry of the complexes and equilibriumconstants can be studied. In addition, AUCallows sample testing to be conducted in theexact liquid formulation or reconstituted li-

quid formulation of the biopharmaceutical inthe vial. AUC is a particularly useful techni-que for the measurement of protein aggrega-tion as it provides an orthogonal measure-ment to other size-based techniques such asSEC, and A4F [319,322–324]. Light scatter-ing, A4F and AUC can measure absolute le-vels of aggregates, excluding artifacts that canbe induced by buffer and column interactionsin the SECmethodology. Studies have demon-strated that SEC can underestimate solubleaggregate relative to measurements by AUCand A4F [325].Biophysical Assessment of Secondary StructureFTIR has been utilized to assess protein sec-ondary structure in a range of environments,including in solution and when adsorbed tosolid surfaces. There is awealth of informationthat can be used to derive structural informa-tion by analyzing the shape and position ofbands in the amide I region of the FTIR spec-trum, including a-helix, b-sheet, and b-turns.As a versatile biophysical characterizationtool FTIR has been utilized to characterizeinclusion bodies within bacterial cells [326],to measure the melting temperature (Tm) ofproteins as an aid to formulation develop-ment [327,328], and as a method that allowsdirect analysis of the conformation of proteinsat high concentration without dilution thatallowed the construction of empirical phasediagrams [329].

CD spectroscopy is based on the differentialabsorption of left- and right-handed circularlypolarized light. Far-UV CD can reveal impor-tant characteristics of protein secondary struc-ture. In particular, CD spectra can be readilyused to estimate the fraction of amolecule thatis in the a-helix, b-sheet, b-turn, and othernonstructured conformations [330,331].Biophysical Assessment of Tertiary Structure andConformational Change Near-UVCDspectracan provide additional detail about the ter-tiary structure of proteins. The signals ob-tained in the near-UV spectra (from 250nmto�300 nm) are due to the absorption, dipoleorientation and the nature of the surround-ing environment of phenylalanine, tyrosine,cysteine, S-S-disulfide bond and tryptophanamino acids. Prolonged exposure of antibo-dies to low pH is unavoidable for protein Apurification and viral inactivation. In order


to minimize structural instability and aggre-gation, CD and other biophysical techniqueshave been used to assess the structural in-tegrity of proteins during these low pH pro-cessing steps [332]. Numerous CD applica-tions have been described in the literaturewith complementary biophysical techniquesin order to characterize the structural integ-rity of antibody therapeutics and confirm thestructural characteristics of Fc and Fabdomains [333,334].

Intrinsic fluorescence spectroscopy de-scribes the ability of tryptophan, tyrosine, andphenylalanine to absorb and fluoresce light ina manner that is characteristic of the localenvironment of these amino acids. Structuralperturbations of proteins can be measured byshifts in fluorescence intensity and wave-length. Intrinsic fluorescence has also provenuseful in analyzing binding interactions be-tween ligands that shifts the fluorescence in-tensity of tryptophan, tyroisine, or phenylala-nine at the interface between the bindingpartners [335].

Extrinsic fluorescence spectroscopy de-scribes the addition of fluorescent tags to pro-teins that produce characteristic fluorescenceemission profiles. Extrinsic fluorescence spec-troscopy has been reported in the literature tocharacterize folding intermediates, measuresurface hydrophobicity, and detect aggrega-tion [336,337]. During protein formulationdevelopment extrinsic fluorescence spectro-scopy has proven useful as a tool to assess theimpact of excipients on the unfolding ofproteins [338].

DSC relies on the ability to measure ther-modynamic parameters, specifically, theamount of heat required to maintain the tem-perature of a given sample relative to a refer-ence. DSC has the potential to measure ther-modynamic parameters including the changein enthalpy (DH) and heat capacity (DCp), aswell as the glass transition temperature (Tg)and the melting transition temperature (Tm).The main application of DSC in biopharma-ceutical analysis is themeasurement of theTg

of lyophilized cakes or the Tm of proteins insolution [339]. Tg, the temperature at which aglassy solid becomes soft upon heating, isoften utilized to optimize formulation buffersand operating conditions for product lyophili-

zation. A thorough understanding of the Tg iscritical in order to reduce the likelihood ofcollapse of a lyophilized “cake” and to retainlong-term product stability. Recent scientificreviews describe the factors involved in stabi-lizing folded proteins during the developmentof lyophilized formulations and lyophilizationprocesses [340,341]. For proteins, consider-able effort has been given to themeasurementof Tm in order to describe protein unfoldingand subsequent propensity to aggregate. Re-cent work with soluble IL-1 receptor and hu-man IgG describes the relationship betweenTm and the kinetics of aggregation [342,343].In these studies the choice of formulationbuffers used in the protein unfolding experi-ments were shown to impact Tm. In anotherstudy from Garber and Demarest, Tm valuesfrom the Fab domains of 17 human or huma-nized antibodies were found to vary from 57 to82�C [344]. In this study, antibodies with low-er Tm values were found to aggregate and toexpress poorly in cell culture. The measure-ment of Tm has also been utilized to evaluatethe impact of antimicrobial preservatives onproteins in solution since antimicrobial pre-servatives may be physically destabilizing toproteins [345].

5.4. Other Support for Process Development

5.4.1. Comparability As mentioned, duringstages of protein product development a pro-cess or formulation is modified in order toincrease production titer, improve purifica-tion yield, make a change to the productionscale or site of production, or to improve thestorage or delivery characteristics of the pro-duct. However, evenminor variations broughtabout by these process changes can lead toclinically relevant changes in efficacy and/orsafety of the end product [346,347]. Analyticalcomparability is used to assure that changesin a process do not change the analytical at-tributes of that product. Appropriate analyti-cal methods for comparability testing includecritical product attributes, such as those de-scribed by biochemical and biophysical char-acterization tests, potency assays, and purityassays [348–350].

Process development scientists publishedresults for thescale-upofa fed-batchbioreactor

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process going from 3L to 2500L scale(Epratuzumab) [351]. In this work, biochem-ical assays including N-linked oligosaccharideprofiling, C-terminal lysine analysis, and tryp-tic peptide mapping showed that the scale-upprocess did not impact the analytical charac-teristics of this compound.Similar results fromMeuwly and coworkers demonstrated analyti-cal comparability following conversion of aCHO cell culture process from perfusion tofed-batch process [352]. Published results withSynagis� (a monoclonal antibody product)showed that there was a different pattern ofglycosylation during the early stages of bior-eactor culture, however, no other changes inmicrogeterogeneity were apparent for theother culture conditions studied [353]. Basedon the results of these experiments criticalprocessing attributes could be controlled toassure that product characteristics were con-sistentduringprocess changesandproduction.Insulin has provided an excellent model sys-tem to illustrate many important considera-tions when dealing with comparability exer-cises for biotechnology products undergoingformulation changes [354]. The bioactivity ofinsulin is known to be impacted by structuralandbiochemical attributes and the influenceofformulation changes must be demonstratedthrough suitable testing methodologies of thedrug product.

Analytical testing has also been carried outin support of the comparability of biosimilarproducts [355]. While this informationmay besupportive of the analytical “quality” of abiosimilar product, since the manufacturingprocess, analytical methods, and formulationof the biosimilar are not the same as theinnovators product, clinical studies will likelybe required to assure suitable product perfor-mance characteristics such as safety/immuno-genicity and efficacy [356].

5.4.2. Process Reagent Clearance and ExcipientTesting During protein process development,analytical methods may be required for deter-mination of the clearance of process reagentswith potential safety concern (PSC) [357].Whether or not process reagents require de-monstration of removal depends on an evalua-tion of their potential impact to product safety,an evaluation of capability of the process to

remove the impurities, and an assessment ofthe overall impact of the impurities to theproduct quality. Generally, the ability of aprocess to clear process reagents is affectedby the process step at which they are intro-duced, their solubility through subsequentprocessing steps, and their relative concentra-tions. Upstream and downstream process re-agents that will likely not require in-processtesting (depending on concentration) includethose substances that are generally recog-nized as safe (GRAS) [358].

Upstream process reagents that may re-quire monitoring include media componentssuchasinsulinandothergrowthfactors, serumproteins,soyproteins,andbuffers(e.g.,HEPESand MES); expression inducers such asisopropyl-B-D-thiogalactopyranoside (IPTG),and methotrexate [359]; antibiotics; cell shearprotectants such as pluronic F-68; and anti-foam agents simethicone [360]. Downstreamprocess reagents that may require testing in-cludebuffers,acids,andsolventssuchasCAPS,MOPS,acetone,andformicacid;proteinrefold-ing agents such as dithiothreitol (DTT), urea,and guanidine [361]; and column leachablessuch as protein A.

Excipients (preservatives, surfactants,salts, buffers, and tonic agents) may also re-quire analytical testing to ensure these arepresent at appropriate levels to achieve theirintended effect on product formulations [362].Preservatives must be routinely identifiedand quantified at release. Excipient assaysare as diverse as the compounds that aremeasured and may rely on HPLC, gas chro-matography or other chemically specificmethodologies [363].

5.5. Future Trends

Analytical technologies will continue to devel-op and evolve, leading to new techniques andmethodologies that will be applied to proteinprocess development. Three specific areaswhere improvements in analytical approacheswill benefit protein process development in-clude (1) greater focus on determining andmeasuring critical quality attributes, (2) de-velopment of higher throughput techniques,and (3) development of higher resolutiontechniques.


While analytical methods are currentlyavailable to measure a myriad of biochemicaland biophysical properties of proteins it ismost important to measure and control thoseattributes that are known to impact productsafety (e.g., immunogenicity), efficacy (e.g.,bioactivity and pharmacokinetics), and stabi-lity (e.g., aggregation). Thenotionof determin-ing and measuring critical product qualityattributes is consistent with the concept ofquality by design (QbD) [364]. Given the cur-rent state of biopharmaceutical developmentit is not possible to assess the impact of allproduct heterogeneities on product safety, eff-icacy and stability; however, work needs to beconducted to do exactly this. For instance, ifthe presence of protein particles in drug pro-duct is indeed a critical product quality attri-bute because of the propensity of particles tobe immunogenic, then quantitative methodsbeyond simple particle visualization need tobe developed and utilized in quality controlenvironments. Conversely, if we can deter-mine that product quality attributes do notimpact product safety, efficacy, or stabilitythen these parameters may not require mea-surement or control during productmanufacturing.

With the advances in high-throughputtechnologies that have been applied to up-stream and downstream process develop-ment, the analytical scientist is faced with anincreasing burden of analyzing large numbersof samples in a rapid manner. High-through-put systems have been developed in practi-cally all areas of analytical testing includingchromatography (ultraperformance liquidchromatography resins) [365], mass spectro-metry (automated sampling from a microwellplate) [366], and capillary electrophoresis (acombinatorial approach to protein separationand peptidemapping) [367]. High-throughputrobotic systems are gaining more widespreaduse as automated liquid sample handlers,especially for ELISA, and multiwell-plate-based applications. Londo and colleagues de-scribe a robotic system used to accelerateprocess development through on-line chroma-tographic analysis of purification yields [368].High-throughput systems are also being de-veloped as a result of the miniaturization ofanalytical techniques, for instance, the use of

disposable microfluidics systems that takeadvantage of computer chip fabrication tech-nology to develop high-throughput devicesthat require small sample volumes, and shorttransit distances [369].

Complementing the high-throughput ap-proaches are the advances in high-resolutionanalytics. Mass spectrometry in particularhas continued to improve in its resolvingpower and mass accuracy. Fourier transformion cyclotron resonance mass spectrometry iscurrently capable of greater than 500,000 re-solution (m/Dm) with less than 1ppm massaccuracy [370,371]. Through evolution of thisfield, what are considered today as lower re-solution detectors are now more economicaland commonly available. These “low re-solution” mass spec decetors are more com-monly used as an adjunct detector for chro-matographic applications, enhancing the uti-lity of many chromatography analyses. Final-ly, although time consuming, the analysis ofprotein crystal structure has the potential toprovide useful structural information on en-gineered proteins [372], and product hetero-geneities. Developments are underway to im-prove the ability to generate protein crystalsthat may bemore widely utilized in the futureto support protein process development.

6. CONCLUSION

The development of processes for the produc-tion of biological compounds is highly complexand requires functional expertise in many di-verse fields including molecular biology,cellular biology, cell physiology, chemical en-gineering, chromatography, filtration science,virology, nucleotide chemistry, protein chem-istry, physical chemistry, and immunochemis-try. To successfully develop protein productionprocesses, experts from each of these fieldsmust work together in an integrated fashionwith an awareness of the impact that indivi-dual processing steps or functionsmayhave onthe final product. For instance, upstream titeror product quality attributesmay significantlyimpact the requirements for downstreampurification processes; other critical productquality attributes delineated by analyticalassessments may dictate the selection of

CONCLUSION 327

upstream clones or cell lines; and downstreampurification limitations may potentially dic-tate upstream processes or formulation pre-sentations. Protein process development is ul-timately an exercise in process optimization,trading variables such as processing speed forproduct titer and product quality.

This reviewsummarizes the currentstateofthe protein process development field. Muchprogress has beenmade toward the productionofbiologicsoverthelast30years.Celllineshavebeen developed that can produce protein titers100-fold higher than a few decades ago, ad-vances in resins and filters have allowed pur-ification processes to handle these titers withrelatively high yields, protein formulationshave improved with respect to stability andconvenience of delivery, and many analyticaltestingdevelopmentshavebeenmadetoenablehigher resolution and higher throughput test-ing and characterization. However; despitethese advances, the development of proteinprocesses can still be empirical and unpredict-able at times. Given the state of the art inprotein process development and the currenteconomic environment in the health care in-dustry, the process development scientist willneed to continue to develop technologies thatwill drive greater efficiencies. In the comingyears, we can look forward to decreasing devel-opment costs, shorter development timelines,and quality improvements for the many pa-tients who benefit from these products.

REFERENCES

1. MurphyA,PerrellaJ.Overviewandbriefhistoryof biotechnology. 1993.Availableathttp://www.woodrow.org/teachers/bi/1993/intro.html.

2. The convention on biological diversity. Article2. Use of terms. 1992. Available at http://www.cbd.int/convention/articles.shtml?¼cbd-02.

3. Biotechnology Industry Orgnization—2007.2007. Available at http://bio.org/speeches/pubs/er/BiotechGuide.pdf.

4. JacobiA,EckermannC,AmbrosiusD.Develop-ing an antibody purification process. In: Subra-manianG, editor.BioseparationandBioproces-sing. Weinheim: Wiley-VCH; 2001. p 431–457.

5. Cohen SN, Chang AC, Boyer HW, Helling RB.Construction of biologically functional bacter-

ial plasmids in vitro. Proc Natl Acad Sci USA1973;70(11):3240–3244.

6. Morrow JF, Cohen SN, Chang AC, Boyer HW,Goodman HM, Helling RB. Replication andtranscription of eukaryotic DNA in Escheri-chia coli. Proc Natl Acad Sci USA 1974;71(5):1743–1747.

7. Yin J, Li G, Ren X, Herrler G. Select what youneed: a comparative evaluation of the advan-tages and limitations of frequently used ex-pression systems for foreign genes. J Biotech-nol 2007;127(3):335–347.

8. Schmidt FR. Recombinant expression systemsin thepharmaceutical industry.ApplMicrobiolBiotechnol 2004;65(4):363–372.

9. Balbas P, Bolivar F. Back to basics: pBR322and protein expression systems in E. coli.Methods Mol Biol 2004;267:77–90.

10. Ernst S, Garro OA, Winkler S, VenkataramanG, Langer R, Cooney CL, Sasisekharan R. Pro-cess simulation for recombinant protein pro-duction: cost estimation and sensitivity analy-sis for heparinase I expressed in Escherichiacoli. Biotechnol Bioeng 1997;53(6):575–582.

11. Graumann K, Premstaller A. Manufacturingof recombinant therapeutic proteins in micro-bial systems. Biotechnol J 2006;1:164–186.

12. Sorensen HP, Mortensen KK. Soluble expres-sionof recombinantproteins in the cytoplasmofEscherichia coli. Microb Cell Fact 2005;4:1–8.

13. Jana S, Deb JK. Strategies for efficient produc-tionofheterologousproteins inEscherichia coli.Appl Microbiol Biotechnol 2005;67(3):289–298.

14. Chou CP. Engineering cell physiology to en-hance recombinant protein production in Es-cherichia coli. Appl Microbiol Biotechnol2007;76(3):521–532.

15. Li P, Anumanthan A, Gao XG, Ilangovan K,SuzaraVV,DuzgunesN, RenugopalakrishnanV. Expression of recombinant proteins in Pi-chia pastoris. Appl Biochem Biotechnol2007;142(2):105–124.

16. Balamurugan V, Reddy GR, SuryanarayanaVVS. Pichia pastoris: a notable heterologousexpression system for the production of foreignproteins—vaccines. Indian J Biotechnol 2007;6:175–186.

17. Reyes-Ruiz JM,Barrera-SaldanaHA.Proteinsin a DNA world: expression systems for theirstudy. Rev Invest Clin 2006;58(1):47–55.

18. Macauley-Patrick S, Fazenda ML, McNeil B,Harvey LM. Heterologous protein productionusing the Pichia pastoris expression system.Yeast 2005;22(4):249–270.


19. JahicM,VeideA,CharoenratT,TeeriT,EnforsSO. Process technology for production and re-covery of heterologousproteinswithPichiapas-toris. Biotechnol Prog 2006;22(6):1465–1473.Erratum: Biotechnol Prog 2007; 23(2):516.

20. Daly R, HearnMT. Expression of heterologousproteins inPichiapastoris: auseful experimen-tal tool in protein engineering and production.J Mol Recog 2005;18(2):119–138.

21. Hamilton SR, Davidson RC, Sethuraman N,NettJH,JiangY,RiosS,BobrowiczP,StadheimTA, LiH,Choi BK,HopkinsD,WischnewskiH,Roser J, Mitchell T, Strawbridge RR, Hoopes J,Wildt S, Gerngross TU. Humanization of yeastto produce complex terminally sialylated glyco-proteins. Science 2006;313(5792):1441–1443.

22. Hu YC. Baculovirus as a highly efficient ex-pression vector in insect andmammalian cells.Acta Pharmacol Sin 2005;26(4):405–416.

23. Donald J, Jarvis DL. Developing baculovirus-insect cell expression systems for humanizedrecombinant glycoproteinproduction.Virology2003;310:1–7.

24. Farid SS. Established bioprocesses for produ-cing antibodies as a basis for future planning.Adv Biochem Eng Biotechnol 2006;101:1–42.

25. Houdebine LM. Transgenic animal bioreac-tors. Transgenic Res 2000;9(4–5):305–320.Comment.

26. KamihiraM,NishijimaK, IijimaS.Transgenicbirds for the production of recombinant pro-teins. Adv Biochem Eng Biotechnol 2004;91:171–189.

27. Sang HM. Transgenics, chickens and thera-peutic proteins. Vox Sang 2004;87(Suppl2):164–166.

28. Lillico SG,McGrewMJ, ShermanA, SangHM.Transgenic chickens as bioreactors for protein-based drugs. Drug Discov Today 2005;10(3):191–196.

29. Fischer R, Stoger E, Schillberg S, Christou P,Twyman RM. Plant-based production of bio-pharmaceuticals. Curr Opin Plant Biol 2004;7(2):152–158.

30. Lienard D, Sourrouille C, Gomord V, Faye L.Pharming and transgenic plants. BiotechnolAnn Rev 2007;13:115–147.

31. Schillberg S, Twyman RM, Fischer R. Oppor-tunities for recombinant antigen and antibodyexpression in transgenic plants–technologyas-sessment. Vaccine 2005;23(15):1764–1769.

32. Ko K, Koprowski H. Plant biopharming ofmonoclonal antibodies. Virus Res 2005;111(1):93–100.

33. Wurm FM. Production of recombinant proteintherapeutics in cultivated mammalian cells.Nat Biotechnol 2004;22(11):1393–1398.

34. ButlerM. Animal cell cultures: recent achieve-ments and perspectives in the production ofbiopharmaceuticals. Appl Microbiol Biotech-nol 2005;68(3):283–291.

35. MorrowKJ Jr. Advances in antibodymanufac-turing using mammalian cells. BiotechnolAnnu Rev 2007;13:95–113.

36. Werner RG, Noe W, Kopp K, Schluter M. Ap-propriate mammalian expression systems forbiopharmaceuticals. Arzneimittelforschung1998;48(8):870–880.

37. Kao FT, Puck TT. Genetics of somatic mamma-lian cells. VII. Induction and isolation of nutri-tional mutants in Chinese hamster cells. ProcNatl Acad Sci USA 1968;60(4):1275–1281.

38. Kao FT, Puck TT. Genetics of somatic mam-malian cells. XIV. Genetic analysis in vitro ofauxotrophic mutants. J Cell Physiol 1972;80(1):41–50.

39. Puck TT, Kao FT. Genetics of somatic mam-malian cells. V. Treatment with 5-bromodeox-yuridine and visible light for isolation of nu-tritionally deficient mutants. Proc Natl AcadSci USA 1967;58(3):1227–1234.

40. Kao F, Chasin L, Puck TT. Genetics of somaticmammalian cells. X. Complementation analy-sis of glycine-requiring mutants. Proc NatlAcad Sci USA 1969;64(4):1284–1291.

41. Kao FT, Puck TT. Induction and isolation ofauxotrophic mutants in mammalian cells.Methods Cell Biol 1974;8(0):23–39.

42. Urlaub G, Chasin LA. Isolation of Chinesehamster cell mutants deficient in dihydrofo-late reductase activity. ProcNatlAcadSciUSA1980;77(7):4216–4220.

43. Urlaub G, Mitchell PJ, Kas E, Chasin LA,Funanage VL, Myoda TT, Hamlin J. Effect ofgamma rays at the dihydrofolate reductaselocus: deletions and inversions. Somatic CellMol Genet 1986;12(6):555–566.

44. Sinacore MS, Charlebois TS, Harrison S,Brennan S, Richards T, Hamilton M, ScottS, Brodeur S, Oakes P, LeonardM, SwitzerM,Anagnostopoulous A, Foster B, Harris A, Jan-kowski M, Bond M, Martin S, Adamson SR.CHO DUKX cell lineages preadapted togrowth in serum-free suspension culture en-able rapid development of cell culture pro-cesses for the manufacture of recombinantproteins. Biotechnol Bioeng 1996;52(4):518–528.

REFERENCES 329

45. Rasmussen B, Davis R, Thomas JN, Reddy P.Isolation, characterization and recombinantprotein expression in Veggie-CHO: a serum-free CHO host cell line. Cytotechnology1998;28:31–42.

46. Kaufman RJ, Sharp PA. Amplification and ex-pressionof sequences cotransfectedwithamod-ular dihydrofolate reductase complementaryDNA gene. J Mol Biol 1982;159(4):601–621.

47. Cockett MI, Bebbington CR, Yarranton GT.The use of engineered E1A genes to transacti-vate the hCMV-MIE promoter in permanentCHO cell lines. Nucleic Acids Res 1991;19(2):319–325.

48. BebbingtonCR,RennerG, ThomsonS,KingD,Abrams D, Yarranton GT. High-level expres-sion of a recombinant antibody from myelomacells using a glutamine synthetase gene as anamplifiable selectable marker. Biotechnology1992;10(2):169–175.

49. CartierM,ChangMW,StannersCP.Use of theEscherichia coli gene for asparagine synthe-tase as a selective marker in a shuttle vectorcapable of dominant transfection and amplifi-cation in animal cells. Mol Cell Biol 1987;7(5):1623–1628.

50. Birch JR, Racher AJ. Antibody production.Adv Drug Del Rev 2006;58(5–6):671–685.

51. Weymouth LA, Barsoum J. Genetic engineer-ing in mammalian cells. In: Thilly WG, editor.Mammalian Cell Technology. Stoneham: But-terworth Publishers; 1986. p 9–62.

52. Lucas BK, Giere LM, DeMarco RA, Shen A,Chisholm V, Crowley CW. High-level produc-tion of recombinant proteins inCHOcells usinga dicistronic DHFR intron expression vector.Nucleic Acids Res 1996;24(9):1774–1779.

53. Aldrich TL, Thomas JN, Morris AE. Improvedbicistronic mammalian expression vectorsusing expression augmenting sequence ele-ment (EASE). Cytotechnology 1998;28:9–17.

54. Gallie DR. The cap and poly(A) tail functionsynergistically to regulatemRNA translationalefficiency. Genes Dev 1991;5(11):2108–2116.

55. Jang SK,Wimmer E. Cap-independent trans-lation of encephalomyocarditis virus RNA:structural elements of the internal ribosomalentry site and involvement of a cellular 57-kDRNA-binding protein. Genes Dev 1990;4(9):1560–1572.

56. Running-Deer J, Allison DS. High-level ex-pression of proteins in mammalian cells usingtranscription regulatory sequences from theChinese hamster EF-1alpha gene. BiotechnolProg 2004;20(3):880–889.

57. Allison DS. Hamster EF-1a transcriptionalregulatory DNA. ICOS Corporation 1999;847218 [US 5888809].

58. Benton T, Chen T, McEnteeM, Fox B, King D,Crombie R, Thomas TC, Bebbington CR. Theuse of UCOE vectors in combination with apreadapted serum free, suspension cell lineallows for rapid production of large quantitiesof protein. Cytotechnology 2002;38:43–46.

59. Kwaks TH, Barnett P, HemrikaW, Siersma T,Sewalt RG, Satijn DP, Brons JF, van BloklandR,KwakmanP,KruckebergAL,KelderA,OtteAP. Identification of anti-repressor elementsthat confer high and stable protein productionin mammalian cells. Nat Biotechnol 2003;21(5):553–558. Erratum: Nat Biotechnol 2003;21(7):822.

60. Kim JM, Kim JS, Park DH, Kang HS, Yoon J,Baek K, Yoon Y. Improved recombinant geneexpression in CHO cells using matrix attach-ment regions. J Biotechnol 2004;107(2):95–105.

61. Beitel LK, McArthur JG, Stanners CP. Se-quence requirements for the stimulation ofgene amplification by a mammalian genomicelement. Gene 1991;102(2):149–156.

62. Hollis G, Mark GE, Homologous recombina-tion antibody expression system for murinecells. PCT/IB95/00014:1995; 23.

63. Bode J, Gotze S, Ernst E, H€usemann Y, BaerA, Seibler J, Mielke C. Architecture and uti-lization of highly expressed genomic sites.In Bernardi G (Ed.). New ComprehensiveBiochemistry. Amsterdam: Elsevier 2003;38:551–572. Makrides S (Volume ed.). GeneTransfer and Expression in MammalianCells, Chap. 20.

64. Wilson TJ, Kola I. The LoxP/CRE system andgenome modification. Methods Mol Biol 2001;158:83–94.

65. Kingston RE, Kaufman RJ, Bebbington CR,Rolfe MR. Amplification using CHO cell ex-pression vectors. In: Ausubel FM (Ed): CurrentProtocols in Molecular Biology. New York:John Wiley & Sons. 2002. Chap. 16: Unit16.23.1–16.12.13.

66. Shen AY, Van de Goor J, Zheng L, Reyes A,Krummen LA. Recombinant DNA technologyand cell line development. In: Ozturk SS, HuW-S, editors. Cell Culture Technology forPharmaceutical and Cell-Based Therapies.New York: CRC Press; 2006. p 15–40.

67. Graham FL, van der Eb AJ. A new techniquefor the assay of infectivity of human adeno-virus 5 DNA. Virology 1973;52(2):456–467.


68. Wilson SP, Smith LA. Addition of glycerolduring DNA exposure enhances calcium phos-phate transfection. Anal Biochem 1997;246(1):148–150.

69. Canatella PJ, Karr JF, Petros JA, PrausnitzMR. Quantitative study of electroporation-mediated molecular uptake and cell viability.Biophys J 2001;80(2):755–764.

70. Felgner PL, Ringold GM. Cationic liposome-mediated transfection. Nature 1989;337(6205):387–388.

71. Wurm FM, Pallavicini MG. Effects of metho-trexate on recombinant sequences in mamma-lian cells. In: Kellems RE,Editor. Gene Ampli-fication in Mammalian Cells: A Comprehen-sive Guide. Boca Raton: CRC press; 1993. p85–94.

72. Weaver JC, Bliss JG, Powell KT, Harrison GI,Williams GB. Rapid clonal growth measure-ments at the single-cell level: gelmicrodropletsand flow cytometry. Biotechnology 1991;9(9):873–877.

73. Gray F, Kenney JS, Dunne JF. Secretion cap-ture and report web: use of affinity derivatizedagarose microdroplets for the selection of hy-bridoma cells. J Immunol Methods 1995;182(2):155–163.

74. SchiffLJ.Review:production, characterization,and testing of banked mammalian cell sub-strates used to produce biological products. InVitro Cell Dev Biol 2005;41(6):5–70.

75. Van Wezel AL. Growth of cell-strains and pri-mary cells on micro-carriers in homogeneousculture. Nature 1967;216:64–65.

76. Hu W-S, Giard DJ, Wang DIC. Serial propa-gation of mammalian cells on microcarriers.Biotechnol Bioeng 1985;27:1466–1476.

77. Hu W-S, Wang DIC. Selection of microcarrierdiameter for the cultivation ofmammalian cellson microcarriers. Biotechnol Bioeng 1987;30:548–557.

78. Sambanis A, HuW-S. Cell culture bioreactors.In: Rehm H, Reed G,Editors. Biotechnology.New York: VCH; 1993. p 106–126.

79. Himes VB,HuW-S. Attachment and growth ofmammalian cells on microcarriers with differ-ent ion exchange capacities. Biotechnol Bioeng1987;29:1155–1163.

80. Warnock JN, Al-Rubeai M. Bioreactor sys-tems for the production of biopharmaceuti-cals from animal cells. Biotechnol Appl Bio-chem 2006;45:1–12.

81. Piret JM, Cooney CL. Mammalian cell andprotein distributions in ultrafiltration hollow

fiber bioreactors. Biotechnol Bioeng 1990;36:902–910.

82. Piret JM, Cooney CL. Model of oxygen trans-port limitations in hollow fiber bioreactors.Biotechnol Bioengin 1991;37:80–92.

83. Fenge C, Lullau E, Cell culture bioreactors. In:Ozturk SS, Hu W-S, editors. Cell Culture Tech-nologyforPharmaceuticalandCell-BasedThera-pies. Boca Raton: CRC Press; 2006. p 155–224.

84. Burgener A, Butler M. Medium development.In: Osturk SS, Hu W-S, editors. Cell Culturetechnology for Pharmaceutical and Cell-BasedTherapies. Boca Raton: CRC Press; 2006.p 41–79.

85. Thomas JN. Nutrients, oxygen and pH In:Thilly WG,Editor. Mammalian Cell Technol-ogy. Stoneham: Butterworth Publishers; 1986.p 109–130.

86. Thomas JN. Mammalian cell physiology. Bio-process Technol 1990;10:93–145.

87. Godia F, Cairo JJ. Cell metabolism. In: OsturkSS, Hu W-S,Editors. Cell Culture Technologyfor Pharmaceutical and Cell-Based Therapies.Boca Raton: CRC Press; 2006. p 81–112.

88. Seth G, Hossler P, Yee JC, HuW-S. Engineer-ing cells for cell culture bioprocessing: physio-logical fundamentals. Adv Biochem Eng Bio-technol 2006;101:119–164.

89. MajorsBS,BetenbaughMJ,ChiangGG. Linksbetween metabolism and apoptosis in mam-malian cells: applications for anti-apoptosisengineering. Metabol Eng 2007;9:317–326.

90. Chiang G, Sisk W. Bcl-xL mediates increasedproduction of humanized monoclonal antibo-dies inChinesehamster ovary cells.BiotechnolBioeng 2005;91(7):779–792.

91. Arden N, Betenbaugh MJ. Life and death inmammalian cell culture: strategies for apopto-sis inhibition. Trends Biotechnol 2004;22(4):174–180.

92. Wlaschin KF, Hu W-S. Engineering cell meta-bolism for high-density cell culture via manip-ulation of sugar transport. J Biotechnol2007;131:168–176.

93. Tan H, LeeM, YapM,Wang DIC. Overexpres-sion of cold-inducible RNA-binding protein in-creases interferon-y production in Chinese-hamster ovary cells. Biotechnol Appl Biochem2008;49:247–257.

94. KorkeR,DeLeonGattiM,LauALY,LimJWE,Seow TK, Chung MCM, Hu W-S. Large scalegene expression profiling of metabolic shift ofmammalian cells in culture. J Biotechnology.2004;107:1–17.

REFERENCES 331

95. Yee JC, de Leon Gatti M, Philp RJ, Yap M, HuW-S. Genomic and proteomic exploration ofCHO and hybridoma cells under sodium buty-rate treatment. Biotechnol Bioeng 2007;99(5):1186–1204.

96. Griffin T, Seth G, Xie H, Bandhakavi S, HuW-S. Advancing mammalian cell culture engi-neering using genome-scale technologies.Trends Biotechnol 2007;25(9):401–408.

97. Wlaschin KF, Seth G, Hu W-S. Toward geno-mic cell culture engineering. Cytotechnol2006;50:121–140.

98. Leser EW, Asenjo JA. Rational design of pur-ification processes for recombinant proteins. JChromatogr A 1992;584(1):43–57.

99. Wolk B, Bezy P, Arnold R, Blank G. Charac-teristics of a good antibodymanufacturing pro-cess. Bioprocess Int 2003;1(9):50–52, 54,56-58.

100. Curling J,GottschalkU. Process chromatogra-phy: five decades of innovation. Biopharm Int2007;1(1):70–94.

101. Farid SS. Process economics of industrialmonoclonal antibody manufacture. J Chroma-togr B 2007;848(1):8–18.

102. Ransohoff TC, Murphy MK, Levine HL. Auto-mation of biopharmaceutical purification pro-cesses. BioPharm 1990;3:20–24.

103. Roque ACA, Silva CSO, Taipa MA. Affinity-based methodologies and ligands for antibodypurification: advances and perspectives. JChromatogr A 2007;1160(1–2):44–55.

104. Farid SS. Economic drivers and trade-offs inantibody purification processes. BioPharm Int2008;21(Suppl 3):37–42.

105. Werner RG. Economic aspects of commercialmanufacture of biopharmaceuticals. JBiotech-nol 2004;113(1–3):171–182.

106. Isaacs IJ. The strategy employed for purifica-tion of protein-based biopharmaceutical pro-ducts. Biotechnol 1996;6(3):88–92.

107. Graslund S, Nordlund P, Weigelt J, HallbergBM, Bray J, Gileadi O, Knapp S, OppermannU, Arrowsmith C, Hui R,Ming J, dhe-PaganonS, Park HW, Savchenko A, Yee A, Edwards A,Vincentelli R, Cambillau C, Kim R, Kim SH,Rao Z, Shi Y, Terwilliger TC, Kim CY, HungLW, Waldo GS, Peleg Y, Albeck S, Unger T,Dym O, Prilusky J, Sussman JL, Stevens RC,Lesley SA,Wilson IA, JoachimiakA, Collart F,Dementieva I, Donnelly MI, Eschenfeldt WH,Kim Y, Stols L, Wu R, Zhou M, Burley SK,Emtage JS, Sauder JM, Thompson D, Bain K,Luz J, Gheyi T, Zhang F, Atwell S, Almo SC,Bonanno JB, Fiser A, Swaminathan S, Studier

FW, Chance MR, Sali A, Acton TB, Xiao R,ZhaoL,MaLC,Hunt JF, TongL, CunninghamK, InouyeM,AndersonS, JanjuaH, ShastryR,HoCK,WangD,WangH, JiangM,MontelioneGT, Stuart DI, Owens RJ, Daenke S, Schutz A,Heinemann U, Yokoyama S, Bussow K, Gun-salus KC. Protein production and purification.Nat Methods. 2008;5(2):135–146.

108. ShuklaAA,Kandula JR.Harvest and recoveryof monoclonal antibodies from large-scalemammalian cell culture. BioPharm Int2008;21(5):34–36 38, 40, 42, 44–45.

109. Yigzaw Y, Piper R, Tran M, Shukla AA. Ex-ploitation of the adsorptive properties of depthfilters for host cell protein removal duringmonoclonal antibody purification. BiotechnolProg 2006;22(1):288–296.

110. Ladisch MR, Velayudhan A. Scale-up techni-ques in bioseparation processes. Food FeedChem J 1995;10:113–138.

111. Scopes RK. The protein purification labora-tory. In: Cantor CR, editor. Protein Purifica-tionPrinciples andPractice. 3rd ed.NewYork:Springer-Verlag; 1994. p 1–21.

112. Shukla AA, Yigzaw Y. Modes of preparativechromatography. In: Shukla AA, EtzelMR,GadamS,Editors. Process scale biosepara-tions for the biopharmaceutical industry.Boca Raton: CRC Press; 2007. p 179–275.

113. Shukla AA, Cramer SM. Bioseparations bydisplacement chromatography. Separ SciTechnol 2000;2:379–415.

114. Swartz JR. Advances in Escherichia coli pro-duction of therapeutic proteins. Curr OpinBiotechnol 2001;12(2):195–201.

115. Baneyx F. Recombinant protein expression inEscherichia coli. Curr Opin Biotechnol 1999;10(5):411–421.

116. Carrio MM, Villaverde A. Construction anddeconstruction of bacterial inclusion bodies.J Biotechnol 2002;96(1):3–12.

117. Lilie H, Schwarz E, Rudolph R. Advances inrefolding of proteins produced in E. coli. CurrOpin Biotechnol 1998;9(5):497–501.

118. Sadana A. Protein refolding and inactivationduring bioseparation: bioprocessing implica-tions. Biotechnol Bioeng 1995;48:481–489.

119. Arakawa T, Tsumoto K, Kita Y, Chang B,Ejima D. Biotechnology applications of aminoacids in protein purification and formulations.Amino Acids 2007;33(4):587–605.

120. Singh SM, Panda AK. Solubilization and re-folding of bacterial inclusion body proteins. JBiosci Bioeng 2005;99(4):303–310.


121. Geng X, Wang C. Protein folding liquid chro-matography and its recent developments. JChromatogr B 2007;849(1–2):69–80.

122. De Bernardez Clark E, Schwarz E, Rudolph R.Inhibition of aggregation side reactions duringin vitro protein folding. Methods Enzymol1999;309:217–236.

123. Cowgill C, Ozturk AG, St John R. Proteinrefolding and scale up. In: Shukla AA, EtzelMR, Gadam S,Editors. Process scale biose-parations for the biopharmaceutical industry.Boca Raton: CRC Press; 2007. p 123–178.

124. Sahdev S, Khattar SK, Saini KS. Production ofactive eukaryotic proteins through bacterialexpression systems: a review of the existingbiotechnology strategies. Mol Cell Biochem2008;307(1–2):249–264.

125. Wong HH, O’Neill BK, Middelberg AP. Cen-trifugal recovery and dissolution of recombi-nant Gly-IGF-II inclusion-bodies: the impactof feed rate and re-centrifugation on proteinyield. Bioseparation 1996;6(3):185–192.

126. Khalilzadeh R, Shojaosadati SA, MaghsoudiN, Mohammadian-Mosaabadi J, MohammadiMR, Bahrami A, Maleksabet N, Nassiri-Kha-lilli MA, Ebrahimi M, Naderimanesh H. Pro-cess development for production of recombi-nant human interferon-gamma expressed inEscherichia coli. J Ind Microbiol Biotechnol2004;31(2):63–69.

127. HutchinsonN,BinghamN,MurrellN,FaridS,HoareM. Shear stress analysis of mammaliancell suspensions for prediction of industrialcentrifugation and its verification. BiotechnolBioengin 2006;95(3):483–491.

128. Maybury JP, Mannweiler K, Titchener-Hoo-kerNJ,HoareM,Dunnhill P. Theperformanceof a scaled down industrial disc stack centri-fuge with a reduced feed material require-ment. Bioprocess Eng 1998;18:191–199.

129. Riske F, Schroeder J, Belliveau J, Kang X,Kutzko J, Menon MK. The use of chitosan asa flocculant inmammalian cell culture drama-tically improves clarification throughputwith-out adversely impacting monoclonal antibodyrecovery. J Biotechnol 2007;128(4):813–823.

130. van Reis R, Zydney A. Membrane separationsin biotechnology. Curr Opin Biotechnol2001;12(2):208–211.

131. Fahrner RL, Knudsen HL, Basey CD, GalanW, Feuerhelm D, Vanderlaan M, Blank GS.Industrial purification of pharmaceutical anti-bodies: development, operation, and valida-tion of chromatography processes. BiotechnolGenet Eng Rev 2001;18:301–327.

132. Shukla AA, Hubbard B, Tressel T, GuhanS, Low D. Downstream processing of mono-clonal antibodies: application of platformapproaches. J Chromatogr B 2007;848(1):28–39.

133. Fahrner RL, Iyer HV, Blank GS. The optimalflow rate and column length for maximumproduction rate of protein A affinity chromato-graphy. Bioprocess Eng 1999;21(4):287–292.

134. Gronberg A, Monie E, Murby M, Rodrigo G,Wallby E, Johansson HJ. A strategy for devel-oping a monoclonal antibody purification plat-form. Bioprocess Int 2007;5(1):48–50, 52–55.

135. Hober S, Nord K, Linhult M, Protein A chro-matography for antibody purification. J Chro-matogr B 2007;848(1):40–47.

136. GE-Healthcare Instructions. 11-0026-01 ABaffinity chromatography MabSelect SuRe�.2005; 1-23.

137. Ghose S, Allen M, Hubbard B, Brooks C, Cra-mer SM.Antibody variable region interactionswith protein A: implications for the develop-ment of generic purification processes. Bio-technol Bioeng 2005;92(6):665–673.

138. Carter-Franklin JN, Victa C, McDonald P,Fahrner R. Fragments of protein A elutedduring protein A affinity chromatography. JChromatography A 2007;1163(1–2):105–111.

139. O’Leary R, FeuerhelmD, PeersD, Xu Y, BlankGS. Determining the useful lifetime of chroma-tography resins. BioPharm 2001;14(9):10–18.

140. Gagnon P. Polishing methods for monoclonalIgC purification. In: Shukla AA, Etzel MR,Gadam S,Editors. Pocess scale bioseparationsfor the biopharmaceutical industry. Boca Ra-ton: CRC Press; 2007. p 491–505.

141. VedanthamG, VunnumS, Tressel T, HubbardB. mAb purification processes: current trendsand future directions. IBC’s 3rd BioProcessInternational� Asia Pacific Conference; 2008.

142. U.S. FDA. Guidance for industry Q5A viralsafety evaluation of biotechnology productsderived from cell lines of humananimal origin.1998. Available at http:///www.fda.gov/cber/guidelines.htm.

143. Curtis S, Lee K, Blank GS, Brorson K, Xu Y.Generic/matrix evaluation of SV40 clearanceby anion exchange chromatography in flow-through mode. Biotechnol Bioeng 2003;84(2):179–186.

144. Zhou JX, Dehghani H. Viral clearance: inno-vative versus classical methods: theoreticaland practical concerns. Am Pharm Rev2006;9(5):74–82.

REFERENCES 333

145. Walter JK, Nothelfer F, WerzW, Validation ofviral safety for pharmaceutical proteins. In:Subramanian G, editor. Bioseparation andBioprocessing. Weinheim: Wiley-VCH; 1998.p 465–496.

146. Wu Y, Ahmed A, Waghmare R, Genest P, Is-sacson S, KrishnanM, KahnDW. Validation ofadventitious virus removal by virus filtration: anovel procedure for monoclonal antibody pro-cesses. BioProcess Int 2008;6(5):54 56, 58–60.

147. Cartwright T. Protein purification applica-tions. National Library 2001;2(2):49–71.

148. Zhou JX, Tressel T. Current practice and fu-ture strategies for viral clearance in mAbdownstream production. Am Pharm Rev2008;11(1):109–110, 112, 114–121.

149. van Reis R. New frontiers in bioseparationtechnology. Adv Filtr Sep Technol 13A:1999;4–7.

150. Coffman JL,KramarczykJF,KelleyBD.High-throughput screening of chromatographic se-parations. I. Method development and columnmodeling. Biotechnol Bioeng 2008;100(4):605–618.

151. Kramarczyk JF,KelleyBD,Coffman JL.High-throughput screening of chromatographic se-parations. II. Hydrophobic interaction. Bio-technol Bioeng 2008;100(4):707–720.

152. Wensel DL, Kelley BD, Coffman JL. High-throughput screening of chromatographic se-parations. III.Monoclonal antibodies on ceramichydroxyapatite. Biotechnol Bioeng 2008;100(5):839–854.

153. Kelley BD, Switzer M, Bastek P, KramarczykJF, Molnar K, Yu T, Coffman J. High-through-put screening of chromatographic separations.IV. Ion-exchange. Biotechnol Bioeng 2008;100(5):950–963.

154. Mehta A, Tse ML, Fogle J, Len A, Shrestha R,Fontes N, Lebreton B, Wolk B, van Reis R.Purifying therapeutic monoclonal antibodies.Chem Eng Prog 2008;104(5):S14–S20.

155. Kelley B. Very large scale monoclonal antibodypurification: the case for conventional unit op-erations.BiotechnolProg2007;23(5):995–1008.

156. Kelley BD, Tobler SA, Brown P, Coffman JL,Godavarti R, Iskra T, Switzer M, Vunnum S.Weak partitioning chromatography for anionexchange purification of monoclonal antibo-dies. Biotechnol Bioeng 2008;101(3):553–566.

157. Shultz J. Large-scale pool-less purification:applications towards reducing overhead costand production time. AIChE National Meet-ing; 2006.

158. Shultz J,Case study: How to develop a 1-tonmicrobial protein process, when the world isnot ready to manufacture it. Presented at Re-covery of Biological Products XIII, Quebec,Canada, Jun 22–26 2008.

159. Datar RV, Cartwright T, Rosen CG. Processeconomics of animal cell and bacterial fer-mentations: a case study analysis of tissueplasminogen activator. Biotechnology 1993;11(3):349–357.

160. Low D, O’Leary R, Pujar NS. Future of anti-body purification. J Chromatogr B 2007;848(1):48–63.

161. VunnumS,VedanthamG,HubbardB. ProteinA based affinity chromatography. InGottschalk U, editor. Process Scale Purifica-tion of Antibodies. John Wiley & Sons, Inc.,2009. p 79–102.

162. Imamoglu S. Simulated moving bed chroma-tography (SMB) for application in biosepara-tion. Adv Biochem Eng Biotechnol 2002;76:211–231.

163. Boi C. Membrane adsorbers as purificationtools for monoclonal antibody purification. JChromatogr B 2007;848(1):19–27.

164. Gottschalk U. Bioseparation in antibody man-ufacturing: the good, the bad and the ugly.Biotechnol Prog 2008;24:496–503.

165. Etzel MR, Riordan WT. Membrane chromato-graphy: breakthrough curves and viral clear-ance. In: Shukla AA, Etzel MR, Gadam S,editors. Process Scale Bioseparations for theBiopharmaceutical Industry. Boca Raton:CRC Press; 2007. p 277–365.

166. Zhou JX, Tressel T. Basic concepts in Q mem-brane chromatography for large-scale anti-body production. Biotechnol Prog 2006;22(2):341–349.

167. Zhou JX, Solamo F, Hong T, Shearer M, Tres-sel T.Viral clearanceusing disposable systemsin monoclonal antibody commercial down-stream processing. Biotechnol Bioeng 2008;100(3):488–496.

168. Przybycien TM, Pujar NS, Steele LM. Alter-native bioseparation operations: life beyondpacked-bed chromatography. Curr Opin Bio-technol 2004;15(5):469–478.

169. Chang BS, Hershenson S. Practical ap-proaches to protein formulation development.Pharm Biotechnol 2002;13:1–25.

170. Daugherty AL, Mrsny RJ. Formulation anddelivery issues for monoclonal antibody thera-peutics. Adv Drug Del Rev 2006;58(5–6):686–706.


171. Krishnan S, Pallito MM, Ricci MS. Develop-ment of formulations for therapeuticmonoclonal antibodies andFc fusionproteins.Formulation and Process DevelopmentStrategies for Manufacturing of a Biophar-maceutical. John Wiley & Sons, Inc., 2010.P. 381–425.

172. Wang W, Singh S, Zeng DL, King K, Nema S.Antibody structure, instability, and formula-tion. J Pharm Sci 2007;96(1):1–26.

173. ManningMC, PatelK, BorchardtRT. Stabilityof protein pharmaceuticals. PharmRes 1989;6(11):903–918.

174. Cleland JL, Powell MF, Shire SJ. The devel-opment of stable protein formulations: a closelook at protein aggregation, deamidation, andoxidation. Crit Rev Ther Drug Carrier Syst1993;10(4):307–377.

175. Volkin DB, Mach H, Middaugh CR. Degrada-tive covalent reactions important to proteinstability. Methods Mol Biol 1995;40:35–63.

176. ChuGC,CheliusD,XiaoG,KhorHK,CoulibalyS, Bondarenko PV. Accumulation of succini-mide in a recombinant monoclonal antibody inmildly acidic buffers under elevated tempera-tures. Pharm Res 2007;24(6):1145–1156.

177. DehartMP,AndersonBD.The role of the cyclicimide in alternate degradation pathways forasparagine-containing peptides and proteins.J Pharm Sci 2007;96(10):2667–2685.

178. Wakankar AA, Borchardt RT. Formulationconsiderations for proteins susceptible to as-paragine deamidation and aspartate isomer-ization. J Pharma Sci 2006;95(11):2321–2336.

179. Patel K, Borchardt RT. Deamidation of aspar-aginyl residues in proteins: a potential path-way for chemical degradation of proteins inlyophilized dosage forms. J Parenter Sci Tech-nol 1990;44(6):300–301.

180. Wakankar AA, Borchardt RT, Eigenbrot C,Shia S, Wang YJ, Shire SJ, Liu JL. Aspartateisomerization in the complementarity-deter-mining regions of two closely related monoclo-nal antibodies. Biochemistry 2007;46(6):1534–1544.

181. Li S, Nguyen TH, Schoneich C, Borchardt RT.Aggregation and precipitation of human relax-in induced by metal-catalyzed oxidation. Bio-chemistry 1995;34(17):5762–5772.

182. Khossravi M, Shire SJ, Borchardt RT. Evi-dence for the involvement of histidine A(12)in the aggregation and precipitation of humanrelaxin induced by metal-catalyzed oxidation.Biochemistry 2000;39(19):5876–5885.

183. Thirumangalathu R, Krishnan S, BondarenkoP, Speed-Ricci M, Randolph TW, CarpenterJF, Brems DN. Oxidation of methionine resi-dues in recombinant human interleukin-1 re-ceptor antagonist: implications of conforma-tional stability on protein oxidation kinetics.Biochemistry 2007;46(21):6213–6224.

184. LiuD,RenD,HuangH,DankbergJ,RosenfeldR, CoccoMJ, Li L, BremsDN, Remmele RL Jr.Structureandstability changesof humanIgG1Fc as a consequence of methionine oxidation.Biochemistry 2008;47(18):5088–5100.

185. Lam XM, Yang JY, Cleland JL. Antioxidantsfor prevention of methionine oxidation in re-combinant monoclonal antibody HER2. JPharm Sci 1997;86(11):1250–1255.

186. YuL,VizelA,HuffMB,YoungM,RemmeleRLJr, He B. Investigation of N-terminal gluta-mate cyclization of recombinant monoclonalantibody in formulation development. JPharm Biomed Anal 2006;42(4):455–463.

187. Kerwin BA, Remmele RL Jr. Protect fromlight: photodegradation and protein biologics.J Pharm Sci 2007;96(6):1468–1479.

188. Gadgil HS, Bondarenko PV, Pipes GD, DillonTM, Banks D, Abel J, Kleemann GR, TreuheitMJ. Identification of cysteinylation of a freecysteine in the Fab region of a recombinantmonoclonal IgG1 antibody using Lys-C limitedproteolysis coupledwithLC/MSanalysis. AnalBiochemistry 2006;355(2):165–174.

189. Banks DD, Gadgil HS, Pipes GD, BondarenkoPV, Hobbs V, Scavezze JL, Kim J, Jiang XR,Mukku V, Dillon TM. Removal of cysteinyla-tion from an unpaired sulfhydryl in the vari-able region of a recombinant monoclonal IgG1antibody improves homogeneity, stability, andbiological activity. J Pharm Sci 2008;97(2):775–790.

190. Andya JD, Maa YF, Costantino HR, NguyenPA, Dasovich N, Sweeney TD, Hsu CC, ShireSJ. The effect of formulation excipients onprotein stability and aerosol performance ofspray-dried powders of a recombinant huma-nized anti-IgE monoclonal antibody. PharmRes 1999;16(3):350–358.

191. Gadgil HS, Bondarenko PV, Pipes G, RehderD, McAuley A, Perico N, Dillon T, Ricci M,Treuheit M. The LC/MS analysis of glycationof IgG molecules in sucrose containing formu-lations. J Pharm Sci 2007;96(10):2607–2621.

192. Quan C, Alcala E, Petkovska I, Matthews D,Canova-Davis E, Taticek R, Ma S. A study inglycation of a therapeutic recombinant huma-nized monoclonal antibody: where it is, how it

REFERENCES 335

got there, and how it affects charge-based be-havior. Anal Biochem 2008;373(2): 179–191.

193. Tzannis ST, HrusheskyWJM,Wood PA, Przy-bycien TM. Adsorption of a formulated proteinon a drug delivery device surface. J ColloidInterface Sci 1997;189(2):216–228.

194. Roberts CJ. Non-native protein aggregationkinetics. Biotechnol Bioeng 2007;98(5):927–938.

195. Chi EY, Krishnan S, Randolph TW, CarpenterJF. Physical stability of proteins in aqueoussolution: mechanism and driving forces in non-nativeprotein aggregation. PharmRes2003;20(9):1325–1336.

196. WangW.Protein aggregationand its inhibitionin biopharmaceutics. International J Pharm2005;289(1–2):1–30.

197. Cromwell ME, Hilario E, Jacobson F. Proteinaggregation and bioprocessing. AAPS J 2006;8(3):E572–E579.

198. Carpenter JF, Kendrick BS, Chang BS, Man-ning MC, Randolph TW. Inhibition of stress-induced aggregation of protein therapeutics.Methods Enzymol 1999;309:236–255.

199. CarpenterJF,RandolphTW,JiskootW,Crom-melin DJ, Middaugh CR, Winter G, Fan YX,Kirshner S, Verthelyi D, Kozlowski S, ClouseKA, Swann PG, Rosenberg A, Cherney B.Overlooking subvisible particles in therapeu-tic protein products: gaps that may compro-mise product quality. J Pharm Sci 2009;98(4):1201–1205.

200. Patten PA, Schellekens H. The immunogeni-city of biopharmaceuticals. Lessons learnedand consequences for protein drug develop-ment. Dev Biol 2003;112:81–97.

201. DemeuleB,GurnyR,ArvinteT.Wherediseasepathogenesismeets protein formulation: renaldeposition of immunoglobulin aggregates. EurJ Pharm Biopharm 2006;62(2):121–130.

202. Gokarn YR, Kosky A, Drad E, McAuley A,Remmele RL. Excipients for protein drugs. In:Ashok Katdare MVC, editor Excipient Devel-opment for Pharmaceutical, Biotechnology,and Drug Delivery Systems. New York: CRCPress; 2006. p 291–332.

203. Arakawa T, Prestrelski SJ, Kenney WC, Car-penter JF. Factors affecting short-term andlong-term stabilities of proteins. Adv Drug DelRev 2001;46(1–3):307–326.

204. Laursen T, Hansen B, Fisker S. Pain percep-tion after subcutaneous injections of mediacontaining different buffers. Basic Clin Phar-macol Toxicol 2006;98(2):218–221.

205. Liu J, Nguyen MD, Andya JD, Shire SJ. Re-versible self-association increases the viscosity

of a concentrated monoclonal antibody inaqueous solution. J Pharm Sci 2005;94(9):1928–1940. Erratum: J Pharm Sci 2006;95(1): 234–235.

206. Wang W, Wang YJ, Wang DQ. Dual effects ofTween 80 on protein stability. Int J Pharm2008;347(1–2):31–38.

207. Roy S, Jung R, Kerwin BA, Randolph TW,Carpenter JF. Effects of benzyl alcohol onaggregation of recombinant human interleu-kin-1-receptor antagonist in reconstituted lyo-philized formulations. J Pharm Sci 2005;94(2):382–396.

208. Ha E, Wang W, Wang YJ. Peroxide formationin polysorbate 80 and protein stability. JPharm Sci 2002;91(10):2252–2264.

209. Stoner MR, Fischer N, Nixon L, Buckel S,Benke M, Austin F, Randolph TW, KendrickBS. Protein-solute interactions affect the out-come of ultrafiltration/diafiltration opera-tions. J Pharm Sci 2004;93(9):2332–2342.

210. Saluja A, Kalonia DS. Nature and conse-quences of protein–protein interactions inhighprotein concentration solutions. Int J Pharm2008;358(1–2):1–15.

211. Kanai S, Liu J, Patapoff TW, Shire SJ. Rever-sible self-association of a concentrated mono-clonal antibody solutionmediated by Fab–Fabinteraction that impacts solution viscosity. JPharm Sci 2008;97(10):4219–4227.

212. Gokarn YR, Kras E, Nodgaard C, Dharmavar-am V, Fesinmeyer RM, Hultgen H, Brych S,Remmele RL Jr, Brems DN, Hershenson S.Self-buffering antibody formulations. J PharmSci 2008;97(8):3051–3066.

213. Chen B, Bautista R, Yu K, Zapata GA,Mulkerrin MG, Chamow SM. Influence ofhistidine on the stability and physical prop-erties of a fully human antibody in aqueousand solid forms. Pharm Res 2003;20(12):1952–1960.

214. ShireSJ, ShahrokhZ,LiuJ.Challenges in thedevelopment of high protein concentrationformulations. J Pharm Sci 2004;93(6):1390–1402.

215. Bhatnagar BS, Bogner RH, Pikal MJ. Proteinstability during freezing: separation of stres-ses and mechanisms of protein stabilization.Pharm Dev Technol 2007;12(5):505–523.

216. Carpenter JF, Pikal MJ, Chang BS, RandolphTW. Rational design of stable lyophilized pro-tein formulations: some practical advice.Pharm Res 1997;14(8):969–975.

217. Nail SL, Jiang S, Chongprasert S, Knopp SA.Fundamentals of freeze-drying. Pharm Bio-technol 2002;14:281–360.


218. Wang W. Lyophilization and development ofsolid protein pharmaceuticals. Int J Pharm2000;203(1–2):1–60.

219. Breen ED, Curley JG, Overcashier DE, HsuCC,ShireSJ.Effect ofmoistureon the stabilityof a lyophilized humanized monoclonal anti-body formulation. Pharm Res 2001;18(9):1345–1353.

220. Iacocca RG, Allgeier M. Corrosive attack ofglass by a pharmaceutical compound. J MatSci 2007;42:801–811.

221. SluzkyV,TamadaJA,KlibanovAM,LangerR.Kinetics of insulin aggregation in aqueous so-lutions upon agitation in the presence of hy-drophobic surfaces. Proc Natl Acad Sci USA1991;88(21):9377–9381.

222. SchwarzenbachMS, Reimann P, Thommen V,Hegner M, Mumenthaler M, Schwob J,Guntherodt HJ. Interferon alpha-2a interac-tionsonglass vial surfacesmeasuredbyatomicforce microscopy. PDA J Pharm Sci Technol2002;56(2):78–89.

223. Qadry SS, Roshdy TH, Char H, Del Terzo S,Tarantino R, Moschera J. Evaluation of CZ-resin vials for packaging protein-based par-enteral formulations. Int J Pharm 2003;252(1–2):207–212.

224. Smith EJ. Siliconization of parenteral packa-ging components. J Parenter Sci Technol1988;42(4S):S1–S13.

225. Baldwin RN. Contamination of insulin bysilicone oil: a potential hazard of plastic in-sulin syringes. Diabet Med 1988;5(8):789–790.

226. Jones LS, Kaufmann A, Middaugh CR. Sili-cone oil induced aggregation of proteins. JPharm Sci 2005;94(4):918–927.

227. GoldbergM,Gomez-Orellana I. Challenges forthe oral delivery of macromolecules. Nat RevDrug Discov 2003;2(4):289–295.

228. Gomez-Orellana I, Paton DR. Advances in theoral delivery of proteins. Expert Opinion onTherapeutic Patents 1998;8(3):223–234.

229. Witschi C, Mrsny RJ. In vitro evaluation ofmicroparticles and polymer gels for use asnasal platforms for protein delivery. PharmRes 1999;16(3):382–390.

230. Smart JD. Recent developments in the use ofbioadhesive systems fordeliveryof drugs to theoral cavity. Criti Rev Ther Drug Carrier Syst2004;21(4):319–344.

231. BrownMB, TraynorMJ,MartinGP, AkomeahFK. Transdermal drug delivery systems: skinperturbation devices. Methods Mol Biol2008;437:119–139.

232. Fineberg SE. Diabetes therapy trials with in-haled insulin. Expert Opin Investig Drugs2006;15(7):743–762.

233. Kumar TR, Soppimath K, Nachaegari SK. No-vel delivery technologies for protein and pep-tide therapeutics. Curr Pharm Biotechnol2006;7(4):261–276.

234. Grainger DW. Controlled-release and local de-livery of therapeutic antibodies. Expert OpinBiol Ther 2004;4(7):1029–1044.

235. Lee KY, Yuk SH. Polymeric protein deliverysystems. Prog Polym Sci 2007;32(7):669–697.

236. SalmasoS,BersaniS, SemenzatoA,Caliceti P.Nanotechnologies in protein delivery. J Na-nosc Nanotechnol 2006;6(9–10):2736–2753.

237. Jorgensen L, Moeller EH, van de Weert M,Nielsen HM, Frokjaer S. Preparing and eval-uating delivery systems for proteins. Eur JPharm Sci 2006;29(3–4):174–182.

238. Bilati U, Allemann E, Doelker E. Protein en-capsulation: protein drugs entrapped withinmicro- and nanoparticles: an overview of ther-apeutic challenges and scientific issues. DrugDeliv Technol 2005;5(8):41–47.

239. Cleland JL. Protein delivery from biodegrad-ablemicrospheres. PharmBiotechnol 1997;10:1–43.

240. Pettit DK, Lawter JR, HuangWJ, Pankey SC,Nightlinger NS, Lynch DH, Schuh JA, Morris-sey PJ, Gombotz WR. Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) mi-crospheres for controlled release of GM-CSF.Pharm Res 1997;14(10):1422–1430.

241. Veronese FM, Mero A. The impact of PEGyla-tion on biological therapies. BioDrugs 2008;22(5):315–329.

242. Farr S, Boyd B, Bridges P, Linn LS. Usingneedle-free injectors for parenteral delivery ofproteins. In: Eugene JEH, McNally J,Editors.Protein Formulation and Delivery. Boca Ra-ton: CRC Press; 2007. p 255–283.

243. Prausnitz MR. Microneedles for transdermaldrug delivery. Adv Drug Del Rev 2004;56(5):581–587.

244. Nayar R, Manning MC. High throughput for-mulation: strategies for rapid development ofstable protein products. Pharm Biotechnol2002;13:177–198.

245. Capelle MA, Gurny R, Arvinte T. Highthroughput screening of protein formulationstability: practical considerations. Eur JPharm Biopharm 2007;65(2):131–148.

246. Jen A, Merkle HP. Diamonds in the rough:protein crystals from a formulation perspec-tive. Pharm Res 2001;18(11):1483–1488.

REFERENCES 337

247. Basu SK, Govardhan CP, Jung CW, MargolinAL.Protein crystals for thedelivery of biophar-maceuticals. Expert Opin Biol Ther 2004;4(3):301–317.

248. GovardhanC, Khalaf N, Jung CW, SimeoneB,Higbie A, Qu S, Chemmalil L, Pechenov S,Basu SK,MargolinAL.Novel long-acting crys-tal formulation of human growth hormone.Pharm Res 2005;22(9):1461–1470.

249. Flatman S, Alam I, Gerard J, Mussa N. Pro-cess analytics for purification of monoclonalantibodies. J Chromatogr B 2007;848(1):79–87.

250. Apostol I, KelnerDN.Managing the analyticallifecycle for biotechnology products: a journeyfrom method development to validation. Part1. Bioprocess Int 2008;6(8):12–19.

251. Apostol I, Schofield T, Koeller G, Powers S,Stawicki M,Wolfe RA. A rational approach forsetting and maintaining specifications for bio-logical and biotechnology-derived products.Part 1. BioPharm Int 2008;21(6):42–55.

252. Wehr T, Rodriguez-Diaz R. Use of size exclu-sion chromatography in biopharmaceutical de-velopment. In: Rodriguez-Diaz R, Wehr T,Tuck S,Editors. Analytical Techniques for Bio-pharmaceutical Development. New York:Marcel Dekker; 2005. p 95–112.

253. Ribarska JT, Jolevska ST, Panovska AP, Di-mitrovska A. Studying the formation of aggre-gates in recombinant human granulocyte-col-ony stimulating factor (rHuG-CSF), lenogras-tim, using size-exclusion chromatography andSDS-PAGE. Acta Pharm 2008;58(2):199–206.

254. Gunturi SR, Ghobrial I, Sharma B. Develop-ment of a sensitive size exclusion HPLCmeth-od with fluorescence detection for the quanti-tation of recombinant human erythropoietin(r-HuEPO) aggregates. J Pharm Biomed Anal2007;43(1):213–221.

255. Rosenberg AS. Effects of protein aggregates:an immunologic perspective. AAPS J 2006;8(3):E501–507.

256. Gagnon P. Practical strategies for protein con-taminant detection by high-performance ion-exchangechromatography. In:Rodriguez-DiazR, Wehr T, Tuck S,Editors. Analytical Techni-ques for Biopharmaceutical Development.New York: Marcel Dekker; 2005. p 67–79.

257. WeitzhandlerM, FarnanD,Horvath J, RohrerJS, Slingsby RW, Avdalovic N, Pohl C. Proteinvariant separations by cation-exchange chro-matography on tentacle-type polymeric sta-tionary phases. J Chromatogr A. 1998;828(1–2):365–372.

258. Weitzhandler M, Farnan D, Rohrer JS, Avda-lovic N. Protein variant separations using ca-tion exchange chromatography on grafted,polymeric stationary phases. Proteomics2001;1(2):179–185.

259. Harris RJ, Kabakoff B, Macchi FD, Shen FJ,KwongM,Andya JD, Shire SJ, BjorkN, TotpalK, Chen AB. Identification of multiple sourcesof charge heterogeneity in a recombinant anti-body. J Chromatogr B 2001;752(2):233–245.

260. DickLWJr,QiuD,MahonD,AdamoM,ChengKC. C-terminal lysine variants in fully humanmonoclonal antibodies: investigation of testmethods and possible causes. BiotechnolBioeng 2008;100(6):1132–1143.

261. Ahrer K, Jungbauer A. Chromatographic andelectrophoretic characterization of protein var-iants. J Chromatog B 2006;841(1–2):110–122.

262. Kundu A, Barnthouse KA, Cramer SM. Selec-tive displacement chromatography of proteins.Biotechnol Bioeng 1997;56(2):119–129.

263. Wypych J, Li M, Guo A, Zhang Z, Martinez T,Allen MJ, Fodor S, Kelner DN, Flynn GC, LiuYD, Bondarenko PV, Ricci MS, Dillon TM,Balland A. Human IgG2 antibodies displaydisulfide-mediated structural isoforms. J BiolChem 2008;283(23):16194–16205.

264. Kannan K, Mulkerrin MG, Zhang M, Gray R,Steinharter T, Sewerin K, Baffi R, Harris R,Karunatilake C. Rapid analytical tryptic map-ping of a recombinant chimeric monoclonalantibody and method validation challenges.J Pharma Biomed Anal 1997;16(4):631–640.

265. Bongers J, Cummings JJ, Ebert MB, FedericiMM, Gledhill L, Gulati D, Hilliard GM, JonesBH, Lee KR, Mozdzanowski J, Naimoli M,Burman S. Validation of a peptide mappingmethod for a therapeuticmonoclonal antibody:what could we possibly learn about a methodwe have run 100 times?. J PharmBiomedAnal2000;21(6):1099–1128.

266. Kroon DJ, Baldwin-Ferro A, Lalan P. Identi-fication of sites of degradation in a therapeuticmonoclonal antibody by peptide mapping.Pharma Res 1992;9(11):1386–1393.

267. Dillon TM, Bondarenko PV, Rehder DS, PipesGD,KleemannGR,RicciMS.Optimizationof areversed-phase high-performance liquid chro-matography/mass spectrometry method forcharacterizing recombinant antibody hetero-geneity and stability. J Chromatogr A 2006;1120(1–2):112–120.

268. Dalmora SL, Masiero SMK, De Oliveira PR,DaSilvaSangoiM,BrumLJr. Validation of anRP-LC method and assessment of rhG-CSF in


pharmaceutical formulations by liquid chro-matography and biological assay. J Liq Chro-matogr Relat Technol 2006;29:1753–1767.

269. Yang J,Wang S, Liu J, Raghani A. Determina-tion of tryptophan oxidation of monoclonalantibody by reversed phase high performanceliquid chromatography. J Chromatogr A 2007;1156(1–2):174–182.

270. Scheich C, Leitner D, Sievert V, Leidert M,SchlegelB,SimonB,Letunic I,BussowK,DiehlA. Fast identification of folded human proteindomains expressed in E. coli suitable for struc-tural analysis. BMC Struct Biol 2004;4:4.

271. Aizawa P, Winge S, Karlsson G. Large-scalepreparation of thrombin from human plasma.Thromb Res 2008;122(4):560–567.

272. Battersby JE, Snedecor B, Chen C, ChampionKM, Riddle L, Vanderlaan M. Affinity-reversed-phase liquid chromatography assayto quantitate recombinant antibodies andantibody fragments in fermentation broth. JChromatogr A 2001;927(1–2):61–76.

273. Ma S. Analysis of protein therapeutics by ca-pillary electrophoresis: applications and chal-lenges. Dev Biol 2005;122:49–68.

274. Han M, Phan D, Nightlinger N, Taylor L,Jankhah S, Woodruff B, Yates Z, FreemanS, Guo A, Balland A, Pettit DK, Optimiza-tion of CE-SDS method for antibody separa-tion based on multi-users experimentalpractices chromatographia 2006;64(5–6):1–8.

275. NunnallyB,ParkS,PatelK,HongM,ZhangX,WangS, Rener B, Reed-BoganA, Salas-SolanoO, Lau W, Girard M, Carnegie H, Garcia-Cañas V, Cheng KC, Zeng M, Ruesch M, Fra-zier R, Jochheim C, Natarajan K, Jessop K,Saeed M, Moffatt F, Madren S, Thiam SA,Altria K. Series of collaborations between var-ious pharmaceutical companies and regula-tory authorities concerning the analysis ofbiomolecules using capillary electrophoresis.Chromatographia 2006;64(5–6):359–368.

276. Salas-Solano O, Tomlinson B, Du S, Parker M,Strahan A, Ma S. Optimization and validationof a quantitative capillary electrophoresis so-diumdodecyl sulfatemethod for quality controland stability monitoring of monoclonal antibo-dies. Anal Chem 2006;78(18):6583–6594.

277. Ma S. Analysis of protein therapeutics by ca-pillary electrophoresis. Chromatographia2001;53(Suppl 1):S75–S89.

278. Guo A, Han M, Martinez T, Ketchem RR,Novick S, Jochheim C, Balland A. Electro-phoretic evidence for thepresence of structural

isoforms specific for the IgG2 isotype. Electro-phoresis 2008;29(12):2550–2556.

279. Amon S, Zamfir AD, Rizzi A. Glycosylationanalysis of glycoproteins and proteoglycansusing capillary electrophoresis-mass spectro-metry strategies. Electrophoresis 2008;29(12):2485–2507.

280. HanM,GuoA, JochheimC, ZhangY,MartinezT, Kodama P, Pettit DK, Balland A. Analysisof glycosylated type II interleukin-1 receptor(IL-1R)by imagedcapillary isoelectric focusing(i-cIEF). Chromatographia 2007;66(11–12):969–976.

281. U.S.FoodandDrugAdministrationCenter forBiologics Evaluation and Research. Points toconsider in the manufacture and testing ofmonoclonal antibody products for human use(1997). J Immun 1997;20(3):214–243.

282. Xu Y, Brorson K. An overview of quantitativePCR assays for biologicals: quality and safetyevaluation. Dev Biol 2003;113:89–98.

283. NissomPM.Specific detection of residualCHOhost cell DNA by real-time PCR. Biologicals2007;35(3):211–215.

284. Lovatt A. Applications of quantitative PCR inthe biosafety and genetic stability assessmentof biotechnology products. J Biotechnol 2002;82(3):279–300.

285. Kubista M, Andrade JM, Bengtsson M, For-ootan A, Jonak J, Lind K, Sindelka R, SjobackR, Sjogreen B, StrombomL, Stahlberg A, ZoricN. The real-time polymerase chain reaction.Mol Asp Med 2006;27(2–3):95–125.

286. EatonLC.Host cell contaminant protein assaydevelopment for recombinant biopharmaceu-ticals. J Chromatogr A 1995;705(1):105–114.

287. ChampionK,MaddenH,DoughertyJ,ShacterE.Defining your product profile and maintainingcontroloverit,Part2.BioprocessInt2005;52–57.

288. Krawitz DC, Forrest W, Moreno GT, KittlesonJ, Champion KM. Proteomic studies supportthe use of multi-product immunoassays tomonitor host cell protein impurities. Proteo-mics 2006;6(1):94–110.

289. Ehrnstrom R. Miniaturization and integra-tion: challenges and breakthroughs in micro-fluidics. Lab Chip 2002;2(2):26N–30N.

290. Poulsen F, Jensen KB. A luminescent oxygenchanneling immunoassay for the determina-tion of insulin in human plasma. J BiomolScreen 2007;12(2):240–247.

291. Kange R, Selditz U, Granberg M, Lindberg U,Ekstrand G, Ek B, Gustafsson M. Comparisonof different IMAC techniques used for enrich-

REFERENCES 339

ment of phosphorylated peptides. J BiomolTech 2005;16(2):91–103.

292. Meager A.Measurement of cytokines by bioas-says: theory and application.Methods 2006;38(4):237–252.

293. Jason-MollerL,MurphyM,BrunoJ.Overviewof Biacore systems and their applications In:Coligan J (Ed). Current Protocols in ProteinScience. New York: John Wiley & Sons 2006.Chapter 19, Unit 19.13.1-19.13.14.

294. Mire-Sluis A. Expression of potency: whyunitsof biological activity, not mass? Pharm Sci1997;3:1–4.

295. Mire-Sluis AR. Progress in the use of biologicalassays during the development of biotechnol-ogy products. Pharm Res 2001;18:1239–1246.

296. Mire-Sluis AR. Setting specifications for po-tency assays: basic principles. Dev Biol 2002;107:107–115.

297. Desjarlais JR, Lazar GA, Zhukovsky EA, ChuSY. Optimizing engagement of the immunesystem by anti-tumor antibodies: an en-gineer’s perspective. Drug Discov Today2007;12(21–22):898–910.

298. Cacia J, Keck R, Presta LG, Frenz J. Isomer-ization of an aspartic acid residue in the com-plementarity-determining regions of a recom-binant antibody to human IgE: identificationand effect on binding affinity. Biochemistry1996;35(6):1897–1903.

299. Remmele RL Jr, Callahan WJ, Krishnan S,ZhouL,BondarenkoPV,NicholsAC,KleemannGR, Pipes GD, Park S, Fodor S, Kras E, BremsDN. Active dimer of Epratuzumab provides in-sight into the complex nature of an antibodyaggregate. J Pharm Sci 2006;95(1):126–145.

300. Kotarsky K, Antonsson L, Owman C, Olde B.Optimized reporter gene assays based ona syn-thetic multifunctional promoter and a secretedluciferase. Anal Biochem2003;316(2):208–215.

301. Srebalus Barnes CA, Lim A. Applications ofmass spectrometry for the structural charac-terization of recombinant protein pharmaceu-ticals.Mass SpectromRev 2007;26(3):370–388.

302. Balland A, JochheimC.Mass spectrometry forbiopharmaceutical development. Anal TechBiopharm Dev 2005; 227–277.

303. Cruz HJ, Conradt HS, Dunker R, Peixoto CM,Cunha AE, Thomaz M, Burger C, Dias EM,Clemente J, Moreira JL, Rieke E, CarrondoMJ. Process development of a recombinantantibody/interleukin-2 fusion protein ex-pressed in protein-free medium by BHK cells.J Biotechnol 2002;96(2):169–183.

304. Field M, Papac D, Jones A. The use of high-performance anion-exchange chromatographyand matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry to moni-tor and identify oligosaccharide degradation.Anal Biochem 1996;239(1):92–98.

305. Yuk IH, Wang DI. Changes in the overallextent of protein glycosylation by Chinesehamster ovary cells over the course of batchculture. Biotechnol Appl Biochem 2002;36(Pt2):133–140.

306. Dillon TM, Ricci MS, Vezina C, Flynn GC, LiuYD, Rehder DS, Plant M, Henkle B, Li Y,Deechongkit S, Varnum B, Wypych J, BallandA, Bondarenko PV. Structural and functionalcharacterization of disulfide isoforms of thehuman IgG2 subclass. J Biol Chem 2008;283(23):16206–16215.

307. Martinez T, Guo A, Allen M, Han M, Pace D,Jones D, Gillespie R, Ketchem RR, Zhang Y,Balland A. Disulfide connectivity of humanimmunoglobulin G2 structural isoforms. Bio-chemistry 2008;47(28):7496–7508.

308. Senderoff RI,KontorKM,KreilgaardL,ChangJJ, Patel S, Krakover J, Heffernan JK, SnellLB,RosenbergGB.Consideration of conforma-tional transitions and racemization duringprocess development of recombinant gluca-gon-like peptide-1. J Pharm Sci 1998;87(2):183–189.

309. Weinberger SR, Boschetti E, Santambien P,Brenac V. Surface-enhanced laser desorption-ionization retentate chromatography massspectrometry (SELDI-RC-MS): a new methodfor rapid development of process chromatogra-phy conditions. J Chromatogr B 2002;782(1–2):307–316.

310. YenTY, JoshiRK,YanH, SetoNO, PalcicMM,Macher BA. Characterization of cysteine resi-dues and disulfide bonds in proteins by liquidchromatography/electrospray ionization tan-dem mass spectrometry. J Mass Spectrom2000;35(8):990–1002.

311. Eng M, Ling V, Briggs JA, Souza K, Canova-DavisE,PowellMF,DeYoungLR.Formulationdevelopment and primary degradation path-ways for recombinant humannerve growth fac-tor. Anal Chem 1997;69(20): 4184–4190.

312. WangL, Amphlett G, Lambert JM, BlattlerW,Zhang W. Structural characterization of a re-combinant monoclonal antibody by electro-spray time-of-flight mass spectrometry.Pharm Res 2005;22(8):1338–1349.

313. Wen J, Arakawa T, Philo JS. Size-exclusionchromatography with on-line light-scattering,


absorbance, and refractive index detectors forstudying proteins and their interactions. AnalBiochem 1996;240(2):155–166.

314. Philo JS. Is anymeasurementmethod optimalfor all aggregate sizes and types?. AAPS J2006;8(3):E564–571.

315. Kendrick BS, Kerwin BA, Chang BS, Philo JS.Online size-exclusion high-performance liquidchromatography light scattering and differen-tial refractometry methods to determine de-gree of polymer conjugation to proteins andprotein–protein or protein–ligand associationstates. Anal Biochem 2001;299(2):136–146.

316. Nobbmann U, Connah M, Fish B, Varley P,Gee C, Mulot S, Chen J, Zhou L, Lu Y, Shen F,Yi J, Harding SE. Dynamic light scattering asa relative tool for assessing the molecular in-tegrity and stability of monoclonal antibodies.Biotechnol Genet EnginRev 2007;24:117–128.

317. Fraunhofer W, Winter G. The use of asymme-trical flow field-flow fractionation in pharma-ceutics and biopharmaceutics. Eur J PharmaBiopharm 2004;58(2):369–383.

318. Litzen A, Walter JK, Krischollek H, WahlundKG. Separation and quantitation of monoclo-nal antibody aggregates by asymmetrical flowfield-flow fractionation and comparison to gelpermeation chromatography. Anal Biochem1993;212(2):469–480.

319. Liu J, Andya JD, Shire SJ. A critical review ofanalytical ultracentrifugation and field flowfractionation methods for measuring proteinaggregation. AAPS J 2006;8(3):E580–589.

320. Schuck P. A model for sedimentation in inho-mogeneous media. I. Dynamic density gradi-ents from sedimenting co-solutes. BiophysChem 2004;108(1–3):187–200.

321. Cole JL, Lary JW, Moody TP, Laue TM. Ana-lytical ultracentrifugation: sedimentation ve-locity and sedimentation equilibrium. Meth-ods Cell Biol 2008;84:143–179.

322. Gabrielson JP, Randolph TW, Kendrick BS,Stoner MR. Sedimentation velocity analyticalultracentrifugation and SEDFIT/c(s): limits ofquantitation for a monoclonal antibody sys-tem. Anal Biochem 2007;361(1):24–30.

323. Berkowitz SA. Role of analytical ultracentri-fugation in assessing the aggregation of pro-tein biopharmaceuticals. AAPS J 2006;8(3):E590–605.

324. Pekar A, Sukumar M. Quantitation of aggre-gates in therapeutic proteins using sedimenta-tion velocity analytical ultracentrifugation:practical considerations that affect precision

and accuracy. Anal Biochem 2007;367(2):225–237.

325. Gabrielson JP, Brader ML, Pekar AH, MathisKB, Winter G, Carpenter JF, Randolph TW.Quantitation of aggregate levels in a recombi-nant humanized monoclonal antibody formu-lation by size-exclusion chromatography,asymmetrical flow field flow fractionation, andsedimentation velocity. J Pharm Sci 2007;96(2):268–279.

326. Doglia SM, Ami D, Natalello A, Gatti-Lafran-coni P, Lotti M. Fourier transform infraredspectroscopy analysis of the conformationalquality of recombinant proteins within inclu-sion bodies. Biotechnol J 2008;3(2):193–201.

327. Matheus S, Mahler HC, Friess W. A criticalevaluation of Tm (FTIR) measurements ofhigh-concentration IgG1 antibody formula-tions as a formulation development tool.Pharm Res 2006;23(7):1617–1627.

328. Matheus S, Friess W, Mahler HC. FTIR andnDSC as analytical tools for high-concentra-tion protein formulations. Pharm Res 2006;23(6):1350–1363.

329. Harn N, Allan C, Oliver C, Middaugh CR.Highly concentrated monoclonal antibody so-lutions: direct analysis of physical structureand thermal stability. J Pharm Sci 2007;96(3):532–546.

330. Whitmore L, Wallace BA. Protein secondarystructure analyses from circular dichroismspectroscopy: methods and reference data-bases. Biopolymers 2008;89(5):392–400.

331. Greenfield NJ. Using circular dichroism col-lected as a function of temperature to deter-mine the thermodynamics of protein unfoldingand binding interactions. Nat Protoc 2006;1(6):2527–2535.

332. Ejima D, Tsumoto K, Fukada H, Yumioka R,Nagase K, Arakawa T, Philo JS. Effects of acidexposure on the conformation, stability, andaggregation of monoclonal antibodies. Pro-teins 2007;66(4):954–962.

333. Kats M, Richberg PC, Hughes DE. Conforma-tional diversity and conformational transi-tions of a monoclonal antibody monitored bycircular dichroism and capillary electrophor-esis. Anal Chem 1995;67(17):2943–2948.

334. VermeerAW,NordeW, vanAmerongenA.Theunfolding/denaturation of immunogammaglo-bulin of isotype 2b and its F(ab) and F(c) frag-ments. Biophys J 2000;79(4):2150–2154.

335. Walker KN, Bottomley SP, Popplewell AG,Sutton BJ, Gore MG. Equilibrium and pre-equilibrium fluorescence spectroscopic studies

REFERENCES 341

of the binding of a single-immunoglobulin-binding domain derived from protein G to theFc fragment from human IgG1. Biochem J1995;310(Pt 1):177–184.

336. HaweA,FriessW,SutterM, JiskootW.Onlinefluorescent dye detection method for the char-acterization of immunoglobulin G aggregationby size exclusion chromatography and asym-metrical flow field flow fractionation. AnalBiochem 2008;378(2):115–122.

337. Hawe A, Sutter M, Jiskoot W. Extrinsic fluor-escent dyes as tools for protein characteriza-tion. Pharm Res 2008;25(7):1487–1499.

338. Shah D, Johnston TP, Heitz S, Mitra AK. Theeffects of various excipients on the unfolding ofbasic fibroblast growth factor. PDA J PharmaSci Technol 1998;52(5):209–214.

339. Krell T. Microcalorimetry: a response to chal-lenges in modern biotechnology. Microb Bio-technol 2008;1(2):126–136.

340. Hill JJ, Shalaev EY, Zografi G. Thermody-namic and dynamic factors involved in thestability of native protein structure in amor-phous solids in relation to levels of hydration. JPharm Sci 2005;94(8):1636–1667.

341. Kett V, McMahon D, Ward K. Thermoanaly-tical techniques for the investigation of thefreeze drying process and freeze-dried pro-ducts. Curr Pharm Biotechnol 2005;6(3):239–250.

342. Remmele RL Jr, Zhang-van Enk J, Dharma-varam V, Balaban D, Durst M, ShoshitaishviliA, RandH. Scan-rate-dependentmelting tran-sitions of interleukin-1 receptor (type II): elu-cidation of meaningful thermodynamic andkinetic parameters of aggregation acquiredfrom DSC simulations. J Am Chem Soc2005;127(23):8328–8339.

343. Ahrer K, Buchacher A, Iberer G, Josic D, Jung-bauer A. Thermodynamic stability and forma-tion of aggregates of human immunoglobulin Gcharacterised by differential scanning calori-metry and dynamic light scattering. J BiochemBiophys Methods 2006;66(1–3): 73–86.

344. Garber E, Demarest SJ. A broad range of Fabstabilities within a host of therapeutic IgGs.Biochem Biophys Res Commun 2007;355(3):751–757.

345. Meyer BK, Ni A, Hu B, Shi L. Antimicrobialpreservativeuseinparenteralproducts:pastandpresent. J Pharm Sci 2007;96(12):3155–3167.

346. SharmaB. Immunogenicity of therapeutic pro-teins. Part 3. Impact of manufacturingchanges. Biotechnol Adv 2007;25(3):325–331.

347. SchellekensH. How to predict and prevent theimmunogenicity of therapeutic proteins. Bio-technol Annu Rev 2008;14:191–202.

348. Schenerman MA, Phillips K. Comparability(bioequivalence) testing of monoclonal antibo-dies. BioPharm 1997;10:20–27.

349. Chirino AJ, Mire-Sluis A. Characterizing bio-logical products and assessing comparabilityfollowing manufacturing changes. Nat Bio-technol 2004;22(11):1383–1391.

350. Chirino AJ, Mire-Sluis AR. State of the artanalytical comparability: a review. Dev Biol2005;122:3–26.

351. Yang JD, Lu C, Stasny B, Henley J, GuintoW,Gonzalez C, Gleason J, Fung M, Collopy B,BenjaminoM, Gangi J, HansonM, Ille E. Fed-batch bioreactor process scale-up from 3-L to2,500-L scale for monoclonal antibody produc-tion from cell culture. Biotechnol Bioeng2007;98(1):141–154.

352. Meuwly F, Weber U, Ziegler T, Gervais A,Mastrangeli R, Crisci C, Rossi M, Bernard A,von Stockar U, Kadouri A. Conversion of aCHO cell culture process from perfusion tofed-batch technology without altering productquality. J Biotechnol 2006;123(1):106–116.

353. SchenermanMA,HopeJN,KletkeC,SinghJK,KimuraR,TsaoEI,Folena-WassermanG.Com-parability testing of a humanized monoclonalantibody (Synagis) to support cell line stability,process validation, and scale-up for manufac-turing. Biologicals 1999;27(3):203–215.

354. Defelippis MR, Larimore FS. The role of for-mulation in insulin comparability assess-ments. Biologicals 2006;34(1):49–54.

355. Combe C, Tredree RL, Schellekens H. Biosimi-lar epoetins: an analysis based on recently im-plemented European medicines evaluationagency guidelines on comparability of biophar-maceutical proteins. Pharmacotherapy 2005;25(7):954–962.

356. Covic A, Kuhlmann MK. Biosimilars: recentdevelopments. Int Urol Nephrol 2007;39 (1):261–266.

357. Simmerman H, Donnelly RP. Defining yourproduct profile and maintaining control overit. Part 1. Bioprocess Int 2005;3(6):32–40.

358. U.S. Food and Drug Administration, HHS.Substances generally recognized as safe. Codeof Federal Regulations. Part 182. Title 21.2004.

359. Huang MM, Penn L, Bongers J, Gurman S.Validation of a shielded-hydrophobic-phasehigh-performance liquid chromatography


method for the determination of residualmeth-otrexate inrecombinantproteinbiopharmaceu-ticals. J Chromatogr 1998;828(1–2):303–309.

360. Kennan JJ, Breen LL, Lane TH, Taylor RB.Methods for detecting silicones in biologicalmatrixes. Anal Chem 1999;71(15):3054–3060.

361. Qiu J, Lee H, Zhou C. Analysis of guanidinein high salt and protein matrices by cation-exchange chromatographyandUVdetection. JChromatogr A 2005;1073(1–2):263–267.

362. Food US, Drug Administration HHS. Interna-tional Conference on Harmonisation; Gui-dance on Q8 Pharmaceutical Development;availability. Notice. Federal Register. 2006;71(98):1–9.

363. Lindemann CJ, Rosolia A. Gas chromato-graphic assay for benzyl alcohol and pheny-lethyl alcohol in pharmaceutical formulations.J Pharma Sci 1969;58(1):118–119.

364. Yu LX. Pharmaceutical quality by design:product and process development, under-standing, and control. Pharm Res 2008;25(4):781–791.

365. Wren SA, Tchelitcheff P. Use of ultra-perfor-mance liquid chromatography in pharmaceu-tical development. J Chromatogr A 2006;1119(1–2):140–146.

366. Zhang B, Foret F, Karger BL. High-through-put microfabricated CE/ESI-MS: automated

sampling from a microwell plate. Anal Chem2001;73(11):2675–2681.

367. KangSH,GongX,YeungES.High-throughputcomprehensive peptidemapping of proteins bymultiplexed capillary electrophoresis. AnalChem 2000;72(14):3014–3021.

368. LondoT,LynchP,KehoeT,MeysM,GordonN.Accelerated recombinant protein purificationprocess development automated, robotics-based integration of chromatographic purifica-tion and analysis. J Chromatogr A 1998;798(1–2):73–82.

369. Haeberle S, ZengerleR.Microfluidic platformsfor lab-on-a-chip applications. LabChip2007;7(9):1094–1110.

370. Johlman CL, White RL, Wilkins CL. Applica-tions of Fourier transformmass spectrometry.Mass Spectrom Rev 2005;2(3):389–415.

371. Bereman MS, Nyadong L, Fernandez FM,Muddiman DC. Direct high-resolution peptideandproteinanalysis bydesorptionelectrosprayionization Fourier transform ion cyclotron re-sonance mass spectrometry. Rapid CommunMass Spectrom 2006;20(22):3409–3411.

372. Morstadt L, Bohm A, Yuksel D, Kumar K,Stollar BD, Baleja JD. Engineering and char-acterization of a single chain surrogate lightchain variable domain. Protein Sci 2008;17(3):458–465.

REFERENCES 343

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