Cho Antibody

96 BioPharm International www.biopharminternational.com October 2007

The first therapeutic mono-clonal antibody (MAb) prod-uct entered the market in1986, but it took another

decade before the potential of this newclass of biologic products began to berealized. From the mid 1990s untiltoday, almost 30 therapeutic mono-clonal antibodies (MAbs) have beenapproved throughout the world alongwith several antibody-related products(e.g., Fc-fusion proteins) making MAbsand related products a dominant com-ponent of the biopharmaceutical mar-ket, generating revenues of severalbillion dollars. The first approved MAbwas a murine antibody. This was fol-lowed by several chimeric MAbs con-taining a mix of murine and humanregions. These early antibody productsposed a moderate risk of immuno-genicity to patients from their residualmurine components, somewhat limit-

ing the development of MAb products.To address this issue, new technologiesfor creating MAbs that were predomi-nately or entirely of human origin weredeveloped. Today, almost all antibodyproducts currently in development arehumanized or fully human.

While MAbs have proven to be valu-able therapeutic products, the typicaldoses of these products required fortreatment are significantly higher thanthose required for most other biologics,resulting in the need for large-scale pro-duction and efficient, cost-effective man-ufacturing processes. In the past fewyears, improvements have been made incritical areas, such as cell line generationand large-scale cell culture production,to maximize specific antibody productiv-ity from a given cell line and improveoverall productivity in bioreactors. Theseadvances include the use of new expres-sion vectors and transfection technology

Advancesinthe Developmentof Therapeutic

Monoclonal AntibodiesABSTRACTMonoclonal antibodies (MAbs) and related products are a dominant component of thebiopharmaceutical market, generating revenues of several billion dollars. While MAbshave proven to be valuable therapeutic products, the typical doses of these productsrequired for treatment are significantly higher than those required for most otherbiologic products, resulting in the need for large-scale production and efficient, cost-effective manufacturing processes. In the past few years, improvements have beenmade in critical areas, such as cell line generation and large-scale cell cultureproduction, to maximize productivity. These advances, coupled with improvements incell culture media and optimized bioreactor processes, have made large-scaleproduction of MAbs economically viable. However, the increasing productionrequirements and the drive to reduce the cost to develop these expensive medicinescontinue to present challenges to the industry to further improve the overall efficiencyof manufacturing processes. This article presents a historical review of the discovery,development, and production of therapeutic antibodies.

Susan Dana Jones, PhD, is a seniorconsultant, Francisco J. Castillo, PhD,

is a senior consultant, and Howard L. Levine, PhD, is a

principal consultant, all atBioProcess Technology Consultants, Inc.,

Acton, MA, 978.266.9159,[email protected].

Susan Dana Jones, Francisco J. Castillo, Howard L. Levine

Listen to a podcast interview with Howard Levine at

biopharminternational.com/biopharmnow

to improve cell line generation; novel parentalcell lines that have been selected or designedto grow to maximum density and productivityunder standard bioreactor conditions; andhigh-throughput, robust screening technolo-gies to select the highest producing clones rap-idly and more effectively. As a result, theproduction of cell lines expressing multigramquantities of antibody per liter of culturemedium is now routine.

These advances, coupled with improve-ments in cell culture media and greatly opti-mized bioreactor processes, have made thelarge-scale production of MAbs economicallyviable. However, the increasing productionrequirements and the drive to reduce thecost to develop these expensive medicinescontinue to present challenges to the indus-try to further improve the overall efficiencyof manufacturing processes. These chal-lenges include the need to streamline down-stream processing to enable the processing ofincreased product quantities; the implemen-tation of Quality by Design (QbD) and othernew regulatory concepts to reduce the costand development timelines for MAb prod-ucts without adversely affecting their qual-ity; the need for high-concentration productformulations with sufficient stability to

address the increasing doses of antibodyproducts; and the development of alterna-tive delivery systems.

DISCOVERY OF ANTIBODY THERAPEUTICSIn 1984, Kohler and Milstein received theNobel Prize in Medicine for their pioneer-ing work on the production of MAbs.1 Oneof the most significant advantages of thisnew technology over traditional tech-niques for producing antibodies was thedevelopment of an immortalized cell linecreating a continuous source of the sameantibody with a single antigen specificity.This enabled the development of highlyspecific antibodies directed toward a singleepitope on the target antigen. Initially,MAbs were used as laboratory reagents, butthey were quickly adopted as clinical diag-nostic reagents, and eventually as thera-peutic agents. The development oftherapeutic MAbs commenced in the early1980s and by 1986 the first monoclonalantibody for human use—OrthocloneOKT3 (Ortho Pharmaceuticals)—was

approved for the prevention of kidney trans-plant rejection. Following the approval ofOKT3, the enthusiasm for MAbs as therapeu-tic products grew with the next wave of anti-body products generally being developed asanticancer agents. Several of these productswere approved in the US and Europe in themid to late 1990s, a trend that continues togrow today. Since the commercialization ofthe first therapeutic MAbs, these productshave become a dominant component of thebiopharmaceutical market, representingapproximately 20% of all biologic products,with combined revenues of over $20 billionin 2006.4 The growth of MAb products overthe past 25 years, as shown in Figure 1, con-firms the importance of these products andalso shows that MAbs represent a significantsubset of all biopharmaceuticals on the mar-ket and in development. With over 300 anti-body products currently in development,this unique and effective category of thera-peutic compounds is poised to grow signifi-cantly in the coming years.

MOLECULAR STRUCTURES OF ANTIBODIES:THEN AND NOWMurine AntibodiesThe initial technology for producing MAbs


Monoclonal Antibodies

Figure 1. Annual approval of recombinant biologic products andmonoclonal antibody products.2,3 The total number of biologics, includingMAb products, approved by FDA for market each year since 1982 isshown in green. MAb product approvals only are shown in black.Antibody-related products such as Fc fusions, engineered antibodyfragments, or other products derived from antibodies but not containingan antibody binding region are not included in the MAb figures. However,those products are included in the total product figures.

0

2

4

6

8

10

12

14

82 84 86 88 90 92 94 96 98 0 2 4 6

Total biologicsincluding MAbs

MAbs

involved fusing individual antibody-secret-ing spleen cells from immunized mice with amurine myeloma cell line to generateimmortalized cell lines that secreted individ-ual, or monoclonal, antibodies. Hence, thefirst MAbs developed for use as potentialhuman therapeutics were murine antibodies.While initial interest in these murine MAbswas high and several companies begandeveloping products based on this technol-ogy, OKT3 was the only murine monoclonalantibody that was approved for human ther-apeutic use. Despite the fact that OKT3 hasbeen moderately successful in the market,the use of murine MAbs as therapeuticagents quickly ran into many roadblocks.One of the potential advantages of MAbs astherapeutic agents is their long circulatinghalf-life, allowing them to provide a thera-peutic effect in patients over several days.However, when murine MAbs were repeat-edly administered to humans during clinicaltrials, it was observed that the half-lifedecreased and the products became lesseffective with each injection. This wasbecause of the immunogenicity of murineproteins in humans and the rapid develop-ment of a human antimurine antibody(HAMA) response in the patients. ThisHAMA response neutralized the effectivenessof the murine antibodies and resulted intheir rapid clearance from the body. Forexample, it has been reported that OKT3 canelicit a HAMA response in up to 86% ofpatients treated, leading to some limitationsin its efficacy.5

Chimeric AntibodiesTo overcome the HAMA responses occuringfrom the usage of murine MAbs as therapeu-tics, several approaches were developed in anattempt to make MAbs more human-like andless immunogenic. In the early 1990s,molecular biology techniques enabled thecreation of “chimeric” antibodies by linkingthe murine genes encoding the antigen-binding portion of the antibody (the variableregion) to the genes encoding the constantregion of human immunoglobulin light andheavy chains. Because over 75% of the pro-tein sequence of the resulting chimeric anti-bodies was of human origin, these chimericMAbs elicited much lower HAMA responsesin patients. Moreover, because the antibody

constant region in these chimeric antibodiesis human, it is capable of activating othercomponents of the human immune systemto potentially create more effective therapeu-tic agents. Many of the MAbs approved forcommercialization in the 1990s and early2000s were chimeric antibodies, includingthe highly successful anticancer antibodiesRituxan (approved in 1997) and Erbitux(approved in 2004), as well as the anti-inflammatory product Remicade (approvedin 1998). Chimeric antibody products aresuperior to murine antibody products butthey still pose a moderate risk of immuno-genicity to patients from their residualmurine components. Therefore, antibodyengineering approaches that further reducethe murine component or that removeimmunogenic portions of the chimeric anti-body, have been developed and used to gen-erate fully “humanized” antibody products.

Humanized AntibodiesIn 1991, Protein Design Labs (PDL) developedand patented the first technology for success-fully humanizing MAbs.6 The antigen bindingspecificity of any antibody is determined bythe amino acids present in three distincthighly variable regions per antibody chain,referred to as complementarity determiningregions (CDRs), and located in a more con-served framework sequence in the variableregions. Therefore, PDL scientists developedmethods for engineering an antibody gene inwhich the CDRs of a human antibody genewere replaced by those from the CDR of amurine MAb gene. The resulting humanizedantibody has the same antigen binding prop-erties as the original murine antibody butcontains minimal murine sequences and,therefore, elicits a lower HAMA response inpatients. The CDR-grafted human antibodycan be used as is or, in cases where affinity ofthe chimeric antibody is slightly reducedfrom the original murine antibody, additional

BioPharm International www.biopharminternational.com October 2007 99


Future therapeutic monoclonal antibody

products will be predominantly

humanized or fully human.

changes can be made in the antibodysequence to regain or enhance its bindingproperties. Like chimeric antibodies, human-ized antibodies can activate other parts of theimmune system to create a more effectiveproduct. Several humanized antibody prod-ucts are currently on the market, includingSynagis (approved in 1998), Herceptin(approved in 1998), Mylotarg (approved in2000), Xolair (approved in 2003), and Avastin(approved in 2004).

In addition to the production of chimericand humanized antibodies, other technolo-gies have been developed to help minimizethe HAMA response in patients. Theseinclude human engineering or deimmuniza-tion, in which amino acids on the surface ofthe murine variable region that are known tobe effective immunogenic sequences arechanged to their non-immunogenic humancounterpart, leaving the other non-immuno-genic murine sequences unchanged.7 Theadvantage of this approach is that the struc-tural integrity of the variable region is bettermaintained and reduction of affinity for thetarget is minimized.

Fully Human AntibodiesThe latest advancement in creating lessimmunogenic therapeutic antibody productsis the ability to generate fully human MAbs.Several technologies exist to develop fullyhuman antibodies, each falling into one ofthe two general classes—in vivo approachesusing a murine system in which theimmunoglobulin genes have been replaced bytheir human counterparts or in vitroapproaches using libraries containing millionsof variations of antibody sequences coupledwith a mechanism to express and screen theseantibodies in vitro. Humira (approved in 2004)is the first fully human antibody to beapproved. This anti-TNF-α antibody was firstidentified by scientists at CambridgeAntibody Technology (CAT, now part of

AstraZeneca) using an in vitromolecular engineering technologyknown as phage display. In the mar-ketplace, this human MAb com-petes with Enbrel, an Fc fusionprotein, and Remicade, a chimericantibody. The power of the fullyhuman antibody platform can beseen in the sales figures for these

three products. Although Humira wasapproved four years later than the other prod-ucts, it has successfully taken a significantmarket share from them, garnering almost16% market share in 2006. Worldwide sales in2006 for all three products are shown in Table 1.

Many antibody products currently in earlyclinical development are fully human,because the technologies that enable thegeneration of human antibodies are nowaccessible through partnerships or licensingfrom the companies that have developedthese approaches. Moreover, the expectationin the medical and regulatory community isthat companies will use the best approachfor their product to achieve humanization.There will be exceptions to this generaliza-tion, for example when a short half life isdesired or when a toxic or radioactive pay-load is linked to the antibody, but forunmodified therapeutic antibody productsthe industry standard has changed; mostfuture antibody products will be humanizedor fully human antibodies.

Most MAb products are naked antibodies,which rely on either blocking an importantbiological function or on activating theimmune system, to elicit a therapeutic effect.However, antibodies are also well suited astargeting agents to deliver potent chemo- orradioactive agents specifically to target cells.For example, Mylotarg contains a cytotoxiccompound conjugated to a monoclonal anti-body. This immunoconjugate product isdesigned to deliver the potent cytotoxiccompound selectively to cancer cells. Theradio-immunoconjugate products Zevalinand Bexxar (both anti-CD20 MAbs), deliverradioisotopes for the treatment of lym-phoma. Both these products are murine anti-bodies because the human or humanizedforms of these products would bind to andtarget not only the CD20 positive target cellsbut also those cells that contain the IgG



Table 1. Comparison of sales for antibody-based anti-inflammatory products

Product CompanyYear

approved2006 sales worldwide

($ million) Market share

Humira Abbott 2002 2,000 15.8%

Remicade Johnson & Johnson 1998 4,253 33.7%

Enbrel Amgen 1998 4,379 34.7%

receptors that function to enable antibodiesto recruit additional immune system compo-nents to the site of a foreign antigen. Byinadvertently targeting these cells, humanantibody-based radio-immunoconjugatescould do more harm to nontarget cell typesthan to the targeted cancer cell.

All of the above technologies now allowthe generation of better designed antibodyproducts with fully human sequences andoptimized function. Combining in vivo and invitro discovery and molecular engineeringtechnologies allows exquisite control of theantibody sequences and properties that wasnot possible 20 years ago. New approaches forthe rapid production of cell lines suitable forlarge-scale commercial production haveenabled the development of MAb therapies totreat myriad diseases and made these productsavailable to an increasing number of patients.In addition to enabling more efficient andeconomic production of MAbs, the aboveantibody engineering technologies, coupledwith advances in cell culture production dis-cussed below, have greatly increased our abil-ity to control or alter the properties of theresulting antibodies. For example, the extentof glycosylation, which can increase effectorfunction and thereby increase product effi-cacy, can be controlled by both cell line engi-neering and cell culture technologies.

MARKET DEMANDS AND CELL LINE PRODUCTIVITYOne challenging feature of most therapeuticantibody products is that the doses requiredfor these products are much higher than forother biologic products. To meet the largeannual production requirements for theseproducts, companies have made substantialprogress in developing more efficient and cost-effective methods for manufacturing antibodyproducts. When antibody products were firstdeveloped and approved, expression levels ofMAbs were typically on the order of 100–500milligrams per liter. Even as recently as fiveyears ago, antibody titers in excess of 1 g/Lwere not common and many MAb productswere launched using production cell lines andmanufacturing processes that producedapproximately 0.5–1.0 g/L antibody.8 As MAbproducts became successful in the marketplaceand as the demands for new productsincreased, newer methods of generating high-

expressing antibody production cell lines andof culturing these cell lines for maximum pro-ductivity have been developed. Today’s tech-nologies are enabling antibody production inthe bioreactor of 5 g/L or more.9 Advances incell line generation over the past decadeinclude new expression vectors and transfec-tion technology to introduce the genes intocells; novel parental cell lines that have beenselected or designed to grow to maximumdensity; and robust screening technologiesthat in combination can enable rapid genera-tion of production cell lines.

ADVANCES IN THE GENERATION OF PRODUCTION CELL LINESToday’s MAbs must be manufactured usingreliable production cell lines capable of pro-ducing sufficient quantities of product tomeet the market demand. For most products,this means that antibody titers in the biore-actor must be greater than 1 g/L in a fed-batch process initially and 3–5 g/L followingprocess optimization. To achieve these levelsof productivity, it is necessary to quicklydevelop a cell line expressing reasonablyhigh quantities of antibody for early preclin-ical, formulation, and analytical validationstudies that can be further optimized toachieve the desired productivity levels. If theproductivity of the initial cell line is highenough, it can even be used to support ini-tial clinical development of the product.Once the initial cell line is established, a pro-duction cell line exhibiting the highest pos-sible level of production of functionalantibody and capable of supporting commer-cial production at a reasonable cost can bedeveloped. In today’s highly competitivemarket, it is important to complete the ini-tial stages of cell line development as quicklyand efficiently as possible to enable earlyentry into human clinical trials but equallyimportant is to devote sufficient time andresources to the full development and opti-mization of the commercial cell line so that



In the future, human cell lines mayreplace CHO and other mammalian

cell lines for the production of MAbs.

a suitable cell line is available for commercialproduction as soon as possible.

High-Expressing Cell LinesTo create a production cell line for a specificantibody, expression vectors containing theheavy- and light-chain genes under controlof strong mammalian promoters are intro-duced into the parental cell line. Usually, aselectable marker is also included so thatcells containing the gene can be easilyselected by adding a drug or substance to theculture that causes the cell to require theactivity of the selectable marker. The drivingfactors behind the selection of a particularcell clone during cell line generation is theexpression level of the recombinant protein,which is measured independently of theselection, and the time that it takes to obtaina cell line that expresses enough product toenable nonclinical and clinical development.Technologies that increase the percentage oftransfectants with high expression levels willreduce the time needed to identify a produc-tion cell line because the high-expressingclones will be easier to select without havingto screen thousands of individual clones.Recent advances in cell line generationinclude technologies that increase this per-centage, as well as sophisticated and auto-mated approaches to screening that enablemore individual transfectants to be screenedfor expression levels.10,12,15,16

Production levels in the bioreactor are afunction of specific productivity—the den-sity to which the cells can grow and thelongevity of the culture. Before actual testingin the bioreactor, expression levels are deter-mined in small culture vessels, from multi-well plates to shake flasks. Levels of 15–20picograms of antibody/cell/day (pcd) areconsidered appropriate for initial transfec-tants, with greater productivity arising fromoptimized cell culture conditions, secondarytransfections, or amplification of the trans-fected antibody genes using selective pres-

sure. Using parental cell lines adapted togrow in suspension and serum-free mediareduces development times and increases thelikelihood of reaching high cell densitiesduring manufacturing and high productyields in the grams-per-liter level.

Selection SystemsOne of the earliest effective methods fortransfection, selection, and amplification offoreign genes in mammalian cells was devel-oped in 1981 by scientists at ColumbiaUniversity using dihydrofolate reductase(DHFR) selection. In this method, a parentalmammalian cell line deficient in the enzymeDHFR is transfected with an expression vec-tor containing the DHFR gene under controlof a relatively weak promoter and the anti-body (or other protein) genes under controlof a strong promoter.11 By performing multi-ple rounds of amplification and selection ofcells in the presence of the folate analogmethotrexate (MTX), a potent inhibitor ofDHFR, production cell lines with relativelyhigh levels of expression of the foreign genescan be obtained. The original patents for thistechnology have now expired but it is stillwidely used to generate antibody productioncell lines. However, because each amplifica-tion cycle requires 12 weeks to complete andup to five cycles or more, about one yeartotal may be necessary to obtain a clonewith acceptably high expression levels.Nevertheless, the DHFR system is effectiveand has been used in conjunction with otheraspects of cell line development to achievemultigram-per-liter expression levels of MAb.Also, alternative systems requiring less timeto reach maximal expression have beendeveloped. For example, the glutamine syn-thetase selection system, developed by scien-tists at Celltech (now Lonza), can achieveproduction clones with higher levels of anti-body or protein expression in 4–6 months.12

Glutamine synthetase (GS) is the enzymeresponsible for the biosynthesis of gluta-mine from glutamate and ammonia. Thisenzymatic reaction provides the only path-way for glutamine formation in a mam-malian cell. Therefore, in the absence ofglutamine in the growth medium, the GSenzyme is essential for the survival of themammalian cells in culture. Some mam-malian cell lines, such as the murine cell



The use of parental cell lines adapted togrow in suspension and serum-free

media can reduce development times.


lines NSO or SP2/0 widely used for anti-body production, do not express sufficientGS to survive without added glutamine.With these cell lines, a transfected GS genecan function as a selectable marker by per-mitting growth in a glutamine-freemedium. Chinese hamster ovary (CHO)cells, also widely used for antibody andother recombinant protein production,contain sufficient active GS to survivewithout exogenous glutamine.13 In thesecases the specific GS inhibitor, methioninesulphoximine (MSX), can be used toinhibit endogenous GS activity such thatonly transfectants with additional GS activ-ity can survive. GS selection can be used toselect high-expressing cell lines withoutamplification, which reduces the time com-pared to the DHFR selection approach. TheGS system has enabled the rapid identifica-tion and selection of production cell linesthat express up to 20–50 pcd and multiplegrams per liter of product as part of anoverall cell culture process developmenteffort. According to Lonza, more than 85global pharmaceutical companies are cur-rently using this technology to create pro-duction cell lines and five products usingthe GS system have been approved forcommercial sale, including Synagis andZenapax. The GS technology is availablefor licensing from Lonza for the use inresearch and commercial applications,

making it widely available for thedevelopment of MAb products.14

Improving Gene ExpressionAnother recent approach to improveexpression of antibody genes in theinitially transfected cells is to ensurethat the genes are integrated intoregions of the chromatin, which areeasily available to the enzymes thattranscribe the gene into RNA, therebyincreasing the rate of transcription.The transfection of a mammalian cellgenerally results in the integration ofthe DNA into the chromatin in one ormore random locations. Because mostof the genome is not transcriptionallyactive, there is a high likelihood thatintegration will occur in regions thatare not able to transcribe high levelsof the antibody genes. Targeting the

expression plasmid to locations on the chro-matin that are known to be transcriptionallyactive and accessible to the necessaryenzymes would increase the expression ofall genes integrated at these sites. Althoughthis is an excellent concept in theory,homologous recombination or targeted inte-gration has not been widely adapted in prac-tice because of the lack of informationabout which sites are good locations forintegration and the need to have uniqueplasmids and cell lines that are able to per-form the recombination.

Rather than targeting a specific site in thechromatin for integration, an alternativeapproach is to include elements on theexpression plasmid. This will cause the ran-dom integration site to become transcrip-tionally active and available to the enzymesthat transcribe the genes. There have beenseveral reports of such genetic elements thatenable the integrated plasmid to create atranscriptionally active region at any integra-tion location on the chromosome and toenable higher transcription levels in a higherpercentage of transfectants. Two types of ele-ments that function to create a region oftranscriptionally active chromatin are theubiquitous chromatin opening elements(UCOE) and the matrix attachment regions(MAR) elements.15,16 These genetic elementshave different mechanisms of action butboth work to increase the expression levels


Figure 2. In matrix attachment region (MAR) technology, MAR elements areinserted into expression vectors surrounding the desired transgene and imposean open chromatin configuration on the nearby chromatin. This open structureallows RNA polymerase and other transcription factors to access thetranscriptional promoters and enhancers found within the expression vector andthereby enables greater levels of transcription. This leads to increased product-specific translation and a higher yield in a greater percentage of transfectedcells. Figure provided courtesy of Selexis SA.

‘Open’ chromatin

‘Closed’ chromatin

‘Closed’

chromatin

Promoters/ enhancers

MAR

of linked genes that are transfected on thesame plasmid as the MAR or UCOE.

The use of MAR elements for improvingexpression has been commercialized bySelexis. The company has developed a set ofexpression vectors and transfection technolo-gies (the “MARtech” technology) that usethese elements to increase the percentage ofcells expressing the desired gene. As shownschematically in Figure 2, the MAR elementsare inserted into an expression vector suchthat the gene for the desired product is sur-rounded by these elements to impose anopen chromatin configuration, therebyallowing RNA polymerase and other tran-scription factors to access the transcriptionalpromoters and enhancers found in theexpression vector. For this reason, MARtechincreases the number of independently trans-formed cells that express the desired proteinand enables expression levels in the initialtransfectants of as much as 50–70 pcd. Selexisclaims that MARtech allows for generation ofclonal mammalian production cell lines inabout 10 weeks. Many companies havebegun exploring the use of MARtech toenable rapid generation of high producingcell lines for their antibody products. Laterthis year the first product using this technol-ogy will enter clinical trials.17

UCOE technology, now available throughMillipore Corporation, provides anapproach to increasing gene expression simi-lar to that of the MARtech technology. TheUCOE elements are functionally similar toMAR elements although their compositionand structure are different.16 UCOE consistsof regions that are rich in the sequence CpG,and that increase the accessibility of the sur-rounding chromatin. Therefore, a singleUCOE element can be included on anexpression vector and can increase theexpression levels of linked genes. There isless commercial experience with UCOE ele-ments than with MAR elements, but theintent is to offer the technology to compa-nies for use in research and in commercialproduction cell line generation.

ADVANCES IN CELL CULTURE TECHNOLOGYHost cell lines currently used to producecommercial MAb products include murinehybridoma and myeloma cell lines, CHO celllines, and one human cell line (Table 2).

Those antibody products produced inhybridoma cell lines generally have lowerdose requirements than others and are alsoolder than those produced using highly engi-neered systems such as CHO, NSO, or SP2/0.The single product produced in a human cellline may represent a trend in coming yearsas others develop human cell lines capable ofproducing antibody products at high levels.While the use of murine cell lines still pre-vails in commercial processes, the use ofCHO cells for producing commercial prod-ucts is growing and most antibody productscurrently in development are produced fromCHO or human cell lines.

Hybridoma TechnologyMAbs were first produced from hybridomasconsisting of a murine B cell producing aspecific antibody fused to an immortalmurine lymphoid cell line. Initially, MAbswere produced by injecting a hybridoma cellline into the abdomen of pristane-primedmice, in which the cells could grow to a sig-nificant level. As the hybridoma cells growin the abdomen, MAb-rich ascites fluid accu-mulates. The ascites fluid can then be col-lected by withdrawing it with needles atseveral day intervals. The collected ascitesfluid is very complex in composition andhighly contaminated, but frequently contains antibody concentrations approach-ing 1 g/L or greater. This process is widelyused for the production of small to moderateamounts of antibodies for multiple applica-tions and one commercial antibody productis produced today using this technology.

The limitations of large-scale productionin the abdomens of mice were quickly real-ized and scientists turned their efforts to use

Table 2. Host cell types used in the manufacture of commercial MAbs



Cell line Species Number of products

Hybridoma Murine 5

SP2/0 myeloma Murine 5

NS0 myeloma Murine 3

Other myeloma Murine 1

Chinese hamster ovary (CHO) Hamster 10

EBV-transformed B cell Human 1

E. coli Microbial 1


in vitro culture as an alternative to replace invivo production in ascites. These initialefforts focused on growing hybridomas inculture, under conditions enabling the samehigh level of antibody expression as seen inthe ascites fluid. Initial studies characterizedand compared the growth of hybridomasand production of antibodies in either batchsuspension cultures using stirred tanks andairlift fermentors or in perfusion culturesusing a variety of methods for cell retention.From simple batch cultures, the use of con-trolled feeding, also know as fed-batch,evolved as extremely successful in increasingmaximum cell concentrations, culturelongevities, and corresponding producttiters. Fed-batch is the primary mode of bio-pharmaceutical production used today, bothfor antibodies and other recombinant pro-tein products.

Hybridoma technology enabled the cre-ation and production of MAbs for research,analytical use, and as limited-dose therapeu-tic products. However, these cell lines aregenerally difficult to engineer for high levelsof protein expression and usually grow toonly moderate densities in bioreactors.Hence, although these cells are designed toproduce antibodies, in many cases they doso at levels that are too low to be optimal formanufacturing today’s MAbs.

Using CHO Cells as Production HostsTo circumvent the limitations of hybridomasfor MAb production, scientists began experi-menting with alternative production hoststhat could be grown to higher densities andtransfected with the antibody genes toenable higher cellular productivity. Themurine myeloma cell lines NSO and SP2/0were among the first used to produce recom-binant MAbs. At the same time, others beganexamining CHO cell lines. The CHO celllines proved to be a suitable production host

for antibodies. Today, the vast majority ofbiologic products made in mammalian cellsare produced using a CHO host cell line.Because of the widespread adoption of thishost cell, the growth characteristics, metabo-lism, behavior in bioreactors, virulence fac-tors, and the likely host-cell relatedimpurities that might be in a process orproduct are well understood. Moreover,because there is a strong regulatory historyof CHO cells, more and more products indevelopment are now made using CHO cells.

Human Cell LinesWhile the use of CHO cells as productionhosts continues, other cell lines, especiallyhuman cell lines, are being developed as alter-native hosts. For example, the PER.C6 cell linedeveloped by Crucell, has been shown to pro-duce antibodies at levels similar to or evengreater than CHO cell lines.18 One potentialadvantage of products produced in these celllines is that the glycosylation patterns andother post-translational modifications of anti-bodies produced in them may be more similarto human antibodies. Therefore, the PER.C6cell line and other human cell lines mayprove to be reliable, safe, scalable, and eco-nomical alternatives to the CHO cell lines cur-rently in use for the production of MAbs.

Chemically Defined MediaCurrent regulatory requirements strongly dis-courage or ban the use of any products in theculture media that are derived from animals,especially from bovine sources. Therefore, theuse of bovine serum, commonly used earlierin mammalian cell culture, has been discon-tinued and significant efforts have beendirected towards the development of cell cul-ture media, that is free from animal-derivedproducts. There is a growing trend toward theuse of chemically defined media. In suchmedia, recombinant proteins such as IGF-1,transferrin, insulin, or others may be includedto provide the necessary signals for cellgrowth. When used, the recombinant humanversions of these proteins are preferred. Tofurther minimize the risk associated with theaddition of animal-derived components, CHOand other production host cell lines used forantibody production are now selected fortheir ability to grow and produce product athigh levels in chemically defined media.


Fed-batch processes are readily scaled-up to commercial

volumes and represent the primarymethod in use today.

Several different chemically defined media arenow commercially available from a variety ofvendors. However, most companies involvedin the development of MAb products todayhave developed proprietary cell culture mediaand growth conditions suitable for produc-tion of their particular monoclonal antibodyat high titers.

Along with improvements and refine-ments in expression systems and cell linesfor MAb production, there have also beensignificant advances in cell culture condi-tions over the past 20 years to further opti-mize antibody production.19,20 Theoptimization of fed-batch processes hasincreased antibody titers in culture orders ofmagnitude so that expression levels ofgreater than 1 g/L are frequently achieved.

Perfusion TechnologyOne initial approach to increase the yield ofantibody products from a single bioreactorwas the use of perfusion technology in whichthe media is continuously removed from thebioreactor and replaced with fresh media.Perfusion technology is based on the ration-ale that cells in culture could continue toproduce antibody over several weeks if theconditioned media, containing the antibodyproduct along with potentially growth limit-ing metabolites, were replaced regularly withfresh media and growth factors. Years of com-parative work have shown that perfusion cul-tures can achieve higher volumetricproductivities than fed-batch cultures at theexpense of lower product titers per liter ofmedium consumed. Moreover, the continu-ously changing media conditions and longculture times required for perfusion produc-tion frequently lead to inconsistent processes,variable glycosylation, and other post-transla-tional modifications in the product over timein culture. The risk of contamination alsoincreases. Perfusion operations tend to becomplex, difficult to scale up, and generallyless robust than fed-batch processes.21,22

Therefore, fed-batch culture is now themethod of choice for robust, reproducible,and reliable manufacturing processes. Whilethe capital investments in a manufacturingfacility using fed-batch culture are higherthan those for a perfusion-based facility, theoverall cost of goods for fed-batch and perfu-sion processes are similar. While both culturetechnologies are successfully used today bycommercial manufacturers, the biopharma-ceutical industry is converging on the use offed-batch suspension cultures in stirred-tankbioreactors with controlled feeding.

FUTURE CHALLENGES IN ANTIBODY MANUFACTURINGThe advances in cell line generation and cellculture described above have enabledcompanies to produce monoclonal antibodiesat very high expression levels. As a result,early concerns that the industry would not beable to meet the growing productiondemands of MAbs have subsided. While thesesignificant improvements in upstreamproduction have resulted in the ability toexpress MAbs at levels approaching 10 g/L,the capacity and ability of downstreamprocesses to handle these high quantities ofantibody has been strained. The competingdemands of growing production requirementsand reduced cost to the patient presentchallenges to the industry to makemanufacturing processes even more efficient.Improvements in chromatography media forantibody purification have resulted in mediawith higher capacities, faster throughput, andimproved contaminant clearance. Significantefforts are currently being devoted todeveloping alternative techniques to improvedownstream processing to enable the efficientprocessing of high levels of antibody, enhanceprocess robustness and yields, and reduceoverall manufacturing costs. Companiestoday are striving to incorporate Quality byDesign and other new regulatory conceptsinto the development of MAb products tofurther reduce the cost and developmenttimelines for these products. Themanufacturers are also striving to developfinal product formulations containing highconcentrations of antibody with sufficientstability to address the increasing doses ofantibody products without adverselyimpacting the quality of these products. u



Significant efforts are being devotedto the continuous improvement

in the safety and quality of MAbs.


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cells secreting antibody of predefined specificity.Nature. 1975;256(5517):495–7.

2. Available from FDA, CDER, 2003. TherapeuticBiological Products Approval. Available onhttp://fda.gov/cder/biologics/biologics_table.htm

3. PhRMA 2006. Report on Biotechnology medicines indevelopment. Available on http://www.phrma.org/files/Biotech%202006.pdf.

4. Das, RC, Morrow, KJ. Antibody technologies rise tonew challenges. Am Biotechnol Lab. 2007;25(8):9-11.

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6. Co MS, Queen C. Humanized antibodies for therapy.Nature. 1991;351(6326):501–2.

7. Studnicka GM, Soares S., Better, M., Williams, RE,Nadell, R., and Horwitz, AH. Human-engineered Mabsretain full specific binding activity by preserving non-CDR complementarity-modulating residues. ProteinEng. 1994; 7(6) 805-14

8. Adamson SR. The role of technology and science inmanufacturing economics. IBC Conference on AntibodyDevelopment and Production; 2007 Feb 28–Mar 2.

9. Butler M. Animal cell cultures: recent achievements andperspectives in the production of biopharmaceuticals.Appl Microbiol Biotechnol. 2005;68(3):283–91.

10. Available from http://www.genetix.com/xhtml/product.aspx?pid=16.

11. Schimke RT, Roos DS, Brown PC. Amplification ofgenes in somatic mammalian cells. Methods Enzymol.1987;151:85–104.

12. Bebbington, CR et al. High level expression of arecombinant antibody from myeloma cells using aglutamine synthetase gene as an amplifiableselectable marker. Biotechnol. 1992;10:169–175.

13. Wilson RH. Glutamine synthetase gene amplificationin Chinese hamster ovary cells. Gene amplification inmammalian cells (ed.) Kellens, RE. Marcel Dekker Inc.(New York) pp 301–311.

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therapeutics in cultivated mammalian cells. NatureBiotech. 2002;22(11):1393–1398.

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How Cell Culture Became King... and May be Usurped

David Estell, vice president of technology at Genencor International,

650.846.7500, [email protected]

In the early 1980s, most recombinant protein production was carriedout in E. coli. The disadvantage of this method was that the proteins

were produced intracellularly and had to be refolded to obtain activeprotein. As a result, at Genentech we were looking for ways to produceproperly folded proteins in other cell systems.

By 1981, Art Levinson’s group had developed techniques to allowselectable, stable expression in mammalian cells. These methods wereinitially applied to our hepatitis B surface antigen and tissue plas-minogen activator (t-PA) expression. The resulting proteins were effi-ciently expressed in a properly folded form. Meanwhile, JamesStramondo’s group had developed large-scale cell culture processesto improve performance.

The biggest concern in using transformed cells was that DNA orviruses could be carried into the final product. The hepatitis B surfaceantigen assembled into 22-nm particles that were similar in size andshape to some viruses, which made the problem particularly difficult. Sothe team proceeded to work toward FDA approval. My group was

responsible for creatingthe initial recovery processand for demonstratingviral clearance and DNAremoval. Several otherresearch and develop-ment groups also put in a

tremendous amount of work to develop other aspects of the new mam-malian-cell-based processes. In the end, it paid off. Within a few years,both the hepatitis B vaccine and the t-PA processes were validated andapproved by the FDA.

This new expression technology rapidly spread through the indus-try to become the standard production system for recombinant pro-teins. Thus, cell culture became king. The fact that most humanproteins are secreted efficiently in properly folded form by mam-malian cells means that the production of test quantities of a newpharmaceutical protein is now straightforward, and many productionprocesses have become highly standardized.

Cell culture may not always keep its crown, however. Mammaliancell expression is highly efficient on a per cell basis, but creating theinitial working cell banks and production trains requires long leadtimes and is expensive, leading to costs of $500–$1,000 per gram ofprotein. The system’s effectiveness, however, has made the industryreluctant to investigate other options, such as bacillus and fungalexpression systems. These alternative systems have been demon-strated to secrete extremely large amounts of protein in fermenta-tions that take only a few days per batch, and produce several metrictons of protein per year. Because these microbial systems can be cre-ated in weeks and produce protein at 1/10,000th of the cost of mam-malian cells, they may replace some of the mammalian cell capacityfor high volume, lower-cost pharmaceutical proteins in the future.

So watch out, cell culture. A microbial coup may be in the making.

Bacterial expression systems can secrete large amounts of

protein in fermentations that takeonly a few days per batch.

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