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ReviewReceived: 1 September 2010 Accepted: 24 September 2010 Published online in Wiley Online Library: 15 February 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2574

New frontiers in cell line development:challenges for biosimilarsJeff Jia Cheng Hou, Joe Codamo, Warren Pilbrough, Benjamin Hughes,Peter P Gray and Trent P Munro∗

Abstract

Worldwide sales of biologic drugs exceeded US$92 billion in 2009. With many biopharmaceutical patents expiring over thenext decade, a wave of second-generation or ‘follow-on’ biologics will be vying for market share and regulatory approval.Patents cover not only the drugs, but also the molecular modalities that facilitate their high-level expression. Companieshave historically relied on gene amplification to create productive cell lines, yet this lengthy and imprecise process usuallyleads to extensive variation and unpredictable stability of expression. Biosimilar manufacturers must therefore decide whethertraditional methods of cell line development will suffice or if emerging technologies can provide greater reproducibility andspeed. Volumetric yields of 1–2 g L−1 are adequate for most production processes and the focus has shifted towards reliableand predicable product quality attributes over maximum possible titres. Recent advances in this area include cell lines withtargeted genetic modifications, alternative production hosts such as PER.C6 or yeast, and engineered expression vectors,including the UCOE and Selexis platforms. Host cell engineering, single-use technologies, and rapid transient gene expressionare also likely to be enablers of biosimilars. Given the well-known biologics industry mantra ‘the process defines the product’,it remains to be seen how novel cell line development strategies will affect product equivalence and regulatory approval in abiosimilars context. Some recent advances in the field and how they relate to biosimilars are explored.c© 2011 Society of Chemical Industry

Keywords: biosimilars; cell line engineering; CHO; monoclonal antibody; biopharmaceutical; biologic; single-use bioreactors; transientgene expression

INTRODUCTIONSince the approval of Escherichia coli-derived recombinant humaninsulin (Humulin) in 1982 and Chinese hamster ovary (CHO)derived tissue plasminogen activator (tPA, Activase) in 1986,recombinant protein therapeutics have revolutionized modernmedicine.1 There are presently over 400 biologic drugs inlate stage clinical development and worldwide sales exceeded$US90 billion in 2009.1 – 3 Despite the global financial crisis,the biopharmaceutical industry is showing continuous marketgrowth, driven mainly by sales of monoclonal antibodies (mAb)and hormones.1 However, the expiration of patents governingthe exclusive rights to produce several high-profile biologicspromises to open up a thriving market for follow-on biologicsor ‘‘biosimilars’’ as they are better known. Crucially, severalof these expiring patents cover blockbuster biologics such asEpogen (erythropoietin) and Remicade (infliximab).4 Biosimilarsare recombinant therapeutics that resemble but are not identicalto the original product, mainly due to the difficulty in preciselymatching the structure and composition of these large complexbiological molecules. Both the European Medicines Agency(EMEA), and more recently, the US Food and Drug Adminstration(FDA) have introduced regulatory frameworks for the potentialapproval of biosimilars, but, rather than providing a highlyabbreviated path, as is the case for chemically synthesized smallmolecules, approval is very much on a case-by-case basis.

With the growing demand for biologics and the end-of-patentprotection for many existing treatments, the focus for biogeneric-equivalents will be speed and/or cost. A range of new expression

and host cell technologies have emerged since approval of theearly blockbusters. These new technologies may also create anavenue for the creation of biosuperiors (or biobetters), whichrepresent enhanced versions of the innovator product. Currentindustry best-practice during early cell line development stillrelies heavily on traditional amplification systems in combinationwith immortalized mammalian cell lines such as CHO and NS0cells.5,6 This leaves ample opportunities for innovation, withnovel mammalian lines such as human embryonic retinoblast cells(PERC6)7 now being explored, as well as alternative eukaryotichosts such as glycoengineered yeast8 and insect cells,9,10 or eventransgenic plants and animals. These new expression systems willlikely catalyse the progression and expansion of the biosimilarmarket in the future.

Product yields from CHO cells have increased almost exponen-tially in the past 30 years, with many modern processes exceeding5 g L−1 in fed-batch mode.6 The refinement of dihydrofolate reduc-tase (DHFR) and glutamine synthetase (GS) amplification systemshas contributed to gains in specific productivity beyond 50 pgper cell day−1. Other drivers for titre increase have been opti-mization of basal media, fed-batch supplementation and culture

∗ Correspondence to: Trent P Munro, The University of Queensland, AustralianInstitute for Bioengineering and Nanotechnology (AIBN), Brisbane, Australia4072. E-mail: t.munro@uq.edu.au

The University of Queensland, Australian Institute for Bioengineering andNanotechnology (AIBN), Brisbane, Australia 4072

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environment. However, there are still many fundamental issuesto be overcome with the traditional CHO system, most notablythe expression stability of production cell lines and consistent,predictable product quality attributes.

New technologies such as site specific integration, ubiqui-tous chromatin opening elements (UCOE) or scaffold/matrixattachment regions (S/MARs) and zinc finger mediated host cellengineering will probably be beneficial for developing productioncell lines for biosimilars. A significant challenge for industry willbe the rapid analysis of potential biosimilar candidates in earlystages of development; this can be effectively achieved using high-yielding transient gene expression (TGE) systems in combinationwith high-throughput screening methods. An important consid-eration for biosimilars is the co-development of high-resolutionanalytical techniques to ensure product equivalence. While thissubject will not be addressed here, an excellent recent articleshows how this might be achieved for mAbs.11 Finally, single-usebioproduction systems may also help shorten timelines and cutcosts for biosimilars manufacturers, and offer increased flexibilityto better adapt to changing market demands over a traditionalstainless steel-based facility.

BIOSIMILARS AND BIOSUPERIORS: REGULA-TORY HURDLES AND ROADBLOCKSRegulatory agencies have long co-operated with the biopharma-ceutical industry to produce appropriate and relevant guidelinesfor biologic product approval. But there has been extensive debateabout how, or if, a suitable regulatory pathway can be establishedfor biosimilars, because of the molecular complexity of these prod-ucts. This stems mainly from the difficulty in providing sufficientanalytics to completely define something that is essentially ‘ac-ceptably heterogeneous’. Thus, developing guidelines to ensuresafety and efficacy while providing a simplified approval pathwayhas been challenging.

Biosimilars are touted for their potential to reduce cost andincrease accessibility to consumers and biosuperiors for theirpotential for enhanced efficacy. The main regulatory unknownswith these follow-on molecules revolve around how best tomeasure bioequivalence and how long to provide productexclusivity. The EMEA has taken the lead in this area releasinginitial guidance documents in 2005 and a number of variantssince.12 This has led to regulatory approval of biosimilarversions of hGH, erythropoietin (EPO) and recombinant humangranulocyte-colony stimulating factor (G-CSF) (including Sandoz’sOmnitrope, Binocrit and Zarzio3,4,13).12 Several biosimilarsare also approved in India, China, and other countries; Table 1shows a list of currently available biosimilars. The FDA however,has been relatively slow to provide a framework on biosimilars evenafter it accepted a court decision for the approval of Omnitrope(Sandoz) to compete with Genotropin from Pfizer. Current EMEAguidelines rely on a case-by-case examination rather than a blanketpathway to approval. In essence, the amount of data required forapproval lies somewhere in between that for a small moleculegeneric and a full new entity.

As part of the recent healthcare reform in the USA, the PatientProtection and Affordable Care Act, enacted on 23 March 2010,lobby groups successfully incorporated legislation (Biologics PriceCompetition and Innovation Act of 2009) outlining a pathway forbiosimilar approval. A key clause in this legislation is that innovatorcompanies are now provided with a period of 12 years exclusivityfrom the date reference material is first produced, irrespective of

the patent landscape. Furthermore, no biosimilar submissions maybe made to the FDA within 4 years of reference product production.This is in contrast to the small molecule Hatch-Waxman act, asthere is no ‘Orange Book’ to list patents that cover the referencebiological product. Interestingly, this legislation also cites twopossible avenues for biosimilar approval. First, a product maybe considered a ‘biosimilar’ if it closely resembles the referenceproduct and if safety, purity, and potency show no clinicallymeaningful differences to the reference product. Alternatively, aproduct can be designated as ‘interchangeable’ if it is expectedto produce the same clinical outcome as the reference product inany given patient. How these pathways will be interpreted in FDAguidance documents remains to be seen.

Another major issue not yet addressed by current legislation is‘patent evergreening’, in which innovator companies can extendoriginal patents in a way that introduces novel intellectual propertywhile still maintaining protection of the original invention. Thisis perhaps best exemplified by the US Patent Office decision togrant the Cabilly II patent to Genentech in early 2009, makingit valid until 2018.14 Essentially, this patent affects all companieslooking to commercially produce mAbs, as it covers the method forexpression of immunoglobulins (IgGs) in mammalian cells. Whilethis was protected by the original Cabilly I patent, a key changewas made just prior to expiry to include the DNA encoding forIgGs rather than simply mentioning IgG heavy and light chains.It is estimated that this patent earns Genentech and its parentcompany Roche over US$235 million per year in license fees. Thisstrategy represents a significant roadblock to the cost-effectivedevelopment of biosimilars, as companies will still be forced topay licensing fees on an ever-expanding royalty stack.

Adding complexity to this issue is the increasing demand forbiosimilars in highly populated, developing countries such asChina and India. Significant concerns exist over the enforcementof laws governing both intellectual property and therapeuticgoods. Pharmacological-equivalence and safety of biologicsproduced in these markets also remains a concern. Dr Reddy’s(http://www.drreddys.com), considered a pioneer in this region,has released a number of biosimilars into the Indian market,including the world’s first biosimilar mAb, Reditux , which is ageneric version of Roche’s rituximab (Rituxan).

Even with the long-awaited approval pathways for biosimilars,it remains an uphill battle for new biosimilar manufacturers toproduce products that will offer any significant price advantageover the innovator product. It is also becoming apparentthat several of the established innovator firms are positioningthemselves to be players in the biosimilars marketplace. It will beinteresting to see how interchangable biosimilars are interpretedby the regulators and also whether biosuperiors can utilize thisabbreviated regulatory approval pathway. It seems inevitablethat some clinical studies and post-approval monitoring will berequired.

BIOSIMILARS: FASTER, CHEAPER, BETTER?Follow-on biologics significantly reduce development risk becausethe biological target and molecular entity are already validated forsafety and efficacy. Yet at the same time, potential revenues arealso reduced due to a lack of market exclusivity. To some extentcosts are already lower because investment in innovator R&D doesnot need to be recouped. However, expression system and othertechnology choices will also play a role. Follow-on biologics maybe an ideal opportunity to move away from traditional CHO6 cell

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Table 1. Currently available biosimilars worldwide

Company Drug class BiosimilarApproval/Launch

(year) Country

Biopartners Recombinant human growthhormone (rhGH)

Valtropin 2006/2007 EU/USA

Baxter Recombinant humanhyaluronidase

Hylenex 2005 USA

CT Arzneimittel Recombinant humangranulocyte colonystimulating factor (rhG-CSF)

Biograstim 2008 EU

Dr. Reddy’s Laboratories (DRL) rhG-CSF Grafeel – India

DRL Chimeric murine/humananti-CD20 monoclonalantibody

Reditux 2007 India

DRL rhEPO Cresp 2010 India

Hexal Recombinant humanerythropoietin (rhEPO)

Epoetin alfa Hexal 2007 EU

Hexal rhG-CSF Filgrastim Hexal 2009 EU

Hospira rhEPO Retacrit 2007 EU

Hospira rhG-CSF Nivestim 2010 EU

Medice rhEPO Abseamed 2007 EU

Novo Nordisk Recombinant glycagon GlucaGen 1998 USA

Ratiopharm rhG-CSF Ratiograstim 2008-2009 EU

Ratiopharm rhG-CSF Filgrastim ratiopharm 2008 EU

Sandoz rhGH Omnitrope 2004/2006/2009 Australia/EU & USA

/Japan & Canada

Sandoz rhEPO Binocrit 2007 EU

Sandoz rhG-CSF Zarzio 2009 EU

Stada rhEPO Silapo 2007 EU

Teva/Ferring rhGH Tev-tropin 2005 USA

Teva rhG-CSF Tevagrastim 2008 EU

Upsher Smith Recombinant salmon calcitonin Fortical 2005 USA

A FDA approval via 505(b)(2) of the Food, Drug, and Cosmetic Act.Source3,115: Data collected from the EMEA and FDA drugs database. DRL data was collected for the DRL website (http://www.drreddys.com).

based and other mammalian expressions systems15 to test thewaters with novel expression systems such as transgenic animalsand plants which can produce large amounts of recombinantprotein very economically;16 or glycoengineered strains of themethylotrophic yeast Pichia pastoris,17,18 that have the potential forboth more economic production and the tuning of glycosylation totherapeutically relevant forms with higher fidelity and uniformity.This approach brings a clear focus on cost and quality measuresassociated with the new versus existing expression systems, safe inthe knowledge that the underlying biological mechanisms for drugactivity have already been established. The main challenge withthis approach is that product comparability may be more difficultto justify, as there may be significant differences with processresiduals or the types and abundances of molecular variants.Atryn, recombinant human antithrombin, produced by GTCBiotherapeutics in goats’ milk, was the first therapeutic proteinmade in a transgenic animal to be approved anywhere in theworld (Europe, in 2006), and the first approved in the USA (2009).The company is now also developing several follow-on biologics,including biosimilar versions of Rituxan, Herceptin, Humira,and Erbitux, with potentially improved antibody-dependentcell-mediated cytotoxicity (ADCC).

More recently, the number of players looking to bring newbiologics to market has exploded, as has the number of products inlate stage clinical trials, particularly in the mAb arena. Predictably

there has been intense interest in making both biosimilars ofexisting approved blockbusters and improving them to makebiosuperiors. This is perhaps best demonstrated by Merck’spurchase of GlycoFi in 2006 for US$400 million and Insmed’sbiosimilar product pipeline for US$130 million in 2008, to establishMerck BioVentures.19 The former was a strategic move designedto harness the power of GlycoFi’s glycoengineered yeast strains.This acquisition has parallels with Roche’s earlier purchase ofGlycArt in 2005 for 235 million Swiss Francs. GlycArt’s technologyplatform enables modification of mAb glycoforms to promoteenhanced ADCC, providing significantly enhanced efficacy. Thesetechnologies are particularly well suited to the creation of second-generation biologics, which through their improved propertiesmay be considered biosuperior, rather than merely biosimilar.

An alternative strategy for follow-ons is to closely match theexpression system used by the innovator firm but to cut coststhrough manufacturing efficiency gains and reduced licensingand patent encumbrances. Slight improvements in formulation orcomposition, such as PEGylation, could also help differentiate thefollow-on product. Since the biosimilars manufacturer does nothave access to the innovator’s original cell line, a new stable cellline expressing the recombinant protein must be established. Themost common selectable, amplifiable markers used in CHO andNS0 cells are the DHFR20 and GS21 genes. The original patents onthese systems have now expired, so it might be conceivable to

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incorporate basic DHFR or GS selection into processes to makebiosimilars, matching the innovator host and selection system,with the hope of minimal licensing costs. However, many ensuingpatents have been granted on improvements to the technology,which may limit use to a rudimentary level, possibly constrainingcell line development speed and productivity, and requiring careto avoid infringement. Clearly the patent landscape is complexand must be navigated carefully.

Some companies have instead chosen to adopt non-proprietaryselection systems. PDL BioPharma, though not a biosimilars manu-facturer, utilizes the xanthine-guanine phosphoribosyl transferasegene (gpt) from E. coli, and an NS0 cell line adapted to choles-terol independence.22 While gpt is not an amplifiable marker, highspecific productivities of 20–60 pg per cell day−1 are achievablefrom a single transgene copy using iterative subcloning, withoutthe need to license the GS NS0 system. An alternative host cellline, which is subject to licensing costs, but with the potentialto reduce manufacturing costs, is PER.C6.23 This immortalizedline was derived from human retinal cells transfected with theAdenovirus 5 E1A gene. These cells are capable of growing tovery high cell densities (160 × 106 cells mL−1) using a concen-trated perfusion process and have been shown to express mAbsat over 25 g L−1.24,25 Transactivation of the CMV promoter by E1Ais thought to contribute to the high productivity of PER.C6 cells.The high titres and low culture volumes facilitate reduction in thefinal cost per dose, and allow deployment of single use technologyfor further cost savings since disposable bioreactors are typicallylimited to ≤2000 L scale.

As already discussed, even if host and selection system areperfectly matched, a biotherapeutic is never going to be truly‘generic’ (identical). Furthermore, the best strategy to adopt forproduction of follow-on biologics is still difficult to predict becauseit depends so fundamentally on the regulatory framework, whichremains complex and not yet fully resolved.

BIOSIMILARS: PRODUCING MORE FOR LONGERProduct differentiation in an increasingly competitive biosimilarmarketplace will require renewed focus on rapid, low costcell line development as well as predictable product qualityattributes. Traditional stable cell line development involvesapplying selection pressure to isolate clones with randomintegration of the gene of interest (GOI) into the host cell’s genome,creating heterogeneity and expression instability. Cell linescapable of maintaining expression levels during prolonged cultureis the goal, and should probably be called a ‘‘stable stable’’ cell lineas suggested by Barnes et al.26 The opportunity exists to explorenew technologies to increase clonal stability while minimizingclonal variation. Factors that contribute to instability includegene copy number, position effect, insertional mutagenesis, post-transcriptional and post-translational modifications. Below wediscuss some of the current technologies available for improvedstable cell line development.

The ability to specify the insertion site of the transgene cassettenot only provides uniformity among the transfectant pool, butalso limits gene suppression effects from anomalies such asrepeat-induced gene silencing (RIGS)27 or repeat-induced DNAmethylation.28 Recombinase-mediated cassette exchange (RMCE)enables site-specific recombination of expression vectors in thehost genome and has been used in many different settings fromthe generation of transgenic Drosophila to gene knockouts inhigher eukaryotes.29 The technology uses a recombinase enzyme,

such as cre or flp, which coordinates a strand exchange eventbetween short cis-acting DNA target sequences, for example loxPor the flp-recognition target (FRT) sites. The cre/loxP system withthe necessary loxP sites in place allows complete replacement ofthe expression cassette, therefore allowing both an excision aswell as insertion reaction to occur at the locus.30,31 The successof these methods relies on insertion into a ‘hot spot’, or regionof the genome which facilitates stable, high-level expression suchas the AAVS1 safe harbour locus.32 While showing considerablepromise, these methods still have technical limitations, includingthe challenge of incorporating the loxP or FRT sites into a genomichot spot or ensuring insertion of only a single gene copy.This technology is currently used commercially by ProBioGen(http://www.probiogen.de) but to our knowledge is yet to beincorporated into an approved product. The ability to pre-definethe transgene insertion point has the potential to improve bothexpression levels and product reproducibility.

Position effect variegation refers to stochastic silencing ofthe transgene due to involvement of the molecular nichesurrounding the genomic point of insertion resulting fromexpansion of heterochromatin. Tandem repeat studies withDrosophila transgenes33 and similar studies in mouse models27,34

have clearly demonstrated the marked degree of transgenesilencing in a heterochromatin-rich environment. However, withrecent developments in our understanding of DNA insulators andlocus control regions (LCR), it may be possible to constitutivelyexpress a transgene independent of the genomic insertion point.It has been hypothesized that insulator elements define thetranscriptional boundaries in chromatin and could potentiallyprovide a ‘barrier effect’ to transgenes.35 – 37 Such elements includethe scaffold matrix attachment region (S/MARs)38 – 40; and theUbiquitous Chromatin Opening Element (UCOE)41,42 which havebeen demonstrated to reduce heterogeneity and enhance stabilityin production cell lines.

S/MAR elements are attachment points within the DNAsequence that allow for anchoring of chromatin structure to thenuclear matrix. Certain S/MARs are also recognized as regulatoryelements similar to the LCRs. Such elements not only provide aregion with a high affinity for transcription factors such as CCCTCbinding factor (CTCF) and nuclear matrix proteins (NMP),39,43,44 butalso govern the order of expression at native genomic locations.Elements such as the 5′ hypersensitive site (HS) 4 of the chickenβ-globin gene and the LCR region of the Igf 2/H12 locus are primeexamples where such a region has provided both regulatoryactivity and insulator function.35,43,45,46 The chicken lysozyme 5′

MAR element was one of the first elements used to constructstable cell lines with CHO and C2C12 cells38 as well as being usedin a number of plant based studies.47 Using S/MAR sequences toflank trangenes, it is possible to generate a greater number of highexpressing clones and increase the stability of production cell linesexpressing complex recombinant proteins such as mAbs.38,39,48

An alternative approach to the use of S/MARs is the UCOE

expression technology marketed by Millipore.49 These chromatin-remodelling elements have the capacity to establish and maintainthe chromatin in an open configuration similar to the highlyactive euchromatic regions in the human genome. Like the S/MARelements, UCOE’s have also been compared to LCRs and theirdominant ability to control gene expression. The current UCOE’savailable are primarily from the human TATA binding protein (TBP)and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1)loci of the human genome.49 Williams et al. (2005) illustrated theeffect of using UCOE to examine recombinant protein production

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in CHO cells.42 The use of both enhanced green fluorescent protein(eGFP) and EPO demonstrated a 20- to 40-fold increase in the levelof expression, with such expression levels maintained for greaterthan 100 generations.42

Even though a better understanding of these elements isneeded, the production of biosimilars will not always follow aplatform approach and will probably be a case-by-case scenarioto develop a superior or improved process. Access to a range ofnew choices for cell line engineering will therefore aid biosimilarmanufacturers to develop more cost-efficient processes and toalso make them potentially more reliable for approval purposes.

CELL LINES MADE TO MEASUREMany less complex recombinant proteins, such as industrialenzymes, are produced using microbial fermentation processes ata fraction of the cost of mammalian cell-derived biologics. This isalso seen in the bio-fuels sector, where bacteria are being usedto create the next generation of fuels and other energy denseproducts.50 This has been achieved because systems engineeringand complex genetic manipulation of bacteria has been possiblefor many years. This has permitted the creation of optimized hostcells with a raft of genetic modifications to allow greatly increasedefficiency and productivity for a given process. Mammalian cell lineengineering has traditionally had only modest success; however,the recent advent of new molecular and ‘omics tools should changethis equation by enabling the field of rational cell engineering tomature. Highly optimized host cell lines would provide an efficientplatform for maximal low cost biosimilar production.

Host cell engineering by zinc finger nucleases (ZFN) has recentlyshown particular promise. These are a custom array of molecularrecognition modules with high DNA sequence specificity linked tohalf of a nuclease.51 In essence, they can be designed to recognizealmost any DNA sequence. The modules function in pairs with eachunit linked to half of the promiscuous nuclease, Fok1. Only whenboth pairs bind their target sequence in close proximity is Fok1activity restored and a double strand DNA break created. This breakis then repaired, often via non-homologous end joining, which inturn creates a genetic lesion at the cut site and potentially ablatesgene function. By adding DNA into the system with overlap regionshomologous to the cut site, genes may also be inserted using thehomologous end joining pathway. This method therefore createsa tool for rapid high-efficiency genetic engineering of complexgenomes either by targeted mutagenesis or insertion.

Several examples of cell line engineering strategies incorpo-rating ZFN technology have recently been reported providingan efficient yet stringent approach for the development of‘tailor-made’ cell lines previously too laborious or difficult togenerate.52 – 54 Santiago et al. (2008) were able to generate abiallelic disruption of the DHFR gene without selection.55 Morerecently, Genentech has utilized ZFN technology in conjunctionwith Sangamo Biosciences to create a cell line deficient in α1,6-fucosyltransferase8 (FUT8).52 The absence of FUT8 allows for theproduction of mAbs with either reduced or absent fucosylation,thereby increasing ADCC of the molecule, potentially providingmore efficacious treatments with a significant reduction in cost.These types of engineered cell lines may be ideal for develop-ment of biosuperior mAbs. A FUT8 mutant CHO cell line createdusing traditional mutagenesis had also been previously isolatedby BioWa.56 BioWa and Lonza have now formed a partnershipallowing widespread access to this technology. Genentech andSangamo have also used ZFNs to delete the proapoptotic genes,

BAX and BAK, in CHO cells to create a cell line with increasedresistance to apoptosis, prolonging the production phase in batchculture and leading to a two- to five-fold increase in mAb titre.54

Finally, a triple gene knockout (DHFR, GS and FUT8) using ZFNs inCHO cells has also been achieved.53 The latter example highlightsthe potential for ZFNs to generate multi-gene knockouts in mam-malian cells lines, creating the possibility for complex rationalegenetic manipulation.

Systems biology and genome-scale modelling of mammaliansystems has also recently become a reality.57,58 These techniquesallow complex in silico modelling to identify key pathways targetsfor modification using technologies such as ZFNs. Previously, amajor barrier to this approach in CHO cells has been the lackof publically available genome data. However, at the recent CellCulture Engineering XII meeting (Banff, Canada, April 2010) itwas announced by Bernhard Palsson that the Beijing GenomicsInstitute in China plans to sequence the CHO genome de novo andmake the data freely available to the public. These emergingtechnologies should soon allow for the creation of designermammalian cell lines as has been achieved through systemsengineering of prokaryotes.

REGULATORY RNAS: NEW OPPORTUNITIESFOR CELL LINE ENGINEERINGRecently, our understanding of how genetic information isregulated has changed radically, primarily due to the explosion ofinformation received on the role of regulatory RNAs in geneexpression. With this new knowledge, it has almost becomeimpossible to disregard the importance of RNA with regard torecombinant protein production. Rational genetic engineeringapproaches are designed to optimize systems to reach peak titresand provide homogeneous products; however, such approachesmay lack the necessary knowledge to achieve these goals.Our ability to adapt cells to suspension growth in serum-free or chemically-defined conditions or the cultivation of cellsunder mild-hypothermic conditions demonstrates the incredibleplasticity of cultured cells. Furthermore, it also highlights thefundamental limits in our understanding of complex biologicalsystems, since we cannot fully explain the titre improvementsachieved when incorporating these environmental changes intoany bioproduction process.

We now know regulatory RNAs play a major role in modulatinggene expression, yet our ability to use these molecules as geneticengineering targets to improve biopharmaceutical productionremains largely unexplored. The significance of non-codingRNAs lies within their ability to govern translational processesvia interaction with the mRNA. The first small RNA, lin-4, wasdiscovered in Caenorhabditis elegans through the cloning of thelin-4.59 This has since been followed by a number of significantfindings including the identification of a novel Cricetulus griseusortholog of the miRNA, miR-21 by Gammell et al, through a seriesof expression studies incorporating hypothermia.60 Koh et al. alsoidentified miRNAs in HEK293 cells which potentially affect cellcycle regulation under bioreactor conditions.61 With the publicrelease of the CHO genome and a further understanding of RNA-based gene regulation, regulatory RNAs will probably prove tobe ideal targets for modulating recombinant protein production.While this area of cell line engineering is still in its infancy, itsimportance should not be understated. Once again, maintainingcost competitiveness in the biosimilars space will require improved

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cell line performance which will be aided by future advances inthe field of regulatory RNA engineering.

RAPID SCREENING OF BIOSIMILAR CANDI-DATES BY HIGH-LEVEL TRANSIENT GENEEXPRESSIONStable expression technologies will continue to be used forthe production of biologics at gram to kilogram quantitiesin mammalian cells. But the time and expense to establish,characterize and optimize such cell lines is considerable, duemainly to the time taken to expand and screen large numbersof clones. In order to maintain speed-to-market and minimizesunk costs there is a ‘kill early and kill quickly’ mentalitywhen screening drug candidates. To develop biosimilars orbiosuperiors, a more rapid and efficient way to assess drugcandidates and to rapidly produce product for analytical andtoxicology studies is needed. Recently, there has been a movetowards the routine use of optimized, large-scale, TGE systems forthe production of milligram to gram quantities of recombinantprotein, with such yields sufficient for the pre-clinical assessmentof potential therapeutics. Several efficient protocols for TGE inHEK293 and CHO cells have recently been published.62 – 64 Criticalto the improvements in TGE have been the implementation ofcarefully optimized methods of transfection which have revolvedaround the use of either calcium phosphate,65 – 68 cationic lipds(lipofection)69 – 71 or cationic polymers such as PEI.72 – 75 Due tocost effectiveness and relatively high efficiency, it is the latter thathas been the focus of much research, making it applicable forlarge-scale transfections of both HEK29362,76,77 and CHO78,79 cells.Additionally, development of novel polymers for gene deliveryderived through nanotechnology may also be a source for futureimprovements in transfection efficiencies and yields achieved withTGE.80 – 82

A significant challenge for TGE is rapid plasmid copy numberdilution during cell division, which results in a significant reductionin recombinant protein titres at the end stages of the process.Approaches taken to address this issue have included theincorporation of mild-hypothermia (27–34 ◦C) post-transfection,which in most cases results in a higher specific productivity.83,84

Such improvements have been attributed to enhanced mRNAstability and an accumulation of cells in G0/G1 phases ofthe cell cycle, leading to slower growth rates and in turna reduced rate of toxic metabolite accumulation.85 – 87 Yieldimprovements remain dependent on key variables includingtemperature, cell line and the protein being expressed.85,88

Alternatively, supplementation with histone deacetylase inhibitors(iHDAC), such as sodium butyrate and valproic acid have providedimprovements in transient expression at 37 ◦C89 and more recentlyunder mild-hypothermia at 32 ◦C.90 iHDACs work by up-regulatingtranscription through histone hyperacetylation which grantstranscription machinery easier access to the genes involved in TGE.

The development of episomal systems to amplify and maintainplasmid copy number after transfection has also been effectivein improving TGE yields. These systems incorporate elementsderived from viruses to facilitate plasmid maintenance. In human-derived cells such as HEK293, elements from the well-characterizedherpesvirus, Epstein-barr virus (EBV) have been widely used. Thebinding of EBV nuclear antigen 1 (EBNA-1) stably expressedin the cell line HEK.EBNA or encoded by expression plasmidssuch as pCEP4 from Invitrogen to plasmid DNA containing

the EBV latent origin of replication (OriP) drives the replicationand maintenance of this plasmid in transfected cells, providingelevated and prolonged expression of the recombinant protein ofinterest.62,91 – 93 Recent reports have described transient mAb titresin excess of 1 g L−1 in 10 days using the HEK.EBNA system.94 Thedevelopment of episomal systems in rodent cell lines such as CHOcells have been more challenging, mainly because rodent cells arenot permissive to EBV based replication.92,95 However, the capacityfor EBNA-1 to retain OriP-containing plasmids does function inrodent cells, which has led to incorporation of these elements intotransient expression vectors for enhanced protein expression.87

More recently, an episomal system with the capacity to replicateand retain plasmid DNA in transfected CHO cells was created usingelements from murine polyomavirus to drive plasmid replicationand EBNA-1/OriP to retain and equally segregate plasmid DNAduring cell division.96 This system generated much higher titres ofrecombinant hGH compared with replication and/or retentiondeficient systems.96 In summary, recent advances in TGE inCHO and HEK cells now allow reliable, efficient production ofsignificant amounts of candidate molecules. It will be critical forbiosimilar manufacturers to adopt these systems for rapid, lowcost assessment of candidates during early stage development.

SINGLE-USE BIOPROCESSING SYSTEMS FORBIOSIMILAR PRODUCTIONBiosimilar manufacturers and indeed biomanufacturers in general,are increasingly looking towards disposable or single-use systemsto meet their clinical, and in some cases, manufacturing demands.Traditionally, large-scale mammalian processes have utilized thewell characterized and ‘‘regulatory-accepted’’ stainless steel stirredtank bioreactor.97 – 100 However, recent advances in single-use sys-tems for bioprocesses have made this technology an increasinglyviable alternative.101 The facility of the future is moving away fromthe multiple 10 000 L or larger reactors, towards more flexible,single-use reactors and warehouse manufacturing.101 – 103 Ware-house or ‘modular’ manufacturing involves large open processingareas where equipment can be rolled in or out as required, andis highly complementary to multi-product or multi-modality facil-ities, in which closed processing and single-use systems combineto greatly reduce the risk of cross-contamination.102,104 An efficientlow-cost biosimilars facility might involve manufacturing a rangeof different products using a common platform process in thesame production building by leveraging single use technologiesand modular manufacturing.

Developments in disposable bag technologies were historicallydriven by the medical devices industry and blood banking. Now,along with recent advances in single-use sensors, disposables havegiven biopharmaceutical manufacturers a wealth of processingoptions.104 Single-use solutions presently exist for practicallyevery process step used in the manufacture of biotherapeutics,from relatively simple transfer and hold steps such as bufferand media preparation, final fill and freezing, to fully definedand scalable unit operations including bioreactors, normal andtangential flow filtration and chromatography.103,104 Eibl et al.105

provide an in-depth review of the many single-use options forcell culture now widely accepted and used at lab scale and up to2000 L. Rocking bioreactors including the BIOSTAT Cultibag RM(Sartorius Stedim), Wave Bioreactor (GE Healthcare) and AppliFlex(Applikon) have frequently been used in both industry andacademia for transient79,106 and stable107 – 109 processes. Thesesystems utilize a pre-sterilized, single-use bag containing the

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cells and medium agitated on a rocking platform. More recently,to capitalize on widespread industry know-how with stainlesssteel stirred tank bioreactors, single-use systems based on thetraditional STR geometry have been developed, including theThermo Hyclone SUB, Xcellerex XDR, or Sartorius BIOSTAT CultibagSTR. Finally, systems utilizing non-instrumented round- or square-shaped bottles on orbital shakers have displayed promise asalternatives for low-cost scale-up.110,111

Intuitively, a biosimilars manufacturer might act to leverageeconomies of scale or mass production to drive down their costof goods; however, the recent push to higher titres, advancedperfusion techniques and designer cell lines are allowing just theopposite with smaller reactor volumes able to meet productionneeds.24,25 Single-use systems are perfectly suited to this scenario,because equipment is typically limited to 2000 L, and because thecost of goods advantage is greatest at lower annual productionvolumes. The single-use-based facility25 also offers other distinctadvantages for a biosimilars manufacturer including reduced cap-ital cost and timeline for manufacture and installation.102,104 Withthe removal or elimination of cleaning/sterilization cycles, both sig-nificant validation savings and reduced utilities requirements canalso be achieved.100,101,105 Finally, one of the major advantages ofsingle-use systems is their flexibility in allowing manufacturers torespond efficiently to changing market demands. Manufacturingprocesses can be scaled efficiently through repetitions of the sameprocess train, since additional procurement, installation, and qual-ification requirements are minimal.101 Conversely, if market shareshrinks, or a product does not progress successfully through clini-cal testing, the excess equipment can be re-distributed efficientlyfor alternative manufacturing or development priorities.

Key challenges of single-use systems include the robustness andreliability of disposable sensors and probes105,112 and the effectof potential extractables and leachables on final products.113,114

Currently, no universally accepted regulatory guidelines for ex-tractable and leachable studies exists, and the testing require-ments can be complicated and extensive. However, significantefforts are underway from end-user organizations to standardizeand accelerate the adoption of single-use manufacturing tech-nologies and encourage their acceptance by regulatory bodiesthrough publication of best-practice documents for testing andvalidation.104 Additionally, the need to complete a sound andin-depth economic analysis is imperative for any user of dispos-able technology.102,104 Any initial capital expenditure savings andreduced process time or increased throughput must always bebalanced against the ongoing consumables cost which can tip thebalance in some areas back to traditional solutions. However, it iswithout question that, together with the development of highlyproductive stable cell lines and rapid TGE technologies discussedearlier, single-use bioprocess technology is and will continue tobe a valuable enabler for the biosimilars industry.

CONCLUSIONThe biologics market continues to grow faster than the broaderpharmaceutical sector.2 Biopharmaceuticals offer life-saving treat-ment options for an ever-expanding number of disease indicationssuch as cancer, autoimmune disorders and viral infections. Avail-ability and affordability are major issues in developing countries,and in developed nations the high cost associated with thesemedications places an ever-increasing financial burden on health-care providers. The development of biopharmaceutical generics or

biosimilars represents an opportunity to address the above issuesand provide broader access to cheaper products.

The development of biosimilars relies heavily on the ever-changing intellectual property landscape and the expiration ofkey patents covering both the underlying production modalitiesand molecular drug targets. From a regulatory perspective, boththe EMEA and the FDA now have approval pathways for biosimilars,although the process and protection provided to the innovatorcompanies is vastly different from that covering traditional small-molecule generics. The medium- to long-term impact of thislegislation remains to be seen. For the majority of productshowever, approval will still be dealt with on a case-by-case basisrather than any semblance of a truly abbreviated pathway as theindustry mantra of ‘‘the process defines the product’’ is expectedto endure. Post-approval monitoring will also be key in ensuringequivalence and safety.

The development needs of biosimilars will probably requirevarious approaches to further enhance and improve the upstreampathway in the production process. Modifying existing cell linedevelopment strategies such as traditional amplification systems,incorporating new technologies or eukaryotic hosts to enhanceexpression stability and product quality, and considering the useof large-scale TGE and single-use bioprocess technology are allapproaches that will help address the issue of balancing speed,cost and product quality.

Furthermore, the challenge for biosimilar manufacturers will notsimply revolve around how to incorporate recent technologicaladvances into product development and production, but whetherthey are commercially viable alternatives. Additionally, thequestion of how such changes will equate to a reduction in costto the consumer will require considerable attention. Addressingthese issues represents the true test for the future of biosimilarsand will directly affect their market size and return on investment.

ACKNOWLEDGEMENTSThe authors would like to thank Karen Hughes for critical readingof the manuscript.

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