Review

Received: 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 biosimilars

Jeff 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 the next 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. Companies have historically relied on gene amplication to create productive cell lines, yet this lengthy and imprecise process usually leads to extensive variation and unpredictable stability of expression. Biosimilar manufacturers must therefore decide whether traditional methods of cell line development will sufce or if emerging technologies can provide greater reproducibility and speed. Volumetric yields of 12 g L1 are adequate for most production processes and the focus has shifted towards reliable and predicable product quality attributes over maximum possible titres. Recent advances in this area include cell lines with targeted genetic modications, 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 expression are also likely to be enablers of biosimilars. Given the well-known biologics industry mantra the process denes the product, it remains to be seen how novel cell line development strategies will affect product equivalence and regulatory approval in a biosimilars context. Some recent advances in the eld 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; transient gene expression

INTRODUCTION
Since the approval of Escherichia coli-derived recombinant human insulin (Humulin) in 1982 and Chinese hamster ovary (CHO) derived tissue plasminogen activator (tPA, Activase) in 1986, recombinant protein therapeutics have revolutionized modern medicine.1 There are presently over 400 biologic drugs in late stage clinical development and worldwide sales exceeded $US90 billion in 2009.1 3 Despite the global nancial crisis, the biopharmaceutical industry is showing continuous market growth, driven mainly by sales of monoclonal antibodies (mAb) and hormones.1 However, the expiration of patents governing the exclusive rights to produce several high-prole biologics promises to open up a thriving market for follow-on biologics or biosimilars as they are better known. Crucially, several of these expiring patents cover blockbuster biologics such as Epogen (erythropoietin) and Remicade (iniximab).4 Biosimilars are recombinant therapeutics that resemble but are not identical to the original product, mainly due to the difculty in precisely matching the structure and composition of these large complex biological molecules. Both the European Medicines Agency (EMEA), and more recently, the US Food and Drug Adminstration (FDA) have introduced regulatory frameworks for the potential approval of biosimilars, but, rather than providing a highly abbreviated path, as is the case for chemically synthesized small molecules, approval is very much on a case-by-case basis. With the growing demand for biologics and the end-of-patent protection for many existing treatments, the focus for biogenericequivalents will be speed and/or cost. A range of new expression

and host cell technologies have emerged since approval of the early blockbusters. These new technologies may also create an avenue for the creation of biosuperiors (or biobetters), which represent enhanced versions of the innovator product. Current industry best-practice during early cell line development still relies heavily on traditional amplication systems in combination with immortalized mammalian cell lines such as CHO and NS0 cells.5,6 This leaves ample opportunities for innovation, with novel mammalian lines such as human embryonic retinoblast cells (PERC6)7 now being explored, as well as alternative eukaryotic hosts such as glycoengineered yeast8 and insect cells,9,10 or even transgenic plants and animals. These new expression systems will likely catalyse the progression and expansion of the biosimilar market in the future. Product yields from CHO cells have increased almost exponentially in the past 30 years, with many modern processes exceeding 5 g L1 in fed-batch mode.6 The renement of dihydrofolate reductase (DHFR) and glutamine synthetase (GS) amplication systems has contributed to gains in specic productivity beyond 50 pg per cell day1 . Other drivers for titre increase have been optimization of basal media, fed-batch supplementation and culture

Correspondence to: Trent P Munro, The University of Queensland, Australian Institute for Bioengineering and Nanotechnology (AIBN), Brisbane, Australia 4072. E-mail: t.munro@uq.edu.au The University of Queensland, Australian Institute for Bioengineering and Nanotechnology (AIBN), Brisbane, Australia 4072

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www.soci.org environment. However, there are still many fundamental issues to be overcome with the traditional CHO system, most notably the expression stability of production cell lines and consistent, predictable product quality attributes. New technologies such as site specic integration, ubiquitous chromatin opening elements (UCOE) or scaffold/matrix attachment regions (S/MARs) and zinc nger mediated host cell engineering will probably be benecial for developing production cell lines for biosimilars. A signicant challenge for industry will be the rapid analysis of potential biosimilar candidates in early stages of development; this can be effectively achieved using highyielding transient gene expression (TGE) systems in combination with high-throughput screening methods. An important consideration for biosimilars is the co-development of high-resolution analytical techniques to ensure product equivalence. While this subject will not be addressed here, an excellent recent article shows how this might be achieved for mAbs.11 Finally, single-use bioproduction systems may also help shorten timelines and cut costs for biosimilars manufacturers, and offer increased exibility to better adapt to changing market demands over a traditional stainless steel-based facility.

JJ Cheng Hou et al.

BIOSIMILARS AND BIOSUPERIORS: REGULATORY HURDLES AND ROADBLOCKS

Regulatory agencies have long co-operated with the biopharmaceutical industry to produce appropriate and relevant guidelines for biologic product approval. But there has been extensive debate about how, or if, a suitable regulatory pathway can be established for biosimilars, because of the molecular complexity of these products. This stems mainly from the difculty in providing sufcient analytics to completely dene something that is essentially acceptably heterogeneous. Thus, developing guidelines to ensure safety and efcacy while providing a simplied approval pathway has been challenging. Biosimilars are touted for their potential to reduce cost and increase accessibility to consumers and biosuperiors for their potential for enhanced efcacy. The main regulatory unknowns with these follow-on molecules revolve around how best to measure bioequivalence and how long to provide product exclusivity. The EMEA has taken the lead in this area releasing initial guidance documents in 2005 and a number of variants since.12 This has led to regulatory approval of biosimilar versions of hGH, erythropoietin (EPO) and recombinant human granulocyte-colony stimulating factor (G-CSF) (including Sandozs Omnitrope, Binocrit and Zarzio3,4,13 ).12 Several biosimilars are also approved in India, China, and other countries; Table 1 shows a list of currently available biosimilars. The FDA however, has been relatively slow to provide a framework on biosimilars even after it accepted a court decision for the approval of Omnitrope (Sandoz) to compete with Genotropin from Pzer. Current EMEA guidelines rely on a case-by-case examination rather than a blanket pathway to approval. In essence, the amount of data required for approval lies somewhere in between that for a small molecule generic and a full new entity. As part of the recent healthcare reform in the USA, the Patient Protection and Affordable Care Act, enacted on 23 March 2010, lobby groups successfully incorporated legislation (Biologics Price Competition and Innovation Act of 2009) outlining a pathway for biosimilar approval. A key clause in this legislation is that innovator companies are now provided with a period of 12 years exclusivity from the date reference material is rst produced, irrespective of

the patent landscape. Furthermore, no biosimilar submissions may be made to the FDA within 4 years of reference product production. This is in contrast to the small molecule Hatch-Waxman act, as there is no Orange Book to list patents that cover the reference biological product. Interestingly, this legislation also cites two possible avenues for biosimilar approval. First, a product may be considered a biosimilar if it closely resembles the reference product and if safety, purity, and potency show no clinically meaningful differences to the reference product. Alternatively, a product can be designated as interchangeable if it is expected to produce the same clinical outcome as the reference product in any given patient. How these pathways will be interpreted in FDA guidance documents remains to be seen. Another major issue not yet addressed by current legislation is patent evergreening, in which innovator companies can extend original patents in a way that introduces novel intellectual property while still maintaining protection of the original invention. This is perhaps best exemplied by the US Patent Ofce decision to grant the Cabilly II patent to Genentech in early 2009, making it valid until 2018.14 Essentially, this patent affects all companies looking to commercially produce mAbs, as it covers the method for expression of immunoglobulins (IgGs) in mammalian cells. While this was protected by the original Cabilly I patent, a key change was made just prior to expiry to include the DNA encoding for IgGs rather than simply mentioning IgG heavy and light chains. It is estimated that this patent earns Genentech and its parent company Roche over US$235 million per year in license fees. This strategy represents a signicant roadblock to the cost-effective development of biosimilars, as companies will still be forced to pay licensing fees on an ever-expanding royalty stack. Adding complexity to this issue is the increasing demand for biosimilars in highly populated, developing countries such as China and India. Signicant concerns exist over the enforcement of laws governing both intellectual property and therapeutic goods. Pharmacological-equivalence and safety of biologics produced in these markets also remains a concern. Dr Reddys (http://www.drreddys.com), considered a pioneer in this region, has released a number of biosimilars into the Indian market, including the worlds rst biosimilar mAb, Reditux , which is a generic version of Roches rituximab (Rituxan). Even with the long-awaited approval pathways for biosimilars, it remains an uphill battle for new biosimilar manufacturers to produce products that will offer any signicant price advantage over the innovator product. It is also becoming apparent that several of the established innovator rms are positioning themselves to be players in the biosimilars marketplace. It will be interesting to see how interchangable biosimilars are interpreted by the regulators and also whether biosuperiors can utilize this abbreviated regulatory approval pathway. It seems inevitable that some clinical studies and post-approval monitoring will be required.

BIOSIMILARS: FASTER, CHEAPER, BETTER?

Follow-on biologics signicantly reduce development risk because the biological target and molecular entity are already validated for safety and efcacy. Yet at the same time, potential revenues are also reduced due to a lack of market exclusivity. To some extent costs are already lower because investment in innovator R&D does not need to be recouped. However, expression system and other technology choices will also play a role. Follow-on biologics may be an ideal opportunity to move away from traditional CHO6 cell

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Table 1. Currently available biosimilars worldwide Company Biopartners Baxter CT Arzneimittel Drug class Recombinant human growth hormone (rhGH) Recombinant human hyaluronidase Recombinant human granulocyte colony stimulating factor (rhG-CSF) rhG-CSF Chimeric murine/human anti-CD20 monoclonal antibody rhEPO Recombinant human erythropoietin (rhEPO) rhG-CSF rhEPO rhG-CSF rhEPO Recombinant glycagon rhG-CSF rhG-CSF rhGH rhEPO rhG-CSF rhEPO rhGH rhG-CSF Recombinant salmon calcitonin Biosimilar Valtropin Hylenex Biograstim Approval/Launch (year) 2006/2007 2005 2008 Country EU/USA USA EU

Dr. Reddys Laboratories (DRL) DRL

Grafeel Reditux

2007

India India

DRL Hexal Hexal Hospira Hospira Medice Novo Nordisk Ratiopharm Ratiopharm Sandoz Sandoz Sandoz Stada Teva/Ferring Teva Upsher Smith
A

Cresp Epoetin alfa Hexal Filgrastim Hexal Retacrit Nivestim Abseamed GlucaGen Ratiograstim Filgrastim ratiopharm Omnitrope Binocrit Zarzio Silapo Tev-tropin Tevagrastim Fortical

2010 2007 2009 2007 2010 2007 1998 2008-2009 2008 2004/2006/2009 2007 2009 2007 2005 2008 2005

India EU EU EU EU EU USA EU EU Australia/EU & USA /Japan & Canada EU EU EU USA EU USA

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 the waters with novel expression systems such as transgenic animals and plants which can produce large amounts of recombinant protein very economically;16 or glycoengineered strains of the methylotrophic yeast Pichiapastoris,17,18 that have the potential for both more economic production and the tuning of glycosylation to therapeutically relevant forms with higher delity and uniformity. This approach brings a clear focus on cost and quality measures associated with the new versus existing expression systems, safe in the knowledge that the underlying biological mechanisms for drug activity have already been established. The main challenge with this approach is that product comparability may be more difcult to justify, as there may be signicant differences with process residuals or the types and abundances of molecular variants. Atryn, recombinant human antithrombin, produced by GTC Biotherapeutics in goats milk, was the rst therapeutic protein made in a transgenic animal to be approved anywhere in the world (Europe, in 2006), and the rst 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-dependent cell-mediated cytotoxicity (ADCC). More recently, the number of players looking to bring new biologics to market has exploded, as has the number of products in late stage clinical trials, particularly in the mAb arena. Predictably

there has been intense interest in making both biosimilars of existing approved blockbusters and improving them to make biosuperiors. This is perhaps best demonstrated by Mercks purchase of GlycoFi in 2006 for US$400 million and Insmeds biosimilar product pipeline for US$130 million in 2008, to establish Merck BioVentures.19 The former was a strategic move designed to harness the power of GlycoFis glycoengineered yeast strains. This acquisition has parallels with Roches earlier purchase of GlycArt in 2005 for 235 million Swiss Francs. GlycArts technology platform enables modication of mAb glycoforms to promote enhanced ADCC, providing signicantly enhanced efcacy. These technologies are particularly well suited to the creation of secondgeneration biologics, which through their improved properties may be considered biosuperior, rather than merely biosimilar. An alternative strategy for follow-ons is to closely match the expression system used by the innovator rm but to cut costs through manufacturing efciency gains and reduced licensing and patent encumbrances. Slight improvements in formulation or composition, such as PEGylation, could also help differentiate the follow-on product. Since the biosimilars manufacturer does not have access to the innovators original cell line, a new stable cell line expressing the recombinant protein must be established. The most common selectable, ampliable markers used in CHO and NS0 cells are the DHFR20 and GS21 genes. The original patents on these systems have now expired, so it might be conceivable to

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www.soci.org incorporate basic DHFR or GS selection into processes to make biosimilars, matching the innovator host and selection system, with the hope of minimal licensing costs. However, many ensuing patents have been granted on improvements to the technology, which may limit use to a rudimentary level, possibly constraining cell line development speed and productivity, and requiring care to avoid infringement. Clearly the patent landscape is complex and must be navigated carefully. Some companies have instead chosen to adopt non-proprietary selection systems. PDL BioPharma, though not a biosimilars manufacturer, utilizes the xanthine-guanine phosphoribosyl transferase gene (gpt) from E. coli, and an NS0 cell line adapted to cholesterol independence.22 While gpt is not an ampliable marker, high specic productivities of 2060 pg per cell day1 are achievable from a single transgene copy using iterative subcloning, without the need to license the GS NS0 system. An alternative host cell line, which is subject to licensing costs, but with the potential to reduce manufacturing costs, is PER.C6.23 This immortalized line was derived from human retinal cells transfected with the Adenovirus 5 E1A gene. These cells are capable of growing to very high cell densities (160 106 cells mL1 ) using a concentrated perfusion process and have been shown to express mAbs at over 25 g L1 .24,25 Transactivation of the CMV promoter by E1A is thought to contribute to the high productivity of PER.C6 cells. The high titres and low culture volumes facilitate reduction in the nal cost per dose, and allow deployment of single use technology for further cost savings since disposable bioreactors are typically limited to 2000 L scale. As already discussed, even if host and selection system are perfectly matched, a biotherapeutic is never going to be truly generic (identical). Furthermore, the best strategy to adopt for production of follow-on biologics is still difcult to predict because it depends so fundamentally on the regulatory framework, which remains complex and not yet fully resolved.

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BIOSIMILARS: PRODUCING MORE FOR LONGER

Product differentiation in an increasingly competitive biosimilar marketplace will require renewed focus on rapid, low cost cell line development as well as predictable product quality attributes. Traditional stable cell line development involves applying selection pressure to isolate clones with random integration of the gene of interest (GOI) into the host cells genome, creating heterogeneity and expression instability. Cell lines capable of maintaining expression levels during prolonged culture is the goal, and should probably be called a stable stable cell line as suggested by Barnes et al.26 The opportunity exists to explore new technologies to increase clonal stability while minimizing clonal variation. Factors that contribute to instability include gene copy number, position effect, insertional mutagenesis, posttranscriptional and post-translational modications. Below we discuss some of the current technologies available for improved stable cell line development. The ability to specify the insertion site of the transgene cassette not only provides uniformity among the transfectant pool, but also limits gene suppression effects from anomalies such as repeat-induced gene silencing (RIGS)27 or repeat-induced DNA methylation.28 Recombinase-mediated cassette exchange (RMCE) enables site-specic recombination of expression vectors in the host genome and has been used in many different settings from the generation of transgenic Drosophila to gene knockouts in higher eukaryotes.29 The technology uses a recombinase enzyme,

such as cre or p, which coordinates a strand exchange event between short cis-acting DNA target sequences, for example loxP or the p-recognition target (FRT) sites. The cre/loxP system with the necessary loxP sites in place allows complete replacement of the expression cassette, therefore allowing both an excision as well as insertion reaction to occur at the locus.30,31 The success of these methods relies on insertion into a hot spot, or region of the genome which facilitates stable, high-level expression such as the AAVS1 safe harbour locus.32 While showing considerable promise, these methods still have technical limitations, including the challenge of incorporating the loxP or FRT sites into a genomic hot 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 be incorporated into an approved product. The ability to pre-dene the transgene insertion point has the potential to improve both expression levels and product reproducibility. Position effect variegation refers to stochastic silencing of the transgene due to involvement of the molecular niche surrounding the genomic point of insertion resulting from expansion of heterochromatin. Tandem repeat studies with Drosophila transgenes33 and similar studies in mouse models27,34 have clearly demonstrated the marked degree of transgene silencing in a heterochromatin-rich environment. However, with recent developments in our understanding of DNA insulators and locus control regions (LCR), it may be possible to constitutively express a transgene independent of the genomic insertion point. It has been hypothesized that insulator elements dene the transcriptional boundaries in chromatin and could potentially provide a barrier effect to transgenes.35 37 Such elements include the scaffold matrix attachment region (S/MARs)38 40 ; and the Ubiquitous Chromatin Opening Element (UCOE )41,42 which have been demonstrated to reduce heterogeneity and enhance stability in production cell lines. S/MAR elements are attachment points within the DNA sequence that allow for anchoring of chromatin structure to the nuclear matrix. Certain S/MARs are also recognized as regulatory elements similar to the LCRs. Such elements not only provide a region with a high afnity for transcription factors such as CCCTC binding factor (CTCF) and nuclear matrix proteins (NMP),39,43,44 but also govern the order of expression at native genomic locations. Elements such as the 5 hypersensitive site (HS) 4 of the chicken -globin gene and the LCR region of the Igf 2/H12 locus are prime examples where such a region has provided both regulatory activity and insulator function.35,43,45,46 The chicken lysozyme 5 MAR element was one of the rst elements used to construct stable cell lines with CHO and C2C12 cells38 as well as being used in a number of plant based studies.47 Using S/MAR sequences to ank trangenes, it is possible to generate a greater number of high expressing clones and increase the stability of production cell lines expressing 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 chromatinremodelling elements have the capacity to establish and maintain the chromatin in an open conguration similar to the highly active euchromatic regions in the human genome. Like the S/MAR elements, UCOE s have also been compared to LCRs and their dominant ability to control gene expression. The current UCOE s available 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 the effect of using UCOE to examine recombinant protein production

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New frontiers in cell line development in CHO cells.42 The use of both enhanced green uorescent protein (eGFP) and EPO demonstrated a 20- to 40-fold increase in the level of expression, with such expression levels maintained for greater than 100 generations.42 Even though a better understanding of these elements is needed, the production of biosimilars will not always follow a platform approach and will probably be a case-by-case scenario to develop a superior or improved process. Access to a range of new choices for cell line engineering will therefore aid biosimilar manufacturers to develop more cost-efcient processes and to also make them potentially more reliable for approval purposes.

www.soci.org BAX and BAK, in CHO cells to create a cell line with increased resistance to apoptosis, prolonging the production phase in batch culture and leading to a two- to ve-fold increase in mAb titre.54 Finally, a triple gene knockout (DHFR, GS and FUT8) using ZFNs in CHO cells has also been achieved.53 The latter example highlights the potential for ZFNs to generate multi-gene knockouts in mammalian cells lines, creating the possibility for complex rationale genetic manipulation. Systems biology and genome-scale modelling of mammalian systems has also recently become a reality.57,58 These techniques allow complex in silico modelling to identify key pathways targets for modication using technologies such as ZFNs. Previously, a major barrier to this approach in CHO cells has been the lack of publically available genome data. However, at the recent Cell Culture Engineering XII meeting (Banff, Canada, April 2010) it was announced by Bernhard Palsson that the Beijing Genomics Institute in China plans to sequence the CHO genome de novo and make the data freely available to the public. These emerging technologies should soon allow for the creation of designer mammalian cell lines as has been achieved through systems engineering of prokaryotes.

CELL LINES MADE TO MEASURE

Many less complex recombinant proteins, such as industrial enzymes, are produced using microbial fermentation processes at a fraction of the cost of mammalian cell-derived biologics. This is also seen in the bio-fuels sector, where bacteria are being used to create the next generation of fuels and other energy dense products.50 This has been achieved because systems engineering and complex genetic manipulation of bacteria has been possible for many years. This has permitted the creation of optimized host cells with a raft of genetic modications to allow greatly increased efciency and productivity for a given process. Mammalian cell line engineering has traditionally had only modest success; however, the recent advent of new molecular and omics tools should change this equation by enabling the eld of rational cell engineering to mature. Highly optimized host cell lines would provide an efcient platform for maximal low cost biosimilar production. Host cell engineering by zinc nger nucleases (ZFN) has recently shown particular promise. These are a custom array of molecular recognition modules with high DNA sequence specicity linked to half of a nuclease.51 In essence, they can be designed to recognize almost any DNA sequence. The modules function in pairs with each unit linked to half of the promiscuous nuclease, Fok1. Only when both pairs bind their target sequence in close proximity is Fok1 activity restored and a double strand DNA break created. This break is then repaired, often via non-homologous end joining, which in turn creates a genetic lesion at the cut site and potentially ablates gene function. By adding DNA into the system with overlap regions homologous to the cut site, genes may also be inserted using the homologous end joining pathway. This method therefore creates a tool for rapid high-efciency genetic engineering of complex genomes either by targeted mutagenesis or insertion. Several examples of cell line engineering strategies incorporating ZFN technology have recently been reported providing an efcient yet stringent approach for the development of tailor-made cell lines previously too laborious or difcult to generate.52 54 Santiago et al. (2008) were able to generate a biallelic disruption of the DHFR gene without selection.55 More recently, Genentech has utilized ZFN technology in conjunction with Sangamo Biosciences to create a cell line decient in 1,6fucosyltransferase8 (FUT8).52 The absence of FUT8 allows for the production of mAbs with either reduced or absent fucosylation, thereby increasing ADCC of the molecule, potentially providing more efcacious treatments with a signicant reduction in cost. These types of engineered cell lines may be ideal for development of biosuperior mAbs. A FUT8 mutant CHO cell line created using traditional mutagenesis had also been previously isolated by BioWa.56 BioWa and Lonza have now formed a partnership allowing widespread access to this technology. Genentech and Sangamo have also used ZFNs to delete the proapoptotic genes,

REGULATORY RNAS: NEW OPPORTUNITIES FOR CELL LINE ENGINEERING

Recently, our understanding of how genetic information is regulated has changed radically, primarily due to the explosion of information received on the role of regulatory RNAs in gene expression. With this new knowledge, it has almost become impossible to disregard the importance of RNA with regard to recombinant protein production. Rational genetic engineering approaches are designed to optimize systems to reach peak titres and provide homogeneous products; however, such approaches may lack the necessary knowledge to achieve these goals. Our ability to adapt cells to suspension growth in serumfree or chemically-dened conditions or the cultivation of cells under mild-hypothermic conditions demonstrates the incredible plasticity of cultured cells. Furthermore, it also highlights the fundamental limits in our understanding of complex biological systems, since we cannot fully explain the titre improvements achieved when incorporating these environmental changes into any bioproduction process. We now know regulatory RNAs play a major role in modulating gene expression, yet our ability to use these molecules as genetic engineering targets to improve biopharmaceutical production remains largely unexplored. The signicance of non-coding RNAs lies within their ability to govern translational processes via interaction with the mRNA. The rst small RNA, lin-4, was discovered in Caenorhabditis elegans through the cloning of the lin-4.59 This has since been followed by a number of signicant ndings including the identication of a novel Cricetulus griseus ortholog of the miRNA, miR-21 by Gammell et al, through a series of expression studies incorporating hypothermia.60 Koh et al. also identied miRNAs in HEK293 cells which potentially affect cell cycle regulation under bioreactor conditions.61 With the public release of the CHO genome and a further understanding of RNAbased gene regulation, regulatory RNAs will probably prove to be ideal targets for modulating recombinant protein production. While this area of cell line engineering is still in its infancy, its importance should not be understated. Once again, maintaining cost competitiveness in the biosimilars space will require improved

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www.soci.org cell line performance which will be aided by future advances in the eld of regulatory RNA engineering.

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RAPID SCREENING OF BIOSIMILAR CANDIDATES BY HIGH-LEVEL TRANSIENT GENE EXPRESSION

Stable expression technologies will continue to be used for the production of biologics at gram to kilogram quantities in mammalian cells. But the time and expense to establish, characterize and optimize such cell lines is considerable, due mainly to the time taken to expand and screen large numbers of clones. In order to maintain speed-to-market and minimize sunk costs there is a kill early and kill quickly mentality when screening drug candidates. To develop biosimilars or biosuperiors, a more rapid and efcient way to assess drug candidates and to rapidly produce product for analytical and toxicology studies is needed. Recently, there has been a move towards the routine use of optimized, large-scale, TGE systems for the production of milligram to gram quantities of recombinant protein, with such yields sufcient for the pre-clinical assessment of potential therapeutics. Several efcient protocols for TGE in HEK293 and CHO cells have recently been published.62 64 Critical to the improvements in TGE have been the implementation of carefully optimized methods of transfection which have revolved around the use of either calcium phosphate,65 68 cationic lipds (lipofection)69 71 or cationic polymers such as PEI.72 75 Due to cost effectiveness and relatively high efciency, it is the latter that has been the focus of much research, making it applicable for large-scale transfections of both HEK29362,76,77 and CHO78,79 cells. Additionally, development of novel polymers for gene delivery derived through nanotechnology may also be a source for future improvements in transfection efciencies and yields achieved with TGE.80 82 A signicant challenge for TGE is rapid plasmid copy number dilution during cell division, which results in a signicant reduction in recombinant protein titres at the end stages of the process. Approaches taken to address this issue have included the incorporation of mild-hypothermia (2734 C) post-transfection, which in most cases results in a higher specic productivity.83,84 Such improvements have been attributed to enhanced mRNA stability and an accumulation of cells in G0/G1 phases of the cell cycle, leading to slower growth rates and in turn a reduced rate of toxic metabolite accumulation.85 87 Yield improvements remain dependent on key variables including temperature, cell line and the protein being expressed.85,88 Alternatively, supplementation with histone deacetylase inhibitors (iHDAC), such as sodium butyrate and valproic acid have provided improvements in transient expression at 37 C89 and more recently under mild-hypothermia at 32 C.90 iHDACs work by up-regulating transcription through histone hyperacetylation which grants transcription machinery easier access to the genes involved in TGE. The development of episomal systems to amplify and maintain plasmid copy number after transfection has also been effective in improving TGE yields. These systems incorporate elements derived from viruses to facilitate plasmid maintenance. In humanderived cells such as HEK293, elements from the well-characterized herpesvirus, Epstein-barr virus (EBV) have been widely used. The binding of EBV nuclear antigen 1 (EBNA-1) stably expressed in the cell line HEK.EBNA or encoded by expression plasmids such as pCEP4 from Invitrogen to plasmid DNA containing

the EBV latent origin of replication (OriP) drives the replication and maintenance of this plasmid in transfected cells, providing elevated and prolonged expression of the recombinant protein of interest.62,91 93 Recent reports have described transient mAb titres in excess of 1 g L1 in 10 days using the HEK.EBNA system.94 The development of episomal systems in rodent cell lines such as CHO cells have been more challenging, mainly because rodent cells are not permissive to EBV based replication.92,95 However, the capacity for EBNA-1 to retain OriP-containing plasmids does function in rodent cells, which has led to incorporation of these elements into transient expression vectors for enhanced protein expression.87 More recently, an episomal system with the capacity to replicate and retain plasmid DNA in transfected CHO cells was created using elements from murine polyomavirus to drive plasmid replication and EBNA-1/OriP to retain and equally segregate plasmid DNA during cell division.96 This system generated much higher titres of recombinant hGH compared with replication and/or retention decient systems.96 In summary, recent advances in TGE in CHO and HEK cells now allow reliable, efcient production of signicant amounts of candidate molecules. It will be critical for biosimilar manufacturers to adopt these systems for rapid, low cost assessment of candidates during early stage development.

SINGLE-USE BIOPROCESSING SYSTEMS FOR BIOSIMILAR PRODUCTION

Biosimilar manufacturers and indeed biomanufacturers in general, are increasingly looking towards disposable or single-use systems to meet their clinical, and in some cases, manufacturing demands. Traditionally, large-scale mammalian processes have utilized the well characterized and regulatory-accepted stainless steel stirred tank bioreactor.97 100 However, recent advances in single-use systems for bioprocesses have made this technology an increasingly viable alternative.101 The facility of the future is moving away from the multiple 10 000 L or larger reactors, towards more exible, single-use reactors and warehouse manufacturing.101 103 Warehouse or modular manufacturing involves large open processing areas where equipment can be rolled in or out as required, and is highly complementary to multi-product or multi-modality facilities, in which closed processing and single-use systems combine to greatly reduce the risk of cross-contamination.102,104 An efcient low-cost biosimilars facility might involve manufacturing a range of different products using a common platform process in the same production building by leveraging single use technologies and modular manufacturing. Developments in disposable bag technologies were historically driven by the medical devices industry and blood banking. Now, along with recent advances in single-use sensors, disposables have given biopharmaceutical manufacturers a wealth of processing options.104 Single-use solutions presently exist for practically every process step used in the manufacture of biotherapeutics, from relatively simple transfer and hold steps such as buffer and media preparation, nal ll and freezing, to fully dened and scalable unit operations including bioreactors, normal and tangential ow ltration and chromatography.103,104 Eibl et al.105 provide an in-depth review of the many single-use options for cell culture now widely accepted and used at lab scale and up to 2000 L. Rocking bioreactors including the BIOSTAT Cultibag RM (Sartorius Stedim), Wave Bioreactor (GE Healthcare) and AppliFlex (Applikon) have frequently been used in both industry and academia for transient79,106 and stable107 109 processes. These systems utilize a pre-sterilized, single-use bag containing the

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New frontiers in cell line development cells and medium agitated on a rocking platform. More recently, to capitalize on widespread industry know-how with stainless steel stirred tank bioreactors, single-use systems based on the traditional STR geometry have been developed, including the Thermo Hyclone SUB, Xcellerex XDR, or Sartorius BIOSTAT Cultibag STR. Finally, systems utilizing non-instrumented round- or squareshaped bottles on orbital shakers have displayed promise as alternatives for low-cost scale-up.110,111 Intuitively, a biosimilars manufacturer might act to leverage economies of scale or mass production to drive down their cost of goods; however, the recent push to higher titres, advanced perfusion techniques and designer cell lines are allowing just the opposite with smaller reactor volumes able to meet production needs.24,25 Single-use systems are perfectly suited to this scenario, because equipment is typically limited to 2000 L, and because the cost of goods advantage is greatest at lower annual production volumes. The single-use-based facility25 also offers other distinct advantages for a biosimilars manufacturer including reduced capital cost and timeline for manufacture and installation.102,104 With the removal or elimination of cleaning/sterilization cycles, both signicant validation savings and reduced utilities requirements can also be achieved.100,101,105 Finally, one of the major advantages of single-use systems is their exibility in allowing manufacturers to respond efciently to changing market demands. Manufacturing processes can be scaled efciently through repetitions of the same process train, since additional procurement, installation, and qualication requirements are minimal.101 Conversely, if market share shrinks, or a product does not progress successfully through clinical testing, the excess equipment can be re-distributed efciently for alternative manufacturing or development priorities. Key challenges of single-use systems include the robustness and reliability of disposable sensors and probes105,112 and the effect of potential extractables and leachables on nal products.113,114 Currently, no universally accepted regulatory guidelines for extractable and leachable studies exists, and the testing requirements can be complicated and extensive. However, signicant efforts are underway from end-user organizations to standardize and accelerate the adoption of single-use manufacturing technologies and encourage their acceptance by regulatory bodies through publication of best-practice documents for testing and validation.104 Additionally, the need to complete a sound and in-depth economic analysis is imperative for any user of disposable technology.102,104 Any initial capital expenditure savings and reduced process time or increased throughput must always be balanced against the ongoing consumables cost which can tip the balance in some areas back to traditional solutions. However, it is without question that, together with the development of highly productive stable cell lines and rapid TGE technologies discussed earlier, single-use bioprocess technology is and will continue to be a valuable enabler for the biosimilars industry.

www.soci.org biosimilars represents an opportunity to address the above issues and provide broader access to cheaper products. The development of biosimilars relies heavily on the everchanging intellectual property landscape and the expiration of key patents covering both the underlying production modalities and molecular drug targets. From a regulatory perspective, both the EMEA and the FDA now have approval pathways for biosimilars, although the process and protection provided to the innovator companies is vastly different from that covering traditional smallmolecule generics. The medium- to long-term impact of this legislation remains to be seen. For the majority of products however, approval will still be dealt with on a case-by-case basis rather than any semblance of a truly abbreviated pathway as the industry mantra of the process denes the product is expected to endure. Post-approval monitoring will also be key in ensuring equivalence and safety. The development needs of biosimilars will probably require various approaches to further enhance and improve the upstream pathway in the production process. Modifying existing cell line development strategies such as traditional amplication systems, incorporating new technologies or eukaryotic hosts to enhance expression stability and product quality, and considering the use of large-scale TGE and single-use bioprocess technology are all approaches that will help address the issue of balancing speed, cost and product quality. Furthermore, the challenge for biosimilar manufacturers will not simply revolve around how to incorporate recent technological advances into product development and production, but whether they are commercially viable alternatives. Additionally, the question of how such changes will equate to a reduction in cost to the consumer will require considerable attention. Addressing these issues represents the true test for the future of biosimilars and will directly affect their market size and return on investment.

ACKNOWLEDGEMENTS
The authors would like to thank Karen Hughes for critical reading of the manuscript.

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CONCLUSION
The biologics market continues to grow faster than the broader pharmaceutical sector.2 Biopharmaceuticals offer life-saving treatment options for an ever-expanding number of disease indications such as cancer, autoimmune disorders and viral infections. Availability and affordability are major issues in developing countries, and in developed nations the high cost associated with these medications places an ever-increasing nancial burden on healthcare providers. The development of biopharmaceutical generics or

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