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Drug Discovery Today: Technologies

Editors-in-Chief Kelvin Lam Pzer, Inc., USA Henk Timmerman Vrije Universiteit, The Netherlands
DRUG DISCOVERY

TODAY

TECHNOLOGIES

Protein Therapeutics

Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects
Mattia Matasci, David L. Hacker, Lucia Baldi, Florian M. Wurm*
cole Polytechnique Fe de rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Institute of Bioengineering, Laboratory of Cellular Biotechnology, E

Recombinant therapeutic proteins produced in mammalian cells represent a major class of biopharmaceuticals. In recent years, their demand has increased dramatically and is now driving the development of a variety of improvements to maximize their expression in mammalian cells. Advances in media- and process optimization have already resulted in more than 100fold improvement in yield, but further insights and subsequent targeted modications with respect to the general biology of cells (genomics, physiology, selection and adaptation) in bioreactors are hoped to further improve protein yields and quality in the near future. Introduction
Over the past two decades recombinant proteins have gained increasing importance for therapeutic applications, and the number of proteins either approved or launched into clinical trials has continually increased over this period. Together their annual global market is now valued to be more than $50 billion with an annual sales increase of about 20%. Currently about 60% of all recombinant therapeutic proteins are produced in mammalian cells, mainly because of the ability of mammalian hosts to generate high-quality proteins that are
*Corresponding author: F.M. Wurm (orian.wurm@ep.ch) 1740-6749/$ 2009 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2008.12.003

Section Editors: Marco van de Weert, Eva Horn Moller

similar in their biochemical properties to the naturally occurring human forms. This growing demand for high-quality recombinant therapeutics is driving the research and development of mammalian-cell-based manufacturing systems for enhanced production yields. In this review we will present relevant strategies developed to enhance the generation of high producer cell lines and discuss how new technology may contribute to the further development of cellular processes for the production of therapeutic proteins.

Current strategy for recombinant cell line development

The generation of recombinant cell lines follows a welldened multistep scheme that begins with the molecular cloning of the gene of interest (GOI) in a mammalian expression vector (Fig. 1). The GOI is then delivered into cells along with a selection gene which may be cloned in the same or different expression vector. Following DNA transfer, cells are subjected to selective conditions to recover those that have stably integrated the exogenous genes into a chromosome. Well-established selection strategies rely upon complementation of a host auxotrophy. In Chinese hamster ovary (CHO) cells, the major mammalian host for protein production, the two most commonly used selection systems are based on the
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Figure 1. Schematic representation of the development of stable cell lines for the production of therapeutic recombinant proteins. Steps that can be improved by the use of new technologies are shown on the right. -Omic technologies can contribute to several aspects of the process including engineering of the parental cell line, identication of markers for screening, development of the manufacturing process and media design. Vials represent banks of cells stored in liquid nitrogen. Spinner asks indicate scale-down systems for cell line evaluation and process optimization, and bioreactors represent large-scale production

dihydrofolate reductase (dhfr) and the glutamine synthetase (gs) genes. Selection is achieved by the introduction into cells of the GOI along with a copy of the gene that complements the auxotrophy. Cells are then cultivated in medium lacking the appropriate metabolite(s) (hypoxanthine and thymidine in the case of DHFR selection and glutamine in the case of GS selection) so that only transformed clones survive. A common alternative to the auxotrophic selection method is the use of genes conferring resistance to antibiotics such as geneticin (G418), hygromycin B, zeocin, blasticidin or puromycin. With this strategy, transfected cells are selected using medium containing the appropriate antibiotic. However, a major advantage of both the DHFR and GS selection systems is that they allow for amplication of the integrated recombinant genes. DNA amplication, which normally results in enhanced productivity, is achieved by exposing selected cells to increasing concentrations of an inhibitor of the selection protein. Methotrexate (MTX) and methionine sulphoximide (MSX) are inhibitors of DHFR and GS, respectively. To survive, cells must produce a high level of the selection protein. This may be achieved by gene amplications of the integrated selection gene. This also results in an increased copy number of the GOI because it is located at the same integration site as the selection gene. The pool of cells recovered after selection is highly heterogeneous in terms of specic protein productivity and cell growth. This necessitates the isolation and evaluation of several hundreds of individual cells to recover a few candidate production cell lines that possess the desired characteristics. This is usually accomplished by one or more rounds of limiting dilution in which the selected cells are transferred to multiwell plates so that on average only one cell is present per well. Each clonal cell population derived from this procedure is evaluated for the level of recombinant protein expression and the highest producers are further studied for the stability of recombinant protein production because a decrease in transgene expression over time is commonly observed in the majority of clonal cell lines. Thus candidate production lines must be cultivated and monitored for several months to ensure a constant level of protein expression over time [1]. Whereas the described procedure for the generation of recombinant cell lines is a well-established process, it remains quite tedious and time-consuming. In an industrial setting, the whole process usually takes more than six months. This is particularly inconvenient especially when multiple candidate therapeutics need to be produced in high amounts to be evaluated for efcacy and safety in preclinical studies or clinical trials.

processes. Abbreviations: methotrexate (MTX), methionine sulphoximide (MSX).

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Optimization of mammalian expression vectors

The choice of the appropriate expression vector is a crucial factor to achieve very high and stable expression of recombinant proteins in mammalian cells, and nonviral expression vectors are usually used for the generation of recombinant cell lines. Strong promoter/enhancer sequences of viral or cellular origin are typically used to drive expression of the GOI, which is typically cloned as a cDNA-lacking intron sequences. However, because the splicing process is known to promote mRNA nuclear export and stability [2], most expression plasmids include an intron between the promoter and the 50 -end of the cloned cDNA. Strong transcription termination sequences added near the 30 -end of the cDNA ensure proper transcript termination. Additional optimization of the transgene coding sequence by changing underrepresented codons, removing cryptic splice sites and increasing the overall G + C content may help to improve levels of recombinant protein expression [3]. The integration site in the host genome has a major effect on both the level of transgene expression and the progressive decrease in transcription over time. These two phenomena, known as position effect and gene silencing, are thought to be associated with epigenetic factors resulting in the condensation and transcriptional inactivation of the chromatin at the site of integration. Flanking of transgenes with DNA boundary sequences able to block the formation of condensed chromatin (heterochromatin) may help to obtain stable transgene expression irrespective of the chromosomal integration site. These barrier elements include scaffold or matrix attachment regions (S/MARs), insulators, antirepressor elements and ubiquitous chromatin opening elements (UCOEs) [4,5].

are retained in a stirred-tank bioreactor while the culture medium is exchanged several times each day by the addition of many bioreactor volumes of fresh medium with the simultaneous removal of the same volume of spent medium from the vessel. The possibility to maintain the perfused cultures for several weeks or months at high cell densities is a major advantage of this technology. Additionally the continuous medium exchange allows the simultaneous recombinant product harvest and the removal of toxic by-products. Processes for the production of recombinant proteins from cells attached to a surface rather than in suspension are also used in industry [1,7,8]. However, adherent cell culture processes are more demanding at large scales owing to the high surface to volume ratio needed to maximize cell densities. With roller bottles cells are grown attached to the inner wall of the vessels lled with medium to 1030% of their nominal volume while a slow rotation assures the regular wetting and proper oxygenation of the cells. To increase the cell growth surface area, adherent cells can also be grown attached to polymer spheres (microcarriers). Microcarriers seeded with cells are then maintained in suspension in conventional stirred-tank bioreactors.

Future prospects for mammalian cell processes Genomics and proteomics

Genome-scale technologies including genomics, transcriptomics and proteomics are expected to contribute to the development of mammalian cell-based production systems [9,10]. Although high-throughput genomic and proteomic tools are readily available for human- and mouse-derived production hosts (such as NSO or HEK-293), the lack of genome sequence information for the major host (CHO cells) has hindered the application of these technologies in the recombinant protein production eld. Early transcriptome studies of CHO cells using DNA microarrays containing mouse sequences have provided limited results because of a relevant divergence between the genetic sequences of these two rodent species. In 2006 the Consortium on CHO cell genomics was founded with the aim of accelerating the development of genomic resources for this host (http:// hugroup.cems.umn.edu/CHO/cho_index.html). To date this cooperation between partners from industry and academia has made possible the sequencing of more than 80,000 expressed sequence tags (ESTs) of CHO origin. This EST library that includes more than 27,000 unique sequences have been used to create tools for the transcriptome analysis of CHO cells [11]. With further development these tools may aid in understanding the overall cellular physiology of this host and its derived recombinant cell lines, eventually allowing the identication of features predictive of the level and stability of transgene expression at production scale [12]. Such predictive markers will be very useful in the early identication of high
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Cell culture format and media

The pharmaceutical manufacture of recombinant proteins most frequently employs single-cell suspension cultures in stirred-tank bioreactors of variable sizes up to 20,000 l [1,6,7]. The cells are typically maintained in media that are optimized for suspension growth at high cell density preferentially in the absence of serum and other animal-derived components. Cells may be cultivated during the entire protein production phase without the addition of nutrient additives (batch culture). Alternatively, nutriments may be periodically added to the culture to prolong cell viability and protein production (extended- or fed-batch culture). In some cases, reduction of the temperature to 30338C, increased osmolarity, or addition of histone deacetylase inhibitors such as sodium butyrate or valproic acid enhance protein productivity in both batch and fed-batch processes. Continuous perfusion is another type of process with suspension cultures, useful for certain products that have a high sensitivity toward degradative impacts from cell released proteases or other physico-chemical inuences. Perfused cells

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producing clonal cell lines. For example, several genes related to cell growth, chromatin modication and protein processing and secretion have been shown to be differentially expressed in cell lines producing high levels of recombinant proteins [13,14]. Other -omic studies have compared host cell responses associated with changes in culture conditions that enhance productivity such as hypothermia, hyperosmosis and exposure to butyric acid. Results from these and similar studies may help in predicting how a cell line will respond to changes in environmental conditions relevant to the bioreactor and thus be valuable for the optimization of process conditions and media design [10,15]. In general it is expected that -omic approaches will provide new insights into the general physiology of cells that are crucial for protein production in mammalian cells thus enhancing cell engineering efforts to genetically redirect the cells metabolism toward superior productivity and growth.

reduced accumulation of ammonium ions has been achieved by the overexpression of the urea cycle enzymes carbamoyl phosphate synthase I and ornithine transcarboxylase [23]. Variations in the glycan content of glycoproteins can significantly affect the stability, activity, immunogenicity and pharmacokinetics of recombinant therapeutic proteins. In this regard, engineering of the glycosylation pathways in mammalian cells with the nal aim to generate proteins with a more humanized glycosylation prole and/or to enhance the biological activity of therapeutic proteins has met with some success [24].

High-throughput recombinant cell line selection methods

The traditional gene transfer, selection and amplication methods used for the generation of stable cell lines are not readily scalable to allow screening of more than a few hundred cell lines. Therefore, current research focuses on the development of high-throughput technologies with increased screening capacities [25,26]. Particularly promising are methods based on ow cytometry and cell sorting technologies. One elegant screening strategy employs the cotransfection of cells with the GOI and a gene coding for the green uorescent protein (GFP). If the two genes are genetically linked, a linear correlation between uorescence intensity and expression of the GOI is usually seen. Single clones with high level of GFP expression are then recovered by uorescence-activated cell sorting (FACS) [27]. Coexpression of a nonuorescent cell surface protein that can be labeled with a uorescent antibody before screening by FACS is an alternative strategy [28]. Strategies to directly measure the level of the recombinant protein rather than a reporter protein are expected to be even more effective in the screening of high producing cell lines. However, because most of the recombinant proteins produced in mammalian cells are secreted, techniques to keep the recombinant protein in the vicinity of the producing cell are required. This can be accomplished by embedding individual recombinant cells in a gel or semisolid matrix to limit the diffusion of the secreted product and allow its detection using standard immunohistochemical methods [29]. Automation of these new screening methods will denitely reduce the time needed to generate recombinant cell lines and increase the probability of recovering high producing clones.

Host cell engineering

Many efforts have been made to genetically modify mammalian cells for the purpose of improving recombinant protein yield and quality. Host cell engineering strategies have mainly focused on (i) control of cell proliferation; (ii) enhancement of cellular viability; (iii) reduction of inhibitory metabolic by-products (i.e. lactate and ammonia); (iv) increase of protein secretion capacity; and (v) modulation of post-translational protein modications (i.e. glycosylation) [16,17]. Strategies to genetically modify mammalian cells mainly rely on the overexpression of exogenous effector genes and the downregulation or elimination of endogenous genes. Downregulation of host gene expression is usually achieved by RNA interference (RNAi) or antisense RNA technology. One of the main targets of host engineering has been the apoptotic pathway. Overexpression of antiapoptotic proteins such as Bcl-2 or Bcl-xL or the targeted inhibition of caspases, a family of apoptosis-promoting proteins, has proved to be effective in maintaining cell viability at high density [18]. Enhancement of the cellular capacity for protein secretion through engineering of the protein folding machinery in the endoplasmic reticulum has also been explored. However, the overexpression of single chaperones or chaperon-like proteins such as binding immunoglobulin protein (BiP) or protein disulde isomerase (PDI) has yielded mixed results [19]. Recently the overexpression of the X-box binding protein-1 (Xbp-1), a transcription factor which regulates the expression of multiple genes involved in the unfolded protein response (UPR), has been shown to enhance recombinant protein production in CHO cells [20]. Strategies to reduce the amount of lactate production during cultivation of CHO cells include the knockdown of lactate dehydrogenase subunit A (LDH-A) mRNA by antisense and RNAi approaches or the overexpression of the pyruvate carboxylase enzyme [21,22]. Similarly,
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Transient gene expression

Transient gene expression (TGE) is a relatively new technology that is being considered for the production of recombinant proteins at large scale [30]. A key advantage of this technology is the speed by which signicant amounts (grams) of recombinant protein can be produced. Thus large-scale TGE has attracted interest for the rapid production of recombinant proteins for basic research and preclinical studies. Usually, the protein production phase for TGE lasts up to

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14 days following transfection, avoiding the time-consuming process of generating stable clonal cell lines. The recombinant gene is usually cloned in a nonviral expression vector and transfected into single-cell suspension cultures by means of a chemical delivery agent such as polyethylenimine (PEI). To date, the largest volumetric scale for TGE has been about 100 l [31]. TGE has been performed in stirred-tank bioreactors, orbitally shaken containers and wave-type bioreactors. Whereas several host cell lines have been used for TGE, CHO and HEK-293 cells are preferred owing to their high transfectability and the ability of these cells to support high-density growth in suspension [32,33]. Recently, volumetric yields of recombinant protein comparable to those obtained for optimized processes with stable lines have been achieved, underlining the potential of TGE as an economic alternative for the manufacture of low-dose therapeutic proteins at moderate volumetric scales [34].

genetic engineering of the host or by a more rationale optimization of the culture conditions.

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Conclusions
Despite recent progress, recombinant protein expression technology using mammalian cells is still in its infancy. Much remains to be done to enhance the productivity of production processes. In highly optimized fed-batch manufacturing processes yields of several grams of per liter of secreted protein are frequently attained today for antibody products and some other well-behaved proteins, a 10100fold increase compared to the yields that were obtained two decades ago. This increase in volumetric productivity has been primarily achieved through optimization of the manufacturing process by improvements in media composition, nutrient feeding strategies, cell line development and cell screening strategies. However, even with these impressive improvements in yield, opportunities still exist to further enhance production levels. Yields of 1020 g/l in fed-batch processes are expected to be reached in the near future. This may be primarily achieved by an increase in viable cell densities and duration of the culture process through optimization of the culture conditions. The cell concentrations for batch and extended-batch processes typically peak at about 10 million cells/ml, meaning the cell biomass only occupies about 34% of the volume of the culture. By contrast microbial cultures may reach a biomass of 2030% of the culture volume. The current strategies for the development of cell lines for the recombinant expression of therapeutic proteins remain empirical to a large extent. This is mainly due the lack of consistent methods to predict production capacity and growth characteristics of clonal cell lines at production scale. In this regard the knowledge gained from -omic studies is expected to provide a much better understanding of the biochemistry and physiology of mammalian cells so that higher product yields and quality may be achieved through

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Muller, N. et al. (2007) Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems. Biotechnol. Lett. 29, 703711 Derouazi, M. et al. (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol. Bioeng. 87, 537545 Meissner, P. et al. (2001) Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol. Bioeng. 75, 197203 Backliwal, G. et al. (2008) Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res. 36, e96

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