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Harbor Press, Cold Spring Harbor, N. Y., 1997, Chapter 95, pp. 95.195.15. 160. E.M. Lockyer, Photonics Spectra 31: 8090 (1997). 161. B. Moomaw, Biophotonics 5: 4853 (1998). 162. T.F. Lynch, Am. Lab. 26: 2632 (1994). 163. L. Marton, Nature (London) 133: 911 (1934). 164. F.W. Doane, G.T. Simon, and J.H.L. Watson, Canadian Contributions to Microscopy, Micros. Soc. Can., Toronto, 1993. 165. M.A. Hayat, Principles and Techniques of Electron Microscopy, Multi vol., Van Nostrand-Reinhold, New York, 19701976. 166. A.M. Glauert, Practical Methods in Electron Microscopy, Multi Vol. Ser., North-Holland/Elsevier, New York, 19721991. 167. M.J. Dykstra, Biological Electron Microscopy: Theory, Techniques and Troubleshooting, Plenum, New York, 1992. 168. J. Bozzola and L. Russell, Electron Microscopy. Principles and Techniques for Biologists, 2nd ed., Jones & Bartlett, Boston, 1998. 169. A.B. Maunsbach and B.A. Afzelius, Biomedical Electron Microscopy, Academic Press, San Diego, Calif., 1998. 170. R.F. Egerton, Electron Energy-Loss Spectroscopy in the Electron Microscope, 2nd ed., Plenum, New York, 1996. 171. M.A. Hayat, Introduction to Biological Scanning Electron Microscopy, University Park Press, Baltimore, Md., 1978. 172. G.H. Haggis, Microsc. Res. Tech. 22: 151159 (1992). 173. R.L. Steere, J. Biophys. Biochem. Cytol. 3: 4560 (1957). 174. H. Moor, Z. Zelforsch. Mikrosk. Anat. 62: 546580 (1964). 175. C. Stolinski and A.S. Breathnach, Freeze-fracture Replication of Biological Tissues: Techniques, Interpretations and Applications, Academic Press, New York, 1975.

of biopharmaceuticals includes vaccines, monoclonal antibodies, hormones, growth factors, cytokines, enzymes, and blood factors. Worldwide annual sales of recombinant therapeutic proteins are worth more than $50 billion with an annual increase of about 20% (1). These statistics reect the fact that recombinant proteins are increasingly dominating the product pipelines in the biopharmaceutical industry. Of the approximately 140 recombinant therapeutic proteins currently approved in the United States and the EU, about half are produced in cultivated mammalian cells while most of the others are produced in microbial hosts (Fig. 1) (2). Among the various mammalian cells used for production, Chinese hamster ovary (CHO) cells are clearly dominant (Fig. 1). In 2006, the top 10 recombinant therapeutic proteins in sales included 7 produced in CHO cells, generating total sales of $24 billion (Table 1). Based on estimates of candidate molecules currently in product pipelines (3), it is anticipated that CHO-derived therapeutic proteins will become increasingly important in the next decade. The major reason for choosing cultivated mammalian cells rather than a microbial host for production is protein complexity (4,5). Antibodies, for example, are glycosylated protein assemblies composed of four individual polypeptides (two light and two heavy immunoglobulin chains) covalently linked by disulde bonds. Their correct protein folding, assembly, and glycosylation can only be achieved efciently in mammalian cells (6). The biological activity and pharmacokinetic properties of therapeutic proteins are dependent on correct processing and assembly. It is also advantageous that recombinant proteins produced in mammalian cells are typically secreted into the cell culture medium, simplifying protein recovery and purication (7,8). The mammalian cell lines used for the production of recombinant proteins are immortalized and can therefore grow continuously in culture. Using immortalized cells for the production of recombinant proteins was not viewed favorably by drug regulatory agencies in the early 1980s

CHINESE HAMSTER OVARY CELLS, RECOMBINANT PROTEIN PRODUCTION

DAVID L. HACKER , CHENUET , and SEBASTIEN FLORIAN M. WURM
Institute of Bioengineering, Laboratory of Cellular Biotechnology, Ecole Polytechnique F ed eral de Lausanne (EPFL), Lausanne, Switzerland

Yeast

16%

Bacteria 30% 33%

CHO cells

4% Other hosts

17% Other mammalian cells

INTRODUCTION Recombinant therapeutic proteins are produced in living cells through the application of recombinant DNA technology and are used to treat or cure disease. This class

Figure 1. Distribution of approved and marketed (in United States and EU) recombinant therapeutic proteins according to host production system. The total number of therapeutic proteins was 140 (compiled from Ref. 2).

CHINESE HAMSTER OVARY CELLS, RECOMBINANT PROTEIN PRODUCTION Table 1. Product Enbrel Top 10 Recombinant Therapeutic Proteins in US Market Sales for 2006 Molecule Tumor necrosis factor (TNF) receptor-IgG fragment crystallizable (Fc) fusion Darbepoetin Anti-CD20 Monoclonal Antibody (MAb) Anti-TNF MAb Epoetin Anti-Her2 receptor MAb Epoetin Pegylated granulocyte-colony stimulating factor (G-CSF) Human insulin Anti-vascular endothelial growth factor (VEGF) MAb Company Amgen, Wyeth, and Takeda Sales ($ in millions) 4.4 Host CHO

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Aranesp Rituxan Remicade Procrit/Eprex Herceptin Epogen Neulasta Human insulin Avastin Total ($ millions)

Amgen Biogen Idec, Genentech, and Roche Johnson & Johnson, Schering-Plough Johnson & Johnson Genentech and Roche Amgen and Kirin Amgen NovaNordisk Genentech and Roche

4.1 3.9 3.6 3.2 3.1 2.9 2.7 2.5 2.4 32.8

CHO CHO Sp2/0 CHO CHO CHO Escherichia coli Saccharomyces cerevisiae CHO

(9). Discussions about the risks associated with these cells were controversial and had been initiated more than two decades earlier when a rst generation of classical biological products (i.e. vaccines and the natural interferons) were recovered from cultivated mammalian cells. Manufacturers of recombinant proteins and regulatory agencies were in agreement that it was extremely important to minimize eventual risks associated with the use of mammalian production hosts. Risks were seen in tumor principles carried by the DNA of the host and in adventitious agents (viruses, mycoplasma, etc.) that could infect the host cell lines and eventually be transmitted to patients receiving products from those hosts (10). The result of a long series of scientic discussions spanning a decade was that stringent controls, regulations, and monitoring procedures were enforced as a prerequisite for the manufacture of proteins from mammalian cells. For the rst product, recombinant tissue plasminogen activator (rtPA or Activase), approval in 1986 was achieved only after a large body of data was provided to the regulatory agencies that showed (i) that only a minute quantity of CHO DNA (less than 10 pg/dose) was present in the nal product, (ii) that the protein could be produced with a high degree of reproducibility, and (iii) that it was produced with a purity not achieved for any prior biological product (11). After 20 years of recombinant therapeutic protein manufacturing in mammalian cells, no safety problems have arisen. ORIGIN AND CHARACTERIZATION OF CHO CELLS CHO cells were chosen as the production host for the rst therapeutic recombinant protein mostly because of the availability of an auxotrophic CHO strain lacking dihydrofolate reductase (DHFR) activity that provided a convenient genetic selection method for recovering recombinant CHO cell lines (1215) (section titled Selection of Recombinant Cell Lines). Other protein manufacturers subsequently chose this cell line because the approval barriers for a second product from the same host were

considerably easier to overcome. Although CHO cells are currently the preferred mammalian host for recombinant protein production, other cell lines including baby hamster kidney (BHK-21), human embryo kidney (HEK-293), and mouse myeloma (NS0, Sp2/0) have gained approval for this purpose. CHO cells have several properties besides the DHFR selection method that contribute to their attractiveness as a production host. First, they are easy to cultivate and are capable of growing at high cell densities (> 1 107 cells/mL) in single-cell suspension culture even at volumetric scales up to 20,000 L. Second, they are easily transfected with plasmid DNA using both chemical and physical methods. Third, the risk of involuntary contamination of the recombinant product with an adventitious viral agent is low since CHO cells do not produce infectious endogenous retroviruses, and they are not a permissive host for most pathogenic human viruses (16,17). Fourth, they are highly productive so that under optimized cell culture conditions, recombinant CHO cell lines have achieved specic productivities up to 50 pg/cell/day for secreted proteins (4,5). Fifth, they yield protein glycoforms that are similar to those produced in human cells (18). The original CHO cell line was isolated in 1957 following a spontaneous immortalization event in a culture of primary cells from the ovary excised from a Chinese hamster (Cricetulus griseus) (19). A derivative of the original cell line, the glycine-dependent strain CHO-K1, was mutagenized with ethane methyl sulfonate and then with -radiation to generate the strain CHO-DXB11 (also referred to as CHO-DUKX or CHO-DUK-XB11), lacking DHFR activity (20). This strain has one deleted dhfr allele and a missense mutation in the second allele. Subsequently, the CHO-pro3-strain, another derivative of the original CHO cell line, was treated with two rounds of -radiation to yield CHO-DG44, a strain with deletions of both dhfr alleles (21). These two DHFR-minus strains require glycine, hypoxanthine, and thymidine (GHT) for growth. Although not initially intended for the purpose of

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recombinant protein production, these cells were used for a number of pioneering experiments in which they were stably transfected with an exogenous dhfr gene and then selected in medium lacking GHT (1215). This genetic selection scheme remains the standard method to establish stably transfected CHO cell lines for the production of recombinant proteins (section titled Selection of Recombinant Cell Lines).

RECOMBINANT CELL LINE GENERATION Introduction The generation of recombinant cell lines is a multistep process that begins with the molecular cloning of the gene of interest in a mammalian expression vector. The gene of interest and the selection gene, also cloned in an expression vector, are then delivered into cells by transfection, and the cells are grown under selective conditions to recover those that have the exogenous genes integrated into the genome. The cells that survive selection eventually form cell colonies that may be individually transferred into a new cultivation vessel. These individual cell lines are analyzed for recombinant protein yield and cell growth rate. The most productive cell lines then undergo one or more rounds of cellular cloning to allow recovery of a clonal recombinant cell line. Each of these steps is discussed in more detail in this section. Mammalian Expression Vectors The recombinant gene of interest is usually cloned in a nonviral expression vector (i.e. plasmid) for subsequent transfer into cells. The major advantages for nonviral vectors over viral vectors include ease of construction, ease of delivery into cells with inexpensive chemical agents, and biosafety. The recombinant gene is typically transcribed from a strong promoter such as the immediate early promoters of human cytomegalovirus (hCMV) and mouse cytomegalovirus (mCMV), the SV40 virus early promoter, the Rous sarcoma virus long-terminal repeat promoter, and the promoters of constitutively expressed housekeeping genes like the human elongation factor-1 alpha (EF-1 ) and the chicken -actin genes (22). The selection gene(s) may be cloned in the same vector as the gene(s) of interest or in a separate vector (23). Both the gene of interest and the selection gene are almost always cloned as a cDNA rather than as a full-length gene due to the size constraints imposed on plasmids. Since splicing of mRNA and its subsequent transport to the cytoplasm are functionally linked, mRNAs transcribed from cDNAs are not efciently transported to the cytoplasm since they do not contain introns (24). For this reason, most expression vectors include an intron sequence between the promoter and the 5 -end of the cloned cDNA (22). Finally, 3 -end processing and polyadenylation of the recombinant mRNA require the appropriate regulatory elements near the 3 end of the cDNA (22). Recombinant gene expression is regulated not only at the level of transcription and mRNA processing but also by factors such as mRNA stability and translation efciency that are controlled in large part by

RNA sequence and structure (2527). To enhance levels of recombinant mRNA and protein, the coding sequence may be altered by removing cryptic splice sites, altering codons having underrepresented cognate tRNAs, and increasing the G + C percentage at the third (wobble) position of codons (28,29). Recombinant protein production from stable recombinant cell lines may diminish with time during cell cultivation due to a decrease in transcription of the recombinant gene, a phenomenon termed gene silencing. A major determinant in gene silencing is the structure of the chromatin at the site of integration of the recombinant gene. In general, heterochromatin is condensed and transcriptionally inactive, whereas euchromatin is relaxed and transcriptionally active (30). The two chromatin states are associated with specic histone modications including acetylation, methylation, and phosphorylation that function to control chromatin condensation and transcriptional activity (31). DNA elements like scaffold or matrix attachment regions (S/MARs), insulators, antirepressor elements, and ubiquitous chromatin opening elements (UCOEs) have been shown to ameliorate the effects of gene silencing in recombinant cell lines (3236). These DNA elements are small enough to allow cloning in expression vectors. Nonviral Gene Delivery Nonviral vectors are delivered into cells by chemical or physical methods. The three main chemical reagents are calcium phosphate (CaPi) (37,38), cationic polymers especially the polyamine polyethylenimine (PEI) (39), and cationic liposomes (40). All of these reagents form complexes with negatively charged DNA. The positively charged complexes readily bind to the negatively charged cell surface. Thereafter, all the complexes face the same physical barriers to delivery of DNA into the nucleus (41). The complexes are endocytosed, but only a fraction of the DNA eventually escapes the endosome (42). Within the cytoplasm, the DNA is transported to the nuclear membrane by unknown mechanism(s). Clearly, plasmid DNA does not readily diffuse within the cell (43), and within the cytoplasm, the half-life of plasmid DNA is less than 90 min because it is a substrate for cytoplasmic DNases (44). The plasmid DNA eventually enters the nucleus either through nuclear pores or during mitosis after the breakdown of the nuclear membrane (4547). Physical methods of DNA delivery include electroporation and microinjection. For the former, a brief electrical charge is applied to suspension cultures of cells in the presence of plasmid DNA (48). Under optimal conditions, nearly 100% of the cells receive DNA. Microinjection has been used to directly deliver plasmid DNA into the nucleus of cells including CHO for the generation of recombinant cell lines (49,50). This is the only DNA delivery method that allows operator control of the quantity and quality of DNA reaching the nucleus of the target cell. The disadvantage of the method, however, is the time required to microinject a relatively small number of cells. For the DNA delivery methods described earlier, usually less than 5% of the cells that receive DNA are eventually recovered as recombinant cell lines (4). The reason(s) for the low rate of cell line recovery is not known,

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but the frequency of integration of plasmid DNA into the cells genome may be a major factor. Digestion of plasmid DNA by cellular DNases decreases the percentage of plasmids having an intact recombinant or selection gene (51) On the other hand, for more efcient generation of recombinant cell lines, it is advantageous to linearize the plasmid DNA with a restriction endonuclease outside the recombinant and selection genes prior to DNA delivery (38). Selection of Recombinant Cell Lines Following DNA transfer, the cells with an integrated recombinant gene must be recovered from the general population of transfected cells. The standard method of selection with CHO cells is via cotransfer of the dhfr gene along with the recombinant gene of interest into a DHFR-decient strain such as DG44 (1215). After DNA transfer, the cells are cultured in the absence of GHT. Only cells with one or more stably integrated copies of a transcriptionally active dhfr gene survive to form colonies on the surface of the culture dish. Each colony is a clonal population of recombinant cells, most of which will have one or more integrated copies of the gene of interest. There are no apparent preferred sites of plasmid DNA integration, and the integrated plasmid copy number varies widely from one cell line to another (23). Nevertheless, there is usually only one integration site per cell even if multiple plasmids are transfected (23). From each transfection, numerous recombinant cell lines are recovered but there is usually extensive heterogeneity among them in terms of protein productivity and growth rate. To obtain a few cell lines with the desired characteristics, it is usually necessary to evaluate several hundred individual cell lines. Once the best candidates are identied, one or more rounds of limiting dilution are preformed to attempt to produce a clonal cell line. To accomplish this, the cells originating from a single colony are diluted and then transferred to a multiwell plate so that, on average, only one cell is present per well (52). Once clonal cell lines are established, they are characterized for the stability of recombinant protein production since expression of the integrated gene is not necessarily maintained at a constant level over time. Instead, gene silencing in the entire clonal cell population or in a subpopulation is frequently observed. Thus, the candidate cell lines must be cultivated for several months to ensure stable protein production over time. The dhfr gene is not the only selection marker available for generating recombinant CHO cell lines. The glutamine synthetase (gs) gene initially considered for the selection of NS0-derived cell lines since their endogenous GS (glutamine synthetase) activity is low, is also used to recover recombinant CHO cells even though they have a higher endogenous GS activity than NS0 cells (53,54). After transfection with the gene of interest and the GS gene, recombinant cells are selected in medium without glutamine. Resistance genes for the antibiotics geneticin (G418), hygromycin B, zeocin, blasticidin, and puromycin and the ampliable metallothionein gene represent other classes of selection markers (55). Following DNA transfer, recombinant cells are recovered in medium containing the appropriate antibiotic.

With both DHFR and GS selection, expression of the recombinant gene can be signicantly increased by exposing the cells to a drug that blocks the enzymatic activity of the selection marker. DHFR and GS are inhibited by methotrexate (MTX) and methionine sulphoximine (MSX), respectively (53,54,5658). For CHO-derived cell lines that express the exogenous dhfr gene, a majority of the cells die after 23 weeks of exposure to increasing concentrations of MTX. The rare survivors have a higher integrated plasmid copy number than the original cell line as the result of amplication of the dhfr gene and the surrounding DNA, including the gene of interest (5658). CHO cells lines containing several hundred copies of integrated plasmid DNA have been established this way (59,60). The higher gene copy number accounts for the increased level of recombinant protein productivity observed in most amplied cell lines. Similar observations have been made following the exposure of recombinant NS0 cell lines to MSX (54). The selection and cloning of recombinant cell lines may be performed in suspension culture. This practice avoids exposure of the recombinant cells to conditions of adherent growth in the presence of animal-derived serum. After exposure to the selective conditions, the survivors are transferred to multiwell plates by limiting dilution. The resulting cell lines are screened for recombinant protein productivity and cell growth, and those with the desired characteristics may be subjected to one or more additional rounds of limiting dilution to increase the likelihood of recovering a clonal cell line. By eliminating steps requiring adherent cultivation in serum, there is no need to adapt the recombinant cell lines to suspension cultivation in serum-free medium since this may result in the loss of recombinant protein productivity.

CELL CULTIVATION SYSTEMS Most recombinant protein production processes for clinical manufacture utilize single-cell suspension cultivation in stirred-tank bioreactors of various sizes up to 20,000 L. The cells are maintained in media that are designed for suspension growth to a high cell density, preferably in the absence of serum or other animal-derived components. The cells may be cultivated during the entire protein production run without nutrient additives (batch culture) or medium components may be periodically added to the culture to prolong cell viability and protein production (extended- or fed-batch culture) (4,5). The latter strategy is used for most high-yielding bioprocesses in the biotechnology industry today. Enhancement of protein productivity in batch and fed-batch processes has been demonstrated for some recombinant CHO cell lines by temperature reduction to 3033 C, increased osmolarity, or addition of sodium butyrate, a histone deacetylase inhibitor (6165). To gain the greatest benet, these treatments are normally performed near the end of the exponential growth phase of the culture since they typically reduce or inhibit cell division. The other major type of production process with suspension cultures is continuous perfusion. In this case, the cells are maintained in a stirred-tank bioreactor, and several bioreactor volumes of fresh medium are fed into the

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culture each day while the same volume is withdrawn from the vessel. Perfused cultures normally achieve higher cell densities than batch or extended-batch cultures, and the cells can remain viable for several weeks, with product harvests occurring repeatedly throughout the cultivation period (66,67). The antihemophilic factor VIII (Kogenate) is manufactured from recombinant (BHK-21) cells with this mode. Factor VIII is the largest therapeutic recombinant protein (2332 amino acids) on the market, and its size and sensitivity to proteases contributed to the decision to produce it in a perfused system rather than in a batch or fed-batch process (68,69). Recombinant proteins are also produced from cells attached to a surface rather than in suspension. For example, Epogen is synthesized from a recombinant CHO cell line grown in roller bottles lled with medium to 10%30% of their capacity (70,71). The cells attach to the surface of the bottle whose slow rotation assures the regular wetting of the cells. Cells can also be grown attached to polymeric spheres (microcarriers) of dextran, polyacrylamide, or polystyrene (72,73). Each bead is seeded with cells, and the beads are maintained in suspension in stirred-tank bioreactors. Eventually, after several rounds of cell division, each bead may hold several hundred cells. The disadvantage of this method is that volumetric scale-up requires the removal of cells from the beads followed by reseeding on a greater quantity of beads in a larger volume of medium.

Host Cell Engineering Many efforts have been made to genetically modify CHO cells for the purpose of improving recombinant protein yield and quality (2123). The main targets for host engineering have been the pathways for apoptosis, lactate production, glycosylation, and protein secretion. The two main strategies for genetic modication are (i) the overexpression of exogenous effector genes and (ii) the reduction or elimination of endogenous gene expression (8082). The latter has been achieved by mutagenesis of one or both alleles of an endogenous gene or by decreasing mRNA levels using RNA interference (RNAi) or antisense RNA. In general, these strategies have met with some success, and it is possible that engineered CHO cell lines have now found their way into production processes for industrial manufacturing. One of the main targets of host engineering has been the apoptotic pathway. Antiapoptotic proteins including Bcl-2, Bcl-xL , and others have been stably overexpressed in CHO cells to enhance cell viability (81,8386). In addition, caspase 3 mRNA has been targeted by antisense RNA to reduce the level of this important apoptotic protein (87). Overexpression of the transcription factor X-box binding protein-1 (Xbp-1) has been shown to enhance recombinant protein production in CHO cells through the regulation of genes involved in the unfolded protein response (88,89). To reduce the amount of lactate production during the cultivation of CHO cells, the mRNA of lactate dehydrogenase subunit A (LDH-A) has been targeted by antisense RNA and RNAi (90,91). In addition, pyruvate carboxylase has been overexpressed in order to reduce lactate levels (92). Protein glycosylation in CHO cells is similar to that in human cells but not identical (18). This is an important consideration since glycosylation is critical for protein stability, activity, immunogenicity, and pharmacokinetics (93). The sialic acid on human N-linked oligosaccharides has both (2,3)- and (2,6)-linkages, but only the former is present on glycoproteins produced in CHO cells (18). In general, the N-linked glycans from CHO cells have extensive heterogeneity, are multiantennary, and frequently lack terminal sialic acid and/or galactose on one or more antennae. Glycoproteins lacking terminal sialic acid are rapidly cleared from the circulatory system (93). Protein quality has been improved in CHO cells by the overexpression of (2,3)-sialyltransferase and (1,4)-galactosyltransferase, enzymes involved in terminal glycosylation of secreted proteins (94). Another target for improving glycosylation in CHO has been (1,6)-fucosyltransferase, an enzyme involved in fucosylation of secreted proteins. It has been shown that antibody-dependent cellular cytotoxicity (ADCC) is reduced by the presence of fucose on therapeutic antibodies (9597). Targeted mutagenesis of both FUT8 alleles or reduction in FUT8 mRNA by RNAi eliminated or reduced fucosylation, respectively, of antibodies produced in CHO cells (98100). High Throughput Recombinant Cell Line Selection Methods The traditional selection and amplication methods described in the section titled Selection of Recombinant

FUTURE PROSPECTS FOR CHO CELLS AND MAMMALIAN CELL PROCESSES CHO Genomics and Proteomics Although CHO cells have been extensively studied for several decades, little is known about the CHO genome. Reciprocal chromosome painting between the laboratory mouse (Mus musculus) and the Chinese hamster has been used to establish chromosome synteny between the two organisms (74). Recently, more than 16,000 expressed sequence tags (ESTs) from the Chinese hamster were mapped onto the mouse genome. The chromosome synteny between the two species was then used to dene conserved regions between the two genomes. In this way, a functional scaffold of the Chinese hamster genome was constructed showing the locations of more than 10,000 transcribed genes (75). In addition, the EST library has been used to construct a Chinese hamster-specic DNA array for transcription studies (76). These resource tools will greatly enhance future efforts to sequence the Chinese hamster genome. CHO proteomics has advanced more quickly than has genomics. These studies have mainly focused on the CHO proteome and the phosphoproteome for different bioprocesses (62,7779). Eventually, it is expected that by combining the proteomic and genomic approaches, a thorough picture of the metabolic pathways that are critical for protein production in CHO cells will be revealed and efforts to genetically modify this host will be enhanced.

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Cell Lines are time-consuming and not readily scalable to allow screening of more than a few hundred individual cell lines. Therefore, the current trend is toward the development of high throughput technologies with increased screening capacity (101). For example, cells can be transfected with the gene of interest and a gene-encoding green uorescent protein (GFP). If expression of the two genes is genetically linked, a linear correlation between GFP-specic uorescence and expression of the protein of interest is observed. Individual cells with a high level of GFP can then be recovered by uorescence-activated cell sorting (FACS) and analyzed for productivity of the gene of interest (102,103). Alternatively, GFP can be replaced with a cell surface protein so that the high producing cells can be selected by FACS after they are labeled with a uorescent antibody against the cell surface protein (104,105). As most recombinant proteins produced in CHO cells are secreted, methods are being developed to measure the level of the recombinant protein directly rather than a reporter protein. This can be accomplished by embedding individual recombinant cells in a gel or semisolid matrix, so that the secreted protein does not diffuse away from the cell. The secreted protein can then be detected using standard immunochemical methods (106). Together, these new technologies, which can be automated in many cases, will undoubtedly reduce the time of recombinant cell line development and increase the probability of generating high producing recombinant cell lines. Transient Gene Expression Transient gene expression (TGE) is a new technology that was only recently considered for large-scale recombinant protein production (107,108). TGE is dened as the production of a recombinant protein over a short period (114 days), following DNA transfer into single-cell suspension cultures. The recombinant gene is usually cloned in a nonviral expression vector and transfected into cells with a chemical delivery agent, especially PEI. In contrast to stable gene expression from recombinant cell lines, a genetic selection method is not applied to the transfected cells during the protein production phase. The process has been developed mainly with CHO and HEK-293 cells (109112). TGE is typically performed in stirred-tank bioreactors or in agitated containers including shake asks, wave-type bioreactors, and plastic or glass bottles (113). The main advantage of TGE over protein production in stable cell lines is time savings. Signicant quantities of protein can be obtained within a few days of transfection. Recently, volumetric yields of recombinant protein compared with those obtained from optimized processes with recombinant cell lines have been achieved (114). To date, the largest volumes for TGE have been about 100 L (115,116).

more straightforward way and to make them more productive. CHO and other production hosts have been developed that express, in highly optimized manufacturing processes, several grams per liter of secreted proteins, usually antibodies or antibody-fusion proteins. Thus, recent claims that cells of lymphoid origin, like NS0 cells, are especially equipped for the secretion of proteins and therefore are preferential high producers, have to be questioned. It appears that immortalized mammalian cells, of whatever origin, have tremendous plasticity, both for the uptake of foreign DNA and for supporting a high level protein production under bioreactor conditions. Even with the recent improvements in yield, one should not feel that there are no further opportunities. Yields of 1020 g/L should be possible in the near future, particularly if one considers the fact that batch and extended-batch processes obtain cell densities of about 10 million cells/mL. This corresponds to a biomass of 3%4% with respect to the total volume of the culture. By comparison, microbial processes achieve a biomass of 20%30% of the total volume. Finally, the knowledge gained from advances in CHO genomics will result in a much better understanding of the biochemistry and physiology of this host, so that higher product yields and quality can be achieved through genetic manipulation of the host or through modication of the culture conditions. REFERENCES
1. Aggarwal S. Nat Biotechnol 2006; 25: 10971104. 2. Rader RA. Biopharmaceutical products in the U.S. and European markets. 6th ed. Rockville (MD): BioPlan Associates, Inc. 3. Walsh G. Nat Biotechnol 2006; 24: 769776. 4. Wurm FM. Nat Biotechnol 2004; 22: 13931398. 5. Butler M. Appl Microbiol Biotechnol 2005; 68: 283291. 6. Andersen DC, Reilly DE. Curr Opin Biotechnol 2004; 15: 456462. 7. Roush DJ, Lu Y. Biotechnol Prog 2008; 24: 488495. 8. Gottschalk U. Biotechnol Prog 2008; 24: 496503. 9. Petricciani J. Dev Biol (Basel) 2006; 123: 1121. 10. Petricciani JC. Dev Biol Stand 1999; 100: 5763. 11. Palladino MA, Levinson AD, Svedersky LP, Obijeski JF. Dev Biol Stand 1987; 66: 1322. 12. Ringold G, Dieckmann B, Lee F. J Mol Appl Genet 1981; 1: 165175. 13. Scahill SJ, Devos R, Van der Heyden J, Fiers W. Proc Natl Acad Sci U S A 1983; 80: 46544658. 14. McCormick F, Trahey M, Innis M, Dieckmann B, Ringold G. Mol Cell Biol 1984; 4: 166172. 15. Kaufman RJ, Wesley LC, Spiliotes AJ, Gossels SD, Latt SA, Larsen GR, Kay RM. Mol Cell Biol 1985; 5: 17501759. 16. Wiebe ME, Becker F, Lazar R, May L, Casto B, Semense M, Fautz C, Garnick R, Miller C, Masover G, Bergman D, Lubiniecki AS. In: Spier RE, editor. Advances in animal cell biology and technology. Oxford: Butterworth- Heinemann; 1989. pp. 6871. 17. Hojman F, Emanoil-Raivier R, Lesser J, P eri es J. Dev Biol Stand 1989; 70: 195202. 18. Goochee CF, Gramer MJ, Andersen DC, Behr JC, Rasmussen JR. In: Todd P, Sikdar SK, Bier M, editors. Frontiers

CONCLUSIONS The technology to use mammalian cells for recombinant protein production is, surprisingly, still in its infancy. Much has to be done to establish production processes in a

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