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1546 CHINESE HAMSTER OVARY CELLS, RECOMBINANT PROTEIN PRODUCTION

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CHINESE HAMSTER OVARY CELLS,RECOMBINANT PROTEIN PRODUCTION

DAVID L. HACKER,SEBASTIEN CHENUET, andFLORIAN M. WURM

Institute of Bioengineering,Laboratory of CellularBiotechnology, EcolePolytechnique Federal deLausanne (EPFL),Lausanne, Switzerland

INTRODUCTION

Recombinant therapeutic proteins are produced in livingcells through the application of recombinant DNA tech-nology and are used to treat or cure disease. This class

of biopharmaceuticals includes vaccines, monoclonal anti-bodies, hormones, growth factors, cytokines, enzymes, andblood factors. Worldwide annual sales of recombinant ther-apeutic proteins are worth more than $50 billion with anannual increase of about 20% (1). These statistics reflectthe fact that recombinant proteins are increasingly dom-inating the product pipelines in the biopharmaceuticalindustry. Of the approximately 140 recombinant therapeu-tic proteins currently approved in the United States andthe EU, about half are produced in cultivated mammaliancells while most of the others are produced in microbialhosts (Fig. 1) (2). Among the various mammalian cellsused for production, Chinese hamster ovary (CHO) cellsare clearly dominant (Fig. 1). In 2006, the top 10 recombi-nant therapeutic proteins in sales included 7 produced inCHO cells, generating total sales of $24 billion (Table 1).Based on estimates of candidate molecules currently inproduct pipelines (3), it is anticipated that CHO-derivedtherapeutic proteins will become increasingly importantin the next decade.

The major reason for choosing cultivated mammaliancells rather than a microbial host for production is proteincomplexity (4,5). Antibodies, for example, are glycosylatedprotein assemblies composed of four individual polypep-tides (two light and two heavy immunoglobulin chains)covalently linked by disulfide bonds. Their correct proteinfolding, assembly, and glycosylation can only be achievedefficiently in mammalian cells (6). The biological activityand pharmacokinetic properties of therapeutic proteinsare dependent on correct processing and assembly. It isalso advantageous that recombinant proteins producedin mammalian cells are typically secreted into thecell culture medium, simplifying protein recovery andpurification (7,8).

The mammalian cell lines used for the production ofrecombinant proteins are ‘‘immortalized’’ and can there-fore grow continuously in culture. Using immortalized cellsfor the production of recombinant proteins was not viewedfavorably by drug regulatory agencies in the early 1980s

16%

33%CHO cells

Yeast

Bacteria

Other hosts Othermammalian cells

17%

30%

4%

Figure 1. Distribution of approved and marketed (in UnitedStates and EU) recombinant therapeutic proteins according tohost production system. The total number of therapeutic proteinswas 140 (compiled from Ref. 2).

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CHINESE HAMSTER OVARY CELLS, RECOMBINANT PROTEIN PRODUCTION 1547

Table 1. Top 10 Recombinant Therapeutic Proteins in US Market Sales for 2006

SalesProduct Molecule Company ($ in millions) Host

Enbrel Tumor necrosis factor (TNF)receptor-IgG fragmentcrystallizable (Fc) fusion

Amgen, Wyeth, and Takeda 4.4 CHO

Aranesp Darbepoetin α Amgen 4.1 CHORituxan Anti-CD20 Monoclonal

Antibody (MAb)Biogen Idec, Genentech, and

Roche3.9 CHO

Remicade Anti-TNF MAb Johnson & Johnson,Schering-Plough

3.6 Sp2/0

Procrit/Eprex Epoetin α Johnson & Johnson 3.2 CHOHerceptin Anti-Her2 receptor MAb Genentech and Roche 3.1 CHOEpogen Epoetin α Amgen and Kirin 2.9 CHONeulasta Pegylated granulocyte-colony

stimulating factor (G-CSF)Amgen 2.7 Escherichia coli

Human insulin Human insulin NovaNordisk 2.5 Saccharomyces cerevisiaeAvastin Anti-vascular endothelial

growth factor (VEGF) MAbGenentech and Roche 2.4 CHO

Total ($ millions) 32.8

(9). Discussions about the risks associated with these cellswere controversial and had been initiated more than twodecades earlier when a first generation of ‘‘classical’’ bio-logical products (i.e. vaccines and the natural interferons)were recovered from cultivated mammalian cells. Manu-facturers of recombinant proteins and regulatory agencieswere in agreement that it was extremely important to min-imize eventual risks associated with the use of mammalianproduction hosts. Risks were seen in ‘‘tumor’’ principlescarried by the DNA of the host and in adventitiousagents (viruses, mycoplasma, etc.) that could infect thehost cell lines and eventually be transmitted to patientsreceiving products from those hosts (10). The result ofa long series of scientific discussions spanning a decadewas that stringent controls, regulations, and monitoringprocedures were enforced as a prerequisite for the man-ufacture of proteins from mammalian cells. For the firstproduct, recombinant tissue plasminogen activator (rtPAor Activase), approval in 1986 was achieved only after alarge body of data was provided to the regulatory agenciesthat showed (i) that only a minute quantity of CHO DNA(less than 10 pg/dose) was present in the final product, (ii)that the protein could be produced with a high degree ofreproducibility, and (iii) that it was produced with a puritynot achieved for any prior biological product (11). After 20years of recombinant therapeutic protein manufacturingin mammalian cells, no safety problems have arisen.

ORIGIN AND CHARACTERIZATION OF CHO CELLS

CHO cells were chosen as the production host for thefirst therapeutic recombinant protein mostly because ofthe availability of an auxotrophic CHO strain lackingdihydrofolate reductase (DHFR) activity that provided aconvenient genetic selection method for recovering recom-binant CHO cell lines (12–15) (section titled ‘‘Selectionof Recombinant Cell Lines’’). Other protein manufactur-ers subsequently chose this cell line because the approvalbarriers for a second product from the same host were

considerably easier to overcome. Although CHO cells arecurrently the preferred mammalian host for recombinantprotein production, other cell lines including baby hamsterkidney (BHK-21), human embryo kidney (HEK-293), andmouse myeloma (NS0, Sp2/0) have gained approval forthis purpose.

CHO cells have several properties besides the DHFRselection method that contribute to their attractivenessas a production host. First, they are easy to cultivateand are capable of growing at high cell densities (> 1×107 cells/mL) in single-cell suspension culture even atvolumetric scales up to 20,000 L. Second, they are easilytransfected with plasmid DNA using both chemical andphysical methods. Third, the risk of involuntary contam-ination of the recombinant product with an adventitiousviral agent is low since CHO cells do not produce infectiousendogenous retroviruses, and they are not a permissivehost for most pathogenic human viruses (16,17). Fourth,they are highly productive so that under optimized cell cul-ture conditions, recombinant CHO cell lines have achievedspecific productivities up to 50 pg/cell/day for secreted pro-teins (4,5). Fifth, they yield protein glycoforms that aresimilar to those produced in human cells (18).

The original CHO cell line was isolated in 1957 fol-lowing a spontaneous immortalization event in a cultureof primary cells from the ovary excised from a Chinesehamster (Cricetulus griseus) (19). A derivative of theoriginal cell line, the glycine-dependent strain CHO-K1,was mutagenized with ethane methyl sulfonate and thenwith γ -radiation to generate the strain CHO-DXB11 (alsoreferred to as CHO-DUKX or CHO-DUK-XB11), lackingDHFR activity (20). This strain has one deleted dhfr alleleand a missense mutation in the second allele. Subse-quently, the CHO-pro3-strain, another derivative of theoriginal CHO cell line, was treated with two rounds ofγ -radiation to yield CHO-DG44, a strain with deletionsof both dhfr alleles (21). These two DHFR-minus strainsrequire glycine, hypoxanthine, and thymidine (GHT) forgrowth. Although not initially intended for the purpose of


recombinant protein production, these cells were used fora number of pioneering experiments in which they werestably transfected with an exogenous dhfr gene and thenselected in medium lacking GHT (12–15). This geneticselection scheme remains the standard method to estab-lish stably transfected CHO cell lines for the production ofrecombinant proteins (section titled ‘‘Selection of Recom-binant Cell Lines’’).

RECOMBINANT CELL LINE GENERATION

Introduction

The generation of recombinant cell lines is a multistepprocess that begins with the molecular cloning of thegene of interest in a mammalian expression vector. Thegene of interest and the selection gene, also cloned in anexpression vector, are then delivered into cells by trans-fection, and the cells are grown under selective conditionsto recover those that have the exogenous genes integratedinto the genome. The cells that survive selection eventu-ally form cell colonies that may be individually transferredinto a new cultivation vessel. These individual cell linesare analyzed for recombinant protein yield and cell growthrate. The most productive cell lines then undergo one ormore rounds of cellular cloning to allow recovery of a clonalrecombinant cell line. Each of these steps is discussed inmore detail in this section.

Mammalian Expression Vectors

The recombinant gene of interest is usually cloned in anonviral expression vector (i.e. plasmid) for subsequenttransfer into cells. The major advantages for nonviral vec-tors over viral vectors include ease of construction, ease ofdelivery into cells with inexpensive chemical agents, andbiosafety. The recombinant gene is typically transcribedfrom a strong promoter such as the immediate early pro-moters of human cytomegalovirus (hCMV) and mousecytomegalovirus (mCMV), the SV40 virus early promoter,the Rous sarcoma virus long-terminal repeat promoter,and the promoters of constitutively expressed housekeep-ing genes like the human elongation factor-1 alpha (EF-1α) and the chicken α-actin genes (22). The selection gene(s)may be cloned in the same vector as the gene(s) of interestor in a separate vector (23).

Both the gene of interest and the selection geneare almost always cloned as a cDNA rather than as afull-length gene due to the size constraints imposed onplasmids. Since splicing of mRNA and its subsequenttransport to the cytoplasm are functionally linked,mRNAs transcribed from cDNAs are not efficientlytransported to the cytoplasm since they do not containintrons (24). For this reason, most expression vectorsinclude an intron sequence between the promoter andthe 5′-end of the cloned cDNA (22). Finally, 3′-endprocessing and polyadenylation of the recombinant mRNArequire the appropriate regulatory elements near the 3′

end of the cDNA (22). Recombinant gene expression isregulated not only at the level of transcription and mRNAprocessing but also by factors such as mRNA stability andtranslation efficiency that are controlled in large part by

RNA sequence and structure (25–27). To enhance levelsof recombinant mRNA and protein, the coding sequencemay be altered by removing cryptic splice sites, alteringcodons having underrepresented cognate tRNAs, andincreasing the G + C percentage at the third (wobble)position of codons (28,29).

Recombinant protein production from stable recom-binant cell lines may diminish with time during cellcultivation due to a decrease in transcription of therecombinant gene, a phenomenon termed gene silencing.A major determinant in gene silencing is the structure ofthe chromatin at the site of integration of the recombinantgene. In general, heterochromatin is condensed and tran-scriptionally inactive, whereas euchromatin is relaxed andtranscriptionally active (30). The two chromatin states areassociated with specific histone modifications includingacetylation, methylation, and phosphorylation thatfunction to control chromatin condensation and transcrip-tional activity (31). DNA elements like scaffold or matrixattachment regions (S/MARs), insulators, antirepressorelements, and ubiquitous chromatin opening elements(UCOEs) have been shown to ameliorate the effects ofgene silencing in recombinant cell lines (32–36). TheseDNA elements are small enough to allow cloning inexpression vectors.

Nonviral Gene Delivery

Nonviral vectors are delivered into cells by chemical orphysical methods. The three main chemical reagentsare calcium phosphate (CaPi) (37,38), cationic polymersespecially the polyamine polyethylenimine (PEI) (39),and cationic liposomes (40). All of these reagents formcomplexes with negatively charged DNA. The positivelycharged complexes readily bind to the negatively chargedcell surface. Thereafter, all the complexes face the samephysical barriers to delivery of DNA into the nucleus (41).The complexes are endocytosed, but only a fraction ofthe DNA eventually escapes the endosome (42). Withinthe cytoplasm, the DNA is transported to the nuclearmembrane by unknown mechanism(s). Clearly, plasmidDNA does not readily diffuse within the cell (43), andwithin the cytoplasm, the half-life of plasmid DNA isless than 90 min because it is a substrate for cytoplasmicDNases (44). The plasmid DNA eventually enters thenucleus either through nuclear pores or during mitosisafter the breakdown of the nuclear membrane (45–47).

Physical methods of DNA delivery include electropora-tion and microinjection. For the former, a brief electricalcharge is applied to suspension cultures of cells in thepresence of plasmid DNA (48). Under optimal conditions,nearly 100% of the cells receive DNA. Microinjection hasbeen used to directly deliver plasmid DNA into the nucleusof cells including CHO for the generation of recombinantcell lines (49,50). This is the only DNA delivery methodthat allows operator control of the quantity and quality ofDNA reaching the nucleus of the target cell. The disad-vantage of the method, however, is the time required tomicroinject a relatively small number of cells.

For the DNA delivery methods described earlier, usu-ally less than 5% of the cells that receive DNA areeventually recovered as recombinant cell lines (4). Thereason(s) for the low rate of cell line recovery is not known,


but the frequency of integration of plasmid DNA into thecell’s genome may be a major factor. Digestion of plas-mid DNA by cellular DNases decreases the percentage ofplasmids having an intact recombinant or selection gene(51) On the other hand, for more efficient generation ofrecombinant cell lines, it is advantageous to linearize theplasmid DNA with a restriction endonuclease outside therecombinant and selection genes prior to DNA delivery(38).

Selection of Recombinant Cell Lines

Following DNA transfer, the cells with an integratedrecombinant gene must be recovered from the generalpopulation of transfected cells. The standard method ofselection with CHO cells is via cotransfer of the dhfrgene along with the recombinant gene of interest into aDHFR-deficient strain such as DG44 (12–15). After DNAtransfer, the cells are cultured in the absence of GHT. Onlycells with one or more stably integrated copies of a tran-scriptionally active dhfr gene survive to form colonies onthe surface of the culture dish. Each colony is a clonal pop-ulation of recombinant cells, most of which will have oneor more integrated copies of the gene of interest. There areno apparent preferred sites of plasmid DNA integration,and the integrated plasmid copy number varies widelyfrom one cell line to another (23). Nevertheless, there isusually only one integration site per cell even if multipleplasmids are transfected (23).

From each transfection, numerous recombinant celllines are recovered but there is usually extensive hetero-geneity among them in terms of protein productivity andgrowth rate. To obtain a few cell lines with the desiredcharacteristics, it is usually necessary to evaluate severalhundred individual cell lines. Once the best candidatesare identified, one or more rounds of limiting dilution arepreformed to attempt to produce a clonal cell line. Toaccomplish this, the cells originating from a single colonyare diluted and then transferred to a multiwell plate sothat, on average, only one cell is present per well (52).Once clonal cell lines are established, they are character-ized for the stability of recombinant protein productionsince expression of the integrated gene is not necessarilymaintained at a constant level over time. Instead, genesilencing in the entire clonal cell population or in a sub-population is frequently observed. Thus, the candidatecell lines must be cultivated for several months to ensurestable protein production over time.

The dhfr gene is not the only selection marker availablefor generating recombinant CHO cell lines. The glutaminesynthetase (gs) gene initially considered for the selec-tion of NS0-derived cell lines since their endogenous GS(glutamine synthetase) activity is low, is also used torecover recombinant CHO cells even though they havea higher endogenous GS activity than NS0 cells (53,54).After transfection with the gene of interest and the GSgene, recombinant cells are selected in medium withoutglutamine. Resistance genes for the antibiotics geneticin(G418), hygromycin B, zeocin, blasticidin, and puromycinand the amplifiable metallothionein gene represent otherclasses of selection markers (55). Following DNA transfer,recombinant cells are recovered in medium containing theappropriate antibiotic.

With both DHFR and GS selection, expression ofthe recombinant gene can be significantly increased byexposing the cells to a drug that blocks the enzymaticactivity of the selection marker. DHFR and GS areinhibited by methotrexate (MTX) and methioninesulphoximine (MSX), respectively (53,54,56–58). ForCHO-derived cell lines that express the exogenous dhfrgene, a majority of the cells die after 2–3 weeks ofexposure to increasing concentrations of MTX. The raresurvivors have a higher integrated plasmid copy numberthan the original cell line as the result of amplificationof the dhfr gene and the surrounding DNA, includingthe gene of interest (56–58). CHO cells lines containingseveral hundred copies of integrated plasmid DNA havebeen established this way (59,60). The higher gene copynumber accounts for the increased level of recombinantprotein productivity observed in most ‘‘amplified’’ celllines. Similar observations have been made following theexposure of recombinant NS0 cell lines to MSX (54).

The selection and cloning of recombinant cell lines maybe performed in suspension culture. This practice avoidsexposure of the recombinant cells to conditions of adherentgrowth in the presence of animal-derived serum. Afterexposure to the selective conditions, the survivors aretransferred to multiwell plates by limiting dilution. Theresulting cell lines are screened for recombinant proteinproductivity and cell growth, and those with the desiredcharacteristics may be subjected to one or more additionalrounds of limiting dilution to increase the likelihood ofrecovering a clonal cell line. By eliminating steps requiringadherent cultivation in serum, there is no need to adaptthe recombinant cell lines to suspension cultivation inserum-free medium since this may result in the loss ofrecombinant protein productivity.

CELL CULTIVATION SYSTEMS

Most recombinant protein production processes for clinicalmanufacture utilize single-cell suspension cultivation instirred-tank bioreactors of various sizes up to 20,000 L.The cells are maintained in media that are designed forsuspension growth to a high cell density, preferably inthe absence of serum or other animal-derived components.The cells may be cultivated during the entire protein pro-duction run without nutrient additives (batch culture) ormedium components may be periodically added to theculture to prolong cell viability and protein production(extended- or fed-batch culture) (4,5). The latter strategy isused for most high-yielding bioprocesses in the biotechnol-ogy industry today. Enhancement of protein productivityin batch and fed-batch processes has been demonstratedfor some recombinant CHO cell lines by temperaturereduction to 30–33◦C, increased osmolarity, or addition ofsodium butyrate, a histone deacetylase inhibitor (61–65).To gain the greatest benefit, these treatments are nor-mally performed near the end of the exponential growthphase of the culture since they typically reduce or inhibitcell division.

The other major type of production process with sus-pension cultures is continuous perfusion. In this case, thecells are maintained in a stirred-tank bioreactor, and sev-eral bioreactor volumes of fresh medium are fed into the


culture each day while the same volume is withdrawn fromthe vessel. Perfused cultures normally achieve higher celldensities than batch or extended-batch cultures, and thecells can remain viable for several weeks, with productharvests occurring repeatedly throughout the cultivationperiod (66,67). The antihemophilic factor VIII (Kogenate)is manufactured from recombinant (BHK-21) cells withthis mode. Factor VIII is the largest therapeutic recombi-nant protein (2332 amino acids) on the market, and its sizeand sensitivity to proteases contributed to the decision toproduce it in a perfused system rather than in a batch orfed-batch process (68,69).

Recombinant proteins are also produced from cellsattached to a surface rather than in suspension. Forexample, Epogen is synthesized from a recombinantCHO cell line grown in roller bottles filled with medium to10%–30% of their capacity (70,71). The cells attach to thesurface of the bottle whose slow rotation assures the reg-ular wetting of the cells. Cells can also be grown attachedto polymeric spheres (microcarriers) of dextran, polyacry-lamide, or polystyrene (72,73). Each bead is seeded withcells, and the beads are maintained in suspension instirred-tank bioreactors. Eventually, after several roundsof cell division, each bead may hold several hundredcells. The disadvantage of this method is that volumet-ric scale-up requires the removal of cells from the beadsfollowed by reseeding on a greater quantity of beads in alarger volume of medium.

FUTURE PROSPECTS FOR CHO CELLS AND MAMMALIANCELL PROCESSES

CHO Genomics and Proteomics

Although CHO cells have been extensively studied forseveral decades, little is known about the CHO genome.Reciprocal chromosome painting between the laboratorymouse (Mus musculus) and the Chinese hamster has beenused to establish chromosome synteny between the twoorganisms (74). Recently, more than 16,000 expressedsequence tags (ESTs) from the Chinese hamster weremapped onto the mouse genome. The chromosome syntenybetween the two species was then used to define conservedregions between the two genomes. In this way, a functionalscaffold of the Chinese hamster genome was constructedshowing the locations of more than 10,000 transcribedgenes (75). In addition, the EST library has been usedto construct a Chinese hamster-specific DNA array fortranscription studies (76). These resource tools will greatlyenhance future efforts to sequence the Chinese hamstergenome.

CHO proteomics has advanced more quickly than hasgenomics. These studies have mainly focused on the CHOproteome and the phosphoproteome for different biopro-cesses (62,77–79). Eventually, it is expected that by com-bining the proteomic and genomic approaches, a thoroughpicture of the metabolic pathways that are critical for pro-tein production in CHO cells will be revealed and effortsto genetically modify this host will be enhanced.

Host Cell Engineering

Many efforts have been made to genetically modify CHOcells for the purpose of improving recombinant proteinyield and quality (21–23). The main targets for host engi-neering have been the pathways for apoptosis, lactateproduction, glycosylation, and protein secretion. The twomain strategies for genetic modification are (i) the overex-pression of exogenous effector genes and (ii) the reductionor elimination of endogenous gene expression (80–82). Thelatter has been achieved by mutagenesis of one or bothalleles of an endogenous gene or by decreasing mRNAlevels 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 nowfound their way into production processes for industrialmanufacturing.

One of the main targets of host engineering has beenthe apoptotic pathway. Antiapoptotic proteins includingBcl-2, Bcl-xL, and others have been stably overexpressedin CHO cells to enhance cell viability (81,83–86). In addi-tion, caspase 3 mRNA has been targeted by antisense RNAto reduce the level of this important apoptotic protein (87).Overexpression of the transcription factor X-box bindingprotein-1 (Xbp-1) has been shown to enhance recombinantprotein production in CHO cells through the regulation ofgenes involved in the unfolded protein response (88,89).To reduce the amount of lactate production during the cul-tivation of CHO cells, the mRNA of lactate dehydrogenasesubunit A (LDH-A) has been targeted by antisense RNAand RNAi (90,91). In addition, pyruvate carboxylase hasbeen overexpressed in order to reduce lactate levels (92).

Protein glycosylation in CHO cells is similar to that inhuman cells but not identical (18). This is an importantconsideration since glycosylation is critical for proteinstability, activity, immunogenicity, and pharmacokinetics(93). The sialic acid on human N-linked oligosaccharideshas both α(2,3)- and α(2,6)-linkages, but only the formeris present on glycoproteins produced in CHO cells(18). In general, the N-linked glycans from CHO cellshave extensive heterogeneity, are multiantennary, andfrequently lack terminal sialic acid and/or galactose onone or more antennae. Glycoproteins lacking terminalsialic acid are rapidly cleared from the circulatorysystem (93). Protein quality has been improved in CHOcells by the overexpression of α(2,3)-sialyltransferaseand β(1,4)-galactosyltransferase, enzymes involvedin terminal glycosylation of secreted proteins (94).Another target for improving glycosylation in CHO hasbeen α(1,6)-fucosyltransferase, an enzyme involved infucosylation of secreted proteins. It has been shownthat antibody-dependent cellular cytotoxicity (ADCC)is reduced by the presence of fucose on therapeuticantibodies (95–97). Targeted mutagenesis of both FUT8alleles or reduction in FUT8 mRNA by RNAi eliminated orreduced fucosylation, respectively, of antibodies producedin CHO cells (98–100).

High Throughput Recombinant Cell Line Selection Methods

The traditional selection and amplification methodsdescribed in the section titled ‘‘Selection of Recombinant


Cell Lines’’ are time-consuming and not readily scalableto allow screening of more than a few hundred individualcell lines. Therefore, the current trend is toward the devel-opment of high throughput technologies with increasedscreening capacity (101). For example, cells can betransfected with the gene of interest and a gene-encodinggreen fluorescent protein (GFP). If expression of the twogenes is genetically linked, a linear correlation betweenGFP-specific fluorescence and expression of the protein ofinterest is observed. Individual cells with a high level ofGFP can then be recovered by fluorescence-activated cellsorting (FACS) and analyzed for productivity of the geneof interest (102,103). Alternatively, GFP can be replacedwith a cell surface protein so that the high producingcells can be selected by FACS after they are labeled witha fluorescent antibody against the cell surface protein(104,105). As most recombinant proteins produced inCHO cells are secreted, methods are being developed tomeasure the level of the recombinant protein directlyrather than a reporter protein. This can be accomplishedby embedding individual recombinant cells in a gel orsemisolid matrix, so that the secreted protein does notdiffuse away from the cell. The secreted protein can thenbe detected using standard immunochemical methods(106). Together, these new technologies, which can beautomated in many cases, will undoubtedly reduce thetime of recombinant cell line development and increasethe probability of generating high producing recombinantcell lines.

Transient Gene Expression

Transient gene expression (TGE) is a new technology thatwas only recently considered for large-scale recombinantprotein production (107,108). TGE is defined as the pro-duction of a recombinant protein over a short period (1–14days), following DNA transfer into single-cell suspensioncultures. The recombinant gene is usually cloned in a non-viral expression vector and transfected into cells with achemical delivery agent, especially PEI. In contrast to sta-ble gene expression from recombinant cell lines, a geneticselection method is not applied to the transfected cellsduring the protein production phase. The process has beendeveloped mainly with CHO and HEK-293 cells (109–112).TGE is typically performed in stirred-tank bioreactors orin agitated containers including shake flasks, wave-typebioreactors, and plastic or glass bottles (113). The mainadvantage of TGE over protein production in stable celllines is time savings. Significant quantities of protein canbe obtained within a few days of transfection. Recently,volumetric yields of recombinant protein compared withthose obtained from optimized processes with recombinantcell lines have been achieved (114). To date, the largestvolumes for TGE have been about 100 L (115,116).

CONCLUSIONS

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

more straightforward way and to make them more produc-tive. CHO and other production hosts have been developedthat express, in highly optimized manufacturing pro-cesses, several grams per liter of secreted proteins, usuallyantibodies or antibody-fusion proteins. Thus, recent claimsthat cells of lymphoid origin, like NS0 cells, are especiallyequipped for the secretion of proteins and therefore arepreferential high producers, have to be questioned. Itappears that immortalized mammalian cells, of whateverorigin, have tremendous plasticity, both for the uptake offoreign DNA and for supporting a high level protein pro-duction under bioreactor conditions. Even with the recentimprovements in yield, one should not feel that there areno further opportunities. Yields of 10–20 g/L should be pos-sible in the near future, particularly if one considers thefact that batch and extended-batch processes obtain celldensities of about 10 million cells/mL. This correspondsto a biomass of 3%–4% with respect to the total volumeof the culture. By comparison, microbial processes achievea biomass of 20%–30% of the total volume. Finally, theknowledge gained from advances in CHO genomics willresult in a much better understanding of the biochemistryand physiology of this host, so that higher product yieldsand quality can be achieved through genetic manipula-tion of the host or through modification of the cultureconditions.

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