Abstract
There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number of approved biotherapeutics increases.
Similar content being viewed by others
References
Andersen DC, Bridges T, Gawlitzek M, Hoy C (2000) Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator. Biotechnol Bioeng 70:25–31
Andersen DC, Goochee CF (1994) The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins. Curr Opin Biotechnol 5:546–549
Andersen DC, Krummen L (2002) Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 13:117–123
Antoniou M, Harland L, Mustoe T, Williams S, Holdstock J, Yague E, Mulcahy T, Griffiths M, Edwards S, Ioannou PA, Mountain A, Crombie R (2003) Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing. Genomics 82:269–279
Arden N, Betenbaugh MJ (2004) Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 22:174–180
Baker KN, Rendall MH, Hills AE, Hoare M, Freedman RB, James DC (2001) Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol Bioeng 73:188–202
Barnes LM, Bentley CM, Dickson AJ (2004) Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells. Biotechnol Bioeng 85:115–121
Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (N Y) 10:169–175
Bibila TA, Robinson DK (1995) In pursuit of the optimal fed-batch process for monoclonal antibody production. Biotechnol Prog 11:1–13
Borth N, Zeyda M, Kunert R, Katinger H (2001) Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting. Biotechnol Bioeng 71:266–273. Erratum in Biotechnol Bioeng (2002) 77:118
Borys MC, Linzer DI, Papoutsakis ET (1993) Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Biotechnology (N Y) 11:720–724
Brekke OH, Sandlie I (2003) Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat Rev Drug Discov 2:52–62
Butler M (2004) Animal cell culture and technology, 2nd edn. Bios Scientific, Oxford
Butler M, Jenkins H (1989) Nutritional aspects of growth of animal cells in culture. J Biotechnol 12:97–110
Butler M, Imamura T, Thomas J, Thilly WG (1983) High yields from microcarrier cultures by medium perfusion. J Cell Sci 61:351–363
Carroll S, Al-Rubeai M (2004) The selection of high-producing cell lines using flow cytometry and cell sorting. Expert Opin Biol Ther 4:1821–1829
Castilho LR, Medronho RA (2002) Cell retention devices for suspended-cell perfusion cultures. Adv Biochem Eng Biotechnol 74:129–169
Castro PM, Hayter PM, Ison AP, Bull AT (1992) Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 38:84–90
Chee Furng Wong D, Tin Kam Wong K, Tang Goh L, Kiat Heng C, Gek Sim Yap M (2005) Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89:164–177
Cruz HJ, Moreira JL, Carrondo MJ (2000) Metabolically optimised BHK cell fed-batch cultures. J Biotechnol 80:109–118
Curling EM, Hayter PM, Baines AJ, Bull AT, Gull K, Strange PG, Jenkins N (1990) Recombinant human interferon-gamma. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture. Biochem J 272:333–337
Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M (2001) Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII. Biotechnol Bioeng 74:288–294
Donahue L (2004) Molecular approaches to cell culture medium development. In: IBC International Conference on Transmissible Spongiform Encephalopathies. IBC Life Sciences, Washington
Egrie JC, Dwyer E, Browne JK, Hitz A, Lykos MA (2003) Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp Hematol 31:290–299
Erbayraktar S, Grasso G, Sfacteria A, Xie QW, Coleman T, Kreilgaard M, Torup L, Sager T, Erbayraktar Z, Gokmen N, Yilmaz O, Ghezzi P, Villa P, Fratelli M, Casagrande S, Leist M, Helboe L, Gerwein J, Christensen S, Geist MA, Pedersen LO, Cerami-Hand C, Wuerth JP, Cerami A, Brines M (2003) Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci U S A 100:6741–6746
Fann CH, Guirgis F, Chen G, Lao MS, Piret JM (2000) Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese hamster ovary cells. Biotechnol Bioeng 69:204–212
Fox SR, Patel UA, Yap MG, Wang DI (2004) Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling. Biotechnol Bioeng 85:177–184
Gasser CS, Simonsen CC, Schilling JW, Schimke RT (1982) Expression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells. Proc Natl Acad Sci U S A 79:6522–6526
Gorfien S, Paul B, Walowitz J, Keem R, Biddle W, Jayme D (2000) Growth of NS0 cells in protein-free, chemically defined medium. Biotechnol Prog 16:682–687
Haines AWS, Wedgwood J, Cliffe S, Simpson D, Mountain A, Irvine A (2004) Rapid antibody production from stable pools of mammalian cells and isolation of high expressing clones using UCOEs. In: Cell culture and upstream processing. IBC Life Sciences, Berlin
Hayter PMCE, Baines AJ, Jenkins N, Salmon I, Strange PG, Tong JM, Bull AT (1992) Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-γ. Biotechnol Bioeng 39:327–335
Jenkins N, Curling EM (1994) Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol 16:354–364
Jenkins N, Parekh RB, James DC (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14:975–981
Jones D, Kroos N, Anema R, van Montfort B, Vooys A, van der Kraats S, van der Helm E, Smits S, Schouten J, Brouwer K, Lagerwerf F, van Berkel P, Opstelten DJ, Logtenberg T, Bout A (2003) High-level expression of recombinant IgG in the human cell line per.c6. Biotechnol Prog 19:163–168
Kim JM, Kim JS, Park DH, Kang HS, Yoon J, Baek K, Yoon Y (2004) Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol 107:95–105
Kim NS, Lee GM (2002) Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression. J Biotechnol 95:237–248
Kim NS, Kim SJ, Lee GM (1998) Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure. Biotechnol Bioeng 60:679–688
Kretzmer G (2002) Industrial processes with animal cells. Appl Microbiol Biotechnol 59:135–142
Kunkel JP, Jan DC, Jamieson JC, Butler M (1998) Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody. J Biotechnol 62:55–71
BK Lucas LM Giere RA DeMarco A Shen V Chisholm CW Crowley (1996) ArticleTitleHigh-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector Nucleic Acids Res 24 1774–1779
Mallik A, Pinkus GS, Sheffer S (2002.) Biopharma’s capacity crunch. McKinsey Q 9
Mastrangelo AJ, Hardwick JM, Zou S, Betenbaugh MJ (2000) Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults. Biotechnol Bioeng 67:555–564
Mercille S, Johnson M, Lanthier S, Kamen AA, Massie B (2000) Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates. Biotechnol Bioeng 67:435–450
Molowa DT, Mazanet R (2003) The state of biopharmaceutical manufacturing. Biotechnol Annu Rev 9:285–302
Nabi IR, Dennis JW (1998) The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time. Glycobiology 8:947–953
Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DI (1999) Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng 62:336–347
Okazaki A, Shoji-Hosaka E, Nakamura K, Wakitani M, Uchida K, Kakita S, Tsumoto K, Kumagai I, Shitara K (2004) Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa. J Mol Biol 336:1239–1249
Pavlou AK (2003) Marketspace: trends in biotherapeutics. J Commer Biotechnol 9:358–363
Pavlou AK (2004) The immunotherapies markets, 2003–2008. J Commer Biotechnol 10:273–278
Perlman S, van den Hazel B, Christiansen J, Gram-Nielsen S, Jeppesen CB, Andersen KV, Halkier T, Okkels S, Schambye HT (2003) Glycosylation of an N-terminal extension prolongs the half-life and increases the in vivo activity of follicle stimulating hormone. J Clin Endocrinol Metab 88:3227–3235
Raju TS, Briggs JB, Borge SM, Jones AJ (2000) Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10:477–486
Raju TS, Briggs JB, Chamow SM, Winkler ME, Jones AJ (2001) Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. Biochemistry 40:8868–8876
Restelli V, Butler M (2002) The effect of cell culture parameters on protein glycosylation. In: Al-Rubeai M (ed) Glycosylation, vol 3. Kluwer, Dordrecht, pp 61–92
Rothman RJ, Warren L, Vliegenthart JF, Hard KJ (1989) Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas. Biochemistry 28:1377–1384
Ryll T, Dutina G, Reyes A, Gunson J, Krummen L, Etcheverry T (2000) Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality. Biotechnol Bioeng 69:440–449
Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–26740
Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278:3466–3473
Shirgaonkar IZ, Lanthier S, Kamen A (2004) Acoustic cell filter: a proven cell retention technology for perfusion of animal cell cultures. Biotechnol Adv 22:433–444
Shriver Z, Raguram S, Sasisekharan R (2004) Glycomics: a pathway to a class of new and improved therapeutics. Nat Rev Drug Discov 3:863–873
Sung YH, Lim SW, Chung JY, Lee GM (2004) Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol 63:527–536
Sunstrom NA, Gay RD, Wong DC, Kitchen NA, DeBoer L, Gray PP (2000) Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells. Biotechnol Prog 16:698–702
Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000) Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng 68:31–43
Thiel KA (2004) Biomanufacturing, from bust to boom...to bubble? Nat Biotechnol 22:1365–1372
Thotakura NR, Desai RK, Bates LG, Cole ES, Pratt BM, Weintraub BD (1991) Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells. Endocrinology 128:341–348
Tinto A, Gabernet C, Vives J, Prats E, Cairo JJ, Cornudella L, Godia F (2002) The protection of hybridoma cells from apoptosis by caspase inhibition allows culture recovery when exposed to non-inducing conditions. J Biotechnol 95:205–214
Umana P, Jean-Mairet J, Moudry R, Amstutz H, Bailey JE (1999) Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17:176–180
Walsh G (2003) Biopharmaceuticals benchmarks—2003. Nat Biotechnol 21:865–887
Weikert S, Papac D, Briggs J, Cowfer D, Tom S, Gawlitzek M, Lofgren J, Mehta S, Chisholm V, Modi N, Eppler S, Carroll K, Chamow S, Peers D, Berman P, Krummen L (1999) Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins. Nat Biotechnol 17:1116–1121
Wen ZY, Teng XW, Chen F (2000) A novel perfusion system for animal cell cultures by two step sequential sedimentation. J Biotechnol 79:1–11
Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398
Xie L, Wang DI (1997) Integrated approaches to the design of media and feeding strategies for fed-batch cultures of animal cells. Trends Biotechnol 15:109–113
Yang M, Butler M (2000) Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol Bioeng 68:370–380
Yoon SK, Kim SH, Lee GM (2003) Effect of low culture temperature on specific productivity and transcription level of anti-4-1BB antibody in recombinant Chinese hamster ovary cells. Biotechnol Prog 19:1383–1386
Zanghi JA, Fussenegger M, Bailey JE (1999) Serum protects protein-free competent Chinese hamster ovary cells against apoptosis induced by nutrient deprivation in batch culture. Biotechnol Bioeng 64:108–119
Zanghi JA, Mendoza TP, Knop RH, Miller WM (1998) Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells. J Cell Physiol 177:248–263
Zanghi JA, Renner WA, Bailey JE, Fussenegger M (2000) The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures. Biotechnol Prog 16:319–325
Acknowledgements
The author would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support in animal cell technology.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Butler, M. Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68, 283–291 (2005). https://doi.org/10.1007/s00253-005-1980-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-005-1980-8