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  • Review Article
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Production of recombinant protein therapeutics in cultivated mammalian cells

Abstract

Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s. This increase in volumetric productivity has resulted mainly from improvements in media composition and process control. Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering.

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Figure 1: Cell line generation and development for cell culture processes for the generation of recombinant proteins of interest (o.i.).

Bob Crimi

Figure 2: Comparison of cell culture processes from 1986 and 2004.

Bob Crimi

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References

  1. Robinson, D.K. & Memmert, K.W. Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture. Biotechnol. Bioeng. 38, 972–976 (1991).

    Article  PubMed  CAS  Google Scholar 

  2. Ringold, G., Dieckmann, B. & Lee, F. Co-expression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells. J. Mol. Appl. Genet. 1, 165–175 (1981).

    PubMed  CAS  Google Scholar 

  3. Gopalkrishnan, R.V. et al. Use of the human EF-1alpha promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells. Nucleic Acids Res. 27, 4775–4782 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Le Hir, H., Nott, A., Moore, M.J. How introns influence and enhance eukaryotic gene expression. Trends in Biochem. Sci. 215–220 (2004).

  5. Makrides, S.C. Components of vectors for gene transfer and expression in mammalian cells. Protein Expr. Purif. 17, 183–202 (1999).

    Article  PubMed  CAS  Google Scholar 

  6. Graham, F.L. & van der Eb, A.J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467 (1973).

    Article  PubMed  CAS  Google Scholar 

  7. Norton, P.A. & Pachuk, C.J. Methods for DNA introduction into mammalian cells. in Gene Transfer and Expression in Mammalian Cells. (ed. Makrides, S.C.) 265–277 (Elsevier Science BV, Amsterdam, 2003).

    Chapter  Google Scholar 

  8. Balland, A. et al. Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector. Eur. J. Biochem. 172, 565–572 (1988).

    Article  PubMed  CAS  Google Scholar 

  9. Finn, G.K., Kurz, B.W., Cheng, R.Z. & Shmookler, R.J. Homologous plasmid recombination is elevated in immortally transformed cells. Mol. Cell. Biol. 9, 4009–4017 (1989).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Perucho, M., Hanahan, D. & Wigler, M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22, 309–317 (1980).

    Article  PubMed  CAS  Google Scholar 

  11. Richards, E.J. & Elgin, S.C. Epigenetic codes for heterochromatin formation and silencing: rounding up of the usual suspects. Cell 108, 489–500 (2002).

    Article  PubMed  CAS  Google Scholar 

  12. Mutskov, V. & Felsenfeld, G. Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine. EMBO J. 23, 138–149 (2004).

    Article  PubMed  CAS  Google Scholar 

  13. Girod, P.-A. & Mermod, N. Use of scaffold/matrix-attachment regions for protein production. in Gene Transfer and Expression in Mammalian Cells. (ed. Makrides, S.C.) 359–379 (Elsevier Science BV, Amsterdam, 2003).

    Chapter  Google Scholar 

  14. Antoniou, M. et al. Transgenese encompassing dual-promoter CpG islands from the human TBP and H NRPA2B1 loci are resistant to heterochromatin-mediated silencing. Genomics 82, 269–279 (2003).

    Article  PubMed  CAS  Google Scholar 

  15. Kwaks, T.H.J. et al. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nat. Biotechnol. 21, 553–558 (2003).

    Article  PubMed  CAS  Google Scholar 

  16. Fernandez, L.A., Winkler, M. & Grosschell, R. Matrix attachment region-dependent function of the immunoglobulin mu enhancer involves histone acetylation at a distance without changes in enhancer occupancy. Mol. Cell. Biol. 21, 196–208 (2001).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Zahn-Zabal, M. et al. Development of stable cell lines for production or regulated expression using matrix attachment regions. J. Biotechnol. 87, 29–42 (2001).

    Article  PubMed  CAS  Google Scholar 

  18. Gorman, C.M., Howard, B.H. & Reeves, R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 11, 7631–7648 (1983).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Cuisset, L., Tichonicky, L. & Delpech, M.A. Protein phosphatase is involved in the inhibition of histone diacetylation by sodium butyrate. Biochem. Biophys. Res. Comm. 246, 760–764 (1998).

    Article  PubMed  CAS  Google Scholar 

  20. Zheng, H. & Wilson, J.H. Gene targeting in normal and amplified cell lines. Nature 344, 170–173 (1990).

    Article  PubMed  CAS  Google Scholar 

  21. Wurm, F.M., Johnson, A., Lie, Y.S., Etcheverry, M.T. & Anderson, K.P. Host cell derived retroviral sequences enhance transfection and expression efficiency in CHO cells. in Animal Cell Technology: Developments, Processes and Products (eds. R.E. Spier, J.B. Griffiths, C. MacDonnald) 35–41 (Butterworth-Heinemann, Oxford, UK, 1992).

    Chapter  Google Scholar 

  22. Bode, J. et al. Architecture and utilization of highly expressed genomic sites. in Gene Transfer and Expression in Mammalian Cells. (ed. Makrides, S.C.) 551–571 (Elsevier Science, BV, Amsterdam, 2002).

    Google Scholar 

  23. Wilson, T.J. & Kola, I. The LoxP/CRE system and genome modification. Methods Mol. Biol. 158, 83–94 (2001).

    PubMed  CAS  Google Scholar 

  24. Arden, N., Nivtchanyong, T. & Betenbaugh, M.J. Cell Engineering blocks stress and improves biotherapeutic production. Bioprocessing 3, 23–28 (2004).

    Google Scholar 

  25. Simpson, N.H., Singh, R.P., Perani, A., Goldenzon, C. & Al-Rubeai, M. In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by expression of the bcl-2 gene. Biotechnol. Bioeng. 59, 90–98 (1999).

    Article  Google Scholar 

  26. Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H. & Bailey, J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).

    Article  PubMed  CAS  Google Scholar 

  27. Pallavicini, M.G. et al. Effects of methotrexate on transfected DNA stability in mammalian cells. Mol. Cell. Biol. 10, 401–404 (1990).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Gandor, C., Leist, C., Fiechter, A. & Asselbergs, F.A. Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells. FEBS Lett. 377, 290–294 (1995).

    Article  PubMed  CAS  Google Scholar 

  29. Wurm, F.M., Gwinn, K.A. & Kingston, R.E. Inducible overexpression of the mouse c-myc protein in mammalian cells. Proc. Natl. Acad. Sci. USA 83, 5414–5418 (1986).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Zettlmeissl, G., Wirth, M., Hauser, H.J. & Küpper, H.A. Isolation of overproducing recombinant mammalian cells by a fast and simple selection procedure. Gene 73, 419–426 (1988).

    Article  PubMed  Google Scholar 

  31. Bebbington, C.R., et al. High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology 10, 169–175 (1992).

    CAS  Google Scholar 

  32. Barnes, L.M., Bentley, C.M. & Dickson, A.J. Advances in animal cell recombinant protein production: GS-NS0 expression system. Cytotechnology 32, 109–123 (2000).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Barnes, L.M., Bentley, C.M. & Dickson, A.J. Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells. Biotechnol. Bioeng. 85, 115–121 (2004).

    Article  PubMed  CAS  Google Scholar 

  34. Jones, D. et al. High level expression of recombinant IgG in the human cell line PER.C6. Biotechnol. Prog. 19, 163–168 (2003).

    Article  PubMed  CAS  Google Scholar 

  35. Mazur, X., Fussenegger, M., Renner, W.A. & Bailey, J.E. Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27. Biotechnol. Prog. 14, 705–713 (1998).

    Article  PubMed  CAS  Google Scholar 

  36. Gorman, C.M., Howard, B.H. & Reeves, R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 11, 7631–7648 (1983).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Hsu, T.C. Chromosomal evolution in cell populations. Int. Rev. Cytol. 12, 69–121 (1961).

    Article  PubMed  CAS  Google Scholar 

  38. Barnes, L.M., Bentley, C.M. & Dickson, A.J. Characterization of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnol. Bioeng. 73, 261–270 (2001).

    Article  PubMed  CAS  Google Scholar 

  39. Wurm, F.M., Pallavicini, M.G. & Arathoon, R. Integration and stability of CHO amplicons containing plasmid sequences. Dev. Biol. Stand. 76, 69–82 (1992).

    PubMed  CAS  Google Scholar 

  40. Kim, S.J. & Lee, G.M. Cytogenetic analysis of chimeric antibody-producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol. Bioeng. 64, 741–749 (1999).

    Article  PubMed  CAS  Google Scholar 

  41. Loumaye, E. et al. Recombinant follicle stimulating hormone: development of the first biotechnology product for the treatment of infertility. Hum. Reprod. Update 4, 862–881 (1998).

    Article  Google Scholar 

  42. Spier, R. & Kadouri, A. The evolution of processes for the commercial exploitation of anchorage-dependent animal cells. Enzyme Microb. Technol. 21, 2–8 (1997).

    Article  CAS  Google Scholar 

  43. Bödecker, B.G.D., Newcomb, R., Yuan, P., Braufman, A. & Kelsey, W. Production of recombinant Factor VIII from perfusion cultures: I. Large Scale Fermentation. in Animal Cell Technology, Products of Today, Prospects for Tomorrow. (eds. R.E. Spier, J.B. Griffiths, and W. Berthold) 580–590 (Butterworth-Heinemann, Oxford, UK, 1994).

    Google Scholar 

  44. Wurm, F.M. & Bernard, A.R. Large scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10, 156–159 (1999).

    Article  PubMed  CAS  Google Scholar 

  45. Sinacore, M.S., Drapeau, D. & Adamson, S.R. Adaptation of mammalian cells to growth in serum-free media. Mol. Biotechnol. 15, 249–257 (2000).

    Article  PubMed  CAS  Google Scholar 

  46. Mather, J.P. Laboratory scale up of cell cultures (0.5–50 liters). Methods Cell Biol. 57, 219–227 (1998).

    Article  PubMed  CAS  Google Scholar 

  47. De Jesus, M., et al. TubeSpin satellites: a fast track approach for process development with animal cells using shaking technology. Biochem. Eng. J. 17, 217–223 (2004).

    Article  CAS  Google Scholar 

  48. Weber, W. et al. Gas-inducible transgene expression in mammalian cells, mice and bioreactor. Nat. Biotechnol. 22, 1440–1444 (2004).

    Article  PubMed  CAS  Google Scholar 

  49. Kaufmann, H. & Fussenegger, M. Metabolic engineering of mammalian cells for higher protein yield. in Gene Transfer and Expression in Mammalian Cells (ed. Makrides, S.C.) 457–569 (Elsevier Science, BV, Amsterdam, 2003).

    Chapter  Google Scholar 

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Acknowledgements

I am very grateful to my colleagues David Hacker and Martin Jordan for discussions and extensive editing in the context of the writing of this review.

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Correspondence to Florian M Wurm.

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Wurm, F. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22, 1393–1398 (2004). https://doi.org/10.1038/nbt1026

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