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Cytotechnology

, Volume 15, Issue 1–3, pp 3–9 | Cite as

Cultural and physiological factors affecting expression of recombinant proteins

  • J. B. Griffiths
  • A. J. Racher
Article

Abstract

The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physicochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR).

Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture. The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode. Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate.

The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line. The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min−1) and dilution rates (1 d−1) and glutamine concentration (2.5 mmol l−1), plus the lowest values for bead size (2 mm) and inoculum density (107 ml−1). Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system.

Key words

Chimeric antibody culture systems glutamine growth rate porous carrier 

Abbreviations

FIBR

fluidised bed bioreactor

FBR

fixed bed reactor

STR

stirred tank reactor

SEAP

secreted alkaline phosphatase

rAb

recombinant antibody

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References

  1. Al-Rubeai M, Emery AN, Chalder S, Jan DC (1992) Specific monoclonal antibody productivity and the cell cycle-comparisons of batch, continuous and perfusion cultures. Cytotechnology 9: 85–97.Google Scholar
  2. Benecke B-J, Ben-Ze'er A and Penman S (1978) The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts. Cell 14: 931–939.Google Scholar
  3. Berger J, Hauber J, Hauber R, Geiger R, Cullen BR (1988) Secreted alkaline phosphatase: a powerful new quantitative indicator in eukaryotic cells. Gene 66: 1–10.Google Scholar
  4. Cacan R, Labiau O, Mir A-M and Verbert A (1993) Effect of cell attachment and growth on the synthesis and fate of dolichollinked oligosaccharides in Chinese hamster ovary cells. Eur. J. Biochem. 215: 873–881.Google Scholar
  5. Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technol 8: 662–667.Google Scholar
  6. Conradt HS, Nimtz M, Dittmar KEJ, Lindenmaier W, Hoppe J, Hauser H (1989) Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide. J. Biol. Chem. 264: 17368–17373.Google Scholar
  7. Folkman J and Moscanna A (1978) Role of cell shape in growth control. Nature 273: 345–349.Google Scholar
  8. Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Bull AT (1991) Chinese hamster ovary cell growth and interferon production kinetics in stirred batch cuiture. Appl. Microbiol. Biotechnol. 34: 559–564.Google Scholar
  9. Hynes RO (1992) Integrins: versatility, modulation, signalling in cell adhesion. Cell 69: 11–25.Google Scholar
  10. Kaluza B, Lenz H, Russmann E, Hock H, Rentrop O, Majdic O, Knapp W and Weidle UH (1991) Synthesis and functional characterisation of a recombinant monoclonal antibody directed against the α-chain of the human interleukin-2 receptor. Gene 107: 297–305.Google Scholar
  11. Lee GM, Chuck AS, Palsson BO (1993) Cell culture conditions determine the enhancement of specific monoclonal antibody productivity of calcium alginate-entrapped S3H5/γ2bA2 hybridoma cells. Biotechnol. Bioeng. 41: 330–340.Google Scholar
  12. Moreira JL, Alves PM, Aunins JG, Carrondo MJT (1992) Aggregate suspension cultures of BHK cells. In: Spier RE, Griffith JB, MacDonald C (eds) Animal Cell Technology: Developments, Processes and Products. Butterworth-Heinemann, Oxford, p411.Google Scholar
  13. Park S and Stephanopoulos G (1993) Packed bed bioreactor with porous ceramic beads for animal cell culture. Biotechnol. Bioeng. 41: 25–34.Google Scholar
  14. Pendse GJ, Karkare S, Bailey JE (1992) Effect of cloned gene dosage on cell growth and hepatitis B surface antigen in recombinant CHO cells. Biotechnol. Bioeng. 40: 119–129.Google Scholar
  15. Pollack R, Osborn M and Weber K (1975) Patterns of organisation of actin and myosin in normal and transformed cultured cells. Pro Natl. Acad. Sci., USA 72: 994–998.Google Scholar
  16. Racher AJ, Looby D, Griffiths JB (1990) Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilised in a fixed bed, porosphere culture system. J. Biotechnol. 15: 129–146.Google Scholar
  17. Racher AJ, Griffiths JB (1993) Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines. Cytotechnology 13: 125–131.Google Scholar
  18. Racher AJ, Moreira JL, Alves PM, Wirth M, Weidle UH, Hauser H, Carrondo MJT, Griffiths JB (1994) Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics. Appl. Microbiol. Biotechnol. 40: 851–856.Google Scholar
  19. Robinson DK, Memmert KW (1991) Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture. Biotechnol. Bioeng. 38: 972–976.Google Scholar
  20. Ryll T, Lucki-Lange M, Jager V, Wagner B (1990) Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium. J. Biotechnol. 14: 377–392.Google Scholar
  21. Tucker RW, Sanford KK and Frankel FR (1978) Tubulin and actin in paired nonneoplastic and spontaneously transformed neoplastic cell linesin vitro: fluorescent antibody studies. Cell 13: 629–642.Google Scholar
  22. Wagner R, Ryll T, Krafft H, Lehmann J (1988) Variation of amino acid concentrations in the medium of HU-β-IFN and HU Il-2 producing cell lines. Cytotechnology 1: 145–150.Google Scholar
  23. Zaccharyl I and Rozengurt E (1992) A focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell 71: 891–894.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • J. B. Griffiths
    • 1
  • A. J. Racher
    • 1
  1. 1.CAMRSalisburyUK

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