Applied Microbiology and Biotechnology

, Volume 34, Issue 5, pp 559–564 | Cite as

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

  • P. M. Hayter
  • E. M. A. Curling
  • A. J. Baines
  • N. Jenkins
  • I. Salmon
  • P. G. Strange
  • A. T. Bull
Biotechnology

Summary

Recombinant human interferon-λ production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ardawi MSM, Newsholme EA (1982) Maximum activities of some enzymes of glycolysis, the tricarboxylic acid cycle and ketone body and glutamine utilization pathways in lymphocytes of the rat. Biochem J 208:743–748Google Scholar
  2. Bebbington C, Hentschel C (1985) The expression of recombinant DNA products in mammalian cells. Trends Biotechnol 3:314–317Google Scholar
  3. Busby S, Kumar A, Joseph M, Halfpap L, Insley M, Berkner K, Kurachi K, Woodbury R (1985) Expression of active human factor IX in transfected cells. Nature 316:271–273Google Scholar
  4. Butler M, Spier RE (1984) The effects of glutamine utilisation and ammonia production on the growth of BHK cells in microcarrier cultures. J Biotechnol 1:187–196Google Scholar
  5. Butler M, Thilly WG (1982) MDCK microcarrier cultures: seeding density effects and amino acid utilisation. In Vitro 18:213–219Google Scholar
  6. Curling EMA, Hayter PM, Baines AJ, Bull AT, Strange PG, Jenkins N (1990) Recombinant human interferon-gamma: differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture. Biochem J 272:333–337Google Scholar
  7. Curriden S, Englesberg E (1981) Inhibition of growth of proline-requiring Chinese hamster ovary cells (CHO-K1) resulting from antagonism by A system amino acids. J Cell Physiol 106:245–252Google Scholar
  8. Dube S, Fisher JW, Powell JS (1988) Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion and biological function. J Biol Chem 263:17516–17521Google Scholar
  9. Fawcett JK, Scott JE (1960) A rapid and precise method for the determination of urea. J Clin Pathol 13:156–159Google Scholar
  10. Fontanelle LJ, Henderson JF (1969) Sources of nitrogen as rate-limiting factors for purine biosynthesis de novo in Ehrlich ascites tumor cells. Biochim Biophys Acta 177:88–93Google Scholar
  11. Glacken MW, Fleischaker RJ, Sinskey AJ (1986) Reduction of waste product excretion via nutrient control: possible strategies for maximising product and cell yields on serum in cultures of mammalian cells. Biotechnol Bioeng 28:1376–1389Google Scholar
  12. Hamilton WG, Ham RG (1977) Clonal growth of Chinese hamster cell lines in protein-free media. In Vitro 13:82–92Google Scholar
  13. Holley RW, Armour R, Baldwin JH (1978) Density dependent regulation of growth of BSC-1 cells in culture: control of growth by low molecular weight nutrients. Proc Natl Acad Sci USA 75:339–341Google Scholar
  14. Hu W-S, Dodge TC, Frame KK, Himes VB (1987) Effect of glucose on the cultivation of mammalian cells. Dev Biol Standard 66:279–290Google Scholar
  15. Imamura T, Crespi CL, Thilly WG, Brunnengraber H (1982) Fructose as a carbohydrate source yields stable pH and redox parameters in microcarrier cell culture. Anal Biochem 18:353–358Google Scholar
  16. Kaufman RJ, Wasley LC, Spiliotes AJ, Gossels SD, Latt SA, Larsen GR, Kay RM (1983) Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol Cell Biol 5:1750–1759Google Scholar
  17. Kaufman RJ, Wasley LC, Dorner AJ (1988) Synthesis, processing and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 263:6352–6362Google Scholar
  18. Lanks KW (1987) End products of glucose and glutamine metabolism in L929 cells. J Biol Chem 262:10093–10097Google Scholar
  19. Levintow L, Eagle H (1961) Biochemistry of cultured mammalian cells. Ann Rev Biochem 30:605–640Google Scholar
  20. Ley KD, Tobey RA (1970) Regulation of DNA synthesis in Chinese hamster cells. II. Induction of DNA synthesis and cell division by isoleucine and glutamine in G1-arrested cells in suspension. J Cell Biol 47:453–459Google Scholar
  21. Lubiniecki AS (1987) Pharmaceutical applications of recombinant DNA-modified mammalian cells. Dev Ind Microbiol 28:133–138Google Scholar
  22. McCormick F, Trahey M, Innis M, Dieckmann B, Ringold G (1984) Inducible expression of amplified human beta interferon genes in CHO cells. Mol Cell Biol 4:166–172Google Scholar
  23. McKeehan WL (1986) Glutaminolysis in animal cells. In: Morgan MJ (ed) Carbohydrate metabolism in cultured cells. Plenum Press, New York, pp 111–150Google Scholar
  24. Miller WM, Wilke CR, Blanch HW (1989) Transient responses of hybridoma cells to nutrient additions in continuous culture. I. Glucose pulse and step changes. Biotechnol Bioeng 33:477–486Google Scholar
  25. Moreadith RW, Lehninger AL (1984) The pathways of glutamate and glutamine oxidation by tumor cell mitochondria: role of mitochondrial NAD(P)+-dependent malic enzyme. J Biol Chem 259:6215–6221Google Scholar
  26. Reitzer LJ, Wice BM, Kennell D (1979) Evidence that glutamine, not sugar is the major energy source for cultured HeLa cells. J Biol Chem 254:2669–2676Google Scholar
  27. Reuveny S, Velez D, Macmillan JD, Miller L (1986) Factors affecting cell growth and monoclonal antibody production in stirred reactors. J Immunol Methods 86:53–59Google Scholar
  28. Schlaeger E-J, Schumpp B (1989) Studies on mammalian cell growth in suspension culture. In: Spier RE, Griffiths JB, Stephenne J, Crooy PJ (eds) Advances in animal cell biology and technology for bioprocesses. Butterworths, Sevenoaks, pp 386–396Google Scholar
  29. Seaver SS, Rudolph JL, Gabriels JE (1984) A rapid HPLC technique for monitoring amino acid utilization in cell culture. Biotechniques 2:254–260Google Scholar
  30. Snell K (1985) Enzymes of serine metabolism in normal and neoplastic rat tissues. Biochim Biophys Acta 843:276–281Google Scholar
  31. Snell K, Natsumeda Y, Weber G (1987) The modulation of serine metabolism in hepatoma 3924A during different phases of cellular proliferation in culture. Biochem J 245:609–612Google Scholar
  32. Stoner GD, Merchant DJ (1972) Amino acid utilisation by LM strain mouse cells in a chemically defined medium. In Vitro 7:330–343Google Scholar
  33. Zetterberg A, Engstrom W (1981) Glutamine and the regulation of DNA replication and cell multiplication in fibroblasts. J Cell Physiol 108:365–373Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • P. M. Hayter
    • 1
  • E. M. A. Curling
    • 1
  • A. J. Baines
    • 1
  • N. Jenkins
    • 1
  • I. Salmon
    • 1
  • P. G. Strange
    • 1
  • A. T. Bull
    • 1
  1. 1.Biological LaboratoryUniversity of KentCanterburyUK

Personalised recommendations