Applied Microbiology and Biotechnology

, Volume 38, Issue 1, pp 84–90 | Cite as

Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells

  • Paula M. L. Castro
  • Paul M. Hayter
  • Andrew P. Ison
  • Alan T. Bull
Applied Genetics and Regulation

Summary

The importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.

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References

  1. Aisen P, Listowsky I (1980) Iron transport and storage proteins. Annu Rev Biochem 49:357–393Google Scholar
  2. Barnes D, Sato G (1980) Methods for growth of cultured cells in serum-free medium. Anal Biochem 10:255–270Google Scholar
  3. Bebbington C, Hentschel C (1985) The expression of recombinant DNA products in mammalian cells. Trends Biotechnol 3:314–317Google Scholar
  4. Berman PW, Lasky LA (1985) Engineering glycoproteins for use as pharmaceuticals. Tibtech 3:51–53Google Scholar
  5. Bialy H (1987) Recombinant proteins: virtual authenticity. Biotechnology 5:883–890Google Scholar
  6. Bull AT, Huck TA, Bushell ME (1990) Optimization strategies in microbial process development and operation. In: Poole RK, Bazin MJ, Keevil CW (eds) Microbial growth dynamics. IRL Press, Oxford, pp 145–168Google Scholar
  7. Chang TH, Steplewski Z, Koprowski H (1980) Production of monoclonal antibodies in serum free medium. J Immunol Methods 39:369–375Google Scholar
  8. Curriden S, Englesberg E (1981) Inhibition of growth of proline-requiring Chinese hamster ovary cells (CHO-K1) resulting from antagonism by A system aminoacids. J Cell Physiol 106:245–252Google Scholar
  9. Darfler FJ, Insel PA (1982) Serum free culture of resting, PHA-stimulated, and transformed lymphoid cells, including hybridomas. Exp Cell Res 138:287–295Google Scholar
  10. De Meo M, Laget M, Phan Tan Luu R, Mathieu D, Dume G (1985) Application of experimental design of experiments for media optimization and culture conditions in fermentations. Biosciences 4:99–102Google Scholar
  11. Duval D, Demangel C, Munier-Jolain K, Miossec S, Geahel I (1991) Factors controlling cell proliferation and antibody production in mouse hybridoma cells: I. Influence of the amino-acid supply. Biotechnol Bioeng 38:561–570Google Scholar
  12. Eagle H (1955a) The specific amino acid requirements of a mammalian cell (strain L) in tissue culture. J Biol Chem 214:839–852Google Scholar
  13. Eagle H (1955b) The specific amino acid requirements of a human carcinoma cell (strain HeLa) in tissue culture. J Biol Chem 116:29–43Google Scholar
  14. Engelsberg E, Bass R, Heiser W (1976) Inhibition of the growth of mammalian cells in culture by amino acids and the isolation and characterization of l-phenylalanine-resistant mutants modifying l-phenylalanine transport. Somatic Cell Genet 2:411–428Google Scholar
  15. Fawcett JK, Scott JE (1960) A rapid and precise method for the determination of urea. J Clin Pathol 13:156–159Google Scholar
  16. Ganne V, Mignot G (1991) Application of statistical design of experiments to the optimization of factor VIII expression by CHO cells. Cytotechnology 6:233–240Google Scholar
  17. Gasser F, Mulsant P, Gillois M (1985) Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium. In Vitro Cell & Dev Biol 6:588–592Google Scholar
  18. Gebert C, Gray PP (1990) A screening experiment of media supplements on CHO cells. In: Spier RE, Griffiths JB, Meignier B (eds) Production of biologicals from animal cells in culture. Butterworths, Sevenoaks, pp 76–78Google Scholar
  19. Greasham R, Inamine E (1986) Nutritional improvements of processes. In: Demain AL, Solomon NA (eds) American Society for Microbiology, Washington, D.C., pp 41–48Google Scholar
  20. Hasegawa K, Yasuhiro A, Michihiro K, Akamatsu Y, Nishijima M (1990) Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose. Biochim Biophys Acta 1051:221–229Google Scholar
  21. Hayter PM, Furlotte D, Wilcox M, Curling EMA, Jenkins N (1989) Recombinant gamma-interferon production by CHO cells in serum-free medium. In: Spier RE, Griffiths JB, Stephenne J, Crooy PJ (eds) Advances in animal cell biology and technology for bioprocesses. Butterworths, Sevenoaks, pp 280–282Google Scholar
  22. 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 culture. Appl Microbiol Biotechnol 34:559–564Google Scholar
  23. Iscove NN, Melchers F (1978) Complete replacement of serum by albumin, transferrin and soybean lipids in cultures of lipopolysaccharides-reactive B lymphocytes. J Exp Med 147:923–933Google Scholar
  24. Kao FT, Puck TT (1967) Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirements of proline. Genetics 55:513–524Google Scholar
  25. Kovar J (1986) Hybridoma cultivation in defined serum-free medium: growth supporting substance. II. Insulin, other hormones and growth factors. Folia Biol (Prague) 32:305–310Google Scholar
  26. Kovar J, Franek F (1984) Serum-free medium for hybridoma and parental myeloma cell cultivation: a novel composition of growth supporting substances. Immunol Lett 7:339–343Google Scholar
  27. Kovar J, Franek F (1985) Hybridoma cultivation in defined serum-free medium: growth supporting substance. I. Transferin. Folia Biol (Prague) 31:167–175Google Scholar
  28. Lambert KJ, Birch JR (1985) Cell growth media. In: Spier RE, Griffiths JB (eds) Animal cell biotechnology, vol 1. Academic Press, New York, pp 85–122Google Scholar
  29. Luan YT, Mutharasan R, Magee WE (1987) Strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies. Biotechnol Lett 9:691–696Google Scholar
  30. Lucki-Lange M, Wagner R (1991) Conditions for the production of recombinant IL-2 in stirred suspension culture using a protein free medium. In: Spier RE, Griffiths JB, Meignier B (eds) Production of biologicals from animal cells in culture. Butterworths, Sevenoaks, pp 180–186Google Scholar
  31. Mather JP, Sato GH (1979) The growth of mouse melanoma cells in hormone-supplemented, serum-free medium. Exp Cell Res 20:191–200Google Scholar
  32. McCormick F, Trahey M, Innis M, Dieckmann B, Ringgold G (1984) Inducible expression of amplified human beta interferon genes in CHO cells. Mol Cell Biol 4:166–172Google Scholar
  33. Mutsaers JHGM, Kamerling JP, Devos R, Guisez Y, Friers W, Vliegenthart JFG (1986) Structural studies of the carbohydrate chains of human gamma-interferon. Eur J Biochem 156:651–654Google Scholar
  34. Parekh RB, Dwek RA, Edge CJ, Rademacker TW (1989) N-Glycosylation and the production of recombinant glycoproteins. Tibtech 7:117–122Google Scholar
  35. Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrica 33:305–325Google Scholar
  36. Shotwell MA, Jayme DW, Killberg MS, Oxender DL (1981) Neutral amino acid transport systems in Chinese hamster ovary cells. J Biol Chem 11:5422–5427Google Scholar
  37. Staheli J (1987) Variable screening and optimization. Dev Biol Stand 66:143–163Google Scholar
  38. Stryer L (1981) Biosynthesis of nucleotides. In: Biochemistry, 2nd edn. Freeman, San Francisco, pp 511–538Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Paula M. L. Castro
    • 1
  • Paul M. Hayter
    • 1
  • Andrew P. Ison
    • 2
  • Alan T. Bull
    • 1
  1. 1.Biological LaboratoryUniversity of KentCanterburyUK
  2. 2.The Advanced Centre for Biochemical EngineeringUniversity College LondonLondonUK

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