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Cytotechnology

, Volume 44, Issue 3, pp 103–114 | Cite as

Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside microcarriers in protein-free media

  • J. S. Ballez
  • J. Mols
  • C. Burteau
  • S. N. Agathos
  • Y. J. Schneider
Article

Abstract

We have recently developed a protein-free medium (PFS) able to support the growth of Chinese hamster ovary (CHO) cells in suspension. Upon further supplementation with some plant protein hydrolysates, medium performances reached what could be observed in serum-containing media [Burteau et al. In Vitro Cell. Dev. Biol.-Anim. 39 (2003) 291]. Now, we describe the use of rice and wheat protein hydrolysates, as non-nutritional additives to the culture medium to support productivity and cell growth in suspension or in microcarriers. When CHO-320 cells secreting recombinant interferon-gamma (IFN-γ) were cultivated in suspension in a bioreactor with our PFS supplemented with wheat hydrolysates, the maximum cell density increased by 25% and the IFN-γ secretion by 60% compared to the control PFS. A small-scale perfusion system consisting of CHO-320 cells growing on and inside fibrous microcarriers under discontinuous operation was first developed. Under these conditions, rice protein hydrolysates stimulated recombinant IFN-γ secretion by 30% compared to the control PFS. At the bioreactorscale, similar results were obtained but when compared to shake-flasks studies, nutrients, oxygen or toxic by-products gradients inside the microcarriers seemed to be the main limitation of the system. An increase of the perfusion rate to maintain glucose concentration over 5.5 mM and dissolved oxygen (DO) at 60% was able to stimulate the production of IFN-γ to a level of 6.6 μg h−1 g−1 of microcarriers after 160 h when a cellular density of about 4 × 108 cell g−1 of carriers was reached.

Keywords

Bioreactor CHO-320 cells Fibra-cel® Interferon-gamma (IFN-γ) Microcarriers Perfusion Plant protein hydrolysates (peptones) Protein-free 

Abbreviations

AU

arbitrary units

CHO

Chinese hamster ovary

DO

dissolved oxygen

LDH

lactate deshydrogenase

IFN-y

interferon-gamma

PFA

protein-free medium for adhesion

PFS

protein-free medium for suspension

RP

rice protein hydrolysates

WP

wheat protein hydrolysates.

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References

  1. Agathos, S.N. 1996Insect cell bioreactorsCytotechnology20173189CrossRefGoogle Scholar
  2. Borys, M.C., Linzer, D.I.H., Papoutsakis, E.T. 1994Ammonia affects the glycisylation patterns of recombinant mouse placental lactogen-1 by Chinese hamster ovary cells in a pH-dependent mannerBiotechnol. Bioeng.43505514CrossRefGoogle Scholar
  3. Bragonzi, A., Distefano, G., Buckberry, L.D., Acerbis, G., Foglieni, C., Lamotte, D., Campi, G., Marc, A., Soria, M.R., Jenkins, N., Monaco, L. 2000A new Chinese hamster ovary cell line expressing a 2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteinsBiochim. Biophys. Acta1474273282PubMedGoogle Scholar
  4. Burteau, C.C., Verhoeye, F.R., Mols, J.F., Ballez, J.-S., Agathos, S.N., Schneider, Y.-J. 2003Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interféron-γ-producing CHO cell lineIn Vitro Cell. Dev. Biol.-Animal39291296CrossRefGoogle Scholar
  5. Castro, P.M., Hayter, P.M., Ison, A.P., Bull, A.T. 1992Application of a statistical design to the optimisation of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cellsAppl. Microbiol. Biotechnol.388490CrossRefPubMedGoogle Scholar
  6. Castro, P.M., Ison, A.P., Hayter, P.M., Bull, A.T. 1995The macroheterogeneity of recombinant human interferon-gamma produced by Chinese-hamster ovary cells is affected by the protein and lipid content of the culture mediumBiotechnol Appl Biochem.2187100PubMedGoogle Scholar
  7. Chu, L., Robinson, D.K. 2001Industrial choices for protein production by large-scale cell cultureCurr. Opin. Biotechnol.12180187CrossRefPubMedGoogle Scholar
  8. Coppen, S.R., Newsam, R., Bull, A.T., Baines, A.J. 1995Heterogeneity within populations of recombinant chinese hamster ovary cells expressing human interferon-γBiotechnol. Bioeng.46147158CrossRefGoogle Scholar
  9. Fekkes, D., van Dalen, A., Edelman, M., Voskuilen, A. 1995Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehydeJ Chromatogr B Biomed Appl.669177186CrossRefPubMedGoogle Scholar
  10. Franek, F., Hohenwarter, O., Katinger, H. 2000Plant protein Hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells culturesBiotechnol. Prog.16688692PubMedGoogle Scholar
  11. Franek, F., Katinger, H. 2002Specific effects of synthetic oligopeptides on cultured animal cellsBiotechnol. Prog.18155158PubMedGoogle Scholar
  12. Goldman, M.H., James, D.C., Rendall, M., Ison, A.P., Hoare, M., Bull, A.T. 1998Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cellsBiotechnol. Bioeng.60596607CrossRefGoogle Scholar
  13. Gu, X., Xie, L., Harmon, B.J., Wang, D.I.C. 1997Influence of primatone RL supplementation on sialylation of recombinant human interferon-γ produced by Chinese hamster ovary cell culture using serum-free mediaBiotechnol. Bioeng.56352360CrossRefGoogle Scholar
  14. Hayter, P.M., Curling, E.M., Gould, M.L., Baines, A.J., Jenkins, N., Salmon, I., Strange, P.G., Bull, A.T. 1993The effect of the dilution rate on CHO cell physiology and recombinant interferon-γ production in glucose-limited chemostat cultureBiotechnol. Bioeng.4210771085CrossRefGoogle Scholar
  15. Hayter, P.M., Curling, E.M., Baines, A.J., Jenkins, N., Salmon, I., Strange, P.G., Bull, A.T. 1991Chinese hamster ovary cell growth and interferon production kinetics in stirred-batch cultureAppl. Microbiol. Biotechnol.34559564CrossRefPubMedGoogle Scholar
  16. Heidemann, R., Zhang, C., Qi, H., Rule, J.L., Rozales, C., Park, S., Chuppa, S., Ray, M., Michaels, J., Konstantinov, K., Naveh, D. 2000The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cellsCytotechnology32157167CrossRefGoogle Scholar
  17. Hu, Y.-C., Kaufman, J., Cho, M.W., Golding, H., Shiloach, J. 2000Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression systemBiotechnol. Prog.16744750PubMedGoogle Scholar
  18. Ikonomou, L., Drugmand, J.-C., Bastin, G., Schneider, Y-J., Agathos, S.N. 2002Microcarrier culture of lepidopteran cell lines : implications for growth and recombinant protein productionBiotechnol. Prog.1813451355CrossRefPubMedGoogle Scholar
  19. Jan, D.C.-H., Jones, S.J., Emery, A.N., Al-Rubeai, M. 1994Peptonea low-cost growth-promoting nutrient for intensive animal cell cultureCytotechnology161726CrossRefPubMedGoogle Scholar
  20. Kaufman, J.B., Wang, G., Zhang, W., Valle, M.A., Shiloach, J. 2000Continuous production and recovery of recombinant Ca2+ binding receptor from HEK 293 cells using perfusion through a packed bed bioreactorCytotechnology33311CrossRefGoogle Scholar
  21. Keen, M.J., Rapson, N.T. 1995Development of a serum-free culture medium for the large-scale production of recombinant protein from a Chinese hamster ovary cell lineCytotechnology17153163CrossRefGoogle Scholar
  22. Kompier, R., Kislev, N., Segal, I., Kadouri, A. 1991Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cellsEnzyme Microb. Technol.13822827CrossRefPubMedGoogle Scholar
  23. Laemmli, U.K. 1970Cleavage of structural proteins during the assembly of the head of bacteriophage T4Nature227680685PubMedGoogle Scholar
  24. Landauer, K., Wiederkum, S., Dürrschmid, M., Klug, H., Simic, G., Blüml, G., Doblhoff-Dier, O. 2003Influence of carboxymethyl dextran and ferric citrate on the adhesion of CHO cells on microcarriersBiotechnol. Prog.192129CrossRefPubMedGoogle Scholar
  25. Lao, M.-S., Toth, D. 1997Effects of ammonium and lactate on growth and metabolism of a recombinant Chinese hamster ovary cell cultureBiotechnol. Prog.13688691CrossRefPubMedGoogle Scholar
  26. Lee, G.M., Chuck, A.S., Palsson, B.O. 1993Cell culture conditions determine the enhancement of specific monoclonal antibody productivity of calcium alginate-entrapped S3H5/γ2bA2 hybridoma cellsBiotechnol. Bioeng.41330340CrossRefGoogle Scholar
  27. Lee, G.M., Palsson, B.O. 1990Immobilization can improve the stability of hybridoma antibody productivity in serum-free mediaBiotechnol. Bioeng.3610491055CrossRefGoogle Scholar
  28. Leelavatcharamas, V., Emery, A.N., Al-Rubeai, M. 1999Use of cell cycle analysis to characterise growth and interferon-γ production in perfusion culture of CHO cellsCytotechnology305669CrossRefGoogle Scholar
  29. Merten, O.-W., Cruz, P.E., Rochette, C., Geny-Fiamma, C., Bouquet, C., Gonçalves, D., Danos, O., Carrondo, M.J. 2001Comparison of different bioreactor systems for the production of high titer retroviral vectorsBiotechnol. Prog.17326335CrossRefPubMedGoogle Scholar
  30. Nishijima, K.I., Fujiki, T., Kojima, H., Iijima, S. 2000The effects of cell adhesion on the growth and protein productivity of animal cellsCytotechnology33147155CrossRefGoogle Scholar
  31. Peshwa, M.V., Kyung, Y.-S., McClure, D.B., Hu, W.-S. 1993Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration on spatial distribution of viabilityBiotechnol. Bioeng.41179187CrossRefGoogle Scholar
  32. Racher, A.J., Fooks, A.R., Griffiths, J.B. 1995Culture of 293 cells in different culture systems: cell growth and recombinant adenovirus productionBiotechnol. Tech.9169174CrossRefGoogle Scholar
  33. Rasmussen, B., Davis, R., Thomas, J., Reddy, P. 1998Isolation, characterization and recombinant protein expression in Veggie-CHO: a serum-free CHO host cell lineCytotechnology283142CrossRefGoogle Scholar
  34. Schneider, Y.-J. 1989Optimization of hybridoma cell growth and monoclonal antibody secretion in chemically defined serum- and protein-free culture mediumJ. Immunol. Meth.1166577CrossRefGoogle Scholar
  35. Wang, G., Zhang, W., Jacklin, C., Freedman, D., Eppstein, L., Kadouri, A. 1992Modified CelliGen-packed bed bioreactor for hybridoma cell culturesCytotechnology94149PubMedGoogle Scholar
  36. Watson, E., Shah, B., Leiderman, L., Hsu, Y.-R., Karkare, S., Lu, H.S., Lin, F.-K. 1994Comparison of N-linked oligosaccharides of recombinant human tissue kalikrein produced by Chinese hamster ovary cells on microcarrier beads and in serum-free suspension cultureBiotechnol. Prog.103944PubMedGoogle Scholar
  37. Yang, M., Butler, M. 2000Effects of ammonia on CHO cell growtherythropoietin production and glycosylationBiotechnol. Bioeng.68370380CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • J. S. Ballez
    • 1
  • J. Mols
    • 1
  • C. Burteau
    • 1
  • S. N. Agathos
    • 2
  • Y. J. Schneider
    • 1
  1. 1.Laboratoire de Biochimie cellulaireInstitut desSciences de la Vie and Université catholique de Louvain
  2. 2.Unité de Génie biologiqueInstitut desSciences de la Vie and Université catholique de LouvainLouvain-la-NeuveBelgium

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