Cytotechnology

, Volume 6, Issue 3, pp 197–208 | Cite as

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies

  • Yasushi Shintani
  • Yoh-Ichiro Kohno
  • Hidekazu Sawada
  • Kazuaki Kitano
Original Research Paper

Abstract

Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 mg/l of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 mg/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo MTM Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilized-perfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.

Key words

growth-associated production hepatitis B virus human-human hybridoma immobilized culture monoclonal antibody perfusion culture 

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References

  1. Altshuler, GL, Dziewulski, DM, Sowek, JA and Belfort, G (1986) Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators. Biotech. Bioeng. 28: 646–658.Google Scholar
  2. Buell, DN and Fahey, JL (1969) Limited period of gene expression in immunoglobulin-synthesizing cells. Science 164: 1524–1525.Google Scholar
  3. Harada, K, Ichimori, Y, Sasano, K, Sasai, S, Kitano, K, Iwasa, S, Tsukamoto, K and Sugino, Y (1989) Human-human hybridomas secreting hepatitis B virus-neutralizing antibodies. Bio/Technology 7: 374–377.Google Scholar
  4. Hopkinson, J (1985) Hollow fiber cell culture systems for economical cell-product manufacturing. Bio/Technology 3: 225–230.Google Scholar
  5. Ichimori, Y, Sasano, K, Itoh, H, Hitotsumachi, S, Kimura, Y, Kaneko, K, Kida, M and Tsukamoto, K (1985). Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus antigen. Biochem. Biophys. Res. Commun. 129: 26–33.Google Scholar
  6. Ichimori, Y, Harada, K, Hitotsumachi, S and Tsukamoto, K (1987). Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen. Biochem. Biophys. Res. Commun. 142: 805–812.Google Scholar
  7. Kitano, K, Shintani, Y, Ichimori, Y, Tsukamoto, K, Sasai, S and Kida, M (1986). Production of human monoclonal antibodies by heterohybridomas. Appl. Microbiol. Biotechnol 24: 282–286.Google Scholar
  8. Kitano, K, Iwamoto, K, Shintani, Y and Akiyama, S (1988). Effective production of a human monoclonal antibody against tetanus toxoid by selection of high productivity clones of a heterohybridoma. J. Immunol. Methods 109: 9–16.Google Scholar
  9. Kitano K, Iwamoto K, Shintani Y and Sasada R (1990). Improvement in cell lines for effective production of human monoclonal antibodies by human-human hybridomas. Proceeding of the 2nd annual meeting of Japanese Association for Animal Cell Technology (JAACT '89) in press.Google Scholar
  10. Knazek, RA, Gullino, PM, Kohler, PO and Dedrick, RL (1972) Cell culture on artificial capillaries: an approach to tissue growthin vitro. Science 178: 65–67.Google Scholar
  11. Kwong, CW, Hsieh, JH, Syu, MJ and Chou, SS (1989). Antihepatitis B surface antigen monoclonal antibody IgM production in suspension and immobilized cell bioreactors. Biotechnology Letters 2: 377–382Google Scholar
  12. Lydersen, BK, Pugh, GG, Paris, MS, Sharma, BP and Noll, LA (1985). Ceramic matrix for large scale animal cell culture. Bio/Technology 3: 63–67.Google Scholar
  13. Merten, OW (1987). Concentrating mammalian cells: 1. largescale animal cell culture. Trends Biotechnol. 5: 230–237.Google Scholar
  14. Merten, OW (1988). Batch production and growth kinetics of hybridomas. Cytotechnology 1: 113–121.Google Scholar
  15. Murata, M, Eto, Y and Shibai, H (1988). Large-scale production of erythroid differantiation factor(EDF) by gene engineering chinese hamster ovary (CHO) cells in suspension culture. J. Ferment. Technol. 66: 501–507.Google Scholar
  16. Ray, NG, Karkare, SB and Runstadler, PW (1989). Cultivation of hybridoma cells in continuous cultures: Kinetics of growth and product formation. Biotechnol. Bioeng. 33: 724–730.Google Scholar
  17. Reuveny, S, Velex, D, Miller, L and Macmillan, JD (1986). Comparison of cell propagation methods for their effect on monoclonal antibodies yield in fermenters. J. Immunol. Method. 86: 61–69.Google Scholar
  18. Shintani, Y, Iwamoto, K and Kitano, K (1988). Polyethylene glycols for promoting the growth of mammalian cells. Appl. Microbiol. Biotechnol. 27: 533–537.Google Scholar
  19. Suzuki, E and Ollis, DF (1989). Cell cycle model for antibody production kinetics. Biotechnol. Bioeng. 34: 1398–1402.Google Scholar
  20. Takazawa, Y, Tokashiki, M, Hamamoto, K and Murakami, H (1988). High cell density perfusion culture of hybridoma cells recycling high molecular weight components. Cytotechnology 1: 171–175.Google Scholar
  21. Takazawa, Y and Tokashiki, M (1989). Production of human-mouse chimeric antibody by high cell density perfusion culture. Cytotechnology 2: 95–101.Google Scholar

Copyright information

© Kluwer Academic Publishers 1991

Authors and Affiliations

  • Yasushi Shintani
    • 1
  • Yoh-Ichiro Kohno
    • 1
  • Hidekazu Sawada
    • 1
  • Kazuaki Kitano
    • 1
  1. 1.Microbiology Research Laboratories, Research & Development DivisionTakeda Chemical Industries Ltd.OsakaJapan

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