Abstract
We compared serum and protein-free cultures ofa ras-amplified recombinant BHK-21 cell line(ras-rBHK-IgG), which hyperproduces a lungcancer specific recombinant human monoclonal antibody. Ras-rBHK-IgG cells were shown to grow well, evenin protein-free medium and to be morphologicallysimilar to cells cultured in serum containing medium. However, the growth rate of ras-rBHK-IgG cellswas considerably slower in protein-free medium, whichresults in a longer maintenance period compared with cells cultured in serum containing medium. In addition, it was found that antibody production in protein-free culture had a ten times higher maximum than cells cultured in serum containing medium. On theother hand, in high density culture, using the hollowfiber bioreactor system, ras-rBHK-IgG cellscould be maintained for a month in protein-freeculture in contrast with serum culture, which onlylasted for half a month. However, the markedincrease of antibody production was not observed. A total amount of about 15 mg of the recombinantantibody, obtained in protein-free culture, was abouttwo times of that obtained in serum culture, and wasshown to be reactive to lung cancer cells in tissue. From these properties in protein-free medium, it isconcluded that protein-free culture of ras-rBHK-IgG cells is suitable for middle scaleproduction of recombinant human monoclonal antibody.
Similar content being viewed by others
References
Bebbington CR, Renner G, Thomason S, King D, Abrams D and Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology 10: 169–175.
Friedman JS, Cofer CL, Anderson CL, Kushner JA, Gray PP, Champman GE, Stuart MC, Lazaus L, Shine J and Kushner PJ (1989) High expression in mammalian cells without amplification. Bio/Technology 7: 359–369.
Goldberg MA, Glass GA, Cunningham JM and Bunn HF (1987) The regulated expression of erythropoietin by two human hepatoma cell lines. Proc Natl Acad Sci USA 84: 7972–7976.
Inoue Y, López LB, Kawamoto S, Arita N, Teruya K, Seki K, Shoji M, Kamei M, Hashizume S, Shiozawa Y, Tachibana H, Ohashi H, Yasumoto K, Nagashima A, Nakahashi H, Suzuki T, Imai T, Nomoto K, Takenouchi M, Katakura Y and Shirahata S (1996a) Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium. Biosci Biotech Biochem 60: 811–817.
Inoue Y, Kawamoto S, Seki K, Teruya K, Mochizuki K, Kato M, Hashizume S, Yasumoto K, Nagashima A, Nakahashi H, Suzuki T, Imai T, Nomoto K and Shirahata S (1996b) Production of a recombinant human monoclonal antibody using a novel hollow fiber bioreactor system. J Ferment Bioeng 81: 466–469.
Kong C, Gentz R and Zhang J (1998) Long-term stable production of monocyte-colony inhibitor factor (M-CIF) from CHO microcarrier perfusion cultures. Cytotechnology 26: 131–138.
McKay IA, Malone P, Marshall CJ and Hall A (1986) Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor. Mol Cell Biol 6: 3382–3387.
Miyazaki Y, Nishimoto S, Sasaki T and Sugahara T (1998) Spermine enhances IgM productivity of human-human hybridoma HB4C5 cells and human peripheral blood lymphocytes. Cytotechnology 26: 111–118.
Murakami F, Sasaki T and Sugahara T (1997) Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes. Cytotechnology 24: 177–182.
Murakami H, Hashizume S, Ohashi H, Shinohara K, Yasumoto K, Nomoto K and Omura H (1985) Human-human hybridomas secreting antibodies specific to lung carcinoma. In Vitro Cell Develop Biol 21: 593–596.
Schlapfer BS, Scheibler M, Holtorf A-P, Nguyen HV and Pluschke G (1995) Development of optimized transfectoma cell lines for production of chimeric antibodies in hollow fiber culture system. Biotechnol Bioeng 45: 310–319.
Shirahata S, Rawson C, Loo D, Chang Y-J and Barnes D (1990) Ras and neu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum-free mouse embryo cells. J Cell Physiol 144: 69–76.
Shoji M, Kawamoto S, Seki K, Teruya Y, Setoguchi Y, Mochizuki K, Kato M, Hashizume S, Hanagiri T, Yoshimatsu T, Nakanishi K, Yasumoto K, Nagashima A, Nakahashi H, Suzuki T, Imai T, Shirahata S, Nomoto K and Murakami H (1996) Lung cancer-reacting human recombinant antibody AE6F4: potential usefulness in the sputum cytodiagnosis. Hum Antibodies Hybridomas 7: 27–36.
Shoji M, Kawamoto S, Setoguchi Y, Mochizuki K, Honjoh T, Kato M, Hashizume S, Hanagiri T, Yoshimatsu T, Nakanishi K, Yasumoto K, Nagashima A, Nakahashi H, Suzuki T, Imai T, Nomoto K and Murakami H (1994) The 14-3-3 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4. Hum Antibodies Hybridomas 5: 123–130.
Sporn MB and Roberts AB (1985) Autocrine growth factors and cancer. Nature 313: 745–747.
Takahashi K, Tereda S, Ueda H, Makishima F and Suzuki E (1994) Growth rate suppression of cultured mammlian cells enhances protein productivities. Cytotechnology 15: 57–64.
Teruya K, Yano T, Shirahata S, Watanabe J, Osada K, Ohashi H, Tachibana H, Kim E-H and Murakami H (1995) Ras amplification in BHK-21 cells produces a host cell line for further rapid establishment of recombinant protein hyper-producing cell lines. Biosci Biotech Biochem 59: 341–344.
Xiao C, Zhang Y, Kong W and Wang H (1992) Pilot production of high-titer interferon and virus with a microcarrier suspension culture system. Chin Med Sci J 7: 67–71.
Yamaguchi M, Shirai Y, Inouye Y, Shoji M, Kamei M, Hashizume S and Shirahata S (1997) Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles. Cytotechnology 23: 5–12.
Yano T, Teruya K, Shirahata S, Watanabe J, Osada K, Tachibana H, Ohashi H, Kim E-H and Murakami H (1994) Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells. Cytotechnology 16: 167–178.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Inoue, Y., Kawamoto, S., Shoji, M. et al. Properties of ras-amplified recombinant BHK-21 cells in protein-free culture. Cytotechnology 33, 21–26 (2000). https://doi.org/10.1023/A:1008163312841
Issue Date:
DOI: https://doi.org/10.1023/A:1008163312841