Differences in Optimal pH and Temperature for Cell Growth and Antibody Production Between Two Chinese Hamster Ovary Clones Derived from the Same Parental Clone

  • Kim, Sung-Hyun (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Lee, Gyun-Min (Department of Biological Sciences, Korea Advanced Institute of Science and Technology)
  • Published : 2007.05.31


To investigate clonal variations of recombinant Chinese hamster ovary(rCHO) clones in response to culture pH and temperature, serum-free suspension cultures of two antibody-producing CHO clones(clones A and B), which were isolated from the same parental clone by the limiting dilution method, were performed in a bioreactor at pH values in the range of 6.8-7.6, and two different temperatures, $33^{\circ}C\;and\;37^{\circ}C$. In regard to cell growth, clone A and clone B displayed similar responses to temperature, although their degree of response differed. In contrast, clones A and B displayed different responses to temperature in regard to antibody production. In the case of clone A, no significant increase in maximum antibody concentration was achieved by lowering the culture temperature. The maximum antibody concentration obtained at $33^{\circ}C$(pH 7.4) and $37^{\circ}C$(pH 7.0) were $82.0{\pm}2.6$ and $73.2{\pm}4.1{\mu}g/ml$, respectively. On the other hand, in the case of clone B, an approximately 2.5-fold increase in maximum antibody concentration was achieved by lowering the culture temperature. The enhanced maximum antibody concentration of clone B at $33^{\circ}C$($132.6{\pm}14.9{\mu}g/ml$ at pH 7.2) was due to not only enhanced specific antibody productivity but also to prolonged culture longevity. At $33^{\circ}C$, the culture longevity of clone A also improved, but not as much as that of clone B. Taken together, CHO clones derived from the same parental clone displayed quite different responses to culture temperature and pH with regards antibody production, suggesting that environmental parameters such as temperature and pH should be optimized for each CHO clone.


  1. Borys, M. C., D. I. H. Linzer, and E. T. Papoutsakis. 1993. Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Bio/Technology 11: 720-724 https://doi.org/10.1038/nbt0693-720
  2. Clark, K. J. R., F. W. R. Chaplin, and S. W. Harcum. 2004. Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA. Biotechnol. Prog. 20: 1888-1892 https://doi.org/10.1021/bp049951x
  3. Davies, J. and M. Reff. 2001. Chromosome localization and gene-copy-number quantification of three random integrations in Chinese hamster ovary cells and their amplified cell lines using fluorescence in situ hybridization. Biotechnol. Appl. Biochem. 33: 99-105 https://doi.org/10.1042/BA20000090
  4. Flintoff, W. F., E. Livingston, C. Duff, and R. G. Worton. 1984. Moderate-level gene amplification in methotrexateresistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene. Mol. Cell. Biol. 4: 69-76 https://doi.org/10.1128/MCB.4.1.69
  5. Fogolin, M. B., G. Forno, M. Nimtz, H. S. Conradt, M. Etcheverrigaray, and R. Kratje. 2005. Temperature reduction in cultures of hGM-CSF-expressing CHO cells: Effect on productivity and product quality. Biotechnol. Prog. 21: 17- 21 https://doi.org/10.1021/bp049825t
  6. Furukawa, K. and K. Ohsuye. 1998. Effect of culture temperature on a recombinant CHO cell line producing a C-terminal ${\alpha}-amidating$ enzyme. Cytotechnology 26: 153- 164 https://doi.org/10.1023/A:1007934216507
  7. Furukawa, K. and K. Ohsuye. 1999. Enhancement of productivity of recombinant ${\alpha}-amidating$ enzyme by lowtemperature culture. Cytotechnology 31: 85-94 https://doi.org/10.1023/A:1008059803038
  8. Jiang, Z., Y. Huang, and S. T. Sharfstein. 2006. Regulation of recombinant monoclonal antibody production in Chinese hamster ovary cells: A comparative study of gene copy number, mRNA level, and protein expression. Biotechnol. Prog. 22: 313-318 https://doi.org/10.1021/bp0501524
  9. Kaufmann, H., X. Mazur, M. Fussenegger, and J. E. Bailey. 1999. Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol. Bioeng. 63: 573-582 https://doi.org/10.1002/(SICI)1097-0290(19990605)63:5<573::AID-BIT7>3.0.CO;2-Y
  10. Kaufman, R. J. 1990. Use of recombinant DNA technology for engineering mammalian cells to produce proteins, pp. 15-69. In Lubiniecki, A. S. (ed.), Large-Scale Mammalian Cell Culture Technology. Marcel Dekker, New York
  11. Kim, J. H., S. W. Bae, H. J. Hong, and G. M. Lee. 1996. Decreased chimeric antibody productivity of KR12H-1 transfectoma during long-term culture results from decreased antibody gene copy number. Biotechnol. Bioeng. 51: 479- 487 https://doi.org/10.1002/(SICI)1097-0290(19960820)51:4<479::AID-BIT11>3.3.CO;2-2
  12. Kim, N. Y., Y. J. Lee, H. J. Kim, J. H. Choi, J. K. Kim, K. H. Chang, J. H. Kim, and H. J. Kim. 2004. Enhancement of erythropoietin production from Chinese hamster ovary (CHO) cells by introduction of the urea cycle enzymes, carbamoyl phosphate synthetase 1 and ornithine transcarbamylase. J. Microbiol. Biotechnol. 14: 844-851
  13. Kim, S. J., N. S. Kim, C. J. Ryu, H. J. Hong, and G. M. Lee. 1998. Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol. Bioeng. 58: 73-84 https://doi.org/10.1002/(SICI)1097-0290(19980405)58:1<73::AID-BIT8>3.0.CO;2-R
  14. Na, K. H., S. C. Kim, K. S. Seo, S. H. Lee, W. B. Kim, and K. C. Choon. 2005. Purification and characterization of recombinant human follicle stimulating hormone produced by Chinese hamster ovary cells. J. Microbiol. Biotechnol. 15: 395-402
  15. Ozturk, S. S. and B. O. Palsson. 1990. Chemical decomposition of glutamine in cell culture media: Effect of media type, pH, and serum concentration. Biotechnol. Prog. 6: 121-128 https://doi.org/10.1021/bp00002a005
  16. Park, J. H., S. R. Yu, J. S. Yoon, and K. H. Baek. 2005. Highlevel expression of recombinant human bone morphogenetic protein-4 in Chinese hamster ovary cells. J. Microbiol. Biotechnol. 15: 1397-1401
  17. Renard, J. M., R. Spagnoli, C. Mazier, M. F. Salles, and E. Mandine. 1988. Evidence that monoclonal antibody production kinetics is related to the integral of viable cells in batch systems. Biotechnol. Lett. 10: 91-96 https://doi.org/10.1007/BF01024632
  18. Sauer, P. W., J. E. Burky, M. C. Wesson, H. D. Sternard, and L. Qu. 2000. A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies. Biotechnol. Bioeng. 67: 585-597 https://doi.org/10.1002/(SICI)1097-0290(20000305)67:5<585::AID-BIT9>3.0.CO;2-H
  19. Tritsch, G. L. and G. E. Moore. 1962. Spontaneous decomposition of glutamine in cell culture media. Exp. Cell. Res. 28: 360-364 https://doi.org/10.1016/0014-4827(62)90290-2
  20. Yoon, S. K., Y. H. Ahn, I. C. Kwon, K. Han, and J. Y. Song. 1998. Influence of reducing agents on the secretion rate of recombinant erythropoietin from CHO cells. Biotechnol. Lett. 20: 101-104 https://doi.org/10.1023/A:1005303802776
  21. Yoon, S. K., S. L. Choi, J. Y. Song, and G. M. Lee. 2005. Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and $37.0^{\circ}C.$ Biotechnol. Bioeng. 22: 345-356
  22. Yoon, S. K., S. L. Choi, J. Y. Song, and G. M. Lee. 2004. Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: Clonal variation. Biotechnol. Prog. 20: 1683-1688 https://doi.org/10.1021/bp049847f