Skip to main content
SpringerLink
Log in

The combined effect of sodium butyrate and low culture temperature on the production, sialylation, and biological activity of an antibody produced in CHO cells

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

Cell cultures containing 0 ∼ 5 mM sodium butyrate (NaBu) and grown at 30 and 37°C were conducted to investigate the combined effect of NaBu and low temperature on the quantity and quality of an antibody production in CHO cells. Although NaBu addition decreased cell viability by apoptosis in a dose-dependent manner at both 30 and 37°C, the onset of significant apoptosis induced by NaBu was delayed by lowering culture temperature. The highest specific antibody productivity (q p) of 23.26 pg/cell/day was obtained in the culture containing 2 mM NaBu at 30°C; however, the highest antibody concentration of 167.84 mg/L was achieved in the culture containing 1 mM NaBu at 30°C, as the detrimental effect of further NaBu addition on cell growth compromised its beneficial effect on q p. Moreover, protein quality with respect to the total sialic acid content and Nglycolylneuraminic acid (Neu5Gc) level was evaluated. There were no apparent changes regarding the total sialic acid content of the antibody, but manipulation of cultures with NaBu treatment or (and) low culture temperature did decrease Neu5Gc levels by 5 ∼ 10%. Biological activity of the antibody was also assessed, and no obvious changes were observed. Collectively, the simultaneous application of NaBu and low culture temperature was an effective way to extend culture period and enhance final antibody concentration, without compromising the sialic acid content or biological activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Wurm, F. M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22: 1393–1398.

    Article  CAS  Google Scholar 

  2. Furukawa, K. and K. Ohsuye (1998) Effect of culture temperature on a recombinant CHO cell line producing a C-terminal α-amidating enzyme. Cytotechnol. 26: 153–164.

    Article  CAS  Google Scholar 

  3. Fogolín, M. B., R. Wagner, M. Etcheverrigaray, and R. Kratje (2004) Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells. J. Biotechnol. 109: 179–191.

    Article  Google Scholar 

  4. Yoon, S. K., J. Y. Song, and G. M. Lee (2003) Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese Hamster Ovary cells. Biotechnol. Bioeng. 82: 289–298.

    Article  CAS  Google Scholar 

  5. Ahn, W. S., J. J. Jeon, Y. R. Jeong, S. J. Lee, and S. K. Yoon (2008) Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells. Biotechnol. Bioeng. 101: 1234–1244.

    Article  CAS  Google Scholar 

  6. Virginie, M. B., S. Hisiger, C. Durand, M. Perrier, and M. Jolicoeur (2009) Na-butyrate sustains energetic states of metabolism and t-PA productivity of CHO cells. J. Biosci. Bioeng. 108: 160–167.

    Article  Google Scholar 

  7. Mimura, Y., J. Lund, S. Church, S. Dong, J. Li, M. Goodall, and R. Jefferis (2001) Butyrate increases production of human chimeric IgG in CHO-K1 cells whilst maintaining function and glycoform profile. J. Immunol. Methods. 247: 205–216.

    Article  CAS  Google Scholar 

  8. Yoon, S. K., J. K. Hong, and G. M. Lee (2004) Effect of simultaneous application of stressful culture conditions on specific productivity and heterogeneity of erythropoietin in Chinese Hamster Ovary cells. Biotechnol. Prog. 20: 1293–1296.

    Article  CAS  Google Scholar 

  9. Sung, Y. H., Y. J. Song, S. W. Lim, J. Y. Chung, and G. M. Lee (2004) Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells. J. Biotechnol. 112: 323–335.

    Article  CAS  Google Scholar 

  10. Kim, N. S. and G. M. Lee (2000) Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production. Biotechnol. Bioeng. 71: 184–193.

    Article  CAS  Google Scholar 

  11. Kim, N. S. and G. M. Lee (2001) Inhibition of sodium butyrateinduced apoptosis in recombinant Chinese Hamster Ovary cells by constitutively expressing antisense RNA of caspase-3. Biotechnol. Bioeng. 78: 217–228.

    Article  Google Scholar 

  12. Sung, Y. H., S. J. Hwang, and G. M. Lee (2005) Influence of down-regulation of caspase-3 by siRNAs on sodium-butyrateinduced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin. Metab. Eng. 7: 457–466.

    Article  CAS  Google Scholar 

  13. Sung, Y. H., J. S. Lee, S. H. Park, J. Koo, and G. M. Lee (2007) Influence of co-down-regulation of caspase-3 and caspase-7 by siRNAs on sodium butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin. Metab. Eng. 9: 452–464.

    Article  CAS  Google Scholar 

  14. Oh, H. K., M. K. So, J. Yang, H. C. Yoon, J. S. Ahn, J. M. Lee, J. T. Kim, J. U. Yoo, and T. H. Byun (2005) Effects of N-Acetylcysteine on butyrate-treated Chinese Hamster Ovary cells to improve the production of recombinant human interferon-β-1a. Biotechnol. Prog. 21: 1154–1164.

    Article  CAS  Google Scholar 

  15. Chuppa, S., Y. S. Tsai, S. Yoon, S. Shackleford, C. Rozales, R. Bhat, G. Tsay, C. Matanguihan, K. Konstantinov, and D. Naveh (1997) Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells. Biotechnol. Bioeng. 55: 328–338.

    Article  CAS  Google Scholar 

  16. 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.

    Article  CAS  Google Scholar 

  17. Kim, N. S., K. H. Chang, B. S. Chung, S. H. Kim, J. H. Kim, and G. M. Lee (2003) Characterization of humanized antibody produced by apoptosis-resistant CHO cells under sodium butyrateinduced condition. J. Microbiol. Biotechnol. 13: 926–936.

    CAS  Google Scholar 

  18. Sethuraman, N. and T. A. Stadheim (2006) Challenges in therapeutic glycoprotein production. Curr. Opin. Biotechnol. 17: 341–346.

    Article  CAS  Google Scholar 

  19. Lamari, F. N. and N. K. Karamnnos (2002) Separation methods for sialic acids and critical evaluation of their biologic relevance. J. Chrom. B. 781: 3–19.

    Article  CAS  Google Scholar 

  20. Irie, A., S. Koyama, Y. Kozutsumi, T. Kawasaki, and A. Suzuki (1998) The molecular basis for the absence of N-glycolylneuraminic acid in humans. J. Biol. Chem. 273: 15866–15871.

    Article  CAS  Google Scholar 

  21. Varki, A. (2001) N-glycolylneuraminic acid deficiency in humans. Biochimie. 83: 615–622.

    Article  CAS  Google Scholar 

  22. Lipscomb, M. L., L. A. Palomares, V. Hernandez, O. T. Ramirez, and D. S. Kompala (2005) Effect of production method and gene amplification on the glycosylation pattern of a secreted reporter protein in CHO cells. Biotechnol. Prog. 21: 40–49.

    Article  CAS  Google Scholar 

  23. Zanghi, J. A., A. E. Schmelzer, T. P. Mendoza, R. H. Knop, and W. M. Miller (1999) Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO2 or pH. Biotechnol. Bioeng. 65: 182–191.

    Article  CAS  Google Scholar 

  24. 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.

    Article  Google Scholar 

  25. Gawlitzek, M., T. Ryll, J. Lofgren, and M. B. Sliwkowski (2000) Ammonium alters N-glycan structures of recombinant TNFRIgG: Degradative versus biosynthetic mechanisms. Biotechnol. Bioeng. 68: 637–646.

    Article  CAS  Google Scholar 

  26. Anumula, K. R. (1995) Rapid quantitative determination of sialic acids in glycoproteins by high-performance liquid chromatography with a sensitive fluorescence detection. Anal. Biochem. 230: 24–30.

    Article  CAS  Google Scholar 

  27. Wagner, A., A. Marc, J. M. Engasser, and A. Einsele (1992) The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors. Biotechnol. Bioeng. 39: 320–326.

    Article  CAS  Google Scholar 

  28. Sunley, K. and M. Butler (2010) Strategies for the enhancement of recombinant protein production from mammalian cells by growth arrest. Biotechnol. Adv. 28: 385–394.

    Article  CAS  Google Scholar 

  29. Hendrick, V., P. Winnepenninckx, C. Abdelkafi, O. Vandeputte, M. Cherlet, T. Marique, G. Renemann, A. Loa, G. Kretzmer, and J. Werenne (2001) Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: A cell cycle phases analysis. Cytotechnol. 36: 71–83.

    Article  CAS  Google Scholar 

  30. Jiang, Z. and S. T. Sharfstein (2008) Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells improving gene accessibility. Biotechnol. Bioeng. 100: 189–194.

    Article  CAS  Google Scholar 

  31. Yee, J. C., M. L. Gatti, R. J. Philp, M. Yap, and W. S. Hu (2008) Genomic and proteomic exploration of CHO and Hybridoma cells under sodium butyrate treatment. Biotechnol. Bioeng. 99: 1186–1204.

    Article  CAS  Google Scholar 

  32. Moore, A., J. Mercer, G. Dutina, C. J. Donahue, K. D. Bauer, J. P. Mather, T. Etcheverry, and T. Ryll (1997) Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultures. Cytotechnol. 23:47–54.

    Article  CAS  Google Scholar 

  33. Takeuchi, M., N. Inoue, T. W. Strickland, M. Kubota, M. Wada, R. Shimizu, S. Hoshi, H. Kozutsumi, S. Takeuchi, and A. Kobata (1989) Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA. 86: 7819–7822.

    Article  CAS  Google Scholar 

  34. Yamaguchi. K., K. Akai, G. Kawanishi, M. Ueda, S. Masuda, and R. Sasaki (1991) Effect of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties. J. Biol. Chem. 266: 20434–20439.

    CAS  Google Scholar 

  35. Malykh, Y. N., L. Shaw, and R. Schauer (1998) The role of CMPN-acetylneuraminic acid hydroxylase in determining the level of N-glycolylneuraminic acid in porcine tissues. Glycoconj. J. 15: 885–893.

    Article  CAS  Google Scholar 

  36. Tangvoranuntakul, P., P. Gagneux, S. Diaz, M. Bardor, A. Varki, and E. Muchmore (2003) Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc. Natl. Acad. Sci. USA. 100: 12045–12050.

    Article  CAS  Google Scholar 

  37. Borys, M. C., N. G. Dalal, N. R. Abu-Absi, S. F. Khattak, Y. Jing, Z. H. Xing, and Z. J. Li (2010) Effects of culture conditions on N-Glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells. Biotechnol. Bioeng. 105: 1048–1057.

    CAS  Google Scholar 

  38. Shaw, L. and R. Schauer (1988) The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid. Biol. Chem. Hoppe Seyler 369: 477–486.

    Article  CAS  Google Scholar 

  39. Shaw, L. and R. Schauer (1989) Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver. Biochem. J. 263: 355–363.

    CAS  Google Scholar 

  40. Grampp, G. E., T. K. Blumen, K. Kelly, P. Derby, L. A. Sleeman, and D. Hettwer (1994) Environmental control of sialic acid composition in glycoproteins secreted by mammalian cells. Presented at the Cell Culture Engineering IV meeting. San Diego, CA.

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200-237, China

    Fei Chen, Tianci Kou, Li Fan, Yan Zhou, Zhaoyang Ye, Liang Zhao & Wen-Song Tan

Authors
  1. Fei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tianci Kou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Li Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhaoyang Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Liang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wen-Song Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Liang Zhao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, F., Kou, T., Fan, L. et al. The combined effect of sodium butyrate and low culture temperature on the production, sialylation, and biological activity of an antibody produced in CHO cells. Biotechnol Bioproc E 16, 1157–1165 (2011). https://doi.org/10.1007/s12257-011-0069-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-011-0069-8

Keywords

Search

Navigation