DOI QR코드

DOI QR Code

Effects of Low-Serum Medium and Various Culture Additives on Production of Recombinant Human Erythropoietin in CHO Cell Cultures

CHO 세포 배양을 통한 Recombinant Human Erythropoietin의 생산에서 저혈청 배지와 배양 첨가물질이 미치는 영향

  • Lee, Kyung-Sun (Department of Biological Engineering, Inha University) ;
  • Cha, Hyun-Myoung (Department of Biological Engineering, Inha University) ;
  • Lim, Jin-Hyuk (Department of Biological Engineering, Inha University) ;
  • Kim, Dong-Il (Department of Biological Engineering, Inha University)
  • 이경선 (인하대학교 공과대학 생물공학과) ;
  • 차현명 (인하대학교 공과대학 생물공학과) ;
  • 임진혁 (인하대학교 공과대학 생물공학과) ;
  • 김동일 (인하대학교 공과대학 생물공학과)
  • Received : 2016.03.16
  • Accepted : 2017.05.04
  • Published : 2017.06.30

Abstract

Mammalian cell cultures have been used extensively to produce proteins for therapeutic agent because of their ability to perform post-translational modification including glycosylation. To produce recombinant protein, many factors and parameter are considered such as media composition, host cell type, and culture process. In this study, recombinant human erythropoietin (rhEPO) producing cell line was established by using glutamine synthetase system. To reduce serum concentration in media, we compared direct adaptation with step adaptation. Cell growth was faster in step adaptation. In low-level serum media, there were insufficient glucose for cell growth. Thus, we added glucose in low-level serum media from 2 g/L to 4.5 g/L. Titer of rhEPO was higher than other conditions at 4.5 g/L of glucose. Additionally, N-methyl-D-aspartate (NMDA), 13-cis-retinal, and pluronic F-68 (PF-68) were added to enhance productivity in CHO cell cultures. In conclusion, we applied CHO cell producing rhEPO to low-level of serum in media using step-adaptation. Also, we confirmed positive effect of NMDA, 13-cis-retinal, and PF-68.

Acknowledgement

Supported by : 한국연구재단

References

  1. Yang, M. and M. Butler (2000) Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol. Bioeng. 68: 370-380. https://doi.org/10.1002/(SICI)1097-0290(20000520)68:4<370::AID-BIT2>3.0.CO;2-K
  2. Spier, R. E. (1980) Recent developments in the large scale cultivation of animal cells in monolayers. Adv. Biochem. Eng. 14: 119-162.
  3. Merk, W. A. (1981) Large-scale production of human fibroblast interferon in cellfermenters. Dev. Biol. Stand. 50: 137-140.
  4. Zhang, H., H. Wang, M. Liu, T. Zhang, J. Zhang, X. Wang, and W. Xiang (2013) Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody. Cytotechnology 65: 363-378. https://doi.org/10.1007/s10616-012-9488-4
  5. Kim, S. H. and G. M. Lee (2009) Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments. Appl. Microbiol. Biotechnol. 83: 639-648. https://doi.org/10.1007/s00253-009-1903-1
  6. Gandor, C., C. Leist, A. Fiechter, and F. A. Asselbergs (1995) Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells. FEBS Lett. 377: 290-294. https://doi.org/10.1016/0014-5793(95)01328-8
  7. Cruz, H. J., J. L. Moreira, G. Stacey, E. M. Dias, K. Hayes, D. Looby, B. Griffiths, and M. J. Carrondo (1998) Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium. Cytotechnology 26: 59-64. https://doi.org/10.1023/A:1007951813755
  8. Byrne, B., G. G. Donohoe, and R. O'Kennedy (2007) Sialic acids: Carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells. Drug Discov. Today 12: 319-326. https://doi.org/10.1016/j.drudis.2007.02.010
  9. Lee, J. S., T. K. Ha, S. J. Lee, and G. M. Lee (2012) Current state and perspectives on erythropoietin production. Appl. Microbiol. Biotechnol. 95: 1405-1416. https://doi.org/10.1007/s00253-012-4291-x
  10. 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. https://doi.org/10.1002/bit.10566
  11. 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 $32.5^{\circ}C$ and $37.0^{\circ}C$. Biotechnol. Bioeng. 89: 345-356. https://doi.org/10.1002/bit.20353
  12. Mates, J. M., C. Perez-Gomez, I. N. de Castro, M. Asenjo, and J. Marquez (2002) Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death. Int. J. Biochem. Cell Biol. 34: 439-458. https://doi.org/10.1016/S1357-2725(01)00143-1
  13. Inoue, Y., M. Fujisawa, M. Shoji, S. Hashizume, Y. Katakura, and S. Shirahata (2000) Enhanced antibody production of human-human hybridomas by retinoic acid. Cytotechnology 33: 83-88. https://doi.org/10.1023/A:1008155609072
  14. Sakai, K., T. Matsunaga, C. Hayashi, H. Yamaji, and H. Fukuda (2002) Effects of phosphatidic acid on recombinant protein production by Chinese hamster ovary cells in serum-free culture. Biochem. Eng. J. 10: 85-92. https://doi.org/10.1016/S1369-703X(01)00171-1
  15. Ghebeh, H., A. Handa-Corrigan, and M. Butler (1998) Development of an assay for the measurement of the surfactant pluronic F-68 in mammalian cell culture medium. Anal. Biochem. 262: 39-44. https://doi.org/10.1006/abio.1998.2750
  16. Palomares, L. A., M. Gonzalez, and O. T. Ramirez (2000) Evidence of Pluronic F-68 direct interaction with insect cells: Impact on shear protection, recombinant protein, and baculovirus production. Enzyme Microb. Technol. 26: 324-331. https://doi.org/10.1016/S0141-0229(99)00176-3
  17. Lieth, E., K. F. LaNoue, D. A. Berkich, B. Xu, M. Ratz, C. Taylor, and S. M. Hutson (2001) Nitrogen shuttling between neurons and glial cells during glutamine synthesis J. Neurochem. 76: 1712-1723. https://doi.org/10.1046/j.1471-4159.2001.00156.x
  18. Achan, V., C. T. Tran, F. Arrigoni, G. S. J. Whitley, J. M. Leiper, and P. Vallance (2002) All-trans-Retinoic Acid increases nitric oxide synthesis by endothelial cells a role for the induction of dimethylarginine dimethylaminohydrolase. Circ. Res. 90: 764-769. https://doi.org/10.1161/01.RES.0000014450.40853.2B
  19. Mehta, K. A. P. I. L., T. McQueen, S. Tucker, R. Pandita, and B. B. Aggarwal (1994) Inhibition by all-trans-retinoic acid of tumor necrosis factor and nitric oxide production by peritoneal macrophages. J. Leukoc. Biol. 55: 336-342. https://doi.org/10.1002/jlb.55.3.336