Skip to main content
SpringerLink
Log in

Effect of Environmental Parameters on Glycosylation of Recombinant Immunoglobulin G Produced from Recombinant CHO Cells

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

Abstract

Site specific glycosylation of immunoglobulin G (IgG) occurs at Asn297 in the Fc region. The heterogeneous ensemble of glycoform occurs due to the degree of terminal galactosylation and sialylation, and these differences in glycosylation affect both the pharmacokinetic behavior and effector functions of the IgG, such as complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In this study, the differential glycosylation of IgG was compared and environmental physical and chemical parameters were evaluated in an attempt to promote glycosylation of recombinant antibodies, thereby creating more humanized glycoform antibodies and increasing their in vivo efficacy as therapeutic drugs. It was shown that cells at late stationary growth phase in batch cultures, cells with increased passage number, and the culture conditions of lowered temperature and pH promoted galactosylation and sialylation of antibodies. Galactose, fructose and mannose were found to elicit galactosylation and sialylation when they were used alone as a substitute of glucose. Mannose showed synergistic effects on glycosylation when used with other sugars, such as glucose and galactose. However when fructose was used with other sugars, the degree of galactosylation mechanism appeared to be decreased. These results support understandings of the glycosylation mechanisms in glycoprotein, particularly recombinant antibodies for therapeutics.

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. Burton, D. R. and R. A. Dwek (2006) Immunology. Sugar determines antibody activity. Scienc. 313: 670–673.

    Google Scholar 

  2. Raju, T. S., J. B. Briggs, S. M. Borge, and A. J. Jones (2000) Species–specific variation in glycosylation of IgG: evidence for the species–specific sialylation and branch–specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiolog. 10: 477–486.

    Article  CAS  Google Scholar 

  3. Raju, T. S., J. B. Briggs, S. M. Chamow, M. E. Winkler, and A. J. Jones (2001) Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N–acetylglucosamine and galactose residues. Biochemistr. 40: 8868–8876.

    Article  CAS  Google Scholar 

  4. Sha, S., C. Agarabi, K. Brorson, D. Y. Lee, and S. Yoon (2016) N–Glycosylation design and control of therapeutic monoclonal antibodies. Trends Biotechnol. 34: 835–846.

    Article  CAS  PubMed  Google Scholar 

  5. Nimmerjahn, F., R. M. Anthony, and J. V. Ravetch (2007) Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity. Proc. Natl. Acad. Sci. US. 104: 8433–8437.

    Article  CAS  Google Scholar 

  6. Hodoniczky, J., Y. Z. Zheng, and D. C. James (2005) Control of recombinant monoclonal antibody effector functions by Fc Nglycan remodeling in vitro. Biotechnol. Prog. 21: 1644–1652.

    Article  CAS  PubMed  Google Scholar 

  7. Scallon, B. J., S. H. Tam, S. G. McCarthy, A. N. Cai, and T. S. Raju (2007) Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol. Immunol. 44: 1524–1534.

    Article  CAS  PubMed  Google Scholar 

  8. Parekh, R., D. Isenberg, G. Rook, I. Roitt, R. Dwek, and T. A. Rademacher (1989) Comparative analysis of disease–associated changes in the galactosyl ation of serum IgG. J. Autoimmun. 2: 101–114.

    Article  CAS  PubMed  Google Scholar 

  9. Dwek, R. A., A. C. Lellouch, and M. R. Wormald (1995) Glycobiology: ‘The function of sugar in the IgG molecule’. J. Anat. 187: 279–292.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Gindzienska–Sieskiewicz, E., P. A. Klimiuk, D. G. Kisiel, A. Gindzienski, and S. Sierakowski (2007) The changes in monosaccharide composition of immunoglobulin G in the course of rheumatoid arthritis. Clin Rheumatol. 26: 685–690.

    Article  PubMed  Google Scholar 

  11. Kaneko, Y., F. Nimmerjahn, and J. V. Ravetch (2006) Antiinflammatory activity of immunoglobulin G resulting from Fc sialylation. Scienc. 313: 627–628.

    Article  CAS  Google Scholar 

  12. Krapp, S., Y. Mimura, R. Jefferis, R. Huber, and P. Sondermann (2003) Structural analysis of human IgG–Fc glycoforms reveals a correlation between glycosylation and structural integrity. J. Mol. Biol. 325: 979–989.

    Article  CAS  PubMed  Google Scholar 

  13. Malhotra, R., M. R. Wormald, P. M. Rudd, P. B. Fischer, R. A. Dwek, and R. B. Sim (1995) Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose–binding protein. Nat. Med. 1:237–243.

    Article  CAS  PubMed  Google Scholar 

  14. Matsumoto, A., K. Shikata, F. Takeuchi, N. Kojima, and T. Mizuochi (2000) Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation. J. Biochem. 128: 621–628

    Article  CAS  PubMed  Google Scholar 

  15. Novak, J., M. Tomana, G. R. Shah, R. Brown, and J. Mestecky (2005) Heterogeneity of IgG glycosylation in adult periodontal disease. J. Dent. Res. 84: 897–901.

    Article  CAS  PubMed  Google Scholar 

  16. Li, H., N. Sethuraman, T. A. Stadheim, D. Zha, B. Prinz, N. Ballew, P. Bobrowicz, B. K. Choi, W. J. Cook, M. Cukan, N. R. Houston–Cummings, R. Davidson, B. Gong, S. R. Hamilton, J. P. Hoopes, Y. Jiang, N. Kim, R. Mansfield, J. H. Nett, S. Rios, R. Strawbridge, S. Wildt, and T. U. Gerngross (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat. Biotechnol. 24: 210–215.

    Article  CAS  PubMed  Google Scholar 

  17. Routier, F. H., M. J. Davies, K. Bergemann, and E. F. Hounsell (1997) The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells. Glycoconj J. 14: 201–207.

    Article  CAS  PubMed  Google Scholar 

  18. Baker, K. N., M. H. Rendall, A. E. Hills, M. Hoare, P. B. Freedman, and D. C. James (2001) Metabolic control of recombinant protein N–glycan processing in NS0 and CHO cells. Biotechnol. Bioeng. 73: 188–202.

    Article  CAS  PubMed  Google Scholar 

  19. Leist, C. H., H. P. Meyer, and A. Fiechter (1990) Potential and problems of animal cells in suspension culture. J. Biotechnol. 15: 1–46.

    Article  CAS  PubMed  Google Scholar 

  20. Lifely, M. R., C. Hale, S. Boyce, M. J. Keen, and J. Phillips (1995) Glycosylation and biological activity of CAMPATH–1H expressed in different cell lines and grown under different culture conditions. Glycobiolog. 5: 813–822.

    Article  CAS  Google Scholar 

  21. Fogolin, 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  CAS  PubMed  Google Scholar 

  22. Furukawa, K. and K. Ohsuye (1998) Effect of culture temperature on a recombinant CHO cell line producing a Cterminal alpha–amidating enzyme. Cytotechnolog. 26: 153–164.

    Article  CAS  Google Scholar 

  23. Trummer, E., K. Fauland, S. Seidinger, K. Schriebl, C. Lattenmayer, R. Kunert, K. Vorauer–Uhl, R. Weik, N. Borth, H. Katinger, and D. Müller (2006) Process parameter shifting: PartI. Effect of DOT, pH, and temperature on the performance of Epo–Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol. Bioeng. 94: 1033–1044.

    Article  CAS  PubMed  Google Scholar 

  24. 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 degrees. C. Biotechnol. Bioeng. 89: 345–356.

    Article  CAS  PubMed  Google Scholar 

  25. 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  PubMed  Google Scholar 

  26. Nam, J. H., F. Zhang, M. Ermonval, R. J. Linhardt, and S. T. Sharfstein (2008) The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells. Biotechnol. Bioeng. 100: 1178–1192.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hossler, P., S. F. Khattak, and Z. J. Li (2009) Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiolog. 19: 936–949.

    Article  CAS  Google Scholar 

  28. Gawlitzek, M., U. Valley, and R. Wagner (1998) Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK–21 cells. Biotechnol. Bioeng. 57: 518–528.

    Article  CAS  PubMed  Google Scholar 

  29. 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  PubMed  Google Scholar 

  30. Altamirano, C., C. Paredes, A. Illanes, J. J. Cairó, and F. Gòdia (2004) Strategies for fed–batch cultivation of t–PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. J. Biotechnol. 110: 171–179.

    Article  CAS  PubMed  Google Scholar 

  31. Swinnen, K., A. Krul, I. Van Goidsenhoven, N. Van Tichelt, A. Roosen, and K. Van Houdt (2007) Performance comparison of protein A affinity resins for the purification of monoclonal antibodies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 848: 97–107.

    Article  CAS  PubMed  Google Scholar 

  32. Tarentino, A. L. and T. H. Plummer Jr. (1994) Enzymatic deglycosylation of asparagine–linked glycans: purification, properties, and specificity of oligosaccharide–cleaving enzymes from Flavobacterium meningosepti cum. Methods Enzymol. 230: 44–57.

    Article  CAS  PubMed  Google Scholar 

  33. Anumula, K. R. and P. Du (1999) Characterization of carbohydrates using highly fluorescent 2–aminobenzoic acid tag following gel electrophoresis of glycoproteins. Anal. Biochem. 275: 236–242.

    Article  CAS  PubMed  Google Scholar 

  34. Dhume, S. T., G. N. Saddic, and K. R Anumula (2008) Monitoring glycosylation of therapeutic glycoproteins for consistency by HPLC using highly fluorescent anthranilic acid (AA) Tag. Methods Mol. Biol. 446: 317–331.

    Article  CAS  PubMed  Google Scholar 

  35. Anumula, K. R. and S. T. Dhume (1998) High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. Glycobiolog. 8: 685–694.

    Article  CAS  Google Scholar 

  36. Anumula, K. R. (2006) Advances in fluorescence derivatization methods for high–performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal. Biochem. 350: 1–23.

    Article  CAS  PubMed  Google Scholar 

Download references

Aknowledgement

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015M3A9135053958).

Author information

Authors and Affiliations

  1. Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, 05006, Korea

    Seong-Min Kim & Duk Jae Oh

  2. Department of Bioengineering, Inha University, Incheon, 22212, Korea

    Kyu-Ho Chang

Authors
  1. Seong-Min Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kyu-Ho Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Duk Jae Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Duk Jae Oh.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, SM., Chang, KH. & Oh, D.J. Effect of Environmental Parameters on Glycosylation of Recombinant Immunoglobulin G Produced from Recombinant CHO Cells. Biotechnol Bioproc E 23, 456–464 (2018). https://doi.org/10.1007/s12257-018-0109-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-018-0109-8

Keywords

Search

Navigation