Skip to main content
SpringerLink
Log in

IgG Aggregation Mechanism for CHO Cell Lines Expressing Excess Heavy Chains

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Aggregates in protein therapeutics like IgG monoclonal antibodies (mAb) are detrimental to product safety and efficacy. It has been reported that aggregates form in Chinese hamster ovary (CHO) cell lines expressing greater amount of heavy chain (HC) than light chain (LC). In this study, we observed that aggregates could form within the cells with excess HC and were partially secreted into the supernatant. The aggregates in the supernatant consisted of mainly HC and were partially dissociated under either reducing or denaturing conditions. Mutation of a predicted free cysteine on HC to prevent disulfide bonding did not reduce aggregation. Re-transfecting CHO cells with excess HC with more BiP, an important IgG molecular chaperone, partially reduced unwanted aggregates and fragments possibly by helping retain more incomplete products within the cell for either proper assembly or degradation. A second transfection of LC into CHO cells with excess HC to increase the LC expression to a level greater than the HC expression successfully removed all aggregates and fragments. mAb product aggregation in CHO cells with excess HC occur due to a combination of limited chaperones and LC:HC ratio. These results provide added insights to aggregate formation and would be useful for development of mAb cell lines with reduced aggregates.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Mahler, H. C., Friess, W., Grauschopf, U., & Kiese, S. (2009). Protein aggregation: Pathways, induction factors and analysis. Journal of Pharmaceutical Sciences, 98, 2909–2934.

    Article  CAS  Google Scholar 

  2. Cromwell, M. E., Hilario, E., & Jacobson, F. (2006). Protein aggregation and bioprocessing. AAPS Journal, 8, E572–E579.

    Article  CAS  Google Scholar 

  3. Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., & Narhi, L. O. (2011). Classification and characterization of therapeutic antibody aggregates. Journal of Biological Chemistry, 286, 25118–25133.

    Article  CAS  Google Scholar 

  4. Phillips, J., Drumm, A., Harrison, P., Bird, P., Bhamra, K., Berrie, E., & Hale, G. (2001). Manufacture and quality control of CAMPATH-1 antibodies for clinical trials. Cytotherapy, 3, 233–242.

    Article  CAS  Google Scholar 

  5. Yoo, S. M., & Ghosh, R. (2012). Simultaneous removal of leached protein-A and aggregates from monoclonal antibody using hydrophobic interaction membrane chromatography. Journal of Membrane Science, 390–391, 263–269.

    Article  Google Scholar 

  6. Zhang, Y. B., Howitt, J., McCorkle, S., Lawrence, P., Springer, K., & Freimuth, P. (2004). Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expression and Purification, 36, 207–216.

    Article  CAS  Google Scholar 

  7. Schröder, M., Schäfer, R., & Friedl, P. (2002). Induction of protein aggregation in an early secretory compartment by elevation of expression level. Biotechnology and Bioengineering, 78, 131–140.

    Article  Google Scholar 

  8. Feige, M. J., Hendershot, L. M., & Buchner, J. (2010). How antibodies fold. Trends in Biochemical Science, 35, 189–198.

    Article  CAS  Google Scholar 

  9. Gonzalez, R., Andrews, B. A., & Asenjo, J. A. (2002). Kinetic model of BiP- and PDI-mediated protein folding and assembly. Journal of Theoretical Biology, 214, 529–537.

    Article  CAS  Google Scholar 

  10. Lee, C. J., Seth, G., Tsukuda, J., & Hamilton, R. W. (2009). A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies. Biotechnology and Bioengineering, 102, 1107–1118.

    Article  CAS  Google Scholar 

  11. Gomez, N., Subramanian, J., Ouyang, J., Nguyen, M. D., Hutchinson, M., Sharma, V. K., et al. (2012). Culture temperature modulates aggregation of recombinant antibody in CHO cells. Biotechnology and Bioengineering, 109, 125–136.

    Article  CAS  Google Scholar 

  12. Ho, S. C. L., Koh, E. Y. C., van Beers, M., Mueller, M., Wan, C., Teo, G., et al. (2013). Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability. Journal of Biotechnology, 165, 157–166.

    Article  CAS  Google Scholar 

  13. Borth, N., Mattanovich, D., Kunert, R., & Katinger, H. (2005). Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line. Biotechnology Progress, 21, 106–111.

    Article  CAS  Google Scholar 

  14. Mohan, C., & Lee, G. M. (2010). Effect of inducible co-overexpression of protein disulfide isomerase and endoplasmic reticulum oxidoreductase on the specific antibody productivity of recombinant Chinese hamster ovary cells. Biotechnology and Bioengineering, 107, 337–346.

    Article  CAS  Google Scholar 

  15. Hayes, N. V. L., Smales, C. M., & Klappa, P. (2010). Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines. Biotechnology and Bioengineering, 105, 770–779.

    CAS  Google Scholar 

  16. Nishimiya, D. (2014). Proteins improving recombinant antibody production in mammalian cells. Applied Microbiology and Biotechnology, 98, 1031–1042.

    Article  CAS  Google Scholar 

  17. Koh, E. Y. C., Ho, S. C. L., Mariati, Song, Z., Bi, X., Bardor, M., & Yang, Y. (2013). An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells. PLoS ONE, 8, e82100.

    Article  Google Scholar 

  18. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−Delta Delta C) method. Methods, 25, 402–408.

    Article  CAS  Google Scholar 

  19. Ho, S. C. L., Bardor, M., Feng, H., Mariati, Tong, Y. W., Song, Z., et al. (2012). IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. Journal of Biotechnology, 157, 130–139.

    Article  CAS  Google Scholar 

  20. Ho, S. C. L., Bardor, M., Li, B., Lee, J. J., Song, Z., Tong, Y. W., et al. (2013). Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells. PLoS ONE, 8, e63247.

    Article  CAS  Google Scholar 

  21. O’Callaghan, P. M., McLeod, J., Pybus, L. P., Lovelady, C. S., Wilkinson, S. J., Racher, A. J., et al. (2010). Cell line-specific control of recombinant monoclonal antibody production by CHO cells. Biotechnology and Bioengineering, 106, 938–951.

    Article  Google Scholar 

  22. Liu, H., & May, K. (2012). Disulfide bond structures of IgG molecules: Structural variations, chemical modifications and possible impacts to stability and biological function. mAbs, 4, 17–23.

    Article  CAS  Google Scholar 

  23. Elkabetz, Y., Argon, Y., & Bar-Nun, S. (2005). Cysteines in CH1 underlie retention of unassembled Ig heavy chains. Journal of Biological Chemistry, 280, 14402–14412.

    Article  CAS  Google Scholar 

  24. Chennamsetty, N., Helk, B., Voynov, V., Kayser, V., & Trout, B. L. (2009). Aggregation-prone motifs in human immunoglobulin G. Journal of Molecular Biology, 391, 404–413.

    Article  CAS  Google Scholar 

  25. Schröder, M., & Kaufman, R. J. (2005). ER stress and the unfolded protein response. Mutation Research—Fundamental and Molecular Mechanisms of Mutagenesis, 569, 29–63.

    Article  Google Scholar 

  26. Lee, Y. K., Brewer, J. W., Hellman, R., & Hendershot, L. M. (1999). BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Molecular Biology of the Cell, 10, 2209–2219.

    Article  CAS  Google Scholar 

  27. Vanhove, M., Usherwood, Y. K., & Hendershot, L. M. (2001). Unassembled Ig heavy chains do not cycle from BiP in vivo but require light chains to trigger their release. Immunity, 15, 105–114.

    Article  CAS  Google Scholar 

  28. Hendershot, L., Bole, D., Köhler, G., & Kearney, J. F. (1987). Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein. The Journal of Cell Biology, 104, 761–767.

    Article  CAS  Google Scholar 

  29. Ho, S. C. L., Bardor, M., Feng, H. T., Mariati, Tong, Y. W., Song, Z. W., et al. (2012). IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. Journal of Biotechnology, 157, 130–139.

    Article  CAS  Google Scholar 

  30. Koh, E. Y. C., Ho, S. C. L., Mariati, Song, Z. W., Bi, X. Z., Bardor, M., & Yang, Y. S. (2013). An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells. PLoS ONE, 8, e82100.

    Article  Google Scholar 

  31. van Berkel, P. H. C., Gerritsen, J., Perdok, G., Valbjorn, J., Vink, T., van de Winkel, J. G. J., & Parren, P. W. H. I. (2009). N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG. Biotechnology Progress, 25, 244–251.

    Article  Google Scholar 

  32. Yang, Y. S., Mariati, Ho, S. C., & Yap, M. G. (2009). Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells. Biotechnology and Bioengineering, 102, 1152–1160.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Biomedical Research Council/Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. The authors wish to thank Kong Meng, Hoi for aiding in purification and analytics. We also thank Phyllicia Toh for providing technical support during use of the HPLC for aggregate analysis.

Author information

Authors and Affiliations

  1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore

    Steven C. L. Ho, Tianhua Wang, Zhiwei Song & Yuansheng Yang

  2. School of Chemical and Biomedical Engineering, Nanyang Technological University, N1.2-B2-33, 62 Nanyang Avenue, Singapore, 637459, Singapore

    Yuansheng Yang

Authors
  1. Steven C. L. Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tianhua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhiwei Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuansheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuansheng Yang.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ho, S.C.L., Wang, T., Song, Z. et al. IgG Aggregation Mechanism for CHO Cell Lines Expressing Excess Heavy Chains. Mol Biotechnol 57, 625–634 (2015). https://doi.org/10.1007/s12033-015-9852-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9852-7

Keywords

Search

Navigation