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Engineered CHO cells for production of diverse, homogeneous glycoproteins

Abstract

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like α2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic potential.

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Figure 1: ZFN knockout screen of glycosyltransferase genes involved in N-glycosylation in CHO cells, using human EPO as recombinant expressed reporter glycoprotein.
Figure 2: Reconstruction of sialylation with de novo α2,6-NeuAc in CHO by ZFN targeted knock-in.

References

  1. Walsh, G. Nat. Biotechnol. 32, 992–1000 (2014).

    CAS  Article  Google Scholar 

  2. Walsh, G. Drug Discov. Today 15, 773–780 (2010).

    CAS  Article  Google Scholar 

  3. Sasaki, H., Ochi, N., Dell, A. & Fukuda, M. Biochemistry 27, 8618–8626 (1988).

    CAS  Article  Google Scholar 

  4. Sinclair, A.M. & Elliott, S. J. Pharm. Sci. 94, 1626–1635 (2005).

    CAS  Article  Google Scholar 

  5. Patnaik, S.K. & Stanley, P. Methods Enzymol. 416, 159–182 (2006).

    CAS  Article  Google Scholar 

  6. Yamane-Ohnuki, N. et al. Biotechnol. Bioeng. 87, 614–622 (2004).

    CAS  Article  Google Scholar 

  7. Malphettes, L. et al. Biotechnol. Bioeng. 106, 774–783 (2010).

    CAS  Article  Google Scholar 

  8. Steentoft, C. et al. Glycobiology 24, 663–680 (2014).

    CAS  Article  Google Scholar 

  9. Xu, X. et al. Nat. Biotechnol. 29, 735–741 (2011).

    CAS  Article  Google Scholar 

  10. Lewis, N.E. et al. Nat. Biotechnol. 31, 759–765 (2013).

    CAS  Article  Google Scholar 

  11. North, S.J. et al. J. Biol. Chem. 285, 5759–5775 (2010).

    CAS  Article  Google Scholar 

  12. Amado, M., Almeida, R., Schwientek, T. & Clausen, H. Biochim. Biophys. Acta 1473, 35–53 (1999).

    CAS  Article  Google Scholar 

  13. Narimatsu, H. Curr. Opin. Struct. Biol. 16, 567–575 (2006).

    CAS  Article  Google Scholar 

  14. Fukuda, M.N., Sasaki, H., Lopez, L. & Fukuda, M. Blood 73, 84–89 (1989).

    CAS  PubMed  Google Scholar 

  15. Maresca, M., Lin, V.G., Guo, N. & Yang, Y. Genome Res. 23, 539–546 (2013).

    CAS  Article  Google Scholar 

  16. El Maï, N., Donadio-Andrei, S., Iss, C., Calabro, V. & Ronin, C. Methods Mol. Biol. 988, 19–29 (2013).

    Article  Google Scholar 

  17. DeFrees, S. et al. Glycobiology 16, 833–843 (2006).

    CAS  Article  Google Scholar 

  18. Hamilton, S.R. et al. Science 313, 1441–1443 (2006).

    CAS  Article  Google Scholar 

  19. Duda, K. et al. Nucleic Acids Res. 42, e84 (2014).

    CAS  Article  Google Scholar 

  20. Zhang, Y. et al. Nucleic Acids Res. 43, e59 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

We would like to express our sincere gratitude to the SAFC Sigma team including K. Kayser and N. Sealover for their help with ZFN targeting constructs. We are also grateful to B. Palsson for help throughout this work and critical comments on the manuscript. We are grateful to M. Uhlen, L.E. Pedersen and B. Voldborg at the Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, for RNA-seq analysis. This work was supported by the Novo Nordisk Foundation, Kirsten og Freddy Johansen Fonden, The Mizutani Foundation, A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, Copenhagen University Excellence Programme for Interdisciplinary Research (CDO2016), the Danish National Research Foundation (DNRF107) and The Danish Councils for Strategic and Independent Research. All reagents and cell lines used in the study are available upon request for research purposes under a material transfer agreement.

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Authors

Contributions

Z.Y. designed, planned and ran most of the ZFN targeting experiments and co-wrote the manuscript. S.W. performed purification of EPO and IgG. A.H. performed the glycoprofiling. M.A.S. contributed to expression of EPO and IgG. M.F. contributed to ZFN design. S.H.R., C.B. and M.B.V.-C. contributed to the design of experiments for homogeneous glycoPEGylation of monoantennary N-glycans. S.Y.V., E.P.B., C.K. and H.H.W. contributed to the design of experiments. H.C. designed, planned and co-wrote the manuscript.

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Correspondence to Zhang Yang or Henrik Clausen.

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A patent application has been filed on which some of the authors are listed as inventors.

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Supplementary Spectra 1–38 and Supplementary Tables 1–4 (PDF 5460 kb)

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Yang, Z., Wang, S., Halim, A. et al. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat Biotechnol 33, 842–844 (2015). https://doi.org/10.1038/nbt.3280

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  • DOI: https://doi.org/10.1038/nbt.3280

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