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Engineered CHO cells for production of diverse, homogeneous glycoproteins


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.


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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 and Affiliations



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 Text and Figures

Supplementary Figures 1–12 (PDF 3698 kb)

Supplementary Additional Spectra Combined PDF

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).

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