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Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins

Nature Biotechnology volume 17, pages 1116–1121 (1999)Cite this article

Abstract

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human β1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or α2,3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of ≥90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of ≥90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

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Figure 1: Northern blot analysis of GT and ST overexpression in cell lines secreting (A) TNFR-IgG and (B).
Figure 2: MALDI-TOF MS in (A and B) negative-ion mode and (C and D) positive-ion mode of oligosaccharides on TNK-tPA produced by (A and C) control cells and (B and D) cells reengineered to overexpress ST alone.
Figure 3: MALDI-TOF MS in negative-ion mode of oligosaccharides on TNFR-IgG produced by (A) control cells and cells reengineered to overexpress (B) GT alone (C) ST alone, and (D) both enzymes together (GT/ST).
Figure 4: MALDI-TOF MS in positive-ion mode of oligosaccharides on TNFR-IgG produced by (A) control cells and cells reengineered to overexpress (B) GT alone (C) ST alone, and (D) both enzymes together (GT/ST).
Figure 5

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Acknowledgements

The authors would like to acknowledge and thank Mary Sliwkowski, Thomas Warner, and Thomas Ryll of the Analytical Chemistry and Cell Culture & Fermentation Research and Development Departments, Genentech, Inc., for their insightful discussions and helpful comments in the planning of these studies and the preparation of this manuscript.

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Authors and Affiliations

  1. Department of Cell Culture and Fermentation Research and Development, Genentech, Inc., South San Francisco, 94080, CA

    S. Weikert, S. Tom, J. Lofgren, S. Mehta, K. Carroll, P. Berman & L. Krummen

  2. Department of Analytical Chemistry, Genentech, Inc., South San Francisco, 94080, CA

    D. Papac, J. Briggs & D. Cowfer

  3. Department of Recovery Sciences, Genentech, Inc., South San Francisco, 94080, CA

    S. Chamow & D. Peers

  4. Department of Molecular Biology, Genentech, Inc., South San Francisco, 94080, CA

    V. Chisholm

  5. Department of Pharmacokinetics and Metabolism, Genentech, Inc., South San Francisco, 94080, CA

    N. Modi & S. Eppler

  6. Department of Manufacturing Sciences, Genentech, Inc., South San Francisco, 94080, CA

    M. Gawlitzek

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Correspondence to L. Krummen.

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Weikert, S., Papac, D., Briggs, J. et al. Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins. Nat Biotechnol 17, 1116–1121 (1999). https://doi.org/10.1038/15104

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