Glycosylation of CHO-Derived Recombinant tPA Produced under Elevated pCO2

Authors

  • Roy Kimura,

    1. Chemical Engineering Department, Northwestern University, Evanston, Illinois 60208–3120
    Current affiliation:
    1. MedImmune, Inc., 35 W. Watkins Mill Rd., Gaithersburg, MD 20878
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  • William M. Miller

    Corresponding author
    1. Chemical Engineering Department, Northwestern University, Evanston, Illinois 60208–3120
    • Chemical Engineering Department, Northwestern University, Evanston, Illinois 60208–3120. Phone: (847) 491–4828. Fax: (847) 491–3728
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Abstract

Carbon dioxide is a metabolic byproduct of mammalian cell metabolism that can accumulate in poorly ventilated cultures. A buildup of CO2 at constant pH will be accompanied by an increase in medium osmolality. We have examined the glycosylation of tissue plasminogen activator (tPA) produced under serum-free conditions by recombinant Chinese hamster ovary (CHO) cells (MT2-1–8 cell line) in response to elevated pCO2 at constant or elevated osmolality. The proportion of sialic acids comprising N-glycolylneuraminic acid decreased from 2.3–4.0% under 36 mmHg pCO2 to 1.5–2.2% under 250 mmHg pCO2. No changes were observed in the total sialic acid content, the content of other monosaccharides, the relative amounts of type I and type II tPAs, the distribution of surface charges, or the proportion of high-mannose oligosaccharides—even though these conditions have previously been shown to inhibit the specific growth rate of MT2-1–8 cells by 30–40% and the specific tPA production rate by as much as 40%. These results suggest robust glycosylation of tPA by CHO cells.

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