Identification of cell culture conditions to control N-glycosylation site-occupancy of recombinant glycoproteins expressed in CHO cells

Authors

  • Martin Gawlitzek,

    Corresponding author
    1. Manufacturing Sciences & Technology, Genentech, Inc., One DNA Way, South San Francisco, California 94080
    2. Late Stage Cell Culture, Process Research & Development, Genentech, Inc., One DNA Way, South San Francisco, California
    • Manufacturing Sciences & Technology, Genentech, Inc., One DNA Way, South San Francisco, California 94080; telephone: 1-650-225-8869; fax: 1-650-225-4436.
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  • Michael Estacio,

    1. Manufacturing Sciences & Technology, Genentech, Inc., One DNA Way, South San Francisco, California 94080
    2. Automation & Production Information Technology, Production Technology, Genentech, Inc., One DNA Way, South San Francisco, California
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  • Tobias Fürch,

    1. Manufacturing Sciences & Technology, Genentech, Inc., One DNA Way, South San Francisco, California 94080
    Current affiliation:
    1. Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstraße 7, D-38124 Braunschweig, Germany.
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  • Robert Kiss

    1. Manufacturing Sciences & Technology, Genentech, Inc., One DNA Way, South San Francisco, California 94080
    2. Late Stage Cell Culture, Process Research & Development, Genentech, Inc., One DNA Way, South San Francisco, California
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Abstract

The effect of different cell culture conditions on N-glycosylation site-occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t-PA) and a recombinant enzyme (glycoprotein 2—GP2). Both molecules contain a N-glycosylation site that is variably occupied. Different environmental factors that affect the site-occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t-PA (type I t-PA) by approximately 2.5–4%. Decreasing the cultivation temperature from 37 to 33°C or 31°C gradually increased site-occupancy of t-PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site-occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site-occupancy of t-PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N-glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site-occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N-glycan site-occupancy within a specific range. An increase in culture pH correlated with a decrease in site-occupancy. Similarly, delaying the timing for butyrate addition also decreased site-occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N-glycan site-occupancy, within a specific range when applied appropriately. Biotechnol. Bioeng. 2009;103: 1164–1175. © 2009 Wiley Periodicals, Inc.

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