Degradation of an Fc-fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

Authors

  • Flavie Robert,

    1. Biotech Process Sciences, Merck Serono Biotech Center, Merck Serono SA Corsier-sur-Vevey, CH-1809 Fenil-sur-Corsier, Switzerland; telephone: +41-21-923-2228; fax: +41-21-923-2013
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  • Horst Bierau,

    1. Analytical Development, Merck Serono S.p.A., Ardea, Italy
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  • Mara Rossi,

    1. Analytical Development, Merck Serono S.p.A., Ardea, Italy
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  • David Agugiaro,

    1. Analytical Development, Merck Serono S.p.A., Ardea, Italy
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  • Thomas Soranzo,

    1. UFR de Médecine et Pharmacie de Grenoble, Université Joseph Fourier, Domaine de la Merci, La Tronche, France
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  • Hervé Broly,

    1. Biotech Process Sciences, Merck Serono Biotech Center, Merck Serono SA Corsier-sur-Vevey, CH-1809 Fenil-sur-Corsier, Switzerland; telephone: +41-21-923-2228; fax: +41-21-923-2013
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  • Christine Mitchell-Logean

    Corresponding author
    1. Biotech Process Sciences, Merck Serono Biotech Center, Merck Serono SA Corsier-sur-Vevey, CH-1809 Fenil-sur-Corsier, Switzerland; telephone: +41-21-923-2228; fax: +41-21-923-2013
    • Biotech Process Sciences, Merck Serono Biotech Center, Merck Serono SA Corsier-sur-Vevey, CH-1809 Fenil-sur-Corsier, Switzerland; telephone: +41-21-923-2228; fax: +41-21-923-2013.
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Abstract

A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132–1141. © 2009 Wiley Periodicals, Inc.

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