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Cetuximab Fab and Fc N-Glycan Fast Characterization Using IdeS Digestion and Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry

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Glycosylation Engineering of Biopharmaceuticals

Part of the book series: Methods in Molecular Biology ((MIMB,volume 988))

Abstract

Antibodies and related products represent one of the fastest growing areas of new drug development within the pharmaceutical industry. Monoclonal antibodies (mAbs) undergo many posttranslational modifications (PTMs) that must be extensively characterized. Here we described a rapid mass spectrometry (MS) method for the characterization of cetuximab glycosylation. The reported analytical technique is based on the use of a cystein protease, immunoglobulin-degrading enzyme of Streptococcus pyogenes that allows a fast limited proteolysis of the mAb with low material consumption. The resulting large fragments are analyzed by ultrahigh-performance liquid chromatography combined to an electrospray ionization mass spectrometer and a time-of-flight analyzer (ESI–TOF). Cetuximab is a potent chimeric mouse/human antibody worldwide approved for the treatment of colon and head and neck cancers. This antibody, produced by SP2/0 murine myeloma cells, is N-glycosylated both in the Fc and Fab moieties, which have been shown to impact on safety and PK/PD and considered as a critical quality attribute. The method can also be applied for biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.

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Author information

Authors and Affiliations

  1. Physico-Chemistry Department, Centre d’Immunologie Pierre-Fabre, Saint Julien-en-Genevois, France

    Marie-Claire Janin-Bussat, Laure Tonini, Céline Huillet, Olivier Colas & Christine Klinguer-Hamour

  2. Centre d’Immunologie Pierre-Fabre, Saint-Julien-en-Genevois, France

    Nathalie Corvaïa

  3. Antibody Physico-Chemistry Department, Centre d’Immunologie Pierre-Fabre, Saint Julien-en-Genevois, France

    Alain Beck

Authors
  1. Marie-Claire Janin-Bussat
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  2. Laure Tonini
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  3. Céline Huillet
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  4. Olivier Colas
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  5. Christine Klinguer-Hamour
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  6. Nathalie Corvaïa
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  7. Alain Beck
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Editors and Affiliations

  1. , Antibody Physico-Chemistry, Centre d'Immunologie Pierre Fabre (CIPF), Avenue Napoléon III 5, St. Julien-en-Genevois, 74164, France

    Alain Beck

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Janin-Bussat, MC. et al. (2013). Cetuximab Fab and Fc N-Glycan Fast Characterization Using IdeS Digestion and Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry. In: Beck, A. (eds) Glycosylation Engineering of Biopharmaceuticals. Methods in Molecular Biology, vol 988. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-327-5_7

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  • DOI: https://doi.org/10.1007/978-1-62703-327-5_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-326-8

  • Online ISBN: 978-1-62703-327-5

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