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Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests

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Abstract

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and 2D retention time opening interesting perspectives for use in QA/QC testing.

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Acknowledgments

The authors would like to acknowledge Agilent Technologies for contributing to this study.

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Authors and Affiliations

  1. Research Institute for Chromatography & Metablys, President Kennedypark 26, 8500, Kortrijk, Belgium

    Gerd Vanhoenacker, Isabel Vandenheede, Frank David, Pat Sandra & Koen Sandra

Authors
  1. Gerd Vanhoenacker
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  2. Isabel Vandenheede
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  3. Frank David
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  4. Pat Sandra
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  5. Koen Sandra
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Corresponding author

Correspondence to Koen Sandra.

Additional information

Published in the topical collection Multidimensional Chromatography with guest editors Torsten C. Schmidt, Oliver J. Schmitz, and Thorsten Teutenberg.

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Vanhoenacker, G., Vandenheede, I., David, F. et al. Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests. Anal Bioanal Chem 407, 355–366 (2015). https://doi.org/10.1007/s00216-014-8299-1

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  • DOI: https://doi.org/10.1007/s00216-014-8299-1

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