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Development of a Two-dimensional Liquid Chromatography High-Resolution Mass Spectrometry Method for the Characterization of Monoclonal Antibodies in Cell-Free Culture Supernatant via FcR Affinity Chromatography

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Abstract

A monoclonal antibody’s (mAb) glycosylation pattern has a significant impact on its binding affinity to the FcγRIIIa receptor and thus on antibody-dependent cell-mediated cytotoxicity. However, to analyze the relation in cell cultures, complex sample preparation is required. In this study, we demonstrate the analytical capability of a two-dimensional liquid chromatography–mass spectrometry (2D-HPLC–HRMS) method to analyze the affinity of mAbs to the FcγRIIIa receptor while simultaneously characterizing the glycoforms involved. Results were comparable in purified samples and cell culture-free supernatants. This was demonstrated with the monoclonal antibodies adalimumab, pertuzumab, ustekinumab, tocilizumab and omalizumab. Our method hence renders extensive purification of mAb samples superfluous.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

Abbreviations

mAbs:

Monoclonal antibodies

FcR:

Fragment crystallisable receptor

HRMS:

High-resolution mass spectrometry

ADCC:

Antibody-dependent cell-mediated cytotoxicity

Fab:

Fragment antigen binding

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Acknowledgements

The authors would like to thank for financial support from the German Federal Ministry of Economic Affairs and Energy within the agenda for the Promotion of industrial cooperative research and development (IGF) based on a decision of the German Bundestag (IGF–Project No. 21280 N).

Funding

The work was financially supported by the IGF Project No. 21280 N.

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Authors and Affiliations

  1. Institut für Energie und Umwelttechnik e. V. (IUTA, Institute of Energy and Environmental Technology), Bliersheimer Str. 58-60, 47229, Duisburg, Germany

    Lars M. H. Reinders, Martin D. Klassen & Thorsten Teutenberg

  2. Hochschule Niederrhein (University of Applied Science), Reinarzstr. 49, 47805, Krefeld, Germany

    Lars M. H. Reinders & Martin Jaeger

  3. Faculty of Chemistry, Instrumental Analytical Chemistry, University Duisburg-Essen, Universitätsstr. 5, 45141, Essen, Germany

    Lars M. H. Reinders & Torsten C. Schmidt

  4. Tosoh Bioscience GmbH, Im Leuschnerpark 4, 64347, Griesheim, Germany

    Patrick Endres & Andrea Krumm

Authors
  1. Lars M. H. Reinders
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  2. Martin D. Klassen
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  3. Patrick Endres
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  4. Andrea Krumm
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  5. Martin Jaeger
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  6. Torsten C. Schmidt
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  7. Thorsten Teutenberg
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Contributions

LMHR: conceptualization, methodology, validation, formal analysis, investigation, data curation, writing—original draft, writing—review and editing, visualization, project administration, and funding acquisition. MDK: conceptualization, methodology, and writing—review and editing, supervision, and funding acquisition. PE: conceptualization, investigation, resources, writing—original draft, and writing—review and editing. AK: writing—review and editing. TT: conceptualization, writing—review and editing, supervision, and funding acquisition. MJ: writing—review and editing, and supervision. TCS: writing—review and editing, and supervision.

Corresponding author

Correspondence to Thorsten Teutenberg.

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Conflict of Interest

Patrick Endres and Andrea Krumm are employees of Tosoh Bioscience, part of the Tosoh Corporation, which markets the TSKgel FcR-IIIA-NPR and SkillPak AF-rProtein A HC-650F 5 mL column affinity columns. Beyond this, the authors are not aware of any affiliations, memberships, funding, or financial holding that might be perceived as affecting the objectivity of this work. All other authors have no conflict of interest.

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Reinders, L.M.H., Klassen, M.D., Endres, P. et al. Development of a Two-dimensional Liquid Chromatography High-Resolution Mass Spectrometry Method for the Characterization of Monoclonal Antibodies in Cell-Free Culture Supernatant via FcR Affinity Chromatography. Chromatographia 86, 79–85 (2023). https://doi.org/10.1007/s10337-022-04228-x

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