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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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).
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The work was financially supported by the IGF Project No. 21280 N.
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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.
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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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DOI: https://doi.org/10.1007/s10337-022-04228-x