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Comparison of originator and biosimilar monoclonal antibodies using HRMS, Fc affinity chromatography, and 2D-HPLC

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Abstract

Due to the complex manufacturing process of therapeutic monoclonal antibodies, it is hardly possible to produce an identical copy of the original product (originator). Consequently, follow-on products (biosimilars) must demonstrate their efficacy being similar to the originator in terms of structure and function. During this process, a variety of analytical methods are required for this purpose. This study focuses on three particularly relevant analytical techniques: high-resolution mass spectrometry, fragment crystallisable (Fc) affinity chromatography, and two-dimensional peptide mapping. Each analytical method proved able to identify specific differences between originator and biosimilar. High-resolution mass spectrometry was used to characterize the glycan pattern. It was shown that a trastuzumab biosimilar did not have the G0:G0F sugar modification identified in the originator. The application of affinity chromatography to rituximab showed that originator and biosimilar interacted differently with the immobilized Fc receptor. Furthermore, 2D-HPLC peptide mapping demonstrated the influence of orthogonality of separation dimensions, leading to differentiation of a rituximab originator and biosimilar.

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Abbreviations

mAbs:

Monoclonal antibodies

CHO:

Chinese hamster ovary cells

EMA:

European Medicines Agency

API:

Active pharmaceutical ingredient

HRMS:

High-resolution mass spectrometry

2D-HPLC:

Two-dimensional liquid chromatography

ADCC:

Antibody-dependent cell-mediated cytotoxicity

Fc:

Fragment crystallisable

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Acknowledgements

We would like to thank the Federal Ministry for Economic Affairs and Energy for funding the INNO-KOM project “Sensitive method for the detection of airborne proteins” (49VF170039). Furthermore, we would like to thank Tosoh Bioscience for providing materials and samples.

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

    Lars M. H. Reinders & Torsten C. Schmidt

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  1. Lars M. H. Reinders
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  2. Martin D. Klassen
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  3. Thorsten Teutenberg
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  4. Martin Jaeger
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  5. Torsten C. Schmidt
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Contributions

Lars M. H. Reinders: conceptualization, methodology, validation, formal analysis, investigation, data curation, writing—original draft, writing—review and editing, visualization, project administration, funding acquisition. Martin D. Klassen: conceptualization, methodology, writing—review and editing, supervision, funding acquisition. Thorsten Teutenberg: conceptualization, writing—review and editing, supervision, funding acquisition. Martin Jaeger: writing—review and editing, supervision. Torsten C. Schmidt: writing—review and editing, supervision.

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Correspondence to Thorsten Teutenberg.

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Reinders, L.M.H., Klassen, M.D., Teutenberg, T. et al. Comparison of originator and biosimilar monoclonal antibodies using HRMS, Fc affinity chromatography, and 2D-HPLC. Anal Bioanal Chem 414, 6761–6769 (2022). https://doi.org/10.1007/s00216-022-04236-8

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  • DOI: https://doi.org/10.1007/s00216-022-04236-8

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