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. 2024 Jul:200:114325.
doi: 10.1016/j.ejpb.2024.114325. Epub 2024 May 15.

Comprehensive analysis and characterization of glycan pairing in therapeutic antibodies and Fc-containing biotherapeutics: Addressing current limitations and implications for N-glycan impact

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Comprehensive analysis and characterization of glycan pairing in therapeutic antibodies and Fc-containing biotherapeutics: Addressing current limitations and implications for N-glycan impact

Maximilian Meudt et al. Eur J Pharm Biopharm. 2024 Jul.
Free article

Abstract

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics.

Keywords: Biotherapeutic; Glycan pairing; Monoclonal antibody; N-glycosylation; Pharmacokinetics.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maximilian Meudt, Julia Baumeister, Michaela Blech and Fabian Higel are employees of Boehringer Ingelheim GmbH & Co. KG, which is developing, manufacturing, and marketing biopharmaceutical products.

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