Low pK a of Lys promotes glycation at one complementarity-determining region of a bispecific antibody

• Published in: Biophysical journal Vol. 121; no. 6; p. 1081
• Main Authors: Xu, Xiaobin, O'Callaghan, Jessica Ann, Guarnero, Zachary, Qiu, Haibo, Li, Ning, Potocky, Terra, Kamen, Douglas E, Graham, Kenneth S, Shameem, Mohammed, Yang, Teng-Chieh
• Format: Journal Article
• Language: English
• Published: United States 15.03.2022
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Chromatography, Liquid; Complementarity Determining Regions - chemistry; Complementarity Determining Regions - metabolism; Glycosylation; Kinetics; Lysine - metabolism
• ISSN: 1542-0086
• DOI: 10.1016/j.bpj.2022.02.002

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8-10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C-45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pK on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.