Similarly, proteins or peptides with susceptible amine groups can undergo glycation in vitro ( 7, 8). For example, human serum albumin and hemoglobin were found to have varying degrees of glycation, depending on the blood glucose level and the amount of time in circulation ( 3, 5, 6).

Protein glycation is a naturally occurring process in vivo. When the reaction occurs in a protein, it requires the condensation between an aldehyde group on the reducing sugar and a de-protonated amine group on the amino acid to form a Schiff base intermediate (imine), followed by the Amadori rearrangement to establish a more stable Amadori product (ketoamine) ( 2, 4). Glycation, a non-enzymatic reaction of a reducing sugar with an amine group, is the first step toward a more complex Maillard reaction ( 1, 2, 3). 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. 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. In addition, this glycation reaction was found to be driven by an unusually low p K a on the ε-amino group of HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5☌–45☌ and can be well predicted in vitro and in a non-human primate. 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. 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.

Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies.

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