Investigation of High Molecular Weight Size Variant Formation in Antibody-Drug Conjugates: Microbial Transglutaminase-Mediated Crosslinking

Microbial transglutaminase (mTG) has become a powerful tool for manufacturing antibody-drug conjugates (ADCs). It enables site-specific conjugation by catalyzing formation of stable isopeptide bond between glutamine (Q) side chain and primary amine. However, the downstream impact of mTG-mediated conjugation on ADC product quality, especially on high molecular weight (HMW) size variant formation has not been studied in a systematic manner. This study investigates the mechanisms underlying the formation of HMW size variants in mTG-mediated ADCs using size exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS). Our findings revealed that the mTG-mediated glutamine and lysine (K) crosslinking is the primary source of the increased level of HMW size variants in the ADCs. In the study, two monoclonal antibodies (mAbs) with glutamine engineered for site-specific conjugation were used as model systems. Based on the LC-MS analysis, a single lysine (K56) in the heavy chain (HC) was identified as the major Q-K crosslinking site in one of the two mAbs. The HC C-terminal K was observed to crosslink to the target Q in both mAbs. Quantitative correlation was established between the percentage of HMW size variants determined by SEC and the percentage of crosslinked peptides quantified by MS peptide mapping. Importantly, it was demonstrated that the level of HMW size variants in the second ADC was substantially reduced by the complete removal of HC C-terminal K before conjugation. The current work demonstrates that crosslinking and other side reactions during mTG-mediated conjugation needs to be carefully monitored and controlled to ensure process consistency and high product quality of the final ADC drug product.