In-depth source characterization and analytical control strategy for the charge variants of antibody drug conjugates:...

Antibody-drug conjugates (ADCs) combine the dual characteristics of antibody targeting and high payload potency. ADCs represent a key technology in targeted therapy and have become a major direction in "precision" medicine. Charge variants are a major source of ADC variability with potential effects on efficacy and safety, which necessitates comprehensive characterization to guide analytical control strategies. We employed two orthogonal methods, ion exchange chromatography (IEX) fraction collection and imaged capillary isoelectric focusing-mass spectrometry (iCIEF-MS), to characterize ADC charge variants via "top-down" and "bottom-up" approaches. Beyond inherent the post-translational modifications and fragmentation profile of the monoclonal antibody, conjugation properties, drug-to-antibody ratio (DAR), DAR distribution, and position isomers, drove ADC charge variants. This is the first systematic study on the origins of ADC charge variants. The method of IEX fraction collection and iCIEF-MS were compared in terms of principle and experimental results. Challenges in interpreting the causes of charge variants and the possible reasons for incomplete interpretation of acidic peaks were discussed. Our study provides an insight into the source, in-depth characterization, and risk-based analytical control strategy of ADC charge variants.