Detailed Structural Elucidation of an Antibody-Drug Conjugate, AZD8205, Biotransformation Species Using LC-MRM with CID and EAD Fragmentation

Antibody-drug conjugates (ADC) are a promising drug modality experiencing substantial expansion in both discovery space and clinical development, due to its targeted delivery and potentially improved therapeutic index. Assessing the biotransformation of ADCs in vitro and in vivo is important in understanding their stability and pharmacokinetic properties. We have previously reported biotransformation pathways for the anti-B7H4 topoisomerase I inhibitor ADC, AZD8205, that underpin its structural stability in vivo using intact protein mass LC-HRMS approach. Herein we employed LC-MRM method with both CID and EAD fragmentation that confirmed our earlier findings. Furthermore, we were able to obtain additional detailed structural information of these biotransformation products expanding on earlier intact mass method analyses. We also highlight the high sensitivity of LC-MRM for successfully identifying minor biotransformation products at low concentrations, that were not distinguishable using the intact mass LC-HRMS workflow. Especially, the EAD fragmentation aided in the confirmation of biotransformation species that contain newly formed disulfide bonds, due to the preferential fragmentation of disulfide bonds using this method. We observed biotransformation reactions that vary between linker-payload (PL) conjugation sites on the antibody, including the trend towards constitutional isomerism in thio-succinimide linker hydrolysis, and linker-payload deconjugation. The reported orthogonal analytical approach highly complements and fortifies the earlier findings from intact protein mass LC-HRMS explaining AZD8205 stability in vivo. This study sheds further light upon detailed structural confirmation of ADC biotransformation and validates our earlier findings that explain AZD8205 stability in vivo.