Detailed Structural Elucidation of Antibody-Drug Conjugate Biotransformation Species Using High Resolution Multiple Reaction Monitoring Mass Spectrometry with Orthogonal Dissociation Methods

Antibody-drug conjugates (ADCs) are a promising drug modality substantially expanding in both the discovery space and clinical development. Assessing the biotransformation of ADCs in vitro and in vivo is important in understanding their stability and pharmacokinetic properties. We previously reported biotransformation pathways for the anti-B7H4 topoisomerase I inhibitor ADC, AZD8205, puxitatug samrotecan, that underpin its structural stability in vivo using an intact protein liquid chromatography-high resolution mass spectrometry (LC-HRMS) approach. Herein, we employed a LC-high resolution multiple reaction monitoring (LC-MRMHR) approach using both collision-induced dissociation (CID) and electron-activated dissociation (EAD) methods, confirming our earlier findings. Furthermore, we were able to obtain additional detailed structural information on the biotransformation products expanding on earlier intact analyses. We also highlight the high sensitivity of LC-MRMHR for successfully identifying minor biotransformation products at low concentrations that were not detectable using the intact protein LC-HRMS workflow. Especially, EAD aided in the confirmation of biotransformation species that contain newly formed disulfide bonds due to the preferential dissociation of disulfide bonds using this method. We observed biotransformation reactions that vary between linker-payload (PL) conjugation sites on the antibody. For example, the trend toward constitutional isomerism in thio-succinimide linker hydrolysis, and the resulting positional isomers from thiol adduct formation following linker-PL deconjugation. The reported orthogonal analytical approaches highly complement and fortify the intact protein LC-HRMS data. This study sheds further light on detailed structural characterization of various ADC species and validates the proposed biotransformation pathways explaining the stability of AZD8205 in vivo.