Detailed Structural\nElucidation of Antibody-Drug Conjugate\nBiotransformation Species Using High Resolution Multiple Reaction\nMonitoring Mass Spectrometry with Orthogonal Dissociation Methods

Antibody-drug conjugates\n(ADCs) are a promising drug\nmodality substantially\nexpanding in both the discovery space and clinical development. Assessing\nthe biotransformation of ADCs <i>in vitro</i> and <i>in vivo</i> is important in understanding their stability and\npharmacokinetic properties. We previously reported biotransformation\npathways for the anti-B7H4 topoisomerase I inhibitor ADC, AZD8205,\npuxitatug samrotecan, that underpin its structural stability <i>in vivo</i> using an intact protein liquid chromatography-high\nresolution mass spectrometry (LC-HRMS) approach. Herein, we employed\na LC-high resolution multiple reaction monitoring (LC-MRM<sup>HR</sup>) approach using both collision-induced dissociation (CID) and electron-activated\ndissociation (EAD) methods, confirming our earlier findings. Furthermore,\nwe were able to obtain additional detailed structural information\non the biotransformation products expanding on earlier intact analyses.\nWe also highlight the high sensitivity of LC-MRM<sup>HR</sup> for\nsuccessfully identifying minor biotransformation products at low concentrations\nthat were not detectable using the intact protein LC-HRMS workflow.\nEspecially, EAD aided in the confirmation of biotransformation species\nthat contain newly formed disulfide bonds due to the preferential\ndissociation of disulfide bonds using this method. We observed biotransformation\nreactions that vary between linker-payload (PL) conjugation sites\non the antibody. For example, the trend toward constitutional isomerism\nin thio-succinimide linker hydrolysis, and the resulting positional\nisomers from thiol adduct formation following linker-PL deconjugation.\nThe reported orthogonal analytical approaches highly complement and\nfortify the intact protein LC-HRMS data. This study sheds further\nlight on detailed structural characterization of various ADC species\nand validates the proposed biotransformation pathways explaining the\nstability of AZD8205 <i>in vivo</i>.