Backbone-matched comparison of two trastuzumab-based antibody–drug conjugates: payload localization and gas-phase heterogeneity of trastuzumab deruxtecan and trastuzumab rezetecan

Antibody–drug conjugates (ADCs) are biotherapeutics that are structurally heterogeneous due to the way each ADC is formed. Therefore, complementary analytical approaches need to be used in order to achieve a complete characterization of all aspects of the structure of these biotherapeutics. This project created a multi-level approach to assess the structural differences between trastuzumab, T-deruxtecan, and T-rezetecan. Using hydrophobic interaction chromatography (HIC)-ultraviolet (UV) detector, native size-exclusion chromatography (SEC)-mass spectrometry (MS), denaturing reverse-phase liquid chromatography (RPLC)-MS, IdeS-based middle-up LC-MS, and native ion mobility spectrometry (IM)-MS, we assessed the hydrophobic heterogeneity, average drug-to-antibody ratio (DAR), payload distribution by chain, and glycosylation status of the Fc region, payload localization by subunit, and gas phase mobility of each ADC. Characterization of the two ADCs revealed that, although both ADCs are based on the same trastuzumab scaffold, their structural characteristics differ significantly. The average DARs of T-deruxtecan and T-rezetecan were determined by native SEC-MS to be 7.89 and 3.83, respectively. Chain-level denaturing RPLC-MS showed that payload was distributed across both the heavy and light chains in T-deruxtecan, whereas conjugation was confined to the heavy chain in T-rezetecan. Middle-up LC-MS confirmed that Fc N-glycosylation was unchanged after conjugation in both ADCs. Native IM-MS showed that T-deruxtecan and T-rezetecan gave distinct arrival time distributions at matched charge states, with the more clearly multimodal profile observed for the conjugate having the lower average DAR. The comparative design also proved informative in a way a single-conjugate study could not: species shifted by approximately + 540 Da relative to a common Fd-region species were detected in both conjugates, corresponding to approximately half the mass of the respective linker-payload and establishing that they are not payload-derived. These results provide the first structural description of T-rezetecan at the intact, chain, and subunit levels and show that drug loading alone does not determine the structural heterogeneity of a cysteine-linked ADC.