Speaker
Description
Abstract:
Background: The formation and accumulation of payload-related catabolites from antibody–drug conjugates (ADCs) in targeted and normal tissues are directly associated with ADC's efficacy and systemic toxicity. Rapid and comprehensive identification of these catabolites in vitro is important for supporting the payload/linker design and facilitating preclinical evaluation of ADCs. Here, we developed a quadrupole–Orbitrap HRMS–based workflow to screen and characterize payload-related catabolites generated in in vitro human plasma and lysosomal incubations.
Methods: Brentuximab vedotin and trastuzumab deruxtecan were incubated at 37°C in human plasma (0, 72, 168 h) and liver lysosome (0, 24, 48 h). Samples were protein precipitated with acetonitrile and analyzed by a single-run LC UV HRMS method combining UV scan, SIM scanning for diagnostic payload-specific in-source-dissociated fragment ions (e.g., m/z 506.3588 for Brentuximab vedotin; m/z 419.1402 for trastuzumab deruxtecan; ± 2 Da), and full scan MS with top 5 data-dependent MS2 in both positive and negative modes.
Results: Extraction of diagnostic ions enabled rapid profiling: more than 10 candidate T DxD-related peaks were detected in plasma and lysosome incubates, seven of which were confirmed and structurally identified (including intact T DxD, Cys linker T DxD, and five related catabolites). For Brentuximab vedotin, more than 7 MMAE-related candidate peaks were observed; five were confirmed and identified, including MMAE, Cys linker MMAE (Mc vc PAB MMAE), and three subsequent MMAE metabolites.
Conclusions: The presented LC UV HRMS workflow on a quadrupole–Orbitrap platform provides rapid, sensitive profiling and structural elucidation of ADC payload-related catabolites. Combined plasma and lysosomal biotransformation data support ADC optimization to minimize systemic toxicity and maximize tumor-targeted efficacy.
