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In this study, we developed a quadrupole–Orbitrap HRMS workflow for rapid screening and structural characterization of payload‑related catabolites derived from ADCs. The workflow was evaluated using two commercially successful ADCs (DS-8201a and SGN‑35) as models, incubated in human plasma and liver lysosomes. Successful screening and characterization of multiple payload‑related catabolites from each incubation validated the approach.

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