Identification and quantitation strategies of circulating cysteine-conjugated linker-payload catabolites and free payload from a cleavable-linker ADC.

Antibody‑drug conjugate (ADC) off‑target toxicity is largely driven by exposure to payloads and related conjugated catabolites. Determining whether to measure free payload or conjugated linker‑payload catabolites for exposure assessment is critical but not straightforward. Typically, only free payload is measured for ADCs with cleavable linkers, whereas conjugated linker‑payload catabolites are monitored for ADCs with non‑cleavable linkers. For ADC‑1, which contains a cleavable linker, both free Payload-1 and the cysteine‑conjugated linker‑payload catabolite (CLP) were detected, adding complexity to the bioanalysis. Triple‑quadrupole LC‑MS/MS and high‑resolution mass spectrometry were used to confirm the identity of CLP. Temperature‑dependent in‑source fragmentation of CLP and its contribution to the Payload-1 chromatographic signal were investigated. Additionally, the interconversion of the major linear CLP to a cyclized form during sample handling was characterized. The inclusion of 500 mM ascorbic acid during sample processing effectively stabilized the linear form. The linear CLP exhibited pharmacokinetics closely matching those of the Payload-1. Overall, this work improves the bioanalytical understanding of cleavable‑linker ADCs and provides a quantitation strategy to support the routine monitoring of CLP alongside free payload in safety and pharmacokinetic assessments.