Upon exposure of free thiols on both heavy chain and light chains following deconjugation, resultant secondary reaction products included cysteine and GSH adducts as well as newly reformed disulfide bonds between spatially close free thiols (Figure ). This observation is supported with observed intact mass, and further confirmed with the MS 2 fragmentation for selected parent ions from bottom-up LC-HRMS data (Figures ). The quantification of these secondary, minor, biotransformation species is displayed in Figure . Furthermore, the deconjugated small molecule linkerpayload has been observed to covalently conjugate to circulating albumin (Table , index 41). The albumin-linker-payload can then also undergo thio-succinimide ring hydrolysis (Table , index 42). It is notable that the HC biotransformation species showed a distinct pattern when comparing the loss of one linker-payload versus the loss of two linker-payloads. After deconjugation of a single linkerpayload, we observed a cluster of peaks with several mass changes (Figure , Table , index 11-24), which are indicative of multiple species formed after the exposed free thiol subsequently reacted with other redox-active molecules in plasma. The observed intact mass matched with the proposed adducts and the structure was further confirmed with CID MS 2 spectra (Figure ). On the contrary, after deconjugation of two linker-payloads, the major observed biotransformed species (Table , index 7-10) had a mass change consistent with the loss of two linker-payloads (e.g. -2296 Da for AZD8205). No additional secondary adducts were observed. The potential reason of the distinct pattern after two payload loss is the reformation of the intra-chain disulfide bond. In the case of the HC-1PL species containing two free exposed thiols and if these thiols are spatially close, reformation of the intra chain disulfide bond becomes the major step 2 reaction. The heavy chain intra-chain disulfide bond was confirmed with bottom-up LC-HRMS with fragmentation (Figure ).