The exposed free thiol group generated from Linker-PL deconjugation can undergo several biotransformation reactions. Other than forming disulfide bond with endogenous thiol-bearing molecules (e.g. cysteine, GSH, homocysteine and Cys-Gly, etc.) from plasma, we also observed the disulfide bond reformation between Lc and Hc through intact mass LC-HRMS. Due to the conjugation strategy, there should be no interchain disulfide bonds present in AZD8205. The observation of disulfide linked Lc + Hc product suggests that upon deconjugation, liberated cysteine can form an interchain disulfide bond. Here, we aim to confirm the presence of biotransformation product that contains disulfide bond between Lc and Hc by using a bottom-up LC-MRM approach. We also explored the possibility of disulfide linking between other antibody subunits. Figure shows the XICs of Lc + Hc disulfide linked biotransformation product after tryptic digestion. The sequence of this product is GEC-SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (the hydrolyzed linker-payloads were conjugated on CPPC with the first two cysteines linked via disulfide bond). The reason why the missed cleavage occurred is because trypsin was less accessible to the antibody backbone in close proximity to linker-payload conjugation. While the product with no missed cleavage theoretically should be present as well, it cannot be identified through our current method possibly due to its low molecular weight and strong hydrophilicity. Alternatively, the missed cleavage could be the dominant species. By comparing the XICs at different incubation times, the Lc + Hc structure was not present in samples without incubation, and its concentration increases with incubation time. Thus, Lc + Hc was confirmed to be a biotransformation product. Figure shows the CID MS/MS spectrum of this product. The labelled b and y ions were from the SCDKTHTCPPCPAPELLGGPSVFLFPPKPK peptide (SCDKTHT peptide), suggesting high sequence coverage. The EAD MS/MS spectrum also provided additional evidence including individual GEC and SCDKTHT + 2 Hydrolyzed Linker-PL fragment ions (in Figure ). The LC-MRM approach enabled both the confirmation of the disulfide bond reformation between Lc and Hc and the elucidation of its biotransformation sites using CID and EAD fragmentation mechanisms. Similarly, we observed the disulfide bond reformation between two Hc. The sequence of this biotransformed product was THT + THT, with two hydrolyzed linker-PL from each Hc. Due to the low concentration of this biotransformed species, the XICs over different incubation times are not shown here. Figure is the mass spectrum of disulfide bond linking two Hc species at 0 and 168 hr, suggesting this product is not endogenous. The CID MS/MS spectrum confirms the sequence coverage of the THT peptide backbone, as shown in Figure . However, we were not able to assign the disulfide locations to specific cysteines from THT peptide, as the abundance of this product is extremely low. EAD MS/MS spectrum in Figure shows a distinct fragment ion, representing THT + 1 Hydrolyzed Linker-PL. This information reveals further details of the structure of the Hc + Hc biotransformation species.