Alternatively, the re-formation of disulfide bonds can happen between the light chain and the heavy chain, following payload deconjugation on both chains. This can be confirmed with the increasing amount of a 76 kDa biotransformation species observed at the 4.9 min retention time (Figure , Figure ). The observed mass matched with the expected mass of light chain and heavy chain conjugated complex, with two linker-payloads remaining on the heavy chain and potential thio-succinimide ring hydrolysis. This inter-chain disulfide bond between light chain-heavy chain was confirmed with bottomup LC-HRMS with fragmentation (Figure ). For ADCs that deconjugated to greater extent (ADC2 and ADC3), the potential of reformation of the interchain disulfide bond in vivo may have contributed to stabilization of the protein scaffold (Figure , 4A, Table ). Interchain disulfide bond reformation was observed for all 4 ADCs, although higher for ADC 2 and 3 (Figure ). This is likely due to the larger degree of deconjugation, catalyzing the re-formation of the disulfide bonds. After deconjugation, the deconjugated linker-payload can re-conjugate to various thiol-containing endogenous proteins . Capture with anti-payload antibody enables detection and characterization of additional proteins that would contain the re-conjugated linker-payload, such as albumin. Interestingly, albumin conjugated linkerpayload continued to hydrolyze over time (Figure ). These data are consistent with the hypothesis that the linker hydrolysis needs sufficient time for the reaction to proceed and the slower elimination half-life of albumin conjugated linker-payload enables this reaction to be observed on non-antibody containing macromolecular species. Further relative quantification analyses were also performed on the data set. The analysis suggested that glycoforms on the heavy chain do not seem to have a significant impact on the biotransformation at the conjugation site (Figure ). Cysteinylation is the major secondary reaction for the exposed free thiol after deconjugation and also gradually increased over time (Figure ).