Using this new workflow, we were able to identify all major biotransformation species previously found in the cumbersome manual process (Table and Figure ). Hydrolysis was the major biotransformation in LC and HC. For HC, we saw gradual increase over time. At 120 h post dose, complete maleimide hydrolysis seemed to be the dominant form. The other biotransformation was cysteine adduct once free thiol was exposed. Interestingly, for HC only DAR2 species forming cysteine adduct at the free thiol was observed. We previously hypothesized the reason we did not observe cysteine adduct in HC DAR1 because if the 2 free thiol sites are spatially close, they could form intrachain disulfide bond or the abundance of this species is below detection limit of this method.