To understand the impact different cleavable linkers would have on in vivo DAR stability we further examined ADC biotransformation pathways using LBA-LC-HRMS approach. In a DAR8 ADC where the interchain disulfide bonds were replaced by the payload conjugation, the light chain and the heavy chain are not covalently bonded and would separate under denaturing conditions of reversed phase chromatography. First, as shown in Figure , the light chain and the heavy chain are well separated, with the associated biotransformed species of the light chain and heavy chain eluting in the area close to the parent light chain and the parent heavy chain. Second, to maximize the identification of the various biotransformed products and to facilitate quantification of these species in an unbiased manner, an automated deconvolution was performed with PeakView (research version), where each spectrum was deconvoluted separately and the m/z-time raw data were converted to a mass-time data. Third, manual identification of the major biotransformed species was performed based on the theoretical intact mass difference between the parent peak and the biotransformed species (Figure , Table ). Then, extracted peak areas from the chromatograms were used for quantification (Table ), Lastly, the chemical structures of these proposed biotransformed species (Figure ) were further confirmed with LC-MS/MS with CID fragmentation using in vitro incubation samples that possessed the same biotransformation species (Figures ).