This approach unveiled various macromolecular biotransformed species from light chain (LC), heavy chain (HC) or half antibody, identified over the 12-day period post-dose of each ADC in Tg32 mice (Figure , Table ). To perform relative quantification of complex biotransformation species, the various analytes were clustered based on the relevant characteristics to provide simplified metrics for profiling the in vivo mixture of ADC and its biotransformed species. There are two assumptions for relative characterization of this data set: 1) capture efficiency and ionization efficiency are reasonably comparable among species used for quantification; 2) data processing is performed uniformly for all species regardless of the signal intensity of the biotransformation species. Thio-succinimide-conjugated payloads go through two competing biotransformation reactions: hydrolysis or deconjugation via retro-Michael reaction . It was previously demonstrated that some linkers can partially deconjugate, resulting in a protein-partially cleaved payload structure . Aside from the thiosuccinimide ring hydrolysis, protein scaffold instability has also been reported when the parent molecule has disrupted disulfide bonds. For ADC1 and ADC4 species with hydrolyzed thio-succinimide ring were the major biotransformation products on both the heavy chain and the light chain (Figure ). The retention time of these species did not alter significantly compared to the parent molecule. At 48 h post dose, the hydrolyzed forms replaced the original species and became the most abundant form of light chain for ADC1 and ADC4 (Figure ). For the heavy chain species, the hydrolysis happened gradually: generating partially hydrolyzed species first, then shifting to fully hydrolyzed species. The kinetics of the thio-succinimide hydrolysis is dependent on the chemistry of the linker: both ADC1 and ADC4 contain linkers with propionyl-PEG8 spacers between the amide and the thio-succinimide, resulting in faster hydrolysis rate compared to ADC2 and ADC3 (Figure , S8) that contain caproyl spacer only. The hydrolyzed species were also confirmed with LBA-LC-HRMS bottom-up identification, through both accurate mass MS1 as well as MS2 spectra (Figure Table ). The LC-conjugated linker-payload deconjugates less compared to the HC-conjugated ones for the four ADCs studied here, as observed by other researchers . In contrast to ADC1 and ADC4, for ADC 2 and ADC 3, the deconjugation on HC was observed as the major form especially for timepoints after 48 h (Figure ). Deconjugation results in lower DAR species. These species usually elute earlier compared to the parent molecule. For heavy chain species, the parent heavy chain (with 3 payloads, HC-3PL) eluted around 5.6 min, with the biotransformed species eluting around 5.4 min (HC-2PL) and 5.3 min (HC-1PL), respectively (Figure ). Note that the degree of deconjugation on LC for all ADCs are consistently low (<0.4 %) compared to HC (Figure ).