For the second biotransformation case, involving the pyrrolobenzodiazepine-conjugated ADC, detailed experimental method was previously described in Huang, et al. Data Analysis with Biologics Explorer v4.0 (SCIEX, Framingham, MA). Raw data in .wiff2 format were processed using intact automated deconvolution workflow in Biologics Explorer (BE). Peak pre-processing was performed prior to deconvolution including spectrum baseline subtraction and noise removal. Deconvolution was performed using proprietary maximum entropy algorithm with separate retention time windows for light chain and heavy chain elution regions. Peak identification was performed in Protein Mapping step where HC and LC amino acid sequence was provided as input. To generate linker-payload biotransformation library, ADC processing method in Metabolite Pilot (MP) software was used (Molecule Profiler is the updated version of Metabolite Pilot software). Linkerpayload structure .mol file was uploaded. Conjugation site was specified at the maleimide ring. Potential compound cleavages were filtered by site of attachment, meaning cleavages considered were all conjugated to cysteine in antibody. Maximum number of bonds that break in the structure was set to 2, and no ring bonds were broken. This generated a list of 46 possible cleavages. For additional biotransformation of the linker-payload, hydrolysis (+H2O), oxidation deamidation to alcohol (R-NH2 to R-OH), as well as combination of the two were added. Combining compound cleavages with biotransformation led to a final exported list of 187 possibilities. This list was then reformatted for compatibility with downstream software such as Protein Metrics Byos or BE. Specifically, the name column was reformatted as loss of a chemical formula with or without additional modification (e.g. hydrolysis, deamidation, or combination). The monoisotopic mass (MH+) exported from MP software was converted to neutral mass. Data Analysis with Protein Metrics Byos v5.4 (Dotmatics, Boston, MA). Raw files were uploaded to ADC workflow in Protein Metrics software. Custom TIC window was selected to cover LC elution from 1.7 to 2.2 min. LC sequence was entered and common IgG modifications (N-terminal Q to pyroGlu and C-terminal K loss) were included. In the delta mass table, the biotransformation library generated from MP software was imported. In addition, the cysteine adduct was also added as delta mass. Mass tolerance was set to 3 Da. Deconvolution mass range was from 1000 to 5000 m/z and 20,000 to 80,000 Da. After analysis, for peaks that had multiple assignment within mass tolerance, they were manually reviewed to select most plausible assignment based on enzymatic cleavage mechanisms. For quantification, intensities of all peaks were exported and plotted for fractional abundance of all LC species. Fractional abundance was calculated by each peak intensity divided by total LC biotransformation peak intensity. Percent difference between new and original values was calculated by subtracting new from previous values over mean value of the two.