Purpose: To completely characterize a special modified cysteine-linked ADC.
Methods: An ADC sample was prepared for peptide mapping (reduction, alkylation, and trypsin digestion) or intact analysis (no treatment). For denaturing LC-MS intact analysis, 2 μg of protein sample was separated using a 4 min gradient of 5-90% ACN in H2O and 0.1% formic acid. For native LC-MS intact analysis, 15 μg of sample was desalted online using size exclusion chromatography (50 mM NH4OAc isocratic elution) and directly introduced to the mass spectrometer. Peptide mapping was performed using 1 μg of sample separated with a 49 min gradient of 3-50% ACN in H2O and 0.1% formic acid. A Q Exactive Plus instrument with the Biopharma option was used for all analysis.
Results: With a unique conjugation sites design, populations with three and four drugs loaded onto the antibody were detected with a mass difference between peaks corresponding to the addition of two payloads (+1396.7642 Da). Each of the clusters of ADCs with various payloads were resolved from their adducts with the increase of resolution setting from 17,500 to 35,000. The mass accuracy also greatly increased with the increase of resolution. Thus, drug –antibody ratio (DAR) can be determined accurately using BioPharma Finder software.
Conclusion: With the denatured MS data, it was easily discernible that the non-covalent bonds between antibody chains were broken, and both light chains and heavy chains linked drugs could be observed. The peptide mapping data not only showed almost 100% sequence coverage, but clearly presented the details of conjugated drug modification sites. This workflow provides a complete characterization method for a designed cysteine-linked ADC, including native MS, denatured MS and peptide mapping.