Bottom-up mass spectrometry approaches in the biopharmaceutical industry typically rely on lysine-specific proteases, such as trypsin and/or Lys-C, for sample preparation. However, molecules which contain modified lysine residues, such as lysine-conjugated antibody-drug conjugates (ADCs) and glycated proteins, pose a significant challenge to these traditional digestion methods. Modification at lysine residues is detrimental to the digestion efficiency of lysine-specific proteases and can result in highly complex digestion patterns due to the heterogeneity of the modifications. Digestion with trypsin resulted in incomplete coverage of lysine-containing peptides during the analysis of trastuzumab emtansine, a model ADC. Also, since conjugation prevents digestion at lysine, the modified and unmodified lysines are digested at different rates, therefore, not generating comparable peptides required for easy label-free quantification of site occupancy.

Here we present an alternative and improved bottom-up strategy for the analysis of molecules containing modified lysine residues. Utilizing an arginine-specific protease, GingisREX® , we were able to achieve full coverage of all lysine conjugated sites for easy quantification of site occupancy in trastuzumab emtansine. As GingisREX is highly specific for arginine residues, with no detectable activity towards lysine, the digestion efficiency is independent of potential lysine modifications and for trastuzumab emtansine, the same set of peptides are generated regardless of conjugation level. In addition to site occupancy determination, we were also able to characterize the changes in drug-antibody ratio (DAR) which may occur during forced degradation studies following incubation of the ADC in serum. Finally, intact reconstruction from peptide mapping data was used to correlate the bottom-up site occupancy measurements with DAR determinations performed at the intact level.