Tryptic Peptide Mapping on Antibody-drug Conjugates: Improvements in Hydrophobic Peptide Recovery and Missed Cleavages

Antibody-drug conjugates (ADCs) have become a promising treatment for various cancers, by combining the specificity of monoclonal antibodies (mAbs) with the potent cytotoxicity of small-molecule drugs. The intricate structure of ADCs necessitates comprehensive analytical characterization to ensure their safety, efficacy, and quality. Reduced peptide mapping, integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS), is essential for assessing critical quality attributes (CQAs) of ADCs, including linker-payload (LP) integrity, post-translational modifications (PTMs) of the mAb backbone, and the distribution of conjugation sites. Traditional tryptic peptide mapping protocols optimized for mAbs are often inadequate for ADCs, because of the hydrophobic nature of LPs. These LPs alter the physicochemical properties of conjugated peptides, thus leading to challenges such as suboptimal recovery because of interactions with other hydrophobic peptides and steric hindrance impeding enzymatic digestion. Herein, we explored strategies to enhance the recovery of hydrophobic peptides by incorporating various surfactants and denaturants. The addition of 2.7 M guanidine hydrochloride significantly increased the recovery of hydrophobic peptides. Digestion conditions were optimized to increase the digestion efficiency of the conjugated peptides. A 3-h incubation at 37 °C with a 1:10 (w/w) ratio of Promega sequencing grade trypsin to ADCs yielded minimal missed cleavages and a minimal increase in non-specific cleavages. The optimized peptide mapping workflow demonstrates high hydrophobic peptide recovery and reproducibility, and provides a robust platform for detailed characterization of ADCs, thereby ensuring their consistent product quality.