Overcoming Incomplete Peptide Mapping of Antibody Complementarity-Determining Regions with Alternate Digestion Workflows

Peptide mapping of antibodies is an essential method to monitor peptide modifications in antibody lots that could affect the safety and efficacy product. Conventional protocols rely on protein digestion using proteases, such as trypsin, before with mass spectrometry (MS). However, trypsin may cause incomplete peptides, especially those include highly hydrophobic peptides. Here, we show how pepsin can be used alternative complementary protease for allows improved sequence coverage, proteins regions. We also guanidine hydrochloride (GuHCl) post-digestion improves results. Overall, these two methods—pepsin GuHCl post-digestion—can provide more comprehensive maps, thereby enabling thorough quality checking biopharmaceutical products.