Improvements on sample preparation and peptide separation for reduced peptide mapping based multi-attribute method analysis of therapeutic monoclonal antibodies using lysyl endopeptidase digestion

The mass spectrometry based multi-attribute method (MAM) has gained popularity in the field of biopharmaceutical analysis as it promises a single method for comprehensive monitoring of multiple product quality attributes (PQAs) and product purity. Sample preparation for protein digestion and peptide separation are critical considerations for a reduced peptide mapping-based MAM. To avoid desalting steps required in most tryptic protein digestion and in order to improve peptide separation for hydrophilic peptides, we developed an improved robust sample preparation using lysyl endopeptidase (Lys-C) for high-throughput MAM testing. Additionally, this method optimizes the peptide retention and separation of a stability-indicating VSNK peptide using a HSS T3 column for comprehensive PQA monitoring. A fully automated sample preparation had similar assay variations for PQAs monitoring compared to manual sample preparation. To the best of our knowledge, this is the first report of a high-resolution MS-based MAM using a streamlined Lys-C digestion without desalting with enhanced PQA monitoring for hydrophilic peptides. The improved, robust MAM workflow for protein digestion and peptide separation will pave the way for broader MAM qualification and its applications for the characterization and quality control of therapeutic monoclonal antibodies.