High-Resolution Ion-Mobility-Enabled Peptide Mapping for High-Throughput Critical Quality Attribute Monitoring

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the m/z-extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.