Microwave‐Accelerated Proteolysis Minimizes Artificial Deamidation and Enhances Speed for Peptide Mapping of Protein Pharmaceuticals

RATIONALE: Microwave-accelerated proteolysis with optimized buffers reduces artificial N/Q deamidation introduced during conventional peptide mapping of monoclonal antibodies. This workflow preserves CQA accuracy while significantly reducing digestion time and improving analytical throughput, offering a reliable, high-efficiency strategy for biopharmaceutical quality control and deamidation assessment in therapeutic protein characterization. METHODS: Synthetic PENNY peptides, a murine mAb peptide (VNSAAFPAPIEK), and human IgG were reduced, alkylated, and trypsin-digested in Tris or ammonium bicarbonate using conventional or microwave-assisted protocols (900 W, 1 min; 100 W, 30 min). Deamidation was quantified by UPLC-ESI-MRM on a triple quadrupole MS. Peptide mapping and PTM localization were achieved by LC-ESI-MS/MS, respectively, using a high-resolution SCIEX TripleTOF 6600 and a low-resolution Thermo LTQ XL with CID, ETD, and CID + ETD. RESULTS: Buffer chemistry and exposure time strongly influenced artificial deamidation. Compared with overnight digestion (16-18 h), microwave-assisted processing reduced deamidation (4.0%-4.2% vs. 16.9%) while shortening preparation time. Optimized Tris conditions lowered PENNY peptide deamidation by ~60%-80%. For VNSAAFPAPIEK, high-power microwaving reduced processing to 11 min while preserving fragmentation coverage. Application to IgG decreased multisite N/Q deamidation, and BSA validation confirmed consistently low deamidation (< 1%), supporting robustness across sequence-diverse proteins. CONCLUSIONS: Microwave-accelerated proteolysis under optimized Tris buffer conditions minimizes artificial N/Q deamidation during peptide mapping while substantially reducing preparation time. Digestion time decreases from 16-18 h to minutes, lowering deamidation by ~60%-80%. Consistent findings in IgG and BSA improve confidence in CQA assessment and support high-throughput biopharmaceutical quality control.