Rapid high-throughput antibody analysis using microwave-assisted digestion

Monoclonal antibodies are a class of biotherapeutic proteins that have been developed over the past decade, leading to improved standards of care for the treatment of multiple diseases. Multi-attribute methods have emerged as powerful tools for critical quality attributes (CQAs). They leverage high-resolution accurate mass spectrometry and automated computational pipelines to identify pre-established modifications using DDA. In this study, we describe the development of a mass spectrometry-based workflow capable of processing up to 96 samples simultaneously while monitoring a broad panel of PTMs. We evaluated microwave-assisted digestion under different buffers and pHs, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and DIA. Raw data were processed in dependent-peptide search(DDA) and PTM-probing search(DIA), enabling PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with control of temperature and pH, provides a fast and reliable alternative for efficiently digesting biotherapeutic proteins. It achieves high sequence coverage while minimizing artificial PTM formation. We also show that DIA combined with MW digestion improved peptide identification, highlighting its potential for comprehensive characterization of antibodies. Among the tested buffers, sodium acetate under MW conditions was the most effective in reducing deamidation and oxidation levels. SIGNIFICANCE: This study presents a detailed and optimized protocol for microwave-assisted (MW) protein digestion, enabling simultaneous reduction and alkylation for antibody samples. The method is rapid and minimizes chemical artifacts typically introduced during sample preparation. By combining MW-assisted digestion with both data-dependent (DDA) and data-independent acquisition (DIA), we performed a comprehensive and unbiased multi-attribute analysis (MAM). Notably, the use of DIA alongside MW digestion allowed for higher reproducibility and more complete peptide and post-translational modification (PTM) detection compared to DDA alone. Compared to conventional overnight digestion, MW-assisted digestion significantly reduced deamidation levels, with evident influences of buffer composition and pH on PTM identification. Although the levels of protein oxidation persisted, indicating that further optimization is necessary, this approach substantially decreased other artifacts, particularly deamidation, highlighting its potential as a fast, reliable, and highly informative strategy for antibody characterization.