Rapid High-Throughput Antibody Analysis Using Microwave-Assisted Digestion

ABSTRACT: Monoclonal antibodies (mAbs) are a major class of biotherapeutic proteins that have been extensively developed over the past decade, resulting in improved standards of care for multiple diseases. Regulatory agencies recommend a Quality by Design (QbD) approach for manufacturing therapeutic proteins, which necessitates a comprehensive understanding of the molecular attributes critical for safety, efficacy, and product quality. In this context, multi-attribute methods (MAMs) have emerged as powerful tools for monitoring critical quality attributes (CQAs) in biotherapeutics, leveraging high-resolution Orbitrap mass spectrometry and automated computational pipelines for identification of pre-established modifications using data-dependent acquisition (DDA). In this study, we describe the development of a 17-minute, mass spectrometry–based workflow capable of monitoring a broad panel of post-translational modifications (PTMs), including key CQAs such as oxidation and deamidation. We evaluated microwave-assisted digestion (MW) under different buffer systems and pH conditions, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and data-independent acquisition (DIA), with raw data processed in MaxQuant (DDA, dependent-peptide search) and Spectronaut (DIA, PTM-probing search), which enabled PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with precise control of temperature and pH, provides a fast and reliable alternative for efficient digestion of complex biomolecules, achieving high sequence coverage while minimizing artificial PTM formation, especially deamidation. DIA outperformed DDA in terms of sequence coverage and the identification of both modified and unmodified peptides, highlighting its potential for comprehensive characterization of therapeutic antibodies. Among the tested buffers, sodium acetate in MW was the most effective in reducing deamidation and oxidation levels.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hellen Valerio  

LAB HEAD: Giuseppe Palmisano

PROVIDER: PXD067893 | Pride | 2026-01-26

REPOSITORIES: Pride