Rapid subunit-level multi-attribute monitoring of succinimide and other PTMs for enhanced quality control of therapeutics antibodies.

Succinimide (Asu), a critical intermediate in asparagine deamidation and aspartic acid isomerization, represents a potential critical quality attribute (CQA) of therapeutic antibodies but remains challenging to accurately quantify due to its pH-dependent instability during conventional peptide mapping (PM)-based analysis. To address this limitation, we developed a novel rapid subunit-based multi-attribute method (MAM) that combines IdeS-mediated antibody cleavage with liquid chromatography-mass spectrometry (LC-MS) for rapid, simultaneous monitoring of Asu and other post-translational modifications (PTMs), including oxidation and glycosylation. This approach significantly reduces sample processing time and minimizes Asu degradation by operating under optimized pH conditions (pH 6.5). The method was rigorously validated, demonstrating excellent specificity, precision and accuracy across multiple antibody isotypes. Forced oxidation studies further confirmed strong correlation with traditional PM results, underscoring the method's reliability for comprehensive PTM profiling. As the first reported rapid subunit-based MAM specifically optimized for Asu analysis, this strategy provides a rapid, accurate, and comprehensive (RAC) tool for enhanced quality control and stability monitoring of therapeutic antibodies, thereby supporting improved biopharmaceutical production efficiency and product safety.