Stress-Induced Antibody Aggregates: Insights from Native SEC-MS with Postcolumn Denaturation.

Characterization of high-molecular-weight species (HMWS) that are formed under forced stress conditions is a key component of degradation pathway studies performed during late-stage development for monoclonal antibody (mAb) therapeutics. Native mass spectrometry (MS) is a powerful technique for probing the structure and composition of HMWS. It offers high mass accuracy (surpassing that of the gold-standard SEC-MALS analysis), good sensitivity, and specificity. However, the routine implementation of this method in biopharmaceutical industry laboratories has been hindered by the special considerations required for data acquisition and interpretation. In this study, we present a systematic evaluation and the development of a platform method for native SEC-MS characterization of large aggregates in stressed mAb samples. Our approach utilizes conventional quadrupole time-of-flight mass spectrometry without hardware modifications to general-purpose instruments, making it widely accessible. We applied this platform to characterize aggregates in a model IgG4 antibody subjected to thermal and low-pH stress conditions. By implementing postcolumn denaturation (PCD), we successfully identified the nature of the formed aggregates, distinguishing between covalent and noncovalent species. Our work provides a practical guideline for incorporating native SEC-MS methods into degradation pathway study workflows, offering a robust and versatile tool for HMWS characterization. This method enhances our understanding of stress-induced antibody aggregation, potentially improving the development of monoclonal antibody therapeutics.