HDX-MS Reveals Regions Prone to Aggregation In a mAb Exposed To pH Stress

Monoclonal antibodies (mAbs) are crucial therapeutic proteins. Their large size and complex structure pose significant pharmaceutical challenges, particularly regarding stability and solubility. Structurally, mAbs consist of two light (LC) and two heavy (HC) chains, held together by disulfide bonds, and are divided into two Fab (antigen-binding) regions and one Fc (constant) region1. Throughout development, mAbs encounter stressors at every stage, including purification, shipping, storage, and administration. A major concern is protein aggregation, as aggregated antibodies can trigger immune responses. Identifying specific regions prone to unfolding and aggregation under pharmaceutically relevant conditions is critical. Understanding the unfolding and aggregation mechanism is essential for optimizing mAb stability and to develop strategies to reduce this form of degradation. Hence, we exposed mAbs to three different stressors: 1) low pH, 2) light, since we have previously seen they can cause aggregation2, and 3) treatment with guanidinium to generate partially unfolded species, potentially prone to aggregation. The resulting conformations were studied by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) that provides detailed insights into conformational changes and aggregation-prone regions at local resolution.