Interdomain Interactions Modulate Refolding Kinetics and Aggregation in a Monoclonal Antibody.

Understanding the structural determinants of antibody stability and aggregation is essential for therapeutic development. In this study, we investigated the unfolding and refolding behavior of bevacizumab under denaturing conditions using dynamic light scattering (DLS), circular dichroism (CD), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Unfolding was induced by incubating the antibody in 4 M guanidine hydrochloride (Gnd-HCl), followed by refolding through dilution with 1 M Gnd-HCl. Each domain exhibited distinct unfolding kinetics: the C_H2 and V_H domains unfolded rapidly, while the C_H3 domain retained its structure until 45 min, consistent with its known thermodynamic stability. Aggregation, detected by DLS, was prevalent only after 120 min and overnight unfolding, coinciding with C_H3 destabilization. Notably, aggregation-prone regions were identified in both the Fc and Fab portions of the antibody. Specifically, interactions at the C_H2-C_H3 and C_H3-C_H3 interfaces appear disrupted upon C_H3 unfolding, leading to misfolded and aggregation-prone states in both domains. In parallel, the V_H CDR H1 region exhibited aberrant protection after refolding, suggesting its involvement in aggregation. These findings highlight the cooperative nature of C_H2-C_H3 refolding and underscore the critical role of the C_H3 stability in preventing aggregation. The involvement of both constant and variable domains emphasizes the complex, interdependent nature of monoclonal antibody aggregation. This work provides mechanistic insights into domain-specific contributions to folding and aggregation, offering guidance for the design of more stable therapeutic antibodies.