Work

Interdomain Interactions Modulate Refolding Kinetics and Aggregation in a Monoclonal Antibody

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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 H 2 and V H domains unfolded rapidly, while the C H 3 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 H 3 destabilization. Notably, aggregation-prone regions were identified in both the Fc and Fab portions of the antibody. Specifically, interactions at the C H 2–C H 3 and C H 3–C H 3 interfaces appear disrupted upon C H 3 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 H 2–C H 3 refolding and underscore the critical role of the C H 3 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.

Source: Journal of the American Society for Mass Spectrometry

DOI: 10.1021/jasms.5c00166

Authors: Philipp Trolese, Andrea Pierangelini, Benedetta Fongaro, Patrizia Polverino de Laureto

Institutions: University of Verona, University of Padua

Language: English

Topic: Protein purification and stability

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funders: European Federation of Pharmaceutical Industries and Associations, European Commission, Università degli Studi di Padova, H2020 Health