Accelerating characterization of conformational stability for biologics in Discovery space by combining SEC‑HDX‑MS with molecular modeling

Conformational stability is a critical attribute for biologics discovery and manufacturability assessment, yet conventional approaches often require substantial material, extensive preparation, or lack sufficient structural resolution for early decision-making. Here, we present a native size-exclusion chromatography hydrogen–deuterium exchange mass spectrometry (SEC-HDX-MS) workflow integrated with molecular modeling to rapidly characterize conformational stability of therapeutic proteins in the discovery space. The workflow was evaluated across monoclonal antibody and bispecific antibody modalities. From a sequence variant comparison, SEC-HDX-MS showed that elevated aggregation did not uniformly reflect global conformational instability; instead, only one variant displayed increased deuterium incorporation, which modeling linked to an elongated, solvent-exposed CDR-H3 loop with increased hydrophobicity. In a representative proprietary mAb (“mAb X”), SEC-HDX-MS revealed that thermally-induced isoaspartate accumulation reduced conformational stability, whereas high-pH stress showed behavior comparable to controls despite higher overall deamidation. Molecular dynamics simulations explained this destabilization through disruption of a backbone Asn43–Ala40 hydrogen bond and altered local coordination after isoAsp formation. Finally, application to a complex bispecific antibody confirmed the workflow’s sensitivity for detecting conformational differences in more complex formats. Collectively, this integrated approach connects global HDX measurements with molecular-level interpretation, requires minimal preparation and limited material, and is compatible with partially purified samples. The workflow enables rapid conformational stability assessment to support candidate ranking, protein engineering, formulation development, and CMC risk assessment during biologics discovery.