Predicting Agitation Stability of Monoclonal Antibodies during Developability Assessment

Developability assessment facilitates the selection of antibody drug candidates with desirable pharmaceutical properties. However, it remains uncertain whether agitation-induced aggregation can be predicted from standard developability parameters. Here, we investigated whether key biophysical parameters predict agitation-induced aggregation of monoclonal antibodies (mAbs). To this end, we generated a benchmark data set by characterizing the aggregation upon agitation in the presence of an air-liquid interface of ten approved mAbs reformulated in a common surfactant-free buffer. The extent of aggregation varied substantially among mAbs and was primarily dependent on antibody identity. Flow imaging microscopy combined with machine learning revealed micrometre-sized aggregates with distinct morphologies, consistent with aggregation at air-liquid interfaces. Examination of thin liquid films and foams confirmed the presence of aggregates directly at the air-liquid interface and, therefore, the critical role of this interface for antibody aggregation during agitation. We then applied fluorescence-based, light scattering, and chromatographic techniques to determine standard developability parameters for each mAb, including apparent melting temperature (T_m), nonreversibility onset temperature (T_nr), aggregation onset temperature (T_agg), diffusion self-interaction parameter (k_D), hydrophobic interaction chromatography retention time, and relative monomer yield after isothermal refolding from chemical denaturants. Notably, none of these parameters correlated with agitation-induced aggregation. Finally, we assessed the surface properties of the mAbs via drop shape analysis and found that the combination of surface pressure and elastic modulus yields a good correlation with the concentration of micrometre-sized aggregates formed due to agitation. Overall, these findings highlight limitations in predicting mAb interfacial stability using standard developability assays and underscore the importance of studying antibody behavior at interfaces.