Deciphering protein aggregation in freeze-thaw process: the roles of cold denaturation and shear stress

• A precise downscale model using 10 mL vials was developed to help mimic freeze–thaw protein aggregation manufacturing-scale containers. • Cold denaturation was suggested to be an important contributing factor in protein aggregation during freeze–thaw. • Protein aggregation increased when the heat transfer geometry allowed ice to enclose the liquid phase, which may be related to higher freezing pressure-induced mechanical stresses. Understanding protein aggregation during freeze–thaw is vital for biopharmaceutical quality assurance. This study investigates monoclonal antibody aggregation linked to stress-time, the time a formulation remains partly frozen above glass transition temperature of the freeze concentrate (T g ’). Stress–time distributions of a 0.5 L bottle were implemented in 10 mL vials by controlling ice-nucleation, cooling, and heating rates. These vials were used as downscale models to explore cold denaturation, by varying times under temperatures from −5 °C to T g ’. Matching stress-times between scales resulted in similar aggregation levels. However, extending the stress-time in a low cold denaturation range (−5 °C to freezing equilibrium temperature (T f )) did not significantly increase aggregation, suggesting its critical role in freeze–thaw stability. Aggregation in vials was about 1/3 lower than in bottles, apparently related to the vials’ bottom-up freezing geometry, which does not enclose unfrozen liquid within the ice. Imposing a similar geometry on the bottle, using top insulation, aligning aggregation levels, suggesting the contribution of shear stress from ice interface percolation. Overall, a 5.5 mL sample (in a 10 mL vial) may serve as a model for larger volumes by accurately controlling heat transfer rates with the assistance of computational fluid dynamics. This approach provides supporting evidence and a step toward improving understanding of biopharmaceutical ‘sensitivity’ to freeze–thaw stresses.