Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding

To investigate the cause of unexpected and erratic increase in aggregation during long-term storage of an IgG2 monoclonal antibody in a trehalose formulation at -20A degrees C. Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time. Aggregation increased with time at -20A degrees C but not at -40A degrees C or -10A degrees C. The cause of the instability was the crystallization of freeze-concentrated trehalose from the frozen solute when the storage temperature exceeds the glass transition temperature of the matrix (-29A degrees C). Crystallization at -20A degrees C deprives the protein of the cryoprotectant, leading to a slow increase in aggregation. Storage at -10A degrees C also leads to crystallization of trehalose but no increase in aggregation. It is hypothesized that significantly higher mobility in the matrix at -10A degrees C allows protein molecules that are unfolded at the ice interface on freezing to refold back before significant aggregation can occur. In contrast, lack of mobility at -40A degrees C prevents crystallization, refolding, and aggregation. Aggregation in the frozen state when stored above the glass transition temperature is a consequence of balance between rate of crystallization leading to loss of cryoprotectant, rate of aggregation of the unfolded protein molecules, and rate of refolding that prevents aggregation.