Frozen state storage instability of a monoclonal antibody: aggregation as a consequence of trehalose crystallization and protein unfolding.

PURPOSE: 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 -20°C.
METHODS: Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.
RESULTS: Aggregation increased with time at -20°C but not at -40°C or -10°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 (-29°C). Crystallization at -20°C deprives the protein of the cryoprotectant, leading to a slow increase in aggregation. Storage at -10°C also leads to crystallization of trehalose but no increase in aggregation. It is hypothesized that significantly higher mobility in the matrix at -10°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 -40°C prevents crystallization, refolding, and aggregation.
CONCLUSIONS: 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.