Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding
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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.
Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.
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.
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.
KEY WORDSaggregation cryoprotectant glass transition temperature monoclonal antibody trehalose
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