Protein aggregation in the frozen state induced by dropping stress

Abstract

The formation of protein aggregates, which can be immunogenic and lower the efficacy and safety of protein drugs, has been an issue in biopharmaceutical development for more than a decade. Although protein drugs are often shipped as frozen material, the effect of the accidental dropping of frozen proteins, which can occur during shipping and handling, on the physical stability has not been studied. Here, a frozen Fc fusion protein was subjected to dropping stress and the increase in the aggregate concentration was evaluated. Significant increases in micron-sized aggregates were observed at –30 °C (p ≤ 0.01), but not at –60 °C. Proteins adsorbed on the vial surfaces were not remarkably detached by the action of dropping and were not the primary cause of the increase in micron-sized aggregates. When the vials were dropped, local heat generation occurred and this led to local freeze-thaw stress that induced protein aggregation. Poloxamer-188, which is known to mitigate aggregation caused by freeze-thaw stress, effectively prevented the aggregation caused by the dropping stress in the frozen state at –30 °C. In addition, rapid freezing could suppress the aggregation caused by the dropping stress. The results demonstrated that dropping stress reduced the stability of proteins even in the frozen state, and they provide new insights into the formulation and freezing processes to prevent protein aggregation caused by dropping stress in the frozen state.