Effect of product temperature during primary drying on the long-term stability of lyophilized proteins.

Our objective was to investigate the effect of performing primary drying at product temperatures below and above Tg' (glass transition temperature of the freeze-concentrated phase) on the long-term stability of lyophilized proteins. Two protective media differing in the nature of the bulking agent used (amorphous or crystalline) were selected. Several lyophilization cycles were performed by using various combinations of shelf temperature and chamber pressure to obtain different values of product temperature during primary drying. The antigenic activity of the proteins was measured after lyophilization and after 6 months of storage at 4 degrees C and 25 degrees C. After 6 months of storage and regardless of the protective medium, the losses of antigenic activity of both toxins increased from 0% when primary drying was performed at a product temperature lower than Tg' and to 25% when the product temperature was higher than Tg'. The use of partially crystalline systems makes it possible to withstand high primary drying temperatures (above Tg'). However, the shelf life of lyophilized proteins may be decreased when the amorphous phase including the protein and the stabilizing molecule changes to the viscous state.

Physical characterisation of formulations for the development of two stable freeze-dried proteins during both dried and liquid storage.

The development of stable freeze-dried proteins requires maintaining the physical and biological integrity of the protein as well as increasing the efficiency of the manufacturing process. Our objective was to study the effects of various excipients on both the physical characterisation and the dried and liquid stability of two proteins. Thermo-physical properties of 13 formulations were determined using both differential scanning calorimetry and freeze-drying microscopy. The antigenic activity was evaluated immediately after freeze-drying and after subsequent storage in both dried and liquid state. From the comparison between glass transition (T'g) and collapse (T coll) temperatures, we concluded that the collapse temperature was a more relevant parameter than T'g for freeze-drying cycle development and optimisation. One crystalline formulation composed of 4% mannitol and 1% of sucrose protected efficiently both proteins during subsequent storage in dried state (6 months at 25 degrees C) and in liquid state (3 months at 4 degrees C after rehydration). However, the freeze-drying behaviour of this crystalline formulation remained difficult to predict and control. On the other hand, two amorphous formulations composed of 4% of maltodextrin and 0.02% of Tween 80, or 5% of BSA preserved antigenic activity during storage in dried state. The glassy character of these formulations as well as their high collapse temperature values (-9 and -12 degrees C, respectively) should allow simplification and shortening of freeze-drying process.