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.