Purpose: (i) To study the crystallization of trehalose in frozen solutions and (ii) to understand the phase transitions during the entire freeze-drying cycle. Method: Aqueous trehalose solution was cooled to -40°C in a custom-designed sample holder. The frozen solution was warmed to -18°C and annealed, and then dried in the sample chamber of the diffractometer. XRD patterns were continuously collected during cooling, annealing and drying. Results: After cooling, hexagonal ice was the only crystalline phase observed. However, upon annealing, crystallization of trehalose dihydrate was evident. Seeding the frozen solution accelerated the solute crystallization. Thus, phase separation of the lyoprotectant was observed in frozen solutions. During drying, dehydration of trehalose dihydrate yielded a substantially amorphous anhydrous trehalose. Conclusions: Crystallization of trehalose, as trehalose dihydrate, was observed in frozen solutions. The dehydration of the crystalline trehalose dihydrate to substantially amorphous anhydrate occurred during drying. Therefore, analyzing the final lyophile will not reveal crystallization of the lyoprotectant during freeze-drying. The lyoprotectant crystallization can only become evident by continuous monitoring of the system during the entire freeze-drying cycle. In light of the phase separation of trehalose in frozen solutions, its ability to serve as a lyoprotectant warrants further investigation.
- in situ freeze-drying XRD
- lyoprotectant crystallization
- phase transformation