Trehalose Crystallization During Freeze-Drying: Implications On Lyoprotection

Lyoprotectants are stabilizers used to prevent denaturation of proteins during freeze-drying and subsequent storage. In order to be effective, lyoprotectants must be retained amorphous. The physical state of the lyoprotectant is usually characterized by power X-ray diffractometry of the dried cake. While trehalose is widely used as a lyoprotectant, we report its crystallization during freeze-drying and point out why it may not become evident from characterizing the final lyophile. When an acqueous trehalose solution was cooled to -40 degrees C, ice was the only crystalline phase observed. However upon annealing at -18 degrees C, crystallization of trehalose dihydrate was evident. During drying, the dihydrate dehydrated to substantially amorphous anhydrate. Therefore, analyzing the final dried product will not reveal crystallization of the lyoprotectant during freeze-drying. In light of the observed phase separation of trehalose in frozen solutions, its ability to serve as a lyoprotectant warrants further investigations.