Influence of water and trehalose on - and -relaxation of freeze-dried lysozyme formulations

Molecular mobility in form of alpha and beta relaxations is considered crucial for characterization of amorphous lyophilizates and reflected in the transition temperatures Tg alpha and Tg beta. Based on an overview of applied methods to study beta relaxations, Dynamic Mechanical analysis was used to measure Tg alpha and Tg beta in amorphous freeze-dried samples. Lysozyme and trehalose as wel l as their mixtures in varying ratios were investigated. Three different residual moistu r e levels, ranging from roughly 0.5-7 % (w/w), were prepared via equilibration of the freeze-dried samples. Known plasticising effects of water on Tg alpha were confirmed, also via differential scanning calorimetry. In addition and contra r y to expectations, an influence of water on the Tg beta also was observed. On the other hand, an increasing amount of trehalose lowered Tg alpha but increased Tg beta showing that Tg alpha and Tg beta are not paired. The findings were interpreted with regard to their underlying molecular mechanisms and a correlation with the known influences of water and trehalose on stability. The results provide encouraging hints for future stability studies of freeze-dried protein formulations, which are urgently needed, not least for reasons of sustainability.