The interaction of trypsin with trehalose: an investigation of protein preservation mechanisms.

Preservation of the native protein structure and biological activity in dry protein/excipient mixtures has been previously attributed to either the glass forming properties of the additives or to their ability to hydrogen bond to the protein. There is evidence that both processes are important but it has not yet been elucidated which is the limiting factor that determines the efficiency of a given molecule as a protectant. In this work, gravimetric measurements together with enzyme activity assays have been employed to investigate the protection of proteins by sugars, through direct interaction via hydrogen bonding and as the result of glass formation. As a model protein, trypsin has been employed and the modes of action of two similar disaccharides, sucrose and trehalose, which offer different levels of protection, evaluated and compared. Data obtained on freeze-dried formulations indicate that protein and sugars interact through hydrogen bonding to protein hydration sites. The extent of interaction is found to change dramatically at elevated temperatures; sucrose showing a significantly decreased, and trehalose a considerably increased level of interaction. Protein preservation is shown to be directly related to the number of hydrogen bonds formed. Possible reasons why trehalose interacts more extensively with the protein than sucrose are discussed in terms of differences in the anhydrous structures and molecular mobilities of the sugar molecules.