Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran

The storage stability of a dry protein depends on the structure of the drie d protein, as well as on the storage temperature relative to the glass tran sition temperature of the dried preparation. Disaccharides are known to pre serve the native conformation of a dried protein; however, the resulting T- g of the sample may be too low ensure adequate storage stability. On the ot her hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserv e native protein conformation. We tested the hypothesis that optimizing bot h protein structure and T-g by freeze-drying actin with mixtures of disacch arides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after st orage at elevated temperatures with infrared spectroscopy, and after rehydr ation by infrared and circular dichroism spectroscopy. Structural results w ere related to the polymerization activity recovered after rehydration. Deg radation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T-g observed in trehalose formulations compared to sucrose formulations did not result in appreciabl e increases in storage stability. Addition of dextran to sucrose or trehalo se increased formulation T-g without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved s torage stability. Also, dextran provides an excellent amorphous bulking age nt, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of d isaccharide and polymeric carbohydrates can optimize protein storage stabil ity. (C) 2000 Wiley-Liss, Inc.