A Solid-State NMR Study of Protein Mobility in Lyophilized Protein–Sugar Powders

The molecular mobility of protein in lyophilized lysozyme–sugar systems stored at different relative humidities was studied using solid-state NMR. Relaxation measurements, T1 of high-frequency (MHz), and T1ρ, of low-frequency (kHz) motions, were performed on lysozyme lyophilized with lactose and trehalose. Molecular aggregation and enzymatic activity of the protein were determined using HPLC and bioassays. An increase in hydration had little effect on the T1ρ values of pure lysozyme, trehalose, lactose, trehalose–lysozyme, and lysozyme at low lactose concentrations. The T1 values of pure sugar increased as moisture content increased. The presence of both sugars led to increased T1 values of the lysozyme but increasing hydration gradually reduced T1 values. When a larger amount of lactose was lyophilized with lysozyme, longer T1 (and T1ρ) values were seen for lactose than for lysozyme. Although longer T1 values were related to an increase in protein stability, the effect of crystallization and sugar type appeared to be major contributing factors. Trehalose and lactose decreased relaxation rates in the lysozyme–sugar systems while hydration increased relaxation rates that were correlated with changes in aggregation and activity of the protein. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91: 943–951, 2002