Significance of local mobility in aggregation of β-galactosidase lyophilized with trehalose, sucrose or stachyose

The purpose of this study is to compare the effects of global mobility, as reflected by glass transition temperature (T(g)) and local mobility, as reflected by rotating-frame spin-lattice relaxation time (T(1 rho)) on aggregation during storage of lyophilized beta-galactosidase (beta-GA). The storage stability of beta-GA lyophilized with sucrose, trehalose or stachyose was investigated at 12% relative humidity and various temperatures (40-90 degrees C). beta-GA aggregation was monitored by size exclusion chromatography (SEC). Furthermore, the T(1 rho) of the beta-GA carbonyl carbon was measured by (13) C solid-state NMR, and T(g) was measured by modulated temperature differential scanning calorimetry. Changes in protein structure during freeze drying were measured by solid-state FT-IR. The aggregation rate of beta-GA in lyophilized formulations exhibited a change in slope at around T(g), indicating the effect of molecular mobility on the aggregation rate. Although the T(g) rank order of beta-GA formulations was sucrose < trehalose < stachyose, the rank order of beta-GA aggregation rate at temperatures below and above T(g) was also sucrose < trehalose < stachyose, thus suggesting that beta-GA aggregation rate is not related to (T-T(g)). The local mobility of beta-GA, as determined by the T(1 rho) of the beta-GA carbonyl carbon, was more markedly decreased by the addition of sucrose than by the addition of stachyose. The effect of trehalose on T(1 rho) was intermediate when compared to those for sucrose and stachyose. These findings suggest that beta-GA aggregation rate is primarily related to local mobility. Significant differences in the second derivative FT-IR spectra were not observed between the excipients, and the differences in beta-GA aggregation rate observed between the excipients could not be attributed to differences in protein secondary structure. The aggregation rate of beta-GA in lyophilized formulations unexpectedly correlated with the local mobility of beta-GA, as indicated by T(1 rho), rather than with (T-T(g)). Sucrose exhibited the most intense stabilizing effect due to the most intense ability to inhibit local protein mobility during storage.