Molecular mobility of protein in lyophilized formulations linked to the molecular mobility of polymer excipients, as determined by high resolution C-13 solid-state NMR

Purpose. The mobility of protein molecules in lyophilized protein formulati ons was compared with that of excipient molecules based on the spin-lattice relaxation time (T-1) of each molecule determined by high resolution C-13 solid-state NMR. The relationship between molecular mobility and protein st ability is discussed. Methods. Protein aggregation of lyophilized bovine serum gamma-globulin (BG G) formulation containing dextran was measured by size exclusion chromatogr aphy. The T-1 of the EGG carbonyl carbon and dextran methin carbon in the f ormulation was determined by high resolution C-13 NMR, and subsequently use d to calculate the correlation time (tau(c)) of each carbon. The spin-spin relaxation time (T-2) Of EGG and dextran protons was measured by pulsed NMR spectrometry, and the critical temperature of appearance of Lorentzian rel axation due to liquid EGG and dextran protons (T-mc) was determined. Results. The tau(c) of dextran methin carbon in BGG-dextran formulations ex hibited a linear temperature dependence according to the Adam-Gibbs-Vogel e quation at lower temperatures, and a nonlinear temperature dependence descr ibed by the Vogel-Tamman-Fulcher equation at higher temperatures. The tempe rature at which molecular motion of dextran changed was consistent with the T-mc. The tau(c) of EGG carbonyl carbon exhibited a similar temperature de pendence to the tau(c) of the dextran methin carbon and substantially decre ased at temperatures above T-mc in the presence of dextran. The temperature dependence of EGG aggregation could be described by the Williams-Landel-Fe ny equation even at temperatures 20 degrees C lower than T-mc. Conclusions. High resolution C-13 solid-state NMR indicated that the molecu lar motion of EGG was enhanced above T-mc in association with the increased global segmental motion of dextran molecules.