Molecular mobility of protein in lyophilized formulations linked to the molecular mobility of polymer excipients, as determined by high resolution 13C solid-state NMR.
PURPOSE: The mobility of protein molecules in lyophilized protein formulations was compared with that of excipient molecules based on the spin-lattice relaxation time (T1) of each molecule determined by high resolution 13C solid-state NMR. The relationship between molecular mobility and protein stability is discussed. METHODS: Protein aggregation of lyophilized bovine serum gamma-globulin (BGG) formulation containing dextran was measured by size exclusion chromatography. The T1 of the BGG carbonyl carbon and dextran methin carbon in the formulation was determined by high resolution 13C NMR, and subsequently used to calculate the correlation time (tauc) of each carbon. The spin-spin relaxation time (T2) of BGG and dextran protons was measured by pulsed NMR spectrometry, and the critical temperature of appearance of Lorentzian relaxation due to liquid BGG and dextran protons (Tmc) was determined. RESULTS: The tauc of dextran methin carbon in BGG-dextran formulations exhibited a linear temperature dependence according to the Adam-Gibbs-Vogel equation at lower temperatures, and a nonlinear temperature dependence described by the Vogel-Tamman-Fulcher equation at higher temperatures. The temperature at which molecular motion of dextran changed was consistent with the Tmc. The tauc of BGG carbonyl carbon exhibited a similar temperature dependence to the tauc of the dextran methin carbon and substantially decreased at temperatures above Tmc in the presence of dextran. The temperature dependence of BGG aggregation could be described by the Williams-Landel-Ferry equation even at temperatures 20 degrees C lower than Tmc. CONCLUSIONS: High resolution 13C solid-state NMR indicated that the molecular motion of BGG was enhanced above Tmc in association with the increased global segmental motion of dextran molecules.[1]References
- Molecular mobility of protein in lyophilized formulations linked to the molecular mobility of polymer excipients, as determined by high resolution 13C solid-state NMR. Yoshioka, S., Aso, Y., Kojima, S., Sakurai, S., Fujiwara, T., Akutsu, H. Pharm. Res. (1999) [Pubmed]
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