(M0930-11-63) The Effect of Annealing Temperature and Composition on Lyophilized Sucrose / Glycine Formulations

Purpose: The purpose of this study was to examine the effects of formulation composition and annealing temperature on the failure point of the lyophilized samples.

Methods: Seven different sucrose / glycine formulations were prepared and sterile filtered through bottle-top filtration devices equipped with 0.2 micron nominal pore size filters.  The formulations were filled into 6 mL, glass, tubing vials at 3 mL per vial, labeled with a marker, and arranged on a lyophilizer tray.  One vial from each formulation was equipped with a thermocouple and the remaining vials were equipped with stoppers that were partially seated.  The samples were transferred to the shelf of a lyophilizer and the bottom of the tray was removed.  All cycles used a cooling rate of 1^oC/minute to -40^oC and were held at -40^oC for 2 hours.  Annealing occurred at -10^oC, -15^oC, -20^oC, -25^oC, -30^oC, or -35^oC and the temperatures were reached by warming at 1^oC/min from -40^oC and holding at the annealing temperature for 3 hours before initiating a vacuum of 100 mTorr at that temperature or proceeding to the primary drying temperature at 1^oC/min and initiating a vacuum of 100 mTorr.  Primary drying continued until the difference between the Pirani gauge and the capacitance manometer (CM) was not more than 7 mTorr.  The shelf temperature was increased to 40^oC at 0.5^oC/min for secondary drying and held for 360 minutes.  The shelf temperature was decreased to 20^oC at 5^oC/min and held at that temperature until the samples could be removed. Solution samples were examined by differential scanning calorimetry (DSC) and freeze-dry microscopy (FDM).  Lyophilized samples were examined for appearance and using x-ray powder diffraction (XRPD).

Results: Solution samples were examined at low temperature using DSC and FDM.  Glycine has a low Tg' with a midpoint of approximately -62^oC and the low Tg' decreases the Tg' of sucrose / glycine mixtures (Table I).  There is no change in the midpoint for the Tg' when glycine is present at concentrations less than 1:1.  FDM was used to determine the temperature at which there was a loss of structural integrity of the dry layer indicating failure (Table I).  The data suggest that formulations containing 1% and 3% glycine should result with poor appearance after freeze-drying even after annealing due to the low critical product temperature.  Formulations prepared with 1% or 3% glycine exhibited poor appearance when annealed at -10^oC, -15^oC, -30^oC, or -35^oC, but appeared acceptable when annealed at the other shelf temperatures (Figures 1 and 2).  The product temperatures during primary drying were in all instances much higher than the temperatures of failure identified by FDM.  XRPD data for the formulation containing 1% glycine show that annealing encouraged crystallization of glycine, but that a mixture of all four polymorphs of gamma, beta, alpha, and glycine anhydride is likely present.  XRPD data for the formulation containing 3% glycine show that annealing at -10^oC likely encouraged crystallization of the glycine anhydride and annealing at the other temperatures encouraged crystallization of a mixture of gamma, beta, and alpha polymorphs.

Conclusion: Formulations containing ratios of amorphous to crystalline components of 1:1 or more crystalline component than amorphous component are expected to exhibit good appearance after lyophilization.  Formulations with less crystalline than amorphous component typically exhibit poor appearance because the midpoint for the Tg' for the formulation decreases and the critical product temperature is difficult to maintain during primary drying.  This study demonstrated that formulations containing less glycine than sucrose could exhibit acceptable appearance depending on the annealing temperature.  The study also demonstrated that glycine in the formulations retained crystallinity after lyophilization.

References: Pyne, Abira, and Suryanarayanan, Raj, “Phase Transitions of Glycine in Frozen Aqueous Solutions and During Freeze-Drying”, Pharmaceutical Research, 18, 10, 1448 – 1454, October 2001. Authors studied formulations at 100 mg scale and using cooling rates capable for laboratory-scale instruments.
Li, Xuhong, Nail, Steven L., “Kinetics of Glycine Crystallization During Freezing of Sucrose/Glycine Excipient Systems”, Journal of Pharmaceutical Sciences, 94, 625-631, 2005.
Kasraian, Kasra, Spitznagel, Thomas M., Juneau, Jennifer A., and Yim, Kalvin, “Characterization of the Sucrose/Glycine/Water System by Differential Scanning Calorimetry and Freeze-Drying Microscopy”, Pharmaceutical Development and Technology, 3, 2, 233-239, September 2008.
Akers, Michael J., Milton, Nathaniel, Byrn, Stephen R., and Nail, Steven L., “Glycine Crystallization During Freezing: The Effects of Salt Form, pH, and Ionic Strength”, Pharmaceutical Research, 12, 10, 1457-1461, 1995.
Chongprasert, Suchart, Knopp, Shawn A., and Nail, Steven L., “Characterization of Frozen Solutions of Glycine”, Journal of Pharmaceutical Sciences, 90, 11, 1720-1728, November 2001.


Table I. Thermal Characterization of Frozen Solutions by DSC and FDM.


Figure 1. Appearance of Sucrose / Glycine Formulations After Annealing and Drying at -25oC. From Left, Sucrose 8%, Sucrose 7% with Glycine 1%, Sucrose 5% with Glycine 3%, Sucrose 4% with Glycine 4%, Sucrose 3% with Glycine 5%, Sucrose 1% with Glycine 7%, and Glycine 8%.


Figure 2. Appearance of Sucrose / Glycine Formulations After Annealing and Drying at -30oC. From Left, Sucrose 8%, Sucrose 7% with Glycine 1%, Sucrose 5% with Glycine 3%, Sucrose 4% with Glycine 4%, Sucrose 3% with Glycine 5%, Sucrose 1% with Glycine 7%, and Glycine 8%.