Characterizing the freeze-drying behavior of model protein formulations.

The freeze-drying behavior of three model proteins, namely, lysozyme, BSA, and IgG, has been studied using a variety of techniques under two different primary drying conditions (shelf temperatures of -25°C and +25°C, respectively) in an amorphous formulation. Manometric temperature measurements were used to characterize product temperature (T (pr)), sublimation rates, and product resistance (R (p)) during primary drying. Biophysical techniques such as circular dichroism, fluorescence, and Fourier transform infrared spectroscopy were used to study protein conformation. Size exclusion chromatography was used to monitor the formation of high-molecular-weight species (HMWS) over time on storage, and cake morphology was studied using scanning electron microscopy. The differences in the freeze-drying behavior of the three proteins were more evident at higher protein concentrations, where the protein significantly influences the behavior of the formulation matrix. However, these differences were minimized in the aggressive mode and were insignificant at lower protein concentrations where excipients dominated the freeze-drying behavior. Differences in cake morphology were observed between the two drying conditions employed as well as between the three proteins studied. The stability and the protein structure, however, were equivalent for the protein cakes generated using the two different primary drying conditions.

Determination of the influence of primary drying rates on the microscale structural attributes and physicochemical properties of protein containing lyophilized products.

This work investigated the impact of primary drying conditions on the microstructure and protein stability of bovine serum albumin (BSA) containing lyophilized cakes. Two primary drying conditions were employed (termed 'conservative', slower drying rate and 'aggressive', higher drying rate) at two protein loadings (5 and 50 mg mL(-1)). The cake attributes were characterized using micro-X-ray computed tomography (micro-CT), scanning electron microscopy, manometric temperature measurements, Fourier transform infrared spectroscopy (FTIR) and size exclusion chromatography (SEC). The combination of analytical techniques revealed that although the aggressive drying conditions produced intact cakes which retained their macrostructure, they had undergone various degrees of microcollapse. The study demonstrates the applicability of micro-CT for resolving microstructural attributes of the freeze-dried cakes such as residual porosity, pore size distribution and connectivity. Micro-CT data showed significant differences in residual porosity and matrix connectivity between aggressively and conservatively dried cakes. The FTIR data show that at each protein loading, there is no observable difference in the secondary structure of the protein and the SEC data show comparable stability in the cakes produced under different primary drying conditions. Good content uniformity was observed with respect to BSA and sucrose distribution in the cakes.

Use of manometric temperature measurements (MTM) to characterize the freeze-drying behavior of amorphous protein formulations.

The freeze-drying behavior and cake morphology of a model protein in an amorphous formulation were studied at varying protein concentrations using conservative (-25 degrees C) and aggressive (+25 degrees C) shelf temperatures at constant chamber pressure during primary drying. The two cycles were characterized by manometric temperature measurements (MTM) in a SMART freeze dryer that estimates the sublimation rate (dm/dt), product temperature at the freeze-drying front (T(p-MTM)) and product resistance (R(p)) during a run. The calculated sublimation rates (dm/dt) were 3-4 times faster in the aggressive cycle compared to the conservative cycle. For conservatively dried cakes R(p) increased with both dry layer thickness and protein concentration. For aggressively dried cakes (where freeze-drying occurs at the edge of microcollapse), R(p) also increased with protein concentration but was independent of the dry layer thickness. The sublimation rate was influenced by R(p), dry layer thickness and T(p-MTM) in the conservative cycle, but was governed mainly by T(p-MTM) in the aggressive cycle, where R(p) is independent of the dry layer thickness. The aggressively dried cakes had a more open and porous structure compared to their conservatively dried counterparts.