Stabilization of freeze dried protein formulations

Shan Jiang, Purdue University

Abstract

The broad objective of this study was to gain a better understanding of the effects of formulation and processing conditions on integrity of proteins after freeze drying and the stabilization of freeze dried protein products during long term storage. Catalase, $\beta$-galactosidase, and lactate dehydrogenase were chosen as model proteins. In the absence of protective solutes, all three proteins exhibited a concentration dependent loss of activity after freezing and freeze drying, with significantly higher recovery at a higher concentration. The freezing method and the type of buffer were also important. With sodium phosphate buffer, freezing by immersion of vials in liquid nitrogen was associated with the lowest recovery of activity. The time course of activity loss for $\beta$-galactosidase and LDH was measured during primary drying. The most important drying process variable affecting recovery of activity was residual moisture. Structural collapse is not necessarily associated with a decrease in the recovery of activity of the model proteins. Modulated DSC was used to measure the T$\rm\sb{g}$'s of maltodextrin formulations. The influence of experimental parameters on the measurement of glass transition temperature was investigated. Since there was an uncertainty in determination of T$\rm\sb{g}$'s of M100 and M40 formulations with conventional scanning methods, a stepwise quasi-isothermal experiment was used to measure the heat capacity change across the transition region. The results indicate that the quasi-isothermal experiment provides better sensitivity for T$\rm\sb{g}$ measurement for materials which exhibit a small heat capacity change at the glass transition. To determine whether the glass transition temperature is a critical attribute of a formulation with respect to stability, the storage stability of freeze dried $\beta$-galactosidase and LDH with different molecular weights of maltodextrins and maltose were compared. Only the stability data of LDH in a maltose formulation support the vitrification hypothesis, where a dramatic increase in the inactivation rate was associated with structural collapse. In general, the stability data support the conclusion that glass formation is a necessary condition, but it is not sufficient for stabilization of the protein. The results indicate that as long as the T$\rm\sb{g}$ of the formulation is higher than the storage temperature, the water activity appears to be more important than (T-T$\rm\sb{g})$ in determining the stability of proteins in the solid state.

Degree

Ph.D.

Advisors

Nail, Purdue University.

Subject Area

Pharmaceuticals|Chemistry

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