Title

Investigation of drying stresses on the physical stability of proteins using the mini-freeze-dryer, controlled hydration calorimetry and spectroscopy

Date of Completion

January 2006

Keywords

Chemistry, Pharmaceutical|Health Sciences, Pharmacy

Degree

Ph.D.

Abstract

Proteins are frequently produced as dry powders to improve stability during storage and shipping. The drying process such as freeze-drying may itself cause physical degradation of the protein, which is manifested as non-native structure in the dried state. Water plays an important role in maintaining the native structure and function of the protein. Removal of water is one of the primary reasons for physical degradation of proteins during drying. While the end result of drying, i.e. physical degradation, is well known, the mechanistic understanding of protein-water interactions and effect of process and formulation parameters affecting in-process stability of proteins remains incomplete. The objective of this research was to, (a) study protein-water interactions and use that information to understand the physical stability of the protein during the drying process; and (b) investigate the critical process and formulation parameters affecting protein stability during lyophilization.^ A perfusion isothermal calorimetry method was used to measure simultaneously water desorption isotherms and heats of water desorption (ΔH desorption) of proteins. Above a critical hydration level the measured ΔHdesorption had an endothermic component, which was attributed to conformational changes in the protein due to increased molecular mobility at high hydration.^ The effect of hydration on the secondary structure of proteins was studied using Fourier transform infrared (FTIR) spectroscopy. An investigation of the application of attenuated total reflection (ATR) technique and sample mulls in fluorolube in collecting spectra of hydrated proteins suggested mulls preparing mulls was the most appropriate method since it was directly comparable with other spectra collected in the transmission mode and the sample preparation was not likely to cause protein degradation as possible in the standard KBr pelletization method. The secondary structure of the proteins in hydrated state was similar to that in the dry state, suggesting that conformational changes in the protein as suggested by calorimetric results were mostly tertiary structure changes.^ A controlled humidity mini-freeze-dryer was developed to study the critical process and formulation parameters affecting protein stability during the drying stages of lyophilization. The stresses of primary and secondary drying were studied separately by conducting primary drying at high relative humidity to prevent water desorption. Primary drying did affect the protein stability even when collapse was induced by drying above the collapse temperature. Secondary drying was found to be the critical stage affecting protein stability. Low drying temperature, low relative humidity, shorter drying duration, and glass forming lyoprotectants resulted in higher protein activity suggesting molecular mobility had an impact on the physical stability of the protein. These results were in agreement with the calorimetric data that a combination temperature and water content determined the conformational flexibility of the protein. ^