Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy:: Dehydration stress in the presence of sucrose

Purpose. First, to investigate the role of sucrose in stabilizing protein structure (as measured by changes in the amide I band of lysozyme) caused by dehydration encountered during lyophilization. Second, to demonstrate the utility of internal reflection spectroscopy as a tool for conducting controlled lyophilization experiments. Methods, A custom-built internal reflection FTIR accessory was used to follow the entire freeze-drying process of solutions consisting of 49.4 mg/mL lysozyme in the presence and absence of 10% sucrose in real-time. Studies were carried out using D2O as a transparent medium in the infrared region of the protein amide bands. Potential self-association of the protein in the presence of sucrose was investigated using dynamic light scattering. Hydration levels were determined using a multiple regression equation. Differential scanning calorimetry (DSC) permitted characterization of the final lyophilized product. Moisture content was determined using Karl Fischer titration. Results, Throughout freezing and drying, minimal changes were observed both in frequency (1647 +/- 1 cm(-1)) and bandwidth (46 +/- 1 cm(-1)) of the amide I band in the presence of sucrose. In contrast, cm greater changes in frequency and band width were seen in the absence of sucrose. A successfully lyophilized cake was obtained which had properties of a glass as measured by DSC, with a Tg of 50 degrees C. The lyophilized product containing sucrose had 4% moisture by weight. Three distinct rates of water desorption were discovered during drying under vacuum (50 mg/hr within the sample temperature range from -35 degrees to -25 degrees C; 30 mg/hr from -10 degrees to 25 degrees C; 1.2 mg/hr from 27 degrees to 38 degrees C). Conclusions. The inclusion of sucrose served to minimize perturbations of protein structure caused by freezing and dehydration stresses encountered during lyophilization (compared to studies conducted in the absence of sucrose). The results support the water replacement hypothesis and underscore the role of the sugar in preserving a native structure in the dried state. This investigation demonstrates the usefulness of infrared spectroscopy in evaluating lyophilization process parameters and formulation design.