Use of subambient thermal analysis to optimize protein lyophilization

Abstract

Subambient thermal analysis with differential scanning calorimetry and thermomechanical analysis has been used successfully to optimize the lyophilization of proteins based on the thermal behavior of solution components. In deionized water, frozen protein solutions exhibit two glass transition temperatures ( T gs): the first ca. − 80 °C ( T g″), and a second ca. − 10 °C ( T g′); the latter appears to be related to the collapse phenomenon. Proteins lyophilized in buffers with low T g values such as Tris (− 81 °C) or sodium acetate (− 80 °C) are collapsed during primary drying at − 20 °C. Substitution of salts which crystallize readily during freezing and have eutectic melting temperatures ( T es) above − 20 °C, e.g., sodium carbonate ( T e = 3 °C), prevents the protein solutions from being collapsed during the same process. The crystallization of these salts during freezing, which may lead to undesirable changes in pH, can be prevented by use of excipients such as sugars. To remove the amorphous fraction in partially crystallizable eutectic salts which exhibit very low glass transition temperature values, an adequate annealing at temperatures between devitrification and eutectic melting is necessary. Annealing is not effective for wholly amorphous glass-forming salts. However, the final T g′ of protein solutions which contain such salts can be raised by adding excipients or by increasing the protein concentration.