Effects of bovine somatotropin (rbSt) concentration at different moisture levels on the physical stability of sucrose in freeze-dried rbSt/Sucrose mixtures

The inherent instability of many proteins during freeze-drying and storage necessitates the addition of excipients to protect the proteins. It is emphasized in the literature that lyophilized sugar/protein composites should be stored at temperatures below their glass transition temperature (T-g) to prevent crystallization of excipients. The influence of bovine somatotropin (rbSt) concentration on inhibition of sucrose crystallization at different relative humidities (RH) was of interest. Thermally modulated differential scanning calorimetry (MDSC) was used to measure T-g and sucrose crystallization temperatures (T-c) of the composites. Sorption isotherms of the various sucrose/rbSt mixtures were determined gravimetrically with a controlled atmosphere microbalance (CAM) and verified by Karl Fischer analysis of selected samples. The CAM was also used to determine lag times and sucrose crystal growth rates by monitoring weight losses resulting from water liberation upon crystallization of sucrose at 23 degrees C. Results obtained by MDSC indicate that the T-c increased linearly from similar to 110 degrees C for pure sucrose to similar to 140 degrees C with 20% rbSt at very low water content (<0.1%). Similarly, at 22% RH (4.4% H2O), T-c, increased from similar to 70 degrees C to 120 degrees C. In neither case was T-g impacted significantly by increasing protein from 0 to 20%. No T-c could be noted for samples with greater than or equal to 30% rbSt in nonisothermal conditions. Plasticization by water decreased both T-g and T-c quite similarly but did'nt impact the noted effect of protein on T-c. Induction time for sucrose crystallization (i.e, nucleation) at similar to 45% RH (23 degrees C) increased almost 10-fold by addition of 10% rbSt, whereas rates of water loss due to crystallization decreased by no more than 2-3-fold. The overall results strongly indicate that formulations of higher protein concentration will be more resistant to sucrose crystallization and thus more robust when transiently exposed to storage temperatures above their T-g.