Isoperibol calorimetry was used to evaluate protein/carbohydrate interactions after freeze drying. rh-DNase, rh-GH, rh-MetGH, and rh-IGF-I were freeze dried with either mannitol, sucrose, trehalose, or dextran at concentrations ranging from 0% to 100%(w/w). Enthalpies of solution for both freeze-dried and physical mixtures were measured in water at 258C. Differential scanning calorimetry was used to monitor changes in the melting or crystallization temperatures of the lyoprotectants. Linear relationships between enthalpies of solution and the percentage of protein in the formulations were observed for all physical mixtures. In contrast, nonlinear relationships between the enthalpies of solution and protein content were observed for the freeze-dried mixtures. Mannitol-containing mixtures were characterized by negative deviation from linearity, while positive deviations were detected for mixtures containing sucrose or trehalose. Using DSC, sucrose was found to be amorphous at low and not detected at high protein content in the freeze-dried mixtures. Melting of mannitol was observed through almost all of the protein concentration range examined. Two melting endotherms, however, were observed for mannitol at most protein/mannitol ratios, indicating the presence of protein/mannitol interactions. This work suggests that direct interactions occur between proteins and carbohydrates in lyophilized mixtures. ß 2002