Purpose. To study the effect of processing conditions on the physical state of mannitol during various stages of the lyophilization cycle of a protein formulation. Materials and Methods. Mannitol and trehalose were used as the bulking agent and lyoprotectant, respectively. The physical state of mannitol during various stages of freeze-drying cycle, in the absence and presence of a model protein, was characterized using low temperature X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC). Results. Mannitol did not crystallize even when the solution for lyophilization was cooled to -45°C at a slow cooling rate of 1°C/min. Annealing facilitated mannitol crystallization, and in the absence of the protein, a mixture of δ-mannitol and mannitol hemihydrate was obtained at both low (-18°C) and high (-8°C) annealing temperatures. However, in the presence of protein, the high annealing temperature promoted δ-mannitol crystallization and inhibited formation of mannitol hemihydrate, while the low annealing temperature facilitated the formation of mannitol hemihydrate. Interestingly, the hemihydrate in the frozen solution was retained in the final lyophile, even when the primary and secondary drying temperatures were as high as -5 and 65°C, respectively. Conclusions. The presence of protein as well as the processing conditions (annealing temperature and time, primary and secondary drying temperatures) influenced the physical form of mannitol in the final lyophile. The protein promoted formation of δ-mannitol while inhibiting the formation of mannitol hemihydrate. Since the physical form of mannitol was greatly influenced by the presence of protein, it will be prudent to conduct the preliminary lyophilization cycle development studies in the presence of the protein. If mannitol hemihydrate is formed during annealing, its dehydration may require high secondary drying temperature.