The effect of mannitol crystallization in mannitol-sucrose systems on LDH stability during freeze-drying.

The objective of this study was to investigate the influence of mannitol crystallization and the effect of annealing on the stability of lactate dehydrogenase (LDH) during freeze-drying. For this purpose, protein formulations with different weight ratios of mannitol to sucrose were freeze-dried with and without annealing. Product crystallinity was calculated based on differential scanning calorimetry data. Protein stability was evaluated both functionally by measuring the activity recovery of the model protein LDH after freeze-drying, and structurally by analyzing the protein secondary structure. LDH showed lower stability in annealed samples, and a correlation could be established between the extent of product crystallinity and the stability of the model protein. The destabilizing effect of mannitol crystallization on LDH during freeze-drying can likely be attributed to removal of mannitol from the amorphous phase containing the protein. In addition, the formation of mannitol crystals seemed to enhance the interfacial denaturation of protein during freeze-drying. The use of 0.01% of Tween 80 in the formulation greatly improved the structural and functional stability of LDH against stresses induced by mannitol crystallization.