Effects of additives on the stability of Humicola lanuginosa lipase duringfreeze-drying and storage in the dried solid

The effects of various classes of additives on the stability of a protein w ith a relatively hydrophobic surface, Humicola lanuginosa lipase (HLL), dur ing lyophilization and storage in the dried solid, were investigated. Prior to lyophilization, it was found that 1 M trehalose or 1% (wt/vol) Tween 20 caused the protein to precipitate. Infrared spectroscopy indicated that tr ehalose "salted-out" native HLL, whereas Tween 20 induced non-native aggreg ates. Optimal recovery of native protein in the initial dried solid was obt ained in the presence of additives which formed an amorphous phase and whic h had the capacity to hydrogen bond to the dried protein (e.g., trehalose a nd sucrose). Additives which crystallized during lyophilization (e.g., mann itol) or which remained amorphous, but were unable to hydrogen bond to the dried protein (e.g., dextran), afforded less stabilization relative to that seen in the absence of additives. Optimal storage stability in the dried s olid required that both protein unfolding during lyophilization was minimiz ed and that the formulation was stored at a temperature below its T-g value . Crystallization of sucrose during storage greatly reduced the storage sta bility of HLL. This was attributed to the increased moisture content and th e reduced T-g value in the remaining amorphous phase containing the protein , Sucrose crystallization and the resulting damage to the protein were inhi bited by decreasing the mass ratio of sucrose:protein.