Effects of Additives on the Stability of Humicola Lanuginosa Lipase During Freeze-Drying and Storage in the Dried Solid

Abstract

The effects of various classes of additives on the stability of a protein with a relatively hydrophobic surface, Humicola lanuginosa lipase (HLL), during 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 trehalose "salted-out" native HLL, whereas Tween 20 induced non-native aggregates. Optimal recovery of native protein in the initial dried solid was obtained in the presence of additives which formed an amorphous phase and which had the capacity to hydrogen bond to the dried protein (e.g., trehalose and sucrose). Additives which crystallized during lyophilization (e.g., mannitol) 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 solid required that both protein unfolding during lyophilization was minimized and that the formulation was stored at a temperature below its Tg value. Crystallization of sucrose during storage greatly reduced the storage stability of HLL. This was attributed to the increased moisture content and the reduced Tg value in the remaining amorphous phase containing the protein. Sucrose crystallization and the resulting damage to the protein were inhibited by decreasing the mass ratio of sucrose:protein.