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.

Optimization of Storage Stability of Lyophilized Actin Using Combinations of Disaccharides and Dextran

“…Previous research has shown that storage in the glassy state, below the sample T g , is necessary for stability. 7,10,13,14 In addition, it has also been shown that the presence of unfolded protein in the dried state can result in decreased storage stability. 10,13,14 If the T g mechanism alone were sufficient to stabilize actin during storage, then Figure 1).…”

“…7,10,13,14 In addition, it has also been shown that the presence of unfolded protein in the dried state can result in decreased storage stability. 10,13,14 If the T g mechanism alone were sufficient to stabilize actin during storage, then Figure 1). The solid line in Figure 2 shows the secondderivative amide I infrared spectrum of actin dried with 1% dextran taken immediately after freeze-drying.…”

“…10,[12][13][14][15] During storage in the dried solid, even at temperatures below T g , formulations containing unfolded protein degrade more rapidly via both chemical and physical pathways than do those in which lyophilization-induced unfolding is minimized. 10,13,14 Disaccharides, such as sucrose and trehalose, are capable of maintaining high levels of native protein conformation during freeze-drying. 10,[12][13][14][15][16] The use of trehalose as a stabilizer has been extensively studied, because its higher T g may confer added storage stability to proteins relative to sucrose.…”

Effect of Lyoprotectant on the Solubility and Structure of Silk Sericin

“…Theoretically, sugars can replace the sublimated water molecules and maintain the hydrogen bonding of the protein second structure (Tanaka et al, 1991;Belton et al, 1994;Dong et al, 1996;Remmele et al, 1997;). Additionally, they decrease protein molecules' mobility by increasing the T g of protein (Kreilgaard et al, 1999;Allison et al, 2000;).…”

Stabilization of Protein Structure in Freeze-Dried Amorphous Organic Acid Buffer Salts

“…Insuffi- cient intermolecular interaction with co-lyophilized protein due to steric hindrance should explain the lower structurestabilizing effect of dextran 10.2k compared to sucrose. 31) No apparent relationship between the structure-stabilizing effect and the glass transition temperatures (T g ) or residual water content of the freeze-dried solid (Table 1) was observed.…”