Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage

Abstract

In order to understand the stabilizing effects of disaccharides on freeze-dried proteins, the enzymatic activity of lactate dehydrogenase (LDH) formulations containing four types of disaccharide (trehalose, sucrose, maltose, and lactose) at two relative humidity (RH) levels (about 0 and 32.8%) was investigated after three processes: freeze-thawing, freeze-drying, and storage at three temperatures (20, 40, and 60 degrees C) above and/or below the glass transition temperature (T(g)). The enzymatic activity was determined from the absorbance at 340 nm, and T(g) of the samples was investigated by differential scanning calorimetry. At each RH condition, T(g) values of sucrose formulations were lower than those of other formulations. Although effects of the disaccharides on the process stability of LDH were comparable, storage stability was dependent on the type of disaccharide. All the formulations were destabilized significantly during storage at temperature above T(g). During storage at temperature below T(g), the LDH activity decreased with increases in the storage temperature and moisture. Maltose and lactose formulations showed significant destabilization with the change of color to browning. Taking the storage stability of freeze-dried proteins under the various conditions (temperature and RH) into consideration, trehalose is better suited as the stabilizer than other disaccharides.