The use of disaccharides in inhibiting enzymatic activity loss and secondary structure changes in freeze-dried β-galactosidase during storage.

Purpose: The purpose of this study is to show how disaccharides differ in their ability to protect lyophilized β-galactosidase from enzymatic activity loss and secondary structure changes during storage.

Methods: β-galactosidase was lyophilized with trehalose, sucrose, cellobiose or melibiose at 2:1, 20:1 and 40:1 excipient/protein weight ratios, and stored up to 90 days at 45 °C. Protein enzymatic activity was studied using o-nitrophenyl-β-D-galactopyranoside cleavage test, and its secondary structure in lyophilizates analyzed using Fourier transform infrared spectroscopy. The crystallization tendencies, glass transition temperatures and water contents of lyophilizates were evaluated using x-ray powder diffractometry, differential scanning calorimetry and thermogravimetry, respectively.

Results: The enzymatic activity of β-galactosidase decreased more slowly in lyophilizates containing trehalose or melibiose at 2:1 excipient/protein weight ratio when compared to those containing sucrose or cellobiose. Similar behavior was observed when analyzing the protein's secondary structure in lyophilizates. In 20:1 and 40:1 excipient/protein weight ratio lyophilizates the decrease of enzymatic activity was less dependent on the excipient, but activity was always amongst the highest in melibiose lyophilizates.

Conclusions: Melibiose was shown to be effective in protecting lyophilized β-galactosidase during storage. The protein secondary structure was shown to change at comparable rate in lyophilizates as its enzymatic activity after rehydration.