The Application and Mechanisms of Polyethylene Glycol 8000 on Stabilizing Lactate Dehydrogenase during Lyophilization

Abstract

The purpose of this paper is to explore the application and mechanisms of polyethylene glycol 8000 (PEG 8000) on stabilizing lactate dehydrogenase (LDH) during lyophilization. In earlier freeze-thawing experiments, different molecular weights and concentrations of PEGs were formulated with LDH, and ultraviolet (UV) enzymatic activity and circular dichroism (CD) wavelength scanning studies were conducted. In lyophilization studies, different molecular weights of saccharides, e.g., glucose, sucrose, dextran 37,000 (D 37K), and dextran 160,000 (D 160K), with or without PEG 8000, were formulated with LDH at various molar ratios. UV assays, size exclusion chromatography–high performance liquid chromatography (SEC-HPLC), CD, fourier transform infrared spectroscopy (FTIR) were conducted for LDH. Upon lyophilization, enzymatic activity and tetrameric structure recoveries of LDH-saccharide formulations reached over 90% with PEG 8000 vs. 60–80% without PEG 8000. LDH-PEG 8000-saccharide formulations shifted the melting temperature (Tm) to higher temperatures than did LDH-saccharide formulations. Most LDH-PEG 8000-saccharide formulations at 1:100:1000 molar ratio showed better preservation of LDH secondary structures than did LDH-saccharide formulations at 1:1000 molar ratio. Since PEG 8000 was confirmed an effective cryoprotectant, saccharides were assumed to be protecting LDH from destabilization during drying. However, LDH-PEG 8000-dextran formulations preserved more LDH secondary structure than did LDH-dextran formulations, but preserved less LDH secondary structures than did LDH-PEG 8000 formulations. This indicated that dextrans not only did not stabilize LDH during drying, but they disrupted the stabilization effect of PEG 8000 on LDH during freezing. After reconstitution, CD wavelength scanning showed that some of the unfolded or denatured structures of LDH were refolded. Based on the steric hindrance of the bulky dextrans and the “water replacement mechanism”, sucrose with PEG 8000 had synergistic protective effects, and dextrans with PEG 8000 had antagonistic effects, on stabilization of LDH during lyophilization.