Evaluation of physicochemical and stability properties of human growth hormone upon enzymatic PEGylation

Abstract

BackgroundPEGylation is an increasingly important strategy for improving stability, pharmacokinetic and pharmacodynamic properties of proteins. ObjectiveIn this study, site-specific PEGylated human growth hormone (hGH) was prepared by microbial transglutaminase and physicochemical and stability properties of bioconjugates were tested. MethodhGH was PEGylated by 20kDa mPEG-NH2. PEG-hGH was purified by size exclusion chromatography method and analyzed by SDS-PAGE, IEF gel and CD spectroscopy methods. Physicochemical properties, size and zeta potentials, of native and PEGylated hGH were evaluated by Dynamic Light Scattering (DLS) method. Physical and chemical stabilities were assayed at different temperatures (37, 25 and 4°C) within three weeks by SEC- and RP-HPLC methods, respectively. ResultsBy this preparation method, mono-PEG-hGH was obtained and the secondary structure was unchanged. The DLS results indicated that by PEGylation the size and zeta potentials of the protein were increased and decreased, respectively. These data indicated that PEG chain covered the protein surface. The isoelectric point (pI) of protein was not altered following PEGylation. The results of stability indicated that, the mono-PEG-hGH was considerably more stable especially in elevated temperatures as compared with the non-PEGylated one. ConclusionPEGylation changed the physicochemical properties of hGH and also enhanced the stability of the protein.