earticle

영어

Despite the therapeutic efficacy of exendin-4 for the treatment of type 2 diabetes, its application is still limited because of twice-a-day injection requirement. PEGylation, covalent conjugation of a PEG (polyethylene glycol) molecule to a peptide or protein, is known to prolong in-serum half-life as well as to improve pharmacokinetic and pharmacodynamic properties of a peptide or protein [1-3]. In this study, we attempted an aqueous-phase, two-step PEGylation process, in which amine groups of exendin-4 were first converted into thiol groups and then PEG-maleimide with different molecular weights (5 and 10kD) was conjugated via disulfide bridge. These reactions were performed in PBS buffer (pH 7.4) at 400C for overnight. The PEGylates were purified by cation exchange chromatography, using HiTrap SP HP column with a running buffer (10 mM citric acid, pH 3.5) and an elution buffer (10 mM citric acid with and 1 M NaCl). Then, conjugated peptides were separated by sizeexclusion chromatography (Superdex 75 PC 3.2/30 column), using 50 mM PBS (pH 7.0) with 0.15 M NaCl as an elution buffer.The overall conjugation efficiency was high, more than 70%. Maintenance of in-vitro bioactivity was measured by ELISA. This study demonstrates that exendin-4 can be more effectively PEGylated by the two-step process.