胰高血糖素样肽-1的聚乙二醇定点修饰

Investigation on Site-specific PEGylation of Glucagon-like Peptide-1

采用分子量为5kDa的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)修饰胰高血糖素样肽-1(GLP-1),建立了GLP-1的定点修饰反应及分离纯化工艺,考察了修饰反应各因素对单修饰产物得率及体外活性的影响,得到优化修饰条件为:pH7.5,50mmol/L Na2HPO4-NaH2PO4缓冲液,GLP-1浓度1mg/mL,PEG/GLP-1摩尔比2:1,4℃下反应1h.在此条件下,单修饰PEG-GLP-1得率达50%.反相高效液相色谱分离纯化所得单修饰产品纯度达98%,体外活性保留为未修饰GLP-1的86%,其在Sprague-Dawley远交群大鼠体内的循环半衰期为60min,比原肽提高了20倍.

In order to obtain a more stable PEGylated glucagon-like peptide-1, and prolong its half life, glucagon-like peptide-1 was site-specifically modified with monomethoxy polyethylene glycol succinimidyl carbonate （mPEG-SC, 5 000 kDa）. It was found that the optimized reaction conditions for maximized bioactivity and the highest modification yield of mono PEGylated GLP-1 were as follows： in 50 mmol/L and pH 7.5 phosphate buffer, concentration of GLP-1 1.0 mg/mL, molar ratio of PEG to GLP-1 2：1, and reaction time 1 h at 4℃. Under the optimized conditions, the mono modification yield reached 50%. Reverse phase high performance liquid chromatography was used to separate and purify mono PEGylated GLP-1 from the reaction mixture. The purity of mono PEGylated glucagon-like peptide-1 was higher than 98%, characterized by gelfiltration chromatography, the bioactivity of mono PEGylated glucagon-like peptide-1 was 86% of the native GLP-1, while its half life in Sprague-Dawley rats was much longer than that of native GLP-1. The mono PEGylated glucagon-like peptide-1 was also stable in aqueous solution.