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胰高血糖素样肽-1的聚乙二醇定点修饰
引用本文:张磊,翟艳琴,雷建都,马光辉,苏志国. 胰高血糖素样肽-1的聚乙二醇定点修饰[J]. 过程工程学报, 2009, 9(6): 1169-1173
作者姓名:张磊  翟艳琴  雷建都  马光辉  苏志国
作者单位:中国科学院过程工程研究所生化工程国家重点实验室,北京,100190;中国科学院研究生院,北京,100049;中国科学院过程工程研究所生化工程国家重点实验室,北京,100190
基金项目:国家自然科学基金资助项目,国家高技术研究发展计划(863)基金资助项目,北京市自然科学基金资助项目 
摘    要:采用分子量为5 kDa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:pH 7.5, 50 mmol/L Na2HPO4-NaH2PO4缓冲液,GLP-1浓度1 mg/mL, PEG/GLP-1摩尔比2:1, 4℃下反应1 h. 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60 min,比原肽提高了20倍.

关 键 词:胰高血糖素样肽-1  聚乙二醇  定点修饰
收稿时间:2009-04-20
修稿时间:2009-06-03

Investigation on Site-specific PEGylation of Glucagon-like Peptide-1
ZHANG Lei,ZHAI Yan-qin,LEI Jian-du,MA Guang-hui,SU Zhi-guo. Investigation on Site-specific PEGylation of Glucagon-like Peptide-1[J]. Chinese Journal of Process Engineering, 2009, 9(6): 1169-1173
Authors:ZHANG Lei  ZHAI Yan-qin  LEI Jian-du  MA Guang-hui  SU Zhi-guo
Abstract: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, 5000 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.
Keywords:glucagon-like peptide-1  monomethoxy polyethylene glycol succinimidyl carbonate  site-specific PEGylation
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