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›› 2009, Vol. 9 ›› Issue (6): 1169-1173.

• 生化工程专栏 • 上一篇    下一篇

胰高血糖素样肽-1的聚乙二醇定点修饰

张磊 翟艳琴 雷建都 马光辉 苏志国   

  1. 中国科学院过程工程研究所 中国科学院过程工程研究所 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室
  • 收稿日期:2009-04-20 修回日期:2009-06-03 出版日期:2009-12-20 发布日期:2009-12-20
  • 通讯作者: 张磊

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

ZHANG Lei ZHAI Yan-qin LEI Jian-du MA Guang-hui SU Zhi-guo   

  1. Multi-phase Reaction Laboratory, Institute of Process Engineering Chinese Academic of Science Multi-phase Reaction Laboratory, Institute of Process Engineering Chinese Academic of Science National Laboratoru of Biochemical Engineering, Insitute of Process Engineering National Laboratoru of Biochemical Engineering, Insitute of Process Engineering National Laboratoru of Biochemical State Key Lab. Biochem. Eng., Inst. Process Eng., Chinese Academy of Sciences State Key Lab. Biochem. Eng., Inst. Process Eng., Chinese Academy of Sciences
  • Received:2009-04-20 Revised:2009-06-03 Online:2009-12-20 Published:2009-12-20
  • Contact: ZHANG Lei

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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

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.

Key words: glucagon-like peptide-1, monomethoxy polyethylene glycol succinimidyl carbonate, site-specific PEGylation

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