胰高血糖素样肽-1和干扰素alpha-2b的聚乙二醇修饰研究
文献类型：学位论文
| 作者 | 王友傲 |
| 学位类别 | 硕士 |
| 答辩日期 | 2010-06-04 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 马光辉 |
| 关键词 | 胰高血糖素样肽-1 聚乙二醇 干扰素alpha-2b 乙醇 |
| 学位专业 | 生物化工 |
| 中文摘要 | 聚乙二醇修饰在蛋白质和多肽药物制剂研究中获得了成功应用，在医药领域受到越来越多的重视。本论文针对现有的胰高血糖素样肽-1和干扰素alpha-2b 的PEG修饰技术中存在的问题进行了以下两方面的研究： 1、GLP-1的PEG修饰研究 目前GLP-1的修饰研究一般采用mPEG-SPA和mPEG-SC在水溶液中进行修饰研究，而这两种修饰剂容易水解，单修饰产物转化率低。为此，本论文采用5 kDa的mPEG-SC修饰剂，探索乙醇中GLP-1的PEG修饰。通过对修饰反应条件的优化，获得了最佳修饰反应条件： GLP-1浓度为1mg/mL，修饰剂与GLP-1的摩尔配比为1:1，37 ℃，反应24 h，Mono-PEG-GLP-1转化率达77%。此外，针对修饰反应液为乙醇溶液的特点，提出了Mono-PEG-GLP-1的分离纯化新策略，先采用低温冷冻离心方法对修饰反应液进行预处理，再有效去除未修饰GLP-1的同时浓缩Mono-PEG-GLP-1，再结合反相液相色谱进一步纯化，获得了纯度高达97%的Mono-PEG-GLP-1。修饰位点主要集中在GLP-1的N末端a-氨基上。Mono-PEG-GLP-1的降血糖效果明显提升，同时具有更好的抗胰蛋白酶水解作用且在血清中的稳定性显著增强。 2、干扰素α-2b的PEG修饰 目前，上市的PEG-Intron是干扰素α-2b的mPEG-SC (Mw:12 kDa) 修饰物，在水溶液中稳定性差只能做成冻干粉保存；循环半衰期不够理想，无法实现一周一次给药。针对这两个问题，本研究采用分子量20 kDa的mPEG-ALD干扰素alpha-2b行修饰研究。在实验室前期干扰素α-2b修饰工作的基础上优化了修饰反应条件。结果表明：在反应液pH为6.5时生成的Mono-PEG-IFN a-2b体外活性保留最高，达到31%；还原剂与修饰剂之间的摩尔配比对Mono-PEG-IFN a-2b的转化率有着重要的影响，但是过高的还原剂不利于溶液中蛋白质的稳定；为了简化操作和保持批次间稳定性，在后续研究中采用分批反应方式。 |
| 英文摘要 | More attention had been paid to PEGylation technology in the past years, due to their successful use in improving the pharmacokinetics and pharmacodynamics of many peptide and protein pharmaceuticals. To solve the pegylation problems of the glucagons-like peptide-1 and interferon α-2b, studies of this dissertation were included as follows. 1 Studies on PEGylation of glucagon-like peptide-1 Currently, methoxy polyethylene glycol succinimidyl propionic acid ester (mPEG-SPA) and methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC) were generally used to modify the GLP-1. Unfortunately, mPEG-SPA and mPEG-SC were easily hydrolyzed in water solution and their half-life was very short, which caused low yield of the Mono-PEG-GLP-1. In this study, the GLP-1 was modified with mPEG-SC in ethanol. The reaction conditions were optimized for obtaining high yield of Mono-PEG-GLP-1, and the optimized reaction conditions were as follows: concentration of GLP-1 1.0 mg/mL, molar ratio of PEG to GLP-1 1:1, and reaction time 24 h at 37 ℃. Under the optimized conditions, the yield of mono-PEG-GLP-1 was up to 77%, which is significantly higher than the yields as references reported (< 50%). This method provides a new idea and technology for the PEGylation of other peptides. Considering the characteristics of the reaction system, a preliminary centrifugation under frozen temperature and a sequential purification by reverse phase chromatography were applied to separate and purify Mono-PEG-GLP-1 from reaction mixture. The purity of Mono-PEG-GLP-1 was higher than 97%. Furthermore, the PEGyalted site was N-terminal amino detected by MALDI-TOF. Compared with GLP-1, the Mono-PEG-GLP-1 showed better stability in plasma, decreased proteolysis to trypsin, and better in vivo activity. 2 Investigation on PEGylation of interferon α-2b PEG-Intron, a commercial PEGylated interferon α-2b using mPEG-SC of 12 kDa, which was made into powder dosage due to its poor stability in water solution; and the half-life in vivo was not long enough to ensure one injection per week during clinical usage because of the low weight of mPEG-SC. To solve these problems, interferon α-2b was modified with methoxy polyethylene glycol propionaldehyde (mPEG-ALD) of mocular weight 20 kDa. Based on the preliminary studies in our group, we further exploited the influence of reaction conditions on the yield of Mono-PEG-IFN α-2b. Results showed that Mono-PEG-IFN α-2b preserved the highest in vitro activity (31%) when the reaction solution pH was 6.5, while the in vitro activity of PEG-Inron was 28%. The yield of Mono-PEG-IFN α-2b and the stability of IFN α-2b were also influenced by the amount of reductant. In addition, fed-batch reaction system were also investigated and compared with bacth reaction for improving the yield of Mono-PEG-IFN α-2b, unfortunately, the yield of Mono-PEG-IFN α-2b could not be improved because there were many PEGylation sites on IFNα-2b. Therefore, in order to simplify operations and maintain the stability of Mono-PEG-IFNα-2b between different bacthes, batch reaction system was used to carry out modification of interferonα-2b. |
| 语种 | 中文 |
| 公开日期 | 2013-09-17 |
| 页码 | 93 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1507]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 王友傲. 胰高血糖素样肽-1和干扰素alpha-2b的聚乙二醇修饰研究[D]. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：过程工程研究所
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