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有机相中聚乙二醇对蛋白质的修饰
Alternative TitlePEGylation of proteins in organic solution
彭飞
Subtype博士
Thesis Advisor苏志国
2013-05-01
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword有机溶剂   聚乙二醇修饰   干扰素beta 1b   粒细胞集落刺激因子
Abstract聚乙二醇修饰(PEGylation)能够有效降低药用蛋白质的免疫原性和抗原性,延长在体内的循环半衰期。但这一过程常常遇到修饰率低和修饰剂水解副反应问题。本论文在现有的水相修饰的基础上,探索在有机相中进行蛋白质的聚乙二醇修饰的可行性,选择的目标蛋白质为干扰素beta 1b及粒细胞集落刺激因子(G-CSF)。 干扰素beta 1b是一种强疏水性蛋白,在多发性硬化症治疗中起重要作用。本论文首先优化了干扰素beta 1b的纯化工艺,得到了高纯度、结构及活性正确的蛋白,然后采用聚乙二醇修饰剂SC-mPEG分别在水溶液和含2-丁醇的有机溶剂中对干扰素beta 1b进行修饰,在修饰剂与蛋白质比例为1:1时,2-丁醇溶剂中的修饰率从水相的2%提高到39%;当修饰剂比例提高到5:1时,总修饰率从水相的13%提高到有机溶剂的94%。原子力显微镜和动态光散射对蛋白聚集行为研究发现,干扰素beta 1b在含2-丁醇的有机溶剂中聚集体颗粒更小,分布更加均一,从而产生更多的蛋白单体与修饰剂反应。圆二色和荧光光谱分析表明,在2-丁醇有机溶剂中蛋白质的二三级结构相比水溶液发生一定程度改变,暴露出更多的修饰位点,从而进一步有利于修饰率的提高。得到的单修饰产物半衰期从3 h提高到了9 h左右。 进一步研究发现在水溶液中添加一定比例的乙醇同样可以有效提高蛋白的修饰率,当乙醇浓度为20%时,干扰素beta 1b的单修饰率可以提高20%。通过结构比较发现,一定比例的乙醇溶剂同样会导致蛋白结构发生可逆性改变,增加修饰反应位点进而提高修饰率。 聚乙二醇修饰剂的水解反应不仅导致修饰率低而且修饰剂的用量很大。本论文以G-CSF为模型,选用对蛋白质有良好相溶性的纯有机溶剂DMSO进行蛋白有机相修饰研究。研究发现,修饰剂SC-mPEG、MAL-mPEG和ALD-mPEG在DMSO中均无水解,并且在低修饰剂用量下,3种修饰剂对G-CSF的修饰率均提高30%以上。采用stop-flow装置研究修饰反应动力学发现,蛋白质在有机相中的反应速率是水相反应速率的103-105倍。与传统水相修饰相比,本论文建立的蛋白质有机相PEG修饰新方法能提高修饰率20%以上,修饰剂用量降低3-10倍,同时修饰反应时间可缩短5-10倍。
Other AbstractPEGylation can effectively reduce the immunogenicity and antigenicity of protein drugs and extend the in-vivo circulating half-life. However, low pegylation yield and hydrolysis of pegylation reagent are often confronted in traditional aqueous reaction. This dissertation used interferon-beta 1b (IFN-β-1b) and granulocyte colony stimulating factor (G-CSF) as model proteins to establish a new PEGylation technique in organic solution to overcome these shortcomings. IFN-β-1b is a highly hydrophobic protein and clinically used to treat Multiple Sclerosis (MS). In this study, the purification protocol was optimized to acquire IFN-β-1b with high purity, correct structure and high activity. Then IFN-β-1b in aqueous and 2-BuOH solution was respectively PEGylated by SC-mPEG. The results showed that the overall modification yields increased from 2% in aqueous solution to 39% in 2-BuOH solution when the molar ratio of protein to PEG was 1:1. The overall yields of pegylation could increase from 13% to 94% when the molar ratio increased to 5:1. Atomic force microscope (AFM) and dynamic light scattering (DLS) analysis revealed that the aggregates were much smaller and more homogeneous in 2-BuOH than in aqueous solution, thereby providing larger accessible protein surfaces, which resulted in a more productive PEGylation process. Circular dichroism (CD) and fluorescence spectra showed that the second and tetiary structures of IFN-β-1b in 2-BuOH differed from those in aqueous solution, which made more PEGylation sites exposed to further increase the PEGylation yields. Furthermore, the PEGylation yields of protein could also be effectively increased by adding ethanol to the aqueous solution of IFN-β-1b. When 20% ethanol was added, the PEGylation yields were increased by 20%. Structure analysis indicated that the protein structure was reversibly changed by ethanol, which exposed and increased the PEGylation sites to facilitate the PEGylation. Some PEG polymers such as SC-mPEG are easy to hydrolyze in aqueous solution, which lead to low efficiency and a waste of PEG. Therefore, this disseration carried out the PEGylation of G-CSF in neat DMSO which was a good solvent for proteins. The results showed that the SC-mPEG, MAL-mPEG and ALD-mPEG would not hydrolyze in DMSO, and the PEGylation yields with the three PEG polymers could be increased by more than 30%. Stopped-flow technique was used to compare the reaction kinetic between in DMSO and in aqueous solution. It was found protein PEGylation in organic solution could increase the PEGylation rate by 103-105. In summery, this new PEGylation technique can increase PEGylation yield, prevent the hydrolysis of PEG polymers, save the amount of modifiers and greatly reduce reaction time.
Pages137
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8246
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
彭飞. 有机相中聚乙二醇对蛋白质的修饰[D]. 中国科学院研究生院,2013.
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