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题名	固相介质辅助聚乙二醇修饰蛋白质的研究
作者	索晓燕
学位类别	博士
答辩日期	2009-05-27
授予单位	中国科学院过程工程研究所
授予地点	过程工程研究所
导师	苏志国
关键词	固相修饰 聚乙二醇 牛血红蛋白 人血清白蛋白 葡激酶
其他题名	PEGylation of proteins assisted by
学位专业	生物化工
中文摘要	聚乙二醇修饰可有效地降低蛋白质药物的免疫原性，延长体内循环半衰期和改善药效学性质。然而，现有的聚乙二醇液相修饰反应存在反应可控性差和修饰产物分子量不均一的缺点。本文探索一种新型的聚乙二醇修饰蛋白质的反应过程固相修饰。其特点是将蛋白质吸附在固相介质上，然后加入修饰剂与蛋白质进行反应。由于介质和蛋白质的相互作用以及空间位阻效应，可以在修饰过程中减少修饰反应的随机性以及提高反应的可控性。 首先，本论文使用离子交换介质对三种不同结构和性质的蛋白质进行固相介质辅助聚乙二醇修饰的研究，包括人血清白蛋白（human serum albumin, HSA）、葡激酶（staphylokinase, SAK）和牛血红蛋白（bovine hemoglobin, bHb）。实验证明，使用聚乙二醇衍生物琥珀酰亚胺碳酸酯-单甲氧基聚乙二醇（SC-mPEG）以及离子交换介质进行固相介质辅助蛋白质的修饰反应，可以提高反应的可控性和单一组分修饰产物的比例，能够较好地保持蛋白质的活性。固相介质辅助聚乙二醇修饰以后，人血清白蛋白和葡激酶的聚乙二醇单修饰率分别达到45%和40%，高于普通液相修饰反应的修饰结果（单修饰率小于 30 %）；葡激酶的活性保持率可达65%，高于常规的液相修饰反应的活性保持率（30%左右）。 为了研究在固相介质辅助聚乙二醇修饰蛋白质的过程中修饰反应与分离纯化集成的可行性，本论文进一步研究了离子交换介质辅助聚乙二醇修饰牛血红蛋白的过程。 蛋白质经过聚乙二醇修饰后等电点会有所变化，牛血红蛋白的等电点（pI=7.2）与其反应条件的pH值（pH=6.8）比较接近，据此可通过选择合适的修饰条件，使修饰和未修饰的血红蛋白在层析的过程中彼此分离。在本实验中，牛血红蛋白的聚乙二醇单修饰产物的产率可达到70%左右。分子量为5 kD、10 kD、20 kD的聚乙二醇衍生物修饰牛血红蛋白产物的P50值分别为19.97 mm Hg、20.23 mm Hg、20.54 mm Hg；Hill系数分别为1.98、1.85、1.92；高铁氧化血红蛋白（MetHb） 的含量分别为15.41%、14.23%、13.75%，单修饰率和活性保持率都高于常规的液相反应。 另一方面，由于离子交换介质的吸附是非特异性的，所以离子交换介质辅助聚乙二醇修饰蛋白质不可避免的具有一定局限性。为此，本论文对共价层析介质辅助聚乙二醇修饰蛋白质做了初步探索，蛋白质上的巯基可以特异性地与共价介质上的巯基发生交联，具有一定的位置选择性。在本实验中，牛血红蛋白单修饰产物的产率为55%、P50值为22.13 mm Hg、Hill系数为2.18、高铁氧化血红蛋白（MetHb）的含量为7.2%。该结果表明，特异性共价介质辅助聚乙二醇修饰蛋白质的方法对于血红蛋白活性的保持具有更大的优势。 综上所述，使用固相介质辅助聚乙二醇修饰蛋白质的方法可提高修饰反应的可控性和聚乙二醇单修饰产物的产率，并能较好地保持蛋白质的生物活性。而且，通过选择合适的修饰条件，可实现蛋白质的修饰和分离纯化的集成。为制备单一组成的聚乙二醇修饰产物提供了一种新型有效方法。
英文摘要	PEGylation, i.e., covalent attachment of polyethylene glycol (PEG) to proteins, is an effective method to improve the therapeutic potentials of proteins by prolonging their in vivo half-life and decreasing their immunogenicity and increasing their pharmacology effects. The popular PEGylation target is the ε-amino groups of lysine residues and the α-amino groups of N-terminal amino acid residues. Due to the presence of multiple amino groups, one protein molecule may be randomly conjugated with multiple copies of PEG chains at different sites. A solid-phase adsorption method was developed for PEGylation of proteins. This method is aimed at circumventing the disadvantage of the conventional liquid-phase PEGylation, i.e., the heterogeneity of the PEGylated products. In brief, the proteins were adsorbed on the solid phase media, followed by PEGylation and elution of the protein from the media. The present work is aimed at providing an improved solid phase adsorption method for preparing the homogeneous PEGylated proteins. Firstly, an ion exchange medium has been used to assist pegylation of three proteins, including human serum albumin (HSA), staphylokinase (SAK) and bovine hemoglobin (bHb). Succinimidyl carbonate-monomethoxypolyethylene glycol (SC-mPEG) 5 kD, 10 kD and 20 kD were employed to react with these three proteins. The results of the PEGylationn depended on the characteristic of proteins, including the isoelectric point, molecular weight and molecular structure. In this part, all three proteins had been explored to improve the controllability of reaction. The mono-PEGylated SAK and mono-PEGylated HSA were obtained with the yield of 45% and 40%, respectively. The biological activity of the mono-PEGylated SAK show higher increase than in the liquid phase (from 30% to 65%). Hemoglobin has a pI about 7.0. We chose pH 6.8 as adsorption condition so that the protein was adsorbed by the medium. Upon pegylation, the pI of the product was reduced due to the reaction of amino group. The pegylated protein was then eluted easily by the washing buffer while unreacted protein was still on the medium. In this way, simultaneous pegylation and product separation was realized. This is certainly the advantage of the solid phase method. The mono-PEGylated bHb was obtained with the yield of 70%. The P50, the Hill coefficient and the MetHb ratio for the mono-PEG5k-bHb were 19.97 mm Hg, 1.98 and 15.41%, respectively; for the mono-PEG10k-bHb were 20.23 mm Hg, 1.85 and 14.23%, respectively; for the mono-PEG20k-bHb were 20.54 mm Hg, 1.92 and 13.35%, respectively. In the second part, a covalent solid phase adsorption method has been used to prepare PEGylated bHb with a covalent chromatography medium (Activated Thiol Sepharose 4B) and SC-mPEG. The reactive sulphydryl group on Cys β93 in hemoglobin could covalently attach to the sulphydryl group of the medium. The yield of mono-PEGylated hemoglobin was 55%. The P50, the Hill coefficient and the MetHb ratio for the hemoglobin were 22.13 mm Hg, 2.18 and 7.2%, respectively. Accordingly, the covalent solid phase adsorption method of bHb has a higher ability to maintain the biological activity of bHb. The solid phase pegylated method could prevent or eliminate the multiple site attachment of PEG molecules due to possible steric hindrance. The advantage of solid phase adsorption is that it makes site-specific PEGylation at specific areas on the protein possible so as to minimize the loss of biological activity while reducing immunogenicity. The adsorption of the protein on the chromatography medium could provide an effective approach to prepare homogenous PEGylated products.
语种	中文
公开日期	2013-09-13
页码	145
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1181]
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	索晓燕. 固相介质辅助聚乙二醇修饰蛋白质的研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2009.

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