聚乙二醇单修饰重组水蛭素的研究
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摘要
水蛭素是迄今为止发现的特异性最好的凝血酶抑制剂，但由于其血浆半衰期很短，严重限制了其在临床上的抗凝应用。聚乙二醇(PEG)修饰是常用的提高蛋白和多肽类药物血浆半衰期的方法，已有多种PEG修饰的蛋白类药物上市，如PEG-Intron~(?) (PEGinterferonα-2b)，PEGasys~(?) (PEGinterferonα-2a)，Neulasta~(?)(pegfilgrastim，PEG G-CSF)等，通过修饰这些药物的体内循环半衰期和生物利用度均有所增加。关于水蛭素的聚乙二醇修饰，国内外也已开展了很多工作，但所用修饰方法多为碱性条件下修饰水蛭素的赖氨酸残基，这将不可避免的导致修饰水蛭素的活性中心(Lys47)的修饰，且修饰反应产物是复杂的混合物(四修饰、三修饰、二修饰和单修饰产物的混合物)。为了避免水蛭素活性中心的修饰、得到均一的单修饰产物，本文应用了两种修饰策略来制备聚乙二醇单修饰重组水蛭素。
首先，采用微酸性修饰策略，选择性的修饰重组水蛭素唯一的组氨酸残基以得到单修饰产物。在pH 6.0条件下把单甲氧基聚乙二醇甲酸琥珀酰亚胺酯(Succinimidylcarbonyl mPEG，SC-mPEG，20kDa)与重组水蛭素偶联，反应混合物经一步阴离子交换色谱(IEC)分离纯化得到单修饰产物，单修饰产物占修饰产物的94.6％。通过中性羟胺法处理，发现单修饰产物中79.71％为组氨酸修饰位置异构体，表明该修饰策略大体上避开了重组水蛭素活性中心(Lys47)的修饰。单修饰产物的纯度大于95％，其体外抗凝活性保留率为34％。
另外，本文利用阴离子交换介质辅助重组水蛭素的PEG修饰，以得到单修饰产物。在pH 6.0，20mM PBS，r-Hir∶SC-mPEG=1∶3的条件下，修饰反应和分离纯化在阴离子交换介质上偶联以直接得到单修饰产物。电泳检测修饰产物为单修饰产物，通过中性羟胺法处理单修饰产物，发现在该微酸性条件下，修饰未发生在组氨酸残基，根据修饰反应理论推断修饰位点为N-末端氨基。单修饰产物的纯度大于95％，单修饰产物的体外抗凝活性保留率为34.85％。
Hirudin, the most potent inhibitor ofthrombin found in nature, is of short half-life inserum, which significantly limits its clinical application. Recently, PEGylation iscommonly used as an effective method to prolong the half-life of protein and peptidedrugs in serum. Many PEGylated protein drugs have come into the market, likePEG-Intron (PEGinterferonα-2b), PEGasys (PEGinterferonα-2a), Neulasta (PEG G-CSF)etc. The PEGylation has greatly improved the half-life in vivo and the bioavailability ofthese drugs. Researches in the area of PEGylation of recombinant hirudin(r-Hirudin)commonly concentrate on the the lysine residue PEGylation under basic conditions, thiswill inevitably lead to the active center(Lys47) PEGylation, and the reaction products areususlly a mixture of quadri-, tri-, di-, mono-PEGylated species. In order to avoid thePEGylation of active center and obtain homogeneous monoPEGylated product, twostrategies were applied in this paper.
First, the mild acidic PEGylation strategy was used to target the only histidineresidue(His51) of r-Hirudin. Succinimidyl carbonyl mPEG(SC-mPEG, 20kDa) wasattached to r-Hirudin at pH 6.0, the reaction mixture was easily separated by a one-stepion-exchange chromatographic(IEC) procedure, and 94.6％of the PEGylation productwas monoPEGylated. About 79.71％of the monoPEGylated r-Hirudin was histidinepositional isomer, which showed the acidic PEGylation operation avoided the PEGylationof Lys47 in principle. The more than 95％pure predominant monoPEGylated productwas obtained and retained 34％of the anticoagulant activity in vitro.
In adition, IEC was used to assisted the PEGylation of r-Hirudin. The reaction wasconducted under the reaction condition of pH 6.0, 20mM PBS, r-Hir:SC-mPEG=1:3, andthe PEGylation and seperation were conducted in one step. Only monoPEGylated productwas shown in SDS-PAGE, and the PEGylation wasn't occurred at His51. According tothe PEGylation reaction theory, it may occurr at the N-terminal amine group. The purityof monoPEGylated product was more than 95％, and 34.85％of the anticoagulant activitywas retained in vitro.
首先，采用微酸性修饰策略，选择性的修饰重组水蛭素唯一的组氨酸残基以得到单修饰产物。在pH 6.0条件下把单甲氧基聚乙二醇甲酸琥珀酰亚胺酯(Succinimidylcarbonyl mPEG，SC-mPEG，20kDa)与重组水蛭素偶联，反应混合物经一步阴离子交换色谱(IEC)分离纯化得到单修饰产物，单修饰产物占修饰产物的94.6％。通过中性羟胺法处理，发现单修饰产物中79.71％为组氨酸修饰位置异构体，表明该修饰策略大体上避开了重组水蛭素活性中心(Lys47)的修饰。单修饰产物的纯度大于95％，其体外抗凝活性保留率为34％。
另外，本文利用阴离子交换介质辅助重组水蛭素的PEG修饰，以得到单修饰产物。在pH 6.0，20mM PBS，r-Hir∶SC-mPEG=1∶3的条件下，修饰反应和分离纯化在阴离子交换介质上偶联以直接得到单修饰产物。电泳检测修饰产物为单修饰产物，通过中性羟胺法处理单修饰产物，发现在该微酸性条件下，修饰未发生在组氨酸残基，根据修饰反应理论推断修饰位点为N-末端氨基。单修饰产物的纯度大于95％，单修饰产物的体外抗凝活性保留率为34.85％。
Hirudin, the most potent inhibitor ofthrombin found in nature, is of short half-life inserum, which significantly limits its clinical application. Recently, PEGylation iscommonly used as an effective method to prolong the half-life of protein and peptidedrugs in serum. Many PEGylated protein drugs have come into the market, likePEG-Intron (PEGinterferonα-2b), PEGasys (PEGinterferonα-2a), Neulasta (PEG G-CSF)etc. The PEGylation has greatly improved the half-life in vivo and the bioavailability ofthese drugs. Researches in the area of PEGylation of recombinant hirudin(r-Hirudin)commonly concentrate on the the lysine residue PEGylation under basic conditions, thiswill inevitably lead to the active center(Lys47) PEGylation, and the reaction products areususlly a mixture of quadri-, tri-, di-, mono-PEGylated species. In order to avoid thePEGylation of active center and obtain homogeneous monoPEGylated product, twostrategies were applied in this paper.
First, the mild acidic PEGylation strategy was used to target the only histidineresidue(His51) of r-Hirudin. Succinimidyl carbonyl mPEG(SC-mPEG, 20kDa) wasattached to r-Hirudin at pH 6.0, the reaction mixture was easily separated by a one-stepion-exchange chromatographic(IEC) procedure, and 94.6％of the PEGylation productwas monoPEGylated. About 79.71％of the monoPEGylated r-Hirudin was histidinepositional isomer, which showed the acidic PEGylation operation avoided the PEGylationof Lys47 in principle. The more than 95％pure predominant monoPEGylated productwas obtained and retained 34％of the anticoagulant activity in vitro.
In adition, IEC was used to assisted the PEGylation of r-Hirudin. The reaction wasconducted under the reaction condition of pH 6.0, 20mM PBS, r-Hir:SC-mPEG=1:3, andthe PEGylation and seperation were conducted in one step. Only monoPEGylated productwas shown in SDS-PAGE, and the PEGylation wasn't occurred at His51. According tothe PEGylation reaction theory, it may occurr at the N-terminal amine group. The purityof monoPEGylated product was more than 95％, and 34.85％of the anticoagulant activitywas retained in vitro.
引文
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