Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation.
Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation.
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通过聚乙二醇化预防苯甲醇诱导的胰凝乳蛋白酶原聚集。
DOI：
10.1111/j.2042-7158.2011.01288.x
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Griebenow,Kai
中科院分区：
文献类型：
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作者：
Rodriguez-Martinez,JoseA;Rivera-Rivera,Izarys;Griebenow,Kai
目的在多剂量蛋白质制剂中添加抗菌防腐剂苯甲醇作为重建缓冲液，可促进不需要的聚集体的形成。因此，我们研究了聚乙二醇化(聚乙二醇化)对苯甲醇诱导的模型蛋白α-胰凝乳蛋白酶原A(ACTGN)聚集的抑制作用。方法在合成过程中，通过改变聚乙二醇与蛋白质的比例，制备了不同分子量的聚乙二醇-ACTGN结合物。当模型蛋白在含有0.9%苯甲醇的缓冲液中重组时，在24 h内形成了大量的不溶于缓冲液的聚集体(10%)。当2~5个相对分子质量为5000 Da的聚乙二醇与蛋白质结合时，苯甲醇诱导的聚集体完全被阻止，而2~4个分子结合的700 Da聚乙二醇则完全不能阻止聚集体的形成。机理研究排除了通过聚乙二醇化来防止结构扰动或增加热力学稳定性，而不是对防止聚集负责。在缓冲液中加入聚乙二醇单用也是无效的，必须将聚乙二醇共价连接到蛋白质上才能起到保护作用。结论5000Da聚乙二醇单分子最可能的保护作用是屏蔽暴露的疏水蛋白质表面积和防止蛋白质-蛋白质接触(分子间隔物效应)。
ObjectivesAddition of the antimicrobial preservative benzyl alcohol to reconstitution buffer promotes the formation of undesirable aggregates in multidose protein formulations. Herein we investigated the efficiency of PEGylation (attachment of poly(ethylene glycol)) to prevent benzyl alcohol-induced aggregation of the model proteinα-chymotrypsinogen A (aCTgn).MethodsVarious PEG-aCTgn conjugates were prepared using PEG with a molecular weight of either 700 or 5000 Da by varying the PEG-to-protein ratio during synthesis and the formation of insoluble aggregates was studied. The effect of benzyl alcohol on the thermodynamic stability and tertiary structure of aCTgn was also examined.Key findingsWhen the model protein was reconstituted in buffer containing 0.9% benzyl alcohol, copious amounts of buffer-insoluble aggregates formed within 24 h (>10%). Benzyl alcohol-induced aggregation was completely prevented when two or five molecules of PEG with a molecular weight of 5000 Da were attached to the protein, whereas two or four molecules of bound 700 Da PEG were completely inefficient in preventing aggregation. Mechanistic investigations excluded prevention of structural perturbations or increased thermodynamic stability by PEGylation from being responsible for the prevention of aggregation. Simple addition of PEG to the buffer was also inefficient and PEG had to be covalently linked to the protein to be efficient.ConclusionsThe most likely explanation for the protective effect of the 5000 Da PEG is shielding of exposed hydrophobic protein surface area and prevention of protein–protein contacts (molecular spacer effect).