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首页> 外文期刊>Separation and Purification Technology >Separation of PEGylated from unmodified ribonuclease A using sepharose media
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Separation of PEGylated from unmodified ribonuclease A using sepharose media

机译：使用琼脂糖凝胶介质从未修饰的核糖核酸酶A分离PEG化

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摘要

PEGylation, used to mitigate some of the problems that affect the effectiveness of therapeutic proteins, often results in a heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. This study presents the use of a mildly amphiphilic support, Tris-capped CH Sepharose 4B as an alternative for separating PEGylated proteins from their unmodified counterparts. The effects of parameters such as pH, salt type and salt concentration upon the chromatographic behavior of native, mono-PEGylated and di-PEGylated ribonuclease A on this media were characterized. The separation of the native protein from the PEGylated species was achieved using a gradient elution between a high ionic strength mobile phase (3 M ammonium sulfate in 25 mM potassium phosphate, pH 7.0 or 2 M potassium phosphate, pH 7.0) and a low ionic strength phase (25 mM potassium phosphate, pH 7.0). The pH of the mobile phases as well as the addition of PEG_(600) (as a potential mobile phase modifier) to the low ionic strength phase had no significant influence on chromatographic behavior of the species. This media provides a simple and practical chromatographic method for the separation of unmodified proteins from their corresponding PEG conjugates.
机译：聚乙二醇化用于缓解一些影响治疗性蛋白质功效的问题，通常会导致异质性的共轭物种和未修饰的蛋白质种群，这对蛋白质分离提出了挑战。这项研究提出了使用轻度两亲性支持物Tris封端的CH Sepharose 4B作为从未修饰的对应物中分离PEG化蛋白的替代方法。表征了诸如pH，盐类型和盐浓度之类的参数对天然，单-PEG化和二-PEG化核糖核酸酶A在该介质上的色谱行为的影响。使用高离子强度流动相（25 mM磷酸钾中的3 M硫酸铵，pH 7.0或2 M磷酸钾，pH 7.0）和低离子强度之间的梯度洗脱实现了从PEG化物种中分离天然蛋白质相（25 mM磷酸钾，pH 7.0）。流动相的pH值以及向低离子强度相中添加PEG_（600）（作为潜在的流动相改性剂）对物质的色谱行为没有重大影响。该介质提供了一种简单实用的色谱方法，可将未修饰的蛋白质与其相应的PEG缀合物分离。

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