Development of a Spectrophotometric Biphasic Assay for the Estimation of mPEG-maleimide in Thiol PEGylation Reaction Mixtures
Development of a Spectrophotometric Biphasic Assay for the Estimation of mPEG-maleimide in Thiol PEGylation Reaction Mixtures
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DOI：
10.1080/00986445.2016.1204604
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发表时间：
2016-07
影响因子：
2.5
通讯作者：
Pooja Nanda;P. E. JagadeeshBabu;Purva Gupta;Aprameya Prasad
中科院分区：
文献类型：
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作者：
Pooja Nanda;P. E. JagadeeshBabu;Purva Gupta;Aprameya Prasad
甲氧基（聚乙二醇）-马来酰亚胺 (mPEG-mal) 是一种 PEG 衍生物，用于蛋白质分子的硫醇聚乙二醇化，并在药物递送研究中得到应用。马来酰亚胺基团在水介质中会发生降解，导致 mPEG-mal 的定量分析变得困难。常规用于分离和估计 mPEG-mal 的方法包括繁琐的色谱方法，如离子交换、带有折射率检测器的高效液相色谱以及质谱和质子核磁共振等技术。我们提出了一种直接且可重复的分光光度法来定量硫醇聚乙二醇化反应混合物中游离和蛋白质结合的 mPEG-mal。该方法基于在 mPEG-mal 存在下，PEG 结合的发色团在异铁硫氰酸铵水相与氯仿相之间的分配。研究了影响发色团分配和稳定性的几个重要参数、液相体积比、反应混合物中乙二胺四乙酸浓度、混合时间和用于分配的氯化溶剂。
Methoxy(polyethylene glycol)-maleimide (mPEG-mal) is a PEG derivative used for thiol PEGylation of protein molecules and finds application in drug delivery studies. The maleimide group undergoes degradation in aqueous media, resulting in the difficult quantitative analysis of mPEG-mal. Routinely employed methods for separation and estimation of mPEG-mal include tedious chromatographic methods like ion exchange, high-performance liquid chromatography with refractive index detector and techniques like mass spectrometry and proton nuclear magnetic resonance. We present a direct and reproducible spectrophotometric method to quantify free and protein bound mPEG-mal in thiol PEGylation reaction mixtures. This method is based on the partitioning of a PEG bound chromophore between an aqueous ammonium isoferrothiocyanate phase to a chloroform phase in the presence of mPEG-mal. Several important parameters influencing the partitioning and stability of the chromophore, volume ratios of liquid phases, ethylenediaminetetraacetic acid concentration in the reaction mixture, mixing time, and chlorinated solvents used for partitioning have been studied.