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Abstract：
In this study, a combined optimization method was developed to optimize the N-terminal site-specific PEGylation of recombinant hirudin variant-2 (HV2) with different molecular weight mPEG-propionaldehyde (mPEG-ALD), which is a multifactor-influencing process. The HV2-PEGylation with 5 kDa mPEG-ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono-PEGylated product using combined statistical methods, including the Plackett-Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG-ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono-PEGylated-HV2 was modified at the N-terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N-terminal site-specific PEGylation process to obtain a homogeneous mono-PEGylated protein with desirable yield.
摘要：
在这项研究中,开发了一种组合优化方法来优化具有不同分子量mPEG-丙醛（mPEG-ALD）的重组水蛭素变体2（HV2）的N端位点特异性PEG化，这是一个多因素影响的过程。首先选择使用5kDamPEG-ALD的HV2-聚乙二醇化，以筛选重要因素并确定使用组合统计方法最大化单聚乙二醇化产物产率的局部优化条件。包括Plackett-Burman设计,最陡上升路径分析，和响应面方法(RSM)的中心组成设计。在局部优化条件下，进一步研究了HV2与5、10和20kDamPEG-ALD的PEG化动力学。使用动力学分析在宽范围内对聚乙二醇与HV2的摩尔比和反应时间（影响聚乙二醇化效率的两个最重要的因素）进行了全局优化。使用RSM和动力学分析的组合优化方法预测的数据与相应的实验数据吻合良好。PEG化位点分析显示,几乎100%的获得的单PEG化-HV2在HV2的N-末端被修饰。这项研究表明，所开发的方法是优化N末端位点特异性聚乙二醇化过程的有用工具，以获得具有所需产量的均质单聚乙二醇化蛋白。
