超大孔离子交换介质辅助聚乙二醇修饰水蛭素的动力学研究

Solid-phase PEGylation of protein therapeutics on conventional ion-exchange media can achieve more selective synthesis of the targeted mono-PEGylated protein and improve its activity retention. However, one problem of this PEGylation strategy is the low yield of mono-PEGylated protein. Here, a new approach of solid-phase PEGylation of hirudin on gigaporous ion exchange media is proposed to solve this problem. To improve the PEGylation efficiency, kinetics of this solid-phase PEGylation process will be systematically studied from the perspectives of the control and optimization of process engineering. Furthermore, the key factors that affect the PEGylation site and degree will be regulated. Firstly, based on the yield and activity retention of mono-PEGylated protein, the parameters of gigaporous ion exchange media and the reaction conditions that influence solid-phase PEGylation of protein will be systematically investigated and the key factors will be determined. Secondly, a reaction kinetic model will be constructed to describe the process of solid-phase PEGylation of protein on gigaporous ion exchange media and the unknown PEGylation kinetics parameters will be determined through combining the experiment and the theoretical computation. The established kinetic model will be used to optimize the various reaction conditions to achieve the desired yield of mono-PEGylated protein, and the corresponding experiments will be carried out to verify the optimal conditions predicted by this model. Finally, protein adsorption on the gigaporous ion exchange media will be investigated by means of molecular simulation combined with experiments verification. The mechanisms of solid-phase PEGylation of protein will be interpreted. The results of this study will reveal the process of solid-phase PEGylation kinetics of hirudin on gigaporous ion exchange media from the two levels of reaction kinetics and molecular dynamics, which will provide a new insight into deep understanding of the PEGylation of protein therapeutics.

常规的离子交换介质辅助固相修饰可以提高聚乙二醇（PEG）修饰的位点特异性和修饰产物的活性保留率，但存在修饰率偏低的问题。本课题提出用超大孔离子交换介质辅助PEG修饰水蛭素，从过程工程的角度对其动力学过程进行优化控制，对影响PEG修饰位点和修饰度的关键因素进行调控，提高固相修饰效率。首先以单修饰产物得率和活性保留率为指标，考察超大孔离子交换介质特性和反应条件对固相修饰的影响，确定关键因素并揭示其影响规律；其次采用实验与计算结合的方法，建立超大孔离子交换介质辅助固相修饰的反应动力学模型并确定其动力学参数；应用该模型优化控制影响固相修饰的关键因素并进行实验验证；最后采用分子模拟从分子水平研究蛋白质在固相介质上的吸附及PEG修饰过程，揭示影响固相修饰的分子机理。本研究的成果将从反应动力学和分子动力学两个层次揭示超大孔离子交换介质辅助PEG修饰水蛭素的过程，为深入认识PEG修饰蛋白药物提供新的视角。

聚乙二醇 (PEG) 修饰是提高药用蛋白体内稳定性和生物利用度的有效方法。本项目提出采用超大孔离子交换介质提高PEG修饰药用蛋白的效率。探究了超大孔离子交换介质类型和反应条件对超大孔离子交换柱辅助PEG修饰重组水蛭素的影响规律，实现了反应与分离的过程集成。建立了PEG定点修饰重组水蛭素N末端氨基和洛塞那肽半胱氨酸巯基的反应动力学模型，该模型能有效描述真实PEG修饰过程中同时发生的多个反应，模型预测值与实验值吻合度高。在此基础上建立了一种基于反应动力学的PEG修饰药用蛋白过程优化控制方法，可实现过程高效控制，最大化提高药用蛋白和PEG修饰剂的原料利用率，减少副反应发生和杂质生成。采用计算模拟方法，建立洛塞那肽和PEG-洛塞那肽的同源模建结构并考察了结构稳定性，比较了洛塞那肽和PEG-洛塞那肽与体内受体（胰高血糖素样肽-1受体）和降解酶（二肽基肽酶IV）结合的作用方式，分析了 PEG 修饰在提高洛塞那肽体内稳定性和延长半衰期方面发挥作用的分子机制。研究结果有利于深入理解PEG修饰的机制，为高效制备PEG修饰药用蛋白奠定了基础。共发表SCI论文5篇，申请专利1项，培养博士后1名、博士生2名和硕士生2名。