【作者】 王旭东；

【导师】 修志龙；

【作者基本信息】 大连理工大学 ， 生物化工， 2015， 博士

【摘要】 药用蛋白质具有活性高、特异性强、生物功能明确等优点,已成为生物医药产业发展的热点,但临床上仍存在半衰期短、免疫原性强、稳定性差等缺陷。因此研发长效剂型成为新的发展趋势,其中聚乙二醇(PEG)修饰是研发长效药用蛋白质最为成功的方法。聚乙二醇修饰具有延长药用蛋白质在体内的半衰期、降低免疫原性和延缓蛋白酶降解、提高稳定性和溶解性等优点。然而,PEG修饰药用蛋白质仍然面临降低修饰剂损失、控制修饰位点和修饰度等难题。为解决上述问题,以重组水蛭素作为模型药用蛋白质,建立了PEG修饰重组水蛭素的反应动力学模型,考察了体系的溶剂环境、离子交换柱原位修饰和不同PEG修饰剂的影响。主要研究结果如下：(1)建立了PEG修饰重组水蛭素的反应动力学模型,并将其应用于修饰反应的条件优化,获得了几个关键参数(ymax、mcrit和tmax)的通用表达式,确定了影响单修饰产物得率的关键因素。结果表明,所构建的反应动力学模型不仅能解释PEG修饰重组水蛭素的反应机理,而且能用于PEG修饰蛋白质过程的优化控制。(2)考察了水-有机溶剂混合溶液对PEG修饰重组水蛭素的影响。通过对重组水蛭素的结构、PEG修饰剂的水解动力学和修饰反应动力学的分析表明,水-有机溶剂混合溶液能通过溶剂化效应(PEG修饰加速和PEG水解抑制)提高PEG修饰的效率。根据占主导的溶剂化效应,可将水-有机溶剂混合溶液反应体系分为三种类型：PEG修饰加速驱动型、PEG水解抑制驱动型、PEG修饰加速与PEG水解抑制共同驱动型。用水-二甲基亚砜混合溶液获得理想得率(约50%)的单修饰产物时,PEG修饰剂用量比纯水溶液降低3-5倍。(3)建立了离子交换柱原位修饰重组水蛭素的工艺,使PEG修饰与产物分离纯化在同一个单元操作中完成,实现反应与分离的过程集成。考察了不同离子交换树脂介质对原位修饰的影响,结果表明树脂的孔径和内部表面结构(如结合高分子的葡聚糖)对PEG修饰的影响较大。考察了不同PEG分子量对原位修饰的影响,结果表明离子交换柱原位修饰(固相修饰)是扩散控制的过程,其单修饰产物的理论得率随PEG分子量增大而降低,这与液相修饰正好相反。单修饰产物的体外、体内活性分析表明,离子交换柱原位修饰能提高PEG修饰的选择性,其单修饰产物的体外和体内抗凝活性比液相修饰更高。(4)采用分枝型mPEG2-NHS制备高得率、高活性保留率的单修饰产物。PEG修饰剂的水解动力学和修饰反应动力学表明,分枝型mPEG2-NHS修饰重组水蛭素的反应速度比直线型mPEG-SC稍慢,而其自身失活速度却比mPEG-SC快的多,获得理想得率的单修饰产物的PEG修饰剂用量更大。单修饰产物的体外、体内活性分析表明,分枝型mPEG2-NHS比线型mPEG-SC更容易保护重组水蛭素的活性位点不被修饰,获得的单修饰产物体外抗凝活性更高,体内的药效更强。(5)应用响应面分析法优化了mPEG-ALD定点修饰重组水蛭素N末端的反应条件。结果表明,mPEG-ALD定点修饰比nPEG-SC随机修饰重组水蛭素得到的单修饰产物的修饰位点更均一,理论得率更高。定点修饰得到的单修饰产物仍能保留重组水蛭素20%左右的体外抗凝活性。综上所述,以PEG修饰药用蛋白质反应动力学模型为基础,通过系统研究微环境因子对PEG修饰药用蛋白质的影响,能减少PEG修饰剂的用量并有效控制药用蛋白的PEG修饰位点和修饰度,为高效制备PEG修饰药用蛋白质提供了新的思路。更多还原

【Abstract】 Therapeutic proteins have several advantages over small-molecule drugs, such as high activity, high specificity, and clear biological function. Development of therapeutic proteins has become a hotspot in the biopharmaceutical industry. However, therapeutic proteins show several limitations in clinical use. such as short plasma half-lives, immunogenicity, and poor stability. Therefore, development of long-effective therapeutic proteins has become a new trend. PEGylation is one of the most successful strategies to develop long-effective therapeutic proteins. The therapeutic efficacy of PEGylated therapeutic proteins can be improved through prolonging their circulating half-life, reducing their immunogenicity and proteolysis, and increasing their stability and solubility. However, PEGylation of therapeutic proteins is still suffering from the problems of the reduction of PEG reagent usage and the control of the PEGylation site and degree. To solve these problems, recombinant hirudin was chosen as a model therapeutic protein to be PEGylated. A reaction kinetics model was constructed for the PEGylation of recombinant hirudin. The effects of solvent environment, in situ PEGylation on ion exchange column, and different PEG agents on the PEGylation of recombinant hirudin were investigated. The main results are as follows:(1) A kinetics model was constructed to describe the PEGylation reaction of recombinant hirudin. Moreover, the established model was used to optimize the reaction conditions of PEGylation. Several important process parameters including ymax, mcrit and tmax, and their mathematical equations were obtained to determine the key factors to achieve the mono-PEGylated recombinant hirudin at the desired yield. The results indicated that the proposed reaction kinetics model can provide a possible interpretation of mechanism for real PEGylation reactions and optimize efficiently the PEGylation process.(2) The effects of mixed aqueous-organic solutions on the PEGylation of recombinant hirudin were investigated. Recombinant hirudin structure, hydrolysis kinetics of PEG reagent, and PEGylation kinetics of recombinant hirudin were analyzed. The results revealed that solvent effects including PEGylation acceleration and PEG hydrolysis inhibition can enhance the PEGylation efficiency in mixed aqueous-organic solutions. According to the dominant solvent effect, the selected mixed aqueous-organic solutions can be divided into three different types (PEGylation-driven, PEG hydrolysis-driven, both PEGylation and PEG hydrolysis-driven). In aqueous-DMSO solutions, the optimal usage of PEG reagent to achieve the desired yield (approximately 50%) of mono-PEGylated recombinant hirudin can decrease 3-5 folds, compared with that of PEGylation in pure aqueous solution.(3) An integrated process was developed for in situ PEGylation of recombinant hirudin on anion exchange column. PEGylation reaction and separation can be efficiently integrated into one unit in this process. Effect of different ion exchange resins on the in situ PEGylation was investigated. The results showed that the pores and internal surface structures such as the combination of polymer dextran of different resins have significant impact on the yield of mono-PEGylated recombinant hirudin. Moreover, effect of different PEG sizes on the in situ PEGylation was also investigated. The results showed that in situ PEGylation on anion exchange column (solid-phase PEGylation) is diffusion-driven process. The yield of mono-PEGylated recombinant hirudin decreases as PEG size increases, which is contrary to liquid-phase PEGylation. In vitro and in vivo activity analysis of mono-PEGylated recombinant hirudin showed that in situ PEGylation on anion exchange column could enhance the selectivity of PEGylation. In vitro and in vivo anticoagulant activities of mono-PEGylated recombinant hirudin derived from in situ PEGylation on anion exchange column are greater than those from liquid-phase PEGylation.(4) PEGylation of recombinant hirudin with branched mPEG2-NHS was performed to achieve the mono-PEGylated recombinant hirudin with high yield and active retention. Compared with linear mPEG-SC, branched mPEG2-NHS displayed higher PEGylation rates, but lower PEG reagent deactivation rate. Thus, higher PEG/recombinant hirudin molar ratio was required to achieve the mono-PEGylated recombinant hirudin at the desired yield. In vitro and in vivo activity analysis of mono-PEGylated recombinant hirudin showed that branched mPEG2-NHS could easier shield the active region of the protein from PEGylation to achieve higher in vitro and in vivo anticoagulant activities of the mono-PEGylated recombinant hirudin compared with those from linear mPEG-SC.(5) Site-specific PEGylation of the N-terminus of recombinant hirudin was performed by using mPEG-ALD. The PEGylation reactions were optimized by response surface analysis. The results showed that mono-PEGylated recombinant hirudin derived from site-specific PEGylation of recombinant hirudin with mPEG-ALD had more homogeneous PEGylation site and higher yield, compared with those of random PEGylation with mPEG-SC. N-terminal mono-PEGylated recombinant hirudin retained approximately 20% of the in vitro anticoagulant activity of unmodified recombinant hirudin.In summary, the reduction of PEG reagent usage and the control of PEGylation site and degree were achieved through the study of the effects of micro-environmental factors on the PEGylation of therapeutic proteins based on the reaction kinetics model. The results of this study demonstrated effective strategies for the development of PEGyalted therapie proteins.更多还原