Anti-IgE单链抗体纯化工艺研究及活性鉴定

The Research on Purification Technology and Activity Identification of Anti-IgE scFv Fragment

目的:建立anti-IgE单链抗体纯化工艺并对其活性进行鉴定。方法:根据protein L亲和层析填料和Ni亲和层析填料的特点分析,经初筛后选用protein L填料作为第一步初纯化填料,Ni亲和层析填料作为第二步精细纯化填料。通过对这两种纯化方式的上样条件和洗脱条件进行研究,建立了anti-IgE单链抗体的纯化工艺。结果:protein L亲和层析的初纯化工艺:最佳杂质洗涤pH=3.5,最佳目标蛋白洗脱pH=2.0,并且pH=2.0洗脱的目标蛋白收集在第二步Ni亲和层析的上样缓冲液中,可直接进行第二步Ni亲和层析纯化。所建立的Ni亲和层析精细纯化工艺:最佳杂质洗涤为50mmol/L咪唑,最佳目标蛋白洗脱为500mmol/L咪唑。经两步亲和纯化,目标产物在SDS-PAGE上纯度为99.0%,CE-SDS上纯度为99.5%,两步总收率为80.0%。纯化后的蛋白经竞争ELISA和Biacore检测,证实该产品特异性识别IgE靶标,与IgE靶标的亲和力达到8.59e^(-9)M,并且竞争Biacore结果显示该抗体对IgE有良好的中和活性,其抑制IgE的EC50值为70n M。结论:建立了一种高效、简洁的大肠杆菌表达的anti-IgE单链抗体纯化工艺,为其进一步规模放大工艺的建立奠定了基础。同时证明了该新型小分子anti-IgE抗体对靶标具有良好的特异性、亲和力以及中和活性,并展示出其在医用中的应用价值。所建立的小分子抗体纯化工艺技术对其他小分子单链抗体的纯化具有参考价值。

Objective： To establish the purification process of anti-IgE single chain antibody and identify its biological activity. Methods： Protein L was screened as the first step, and the nickel affinity chromatography was used as the second step of the purification according to the characteristics of protein L affinity chromatography and Ni affinity chromatography. The purification process of anti-IgE single chain antibody was established basing on the study of the loading and the eluting conditions of the two kinds of purification methods. Results： The purification process of protein L ： pH = 3.5 was the best pH value of washing impurity and pH = 2.0 was the best pH value of eluting the target protein. The target protein was collected in the loading buffer of Ni affinity chromatography. The polishing process of Ni affinity chromatography was： Impurity was washed by 50mmol/L imidazole solution and the target protein was eluted by 500mmol/L imidazole solution. The purity of the target protein was 99.0% by SDS-PAGE electrophoresis analysis and 99.5% by CE-SDS electrophoresis analysis and the total yield came up to 80. 0% after the two purification steps. The purified protein was detected by competitive ELISA and Biacore analysis, which confirmed that the product could recognize IgE specifically and its affinity was 8.59e9M ,The product showed good neutralizing IgE activity by competitive Biacore analysis ,its ECs0 which inhibited IgE was 70nM. Conclusions ： An efficient and simple purification process of anti-IgE single chain antibody which was expressed in E. coli has been established and would be the basis of further scale-up process study. At the same time,it also proved that the novel small molecule anti-IgE antibody had good specificity and affinity to the target and displayed medical value. The purification technology of small molecule antibody could be insight to the purification of other small molecules.