一种抗体融合蛋白Ni柱亲和层析纯化效果的研究

Analysis of Ni Affinity Chromatography Purification Effect of Antibody Fusion Protein

随着蛋白表达技术的不断发展与优化,发酵液上清中蛋白表达量不断提升,对下游纯化工艺提出了更高的要求,亟需开发高质高效的纯化工艺。Ni亲和层析是一种传统捕获目的蛋白的常用纯化方法。目前市面已有多种Ni亲和层析介质,其配基为螯合剂共价偶联到4%交联的琼脂糖介质上,再螯合Ni2+制备而成,但不同类别的Ni亲和层析介质配基有各自的基质和配位螯合技术,不同蛋白也有自身特性,从而造成介质对目标蛋白亲和力以及洗脱纯度有所差异。针对某抗体药物的纯化工艺探索,比较了3种不同的Ni亲和层析介质动态载量,从上样体积和平衡液咪唑浓度两方面进行该抗体融合蛋白Ni柱纯化条件筛选,并分析纯化后样品SEC纯度、总量、得率。综合考量除杂效果、总量、得率三个因素可知,Ni-IDA 6FF介质最适合该抗体融合蛋白的纯化,且纯化效率最高。

With the continuous development and optimization of protein expression technology,the protein expression level in the supernatant of fermentation liquid is constantly increasing,which puts forward higher requirements on the downstream purification process,and it is urgent to develop high quality and efficient purification process.Ni affinity chromatography is a traditional purification method for capturing target proteins.Currently,there are a variety of Ni affinity chromatography media on the market,whose ligands are prepared by chelating agent covalently coupled to 4%cross-linked agarose media and then chelating Ni2+.However,different classes of Ni affinity chromatography ligands have their own matrix and coordination chelation technology,and different proteins have their own properties,resulting in differences in affinity and eluting purity of the media for target proteins.In order to explore the purification process of a antibody drug,three different dynamic loads of Ni affinity chromatography media were compared,and the Ni affinity chromatography purification conditions of the antibody fusion protein were screened from two aspects of sample volume and imidazole concentration of washing buffer,and the purity of SEC,total amount and yield were analyzed.Considering the three factor of purity,total amount and yield,the Ni-IDA 6FF medium was the most suitable for the purification of the antibody fusion protein,and the purification efficiency was the highest.