以蛋白A结构中耐碱性更好的C区为基础，构建一种新型耐碱蛋白A，并偶联至琼脂糖基质，获得新型耐碱蛋白A亲和层析介质，分别用激光共聚焦显微镜和石英晶体微天平研究了人免疫球蛋白(hIgG)与蛋白A介质的结合过程。结果表明，该介质具有较商品配基更高的结合力和稳定的再生性能，hIgG动态载量达62.0 mg/mL，40次再生操作后载量为初始载量的84%。该介质在抗体纯化领域具有较好的应用前景。
Monoclonal antibodies (mAb) have been applied for curing a wide range of diseases and is considered to be a major source of new therapies in the next decades. mAb?s production has been received worldwide attention and efficient purification strategies have been always explored. Protein A chromatography was one of the most popular methods for mAb purification. Cleaning-in-place has been widely used in protein A chromatography for meeting the demand of high quality, and therefore an alkali-resistant ligand is necessary. Since protein A from natural sources is not alkali-resistant, it should be genetically modified. In this study, a new alkali-resistant rProtein A was constructed based on C-region gene construction followed by being coupled to epoxy-activated agarose-based microspheres under optimized conditions, and a new rProtein A chromatographic medium was prepared. Both confocal laser scanning microscopy and quartz crystal microbalance were used for analyzing the binding of hIgG to rProtein A chromatographic medium. The results showed that it was transparent and full bright. It had a uniform particle size distribution with an average diameter of 84 μm. This medium had good hydraulics properties with a maximum flow rate of 1400 cm/h. This new rProtein A chromatographic medium had a higher hIgG dynamic binding capacity of 62.0 mg/mL than that of commercial ligand. Also, this medium had a better cleaning-in-place performance and the dynamic binding capacity was kept to 84% of the initial value after 40 cycles, as beneficial for its application on an industrial scale. At the beginning of adsorption to the medium, hIgG was bound quickly to the surface coupled with rProtein A, however, the binding rate decreased gradually due to mass transfer resistance from the inner part of the medium. The desorption rate had a similar tendency to that of the adsorption. The new rProtein A chromatographic medium had great prospects in mAb purification and gave a good basis for its application in the future.
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