The Preparation Of High Performance Affinity Chromatography Resin

Protein A affinity chromatography is a classical purification technology with many advantages including high capacity, simple operation and good reproducibility. In most cases high purity antibodies can be harvested after one step protein A purification. It is most frequently used in the first step of antibody purification around the world. But the price of this resin is more than $10,000 per liter, which is too expensive for most companies. In order to reduce the cost of antibody production we have done a lot of work on the development of protein A affinity chromatography resin with high quality.Firstly, we selected three matrixes as the backbone candidates and compared their background pressure under different flow velocity, including Sepharose 4FF 、 Profinity Epoxide、Polar MC60-Epoxy. The Sepharose 4FF was ruled out due to it’s high background pressure and low pressure resistance. Next, we further evaluated the rest of matrixes on mass transfer rate. The Profinity Epoxide was chosen as it had better performance than Polar MC60 in our assays.Secondly, we studied the following key parameters in Protein A reactions by a series of experiments: 1) The appropriate range of salt concentration based on the stability of the protein A by turbidimetric method. 2) The optimal pH and temperature by orthogonal experiment. 3) The optimal salt concentration and reaction time by all factors methods. 4)The optimal amount of protein A according the difference in binding capacity. Based on the above experiments the optimal experiment parameters were selected as pH 8.5, 1.3 mol/L Na2SO4, 37℃, 4 hours, 8 mg protein A/ml matrix. Furthermore, more experiments showed that the process was scalable and stable.Finally, the protein A affinity chromatography resin produced in our laboratory was evaluated. The capacity was 34.5mg/mL when retention time is 8 minutes, the background pressure was 2.7bar under 1000cm/h linear flow velocity, and the dropped protein A was10 ppm. To further evaluate the actual performance our new protein A resin was applied to purify three antibodies. It showed that both the yield and purity of antibodies were in accordance with the industry standards.In this paper, an effective method of preparation of protein A resin was established.The homemade resin showed that the equal or ever better performance in antibody purification compared to the top international brand. Therefore, it was very helpful toproduce the protein A resin with Chinese brand in the future and finally reduce the cost of antibody purification.