Binary Adsorption Of Proteins Onto Ion-exchange Chromatographic Media

Ion-exchange chromatography is the most widely used liquid chromatographic technique. In order to provide a practical guidance to the development of chromatographic process for the purification of protein, binary adsorption of proteins on anion-exchanger, Q Sepharose FF, was investigated in the well-mixed container and chromatographic column using bovine serum albumin (BSA) and bovine hemoglobin (Hb) as model proteins. The following research works were carried out:1. Proteins adsorption equilibria were conducted at different buffer pHs, ionic strengths, buffer constituents and amount ratios of proteins, respectively. It was confirmed that the competitive adsorption occurred in the binary adsorption of BSA and Hb on Q Sepharose FF. In this pair of proteins, adsorption of BSA was dominant and was mainly influenced by buffer while adsorption of Hb was mainly determined by BSA. Experimental evidence of sequential adsorption of binary proteins indicated that competitive adsorption behavior of BSA and Hb was influenced by order of adsorption.2. Parameters of steric mass-action (SMA) model obtained from the adsorption equilibria of single-component protein were used to fit the isotherms of binary-component adsorption to ion-exchange media at different conditions. The result indicated that SMA model could be used to describe binary adsorption equilibria, and a better fitting curve could be obtained for stronger adsorbed BSA. For a single protein component, better fitting curves could be obtained in conditions in favor of its competitive adsorption.3. Dynamic results of both single and binary components showed BSA had a higher diffusivity inside gel pores than Hb. Furthermore, BSA exhibited similar intraparticle diffusivity in the adsorption of both single and binary proteins, while the intraparticle diffusive rate of Hb was further restrained by BSA. In the binary adsorption of proteins, BSA bound favorably to adsorption sites on anion exchanger, and Hb only interacted with the adsorption sites left by BSA.4. Breakthrough experiments on chromatography realized high performance separation of binary component proteins (BSA and Hb) at different protein concentration ratios. Hb solution was collected with high purity and high recovery. The competition of binary adsorption and its influence facts were better understood by investigating of binary adsorption of model proteins adsorption to ion-exchange media at different conditions with comprehensive researches. Researches on adsorption equilibrium and adsorption dynamics of binary adsorption can guide the separation of ion-exchange chromatography well.