Fabrication Of Cation-exchangers With Polymer Grafting And Protein Adsorption Chromatography

Polymer-grafted ion exchange resins have great potential of development and application for protein chromatography because of their high adsorption capacities and mass transfer rates caused by the three-dimensional adsorption space.Therefore,the exploitation of novel polymeric ligands for protein purification and the more in-depth understanding on the protein adsorption behavior are crucial for the optimization of separation processes of biotechnology products.For these purposes,this paper takes the investigation of alginate(Alg)-grafted Sepharose FF(Alg-FF)as a breakthrough point to explore the novel polymeric ligand-based cation-exchangers for protein chromatography including sulfonate Alg-FF resins and sodium methacrylate(MA)-grafted gels,and systematically investigate protein adsorption and chromatographic behaviors.Firstly,three Alg-grafted cation exchangers of different ionic capacities(ICs,140-240 mmol/L)were synthesized and studied for lysozyme adsorption and chromatographic performance.It was found that the adsorption capacity and mass transfer of lysozyme increased with increasing Alg-grafting density.Dynamic binding capacity of lysozyme on the resin with the highest IC(240 mmol/L,named as Alg-FF-240)were remarkably higher than CM Sepharose FF at Na Cl concentrations lower than 100 mmol/L.Then,Alg-grafted Sepharose FF with an IC of 230 mmol/L(Alg-FF-230)was first prepared and then sulfonated to synthesize two cation exchangers of higher ICs(270 and 370 mmol/L,denoted as S-Alg-FF-IC).It was found that high-charge-density resin showed high salt-tolerance in protein adsorption.The DBC of S-Alg-FF-270 kept high at flow rate up to 1350 cm/h at 50 mmol/L Na Cl,while S-Alg-FF-370 presented high DBC in the same flow rate range at 150 mmol/L Na Cl.The results provide a guideline for selection of resins suitable for different separation conditions.Inspired by the above-mentioned enhanced adsorption performance of lysozyme,we have herein investigated the static and dynamic adsorption behaviors of γ-globulin on a series of Alg-grafted resins of different grafting densities and sulfonation degrees.The adsorption capacity of γ-globulin decreased with increasing Alg-grafting density at 0 mmol/L Na Cl.Protein adsorption capacities on the resins with the IC value of230-370 mmol/L were much higher than that on CM Sepharose FF at 50-100 mmol/L Na Cl.Finally,MA-grafted cation exchange resins of the same grafting density but different chain lengths were fabricated to investigate the adsorption behaviors of lysozyme and γ-globulin.It was found that the two proteins showed the maximal adsorption capacities on the resins of different chain lengths,but the mass transfer rates of them decreased with increasing chain length.Protein mass transfer rates increased with increasing salt concentration,leading to their high DBC values in the salt concentration range of 0-100 mmol/L.Altogether,the findings indicate that the two types of polymer-grafted cation exchangers developed in this work are promising for use as high-performance chromatography.