Preparation And Evaluation Of Macroporous Polyacrylate Strong Anion Exchange Chromatography Medium

The rapid growth of biopharmaceutical scale puts forward higher and higher requirements for chromatography technology.The improvement of separation and purification ability is inseparable from the development of high-performance chromatography medium.Macroporous polyacrylate chromatography medium,as a kind of rigid gel,has the characteristics of large pore diameter,strong pressure resistance and easy surface derivatization,which can meet the needs of high-throughput separation and purification of biological macromolecules.In this paper,three kinds of high-performance macroporous strong anion exchange chromatography medium were developed by grafting polymer ligands on macroporous polyacrylate microspheres,and their preparation conditions and chromatography performance were systematically studied.(1)Polyethylenimine(PEI)was grafted on the surface of polyacrylate microspheres by “ grafting to ” methods,and then coupled with 2,3-epoxypropyl trimethyl ammonium chloride(EPTAC)to prepare strong anion exchange chromatography medium.The effects of various reaction factors on ionic capacity were investigated,and the optimum preparation process was obtained.The PEI ligand density of the medium had a great influence on the protein binding capacity,while the quaternary ammonium group content had no significant effect on the protein binding capacity,and the highest protein binding capacity was 94.3 mg/m L.The PEI ligand density of the medium had little effect on the protein recovery(96% ± 4%),but the protein recovery increased first and then remained unchanged with the increase of quaternary ammonium group content,and the highest recovery reached 99.3%.(2)Polyallylamine(PAA)was grafted on the surface of polyacrylate microspheres by “ grafting to ” method,and then EPTAC was coupled to prepare strong anion exchange chromatography medium.The effects of various reaction factors on ionic capacity were investigated,and the optimal preparation process was obtained.The PAA ligand density of the medium had a great influence on the protein binding capacity,while the quaternary ammonium group content had no significant effect on the protein binding capacity,and the highest protein binding capacity was112.5 mg/m L.The PAA ligand density of the medium had little effect on the protein recovery(95% ± 5%),but the protein recovery increased first and then remained unchanged with the increase of quaternary ammonium group content,and the highest recovery reached 98.2%.The protein binding capacity and mass transfer performance of PAA grafted medium with different pore diameter were different.The specific surface area of microspheres(400 nm pore diameter)was higher,and the dynamic binding capacity was the highest at 147 cm/h velocity,which was 63.4mg/m L.The mass transfer performance of microspheres(1000 nm pore diameter)was better,and the dynamic binding capacity was the highest at737 cm/h velocity,which was 44.9 mg/m L.(3)The strong anion exchange chromatography medium was prepared by grafting methacryloxyethyl trimethyl ammonium chloride(DMC)monomer by “ grafting from ” method.The effects of various reaction factors on ionic capacity were investigated,and the optimal preparation process was obtained.The protein binding capacity of the medium increased with the increase of PDMC ligand density,and the maximum values were 295.4 mg/m L(static adsorption)and 264.9 mg/m L(dynamic adsorption).There was no significant difference in the dynamic binding capacity of the medium at different velocities,but the permeability of the grafted medium decreased,and the flying pressure phenomenon appeared at the velocity of 644 cm/h.The salt concentration required for the elution of the medium was low,and there was basically no adsorption of protein molecules at the salt concentration of 100 m M Na Cl.The PDMC ligand density of the medium had little effect on the protein recovery,and the highest recovery reached 96.8%.In summary,this paper established a preparation method of strong anion exchange chromatography medium,which can be regulated and designed for different ligand density and different quaternary ammonium group content requirements,and systematically studied its effect on protein binding capacity and protein recovery.The investigation of the pore diameter,pressure resistance and salt tolerance of the medium provided certain theoretical guidance for its practical application.At the same time,it is necessary to further study its actual separation and purification ability in the actual separation and purification systems such as viruses and virus-like particles.