Preparation And Protein Adsorption Performance Evaluation Of Cation Exchange Chromatography Medium

In recent years,with the increase of production scale of biopharmaceutical,purification of proteins is facing a large challenge.How to realize rapid and efficient purification of biological macromolecules is the key factor restricting the yield of biological products.Cation exchange chromatography,as one of the efficient purification technologies,is widely used in the downstream process of biological products.Traditional ion exchange medium based on agarose has good bio-compatibility.The application for fast separation of protein is limited due to its soft texture,low tolerance pressure and small average pore size.Macroporous polymethacrylate microspheres have the characteristics of high mechanical strength and large pore size,which are conducive to the rapid separation of proteins.In this study,three preparation methods for cation exchange medium were established using these microspheres as the matrix.The influences of reaction conditions on medium performance was studied.The results are shown in the following:1)A strong cation exchange chromatographic medium with sulfonic acid group was prepared by coupling Na₂S₂O₅ on the surface of the microsphere through multi-step reaction.The morphology of the microspheres was characterized by the instrument.The date showed that microspheres had good spherical structure and uniform particle size.The average particle size was about 30μm and pore size ranged from 100 nm to 1000 nm.The specific surface area ranged from 40 m²/g to 100 m²/g The ligand density of medium was controlled from 0.126 mmol/m L to0.370 mmol/m L.The static binding capacity we measured ranged from37.1 mg/m L to 81.0 mg/m L,and the dynamic binding capacity was up to37.7 mg/m L.It was found that the static and dynamic binding capacity increased with the increase of the density of the coupled ligands.2)Microspheres with a bromine content of 2.31～4.62 mmol/g were prepared,which afforded complete structure and basically uniform particle size.The structure of microspheres was optimized.It was found that the increase of initiators led to the decrease of the pore size of microspheres.With the decrease of the proportion of good solvent in the binary pore-forming agent,the pores of the microsphere increased gradually.The adjustable range of the pore size was from 100 nm to 2000nm.Based on bromine groups contained in the microspheres,strong cation exchange chromatography medium was prepared by grafting methyl allyl sulfonate onto microspheres through surface-initiated atomic transfer radical polymerization.The ion exchange capacity increased with the increase of bromine content in the microspheres.We measured the static binding capacity with lysozyme as a probe,which increased with the ion-exchange capacity.The result showed that ion-exchange capacity ranged from 0.123 mmol/m L to 0.233 mmol/m L,and the corresponding static binding capacity ranged from 38.1 mg/m L to 100.6 mg/m L.3)A weak cation exchange chromatography medium was prepared through grafting polymerization of glycidyl methacrylate modified by iminodiacetic acid initiated by cerium（IV）.We investigated some factors that might affect ion exchange capacity.The relationship between ion exchange capacity and static adsorption performance was summarized.The static binding capacity of lysozyme was measured,and it was found that the static binding capacity increased with the ion-exchange capacity.The ion-exchange capacity was in the range from0.055 mmol/m L to 0.281 mmol/m L and the corresponding static binding capacity was in the range from 20.5 mg/m L to 130.1 mg/m L.