Study On Relationship Between Structure And Chromatographic Performance Of Polyacrylate Microspheres

As one of the important products in biomedical industry,the separation and purification efficiency of biological products has attracted widely attention.As the main technology of protein separation and purification,chromatography medium is the core technology of chromatography.The structure of chromatography medium has a crucial impact on the efficiency of chromatography.Therefore,studying the relationship between chromatographic medium structure and chromatographic performance has great significance for improving protein separation efficiency.In this study,polyacrylate microspheres was used as research object,the relationship between structure and chromatographic performance was studied.The details in this work are summarized as follows.(1)Microspheres based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate were prepared by suspension polymerization.The effects of ratio of porogen components and monomer concentration on the pore size of microspheres were systematically investigated.Polyacrylate microspheres with specific surface area ranging from 2.5 m~2/g to 207.4 m~2/g were prepared by adjusting the ratio of ternary porogen components and monomer concentration.The weak anion exchange chromatography medium was prepared by coupling polyethyleneimine(PEI)with the microspheres.The effect of PEI addition on PEI coupling capacity(ion exchange capacity)was investigated,and it was found that PEI coupling capacity increased with the increase of PEI addition.(2)The weak anion exchange chromatography medium as described as the above were evaluated.The effects of ion exchange capacity and PEI molecule on the protein adsorption capacity and structure of the medium were systematically investigated.The results were showed as the following:1)When the ion exchange capacity was less than 165.5 mmol/L,the static binding capacity and dynamic binding capacity of proteins increased rapidly with the increase of ion exchange capacity.When the ion exchange capacity was greater than 165.5 mmol/L,with the increase of ion exchange capacity,the static binding capacity slowly increases,while the dynamic binding capacity shows a downward trend.2)PEI has different influences on the specific surface area of microspheres with different pore size distribution.When the specific surface area of microspheres before coupling is less than 65 m~2/g,PEI has less influence on the specific surface area of medium.When the specific surface area of microspheres before coupling is more than 65 m~2/g,the proportion of microspheres pores is larger;PEI will cause 1～4 nm pore blockage and the specific surface area of medium decrease.3)It was also found that with the increase of specific surface area of microspheres,the static binding capacity of medium increased and then decreased,and reached maximum when the specific surface area was 170 m~2/g.4)After coupling PEI,the pore volume within the range of 1～4 nm in medium was significantly reduced.However,it had little effect on the pore size above 64 nm.(3)The structure of the polyacrylate microspheres coupled with PEI was determined by ISEC,and the experimental results were fitted with the log-normal distribution function,and a good fitting effect was obtained.It was found that the static binding capacity and dynamic binding capacity of proteins were directly affected by the phase ratio.The static binding capacity of bovine serum albumin and bovine thyroglobulin on different media were also studied.It was found that the small pore size in media is beneficial to the adsorption of bovine serum albumin(1.9688 mmol/m L),while the large pore size media is more conducive to the adsorption of bovine thyroglobulin(0.2110 mmol/m L).(4)The model protein and the VP2 protein of infectious bursal disease virus were separated by polyacrylate microspheres coupled with PEI.Effects of the three pore structures and the differences on the separation degrees of the media at different flow rates were investigated.The results showed that the media with an average pore size of 83.83 nm,14.22 nm,and 8.74 nm all had a good separation effect on the model protein.The separation degree of the medium with an average pore diameter of 83.83nm is better than that of the other two media,and the decrease in the separation degree was the smallest with the increase of flow rate.