Preparation And Evaluation Of Heparin Affinity Chromatography Medium Based On Macroporous Polymer

The binding capacity of proteins is considered an essential parameter for evaluating the performance of the chromatographic medium.The separation efficiency can be improved by increasing the binding capacity of proteins.Therefore,increasing the binding capacity of proteins has always been the focus of attention.The binding capacity of proteins can be improved by increasing the accessible binding sites for proteins.This method will be explored to improve the binding capacity in heparin affinity chromatography in this study.The heparin affinity chromatography medium was prepared through the Schiff-base method using heparin as the ligand,which was based on the macroporous polyacrylate microspheres.Heparin was covalently bound to aldehyde microspheres prepared by oxidized hydroxyl in microspheres.The effects of reaction conditions on aldehyde content were investigated and the reaction were optimized.The amount of aldehyde group of microspheres was 0.2092 mmol/m L.The effects of operating conditions on the coupling heparin reaction were investigated,and the coupling reaction was optimized.This heparin affinity chromatography medium had a higher static binding capacity of lysozyme(Lys)(40.3mg/m L),about 36% higher than that of the commercial GP-heparin.The protein recovery reached 95.6%.The morphology of the microspheres was observed by scanning electron microscopy.The result showed that the throughput pores were maintained in the affinity medium.The dynamic binding capacity of Lys on the affinity support was determined under different retention times(0.5 min-5 min).The result indicated the dynamic binding capacity at 0.5 min decreased by about 12% compared to that at 5min.After 10 cycles,the dynamic binding capacity remained at 81% of the initial value.The synthesized affinity medium was used to isolate lactoferrin from mixtures.The results showed that it had a high separation efficiency.In order to improve the binding capacity of proteins on gigaporous microspheres,the grafted gigaporous heparin affinity chromatography medium was prepared by grafting glycidyl methacrylate(GMA)to the microspheres based matrix,followed by aldehyde-activation and heparin coupling.The microspheres were modified by coupling trimethylolpropane tris(3-mercapto propionate)on the surfaces of gigaporous polyacrylate microspheres.A surface-initiating system was constituted by the mercapto group on the surfaces of modified microspheres and dibenzoyl peroxide(BPO)in the solution.Through the proton transfer of mercapto groups to the decomposition product of BPO,a great deal of primary sulfur free radicals was produced on the surfaces of the microspheres,leading to the graft-polymerization of GMA on modified microspheres,obtaining the grafted PGMA microspheres.In order to prepare the grafted microspheres with a high grafting degree,60 ℃ should be selected as a suitable reaction temperature,the used amount of BPO in the solution should be selected as1.25 wt%,and the appropriate monomer concentration is 8 wt% of the solution mass,8 h should be selected as a suitable reaction time.The maximum epoxy density of the grafted microspheres was 0.3623 mmol/m L,about ten times higher than non-grafted microspheres(0.0396 mmol/m L).The grafted PGMA microspheres was characterization by Fourier transform infrared spectra and the morphology of microspheres was observed by scanning electron microscopy.The result indicated that the grafting polymerization was successful and the gigaporous structure was maintained in the resulted microspheres.The functionalization transformations of the grafted microspheres were conducted via a ringopening reaction of the epoxy groups with heparin as reagents,obtaining a functional composite adsorbent.An enhancement of both the binding performance and the stability was achieved for this PGMA-grafted heparin chromatographic medium.Its static binding capacity was 10.7 mg/m L,which increased by 234% compared with the non-grafted medium(3.2mg/m L),and the protein recovery reached 97.5%.The dynamic binding capacity of Lys on the affinity support was determined under different retention times(0.5 min-5 min).The result indicated that the dynamic binding capacity at 0.5 min decreased by about 4% compared to that at 5min.The result showed that the grafted medium has a high biomacromolecule mass transfer rate.The dynamic binding capacity in grafted adsorbent remained at 95% of the initial value after 10 cycles.