Preparation And Performance Study Of High-Capacity Metal Chelating Affinity Chromatography Media Based On Agarose Microspheres

Agarose is the most widely used polysaccharide chromatography medium.Due to its good hydrophilicity,biocompatibility,and the presence of hydroxyl groups on its molecular structure,agarose plays an important role in the separation and purification of biological macromolecules such as enzymes,proteins,polysaccharides,and nucleic acids.Metal-chelating affinity chromatography medium based on agarose microspheres is a commonly used affinity chromatography medium.Stable chelates formed by the interaction between transition metal ions and some amino acids on the protein surface can efficiently and rapidly separate proteins.Domestic agarose microsphere chromatography medium products are currently monopolized by several overseas suppliers in the domestic market due to their low mechanical strength and low protein loading.Therefore,exploring the preparation,crosslinking,and ligand connection methods of agarose microspheres is of great significance for the development of high load chromatography media.Currently,mechanical stirring is the main method for industrial production of agarose microspheres.However,this method has lots of problems.In this article,a lowcost technology for preparing agarose microspheres is explored.First,the performance of microspheres prepared from several different sources of agarose raw materials was compared,and GS001 agarose was ultimately selected as the raw material.Small and uniform agarose microspheres was obtained under the optimal conditions of using turbine stirring method,toluene:water=1:1(w/w),at a speed of 300～400 rpm,an emulsifier concentration of 1%,a temperature of 60～70℃,and an emulsifier HLB value of 6～7.Most of the agarose microspheres prepared had a particle size distribution of 45～160 μm,an average particle size of 80～100 μm,and a yield of over 95%.The maximum flow rate of the prepared agarose microspheres 4B was about 218 cm/h,and that of 6B microspheres was about 328 cm/h.Based on the self-made agarose microspheres,the effect of different reaction parameters on the flow properties of cross-linked agarose microspheres was explored.Under the optimal conditions of using water:acetone=1:1(v/v)as the solvent,adding an amount of epichlorohydrin equal to 1/5 of the agarose microspheres,a NaOH concentration of 2.0 mol/L,a cross-linking time of 24 h(4 h at room temperature and 20 h at constant temperature),and a cross-linking temperature of 45℃,the flow properties of the cross-linked agarose microspheres 4FF and 6FF were 1190 cm/h and 2228 cm/h.Using the cross-linked agarose microspheres 6FF as the matrix,the effects of different activators on the grafting amount of dextran were compared,with 15.9 mg/mL gel being the highest when using bromopropyl as the activator.The dextran-grafted metal chelating chromatography mediums were prepared to investigate the effects of different molecular weight dextran,chelating agents,and metal ions on the dynamic protein loading capacity of the chromatography mediums.The highest dynamic protein loading capacity of the chromatography medium was achieved using Dextran150000 as the grafted dextran,IDA as the chelating agent,and Ni2+ as the chelating metal,with a value of 44.8 mg/mL gel.Finally,the non-specific adsorption of the prepared chromatography medium was tested,and the unit protein non-specific adsorption amount Wn under the test conditions was 0.401 mg/mL.This article optimized the preparation process and related parameters of agarose microspheres,reducing the amount of oil phase and emulsifiers,which is suitable for scaling up the production of agarose microspheres.It also improved the addition method of crosslinker and alkali solution,and added acetone to the reaction system to improve the crosslinking reaction efficiency.The flow properties of the crosslinked agarose microspheres 4FF and 6FF were superior to similar commercial products.Based on this,the prepared glucose-grafted metal chelating chromatography media has a protein loading capacity equivalent to similar commercial products,which is of guiding significance for the industrial production of agarose chromatography media.