Strong anion exchange chromatography filler as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological separation and purification, and particularly relates to a strong anion exchange chromatography filler as well as a preparation method and application thereof. The surface of the porous polyglycidyl methacrylate-divinylbenzene microsphere is modified with a layer of branched polyethyleneimine, so that the hydrophilic property of the material is greatly improved, and the porous polyglycidyl methacrylate-divinylbenzene microsphere has good biocompatibility; the high-density branched polyethyleneimine is added into the filler, non-specific adsorption caused by hydrophobic interaction in the protein separation and purification process is avoided, and meanwhile, the high-density branched polyethyleneimine is convenient for modifying a high-density quaternary ammonium group, so that the surface charge density of the filler is greatly improved, and the protein adsorption capacity of the filler is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological separation and purification, and specifically relates to a strong anion exchange chromatographic filler and a preparation method and application thereof. Background Art

[0002] Ion exchange chromatography technology is based on the purpose of separation and purification based on the difference in binding ability of ion-based compounds during the interaction with ion exchange chromatography fillers. It is commonly used for the separation and purification of biological molecules such as proteins and nucleic acids, as well as various ions. Ion exchange chromatography fillers are high-performance chromatographic media derived from ion exchange chromatography technology. Commonly used ion exchange chromatography media are divided into strong anionic ion exchange chromatography media (SAX, with quaternary ammonium Q as the surface ligand), weak anionic ion exchange chromatography media (WAX, with diethylamine ethyl DEAE as the surface ligand), strong cationic ion exchange chromatography media (SCX, with sulfopropyl SP as the surface ligand), and weak cationic ion exchange chromatography media (WCX, with carboxyl CM as the surface ligand).

[0003] The commonly used matrices for anion exchange chromatography fillers are carbohydrates and organic polymers. Carbohydrates such as dextran, agarose and sodium alginate are also the most widely used in industry. The carbohydrate matrix has good hydrophilicity. In the process of protein separation and purification, the nonspecific adsorption caused by hydrophobic effect is very low. At the same time, the protein loading capacity of the carbohydrate matrix is ​​high, and the efficiency is relatively high in the separation process; however, this type of material has poor pressure resistance and uneven particle size distribution. As a chromatographic filler, poor pressure resistance will greatly reduce the service life of the filler. Organic polymers are mainly styrene-divinylbenzene (St-DVB), methyl methacrylate-divinylbenzene (PMMA-DVB), glycidyl methacrylate-divinylbenzene (PGMA-DVB) and polyvinyl alcohol. Usually, porous polymers with uniform particle size are selected. Organic polymers are used as matrices. The filler has excellent pressure resistance and can achieve more than a hundred cycles. The porous microspheres have a high loading capacity, which has become the focus of attention in the field of chromatographic fillers. However, the synthesis of traditional polymers mostly adopts suspension polymerization, and the particle size of the microspheres is uneven, which makes the separation efficiency of the filler unsatisfactory.

[0004] Therefore, developing a strong anion exchange chromatography filler with uniform particle size, high protein loading capacity, low nonspecific adsorption and good mechanical properties has become an urgent problem to be solved by technicians in this field. Summary of the invention

[0005] In view of this, the first object of the present invention is to provide a strong anion exchange chromatography filler with uniform particle size, high protein loading capacity, low nonspecific adsorption and good mechanical properties in order to solve the problems existing in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strong anion exchange chromatography filler is based on monodisperse porous poly(glycidyl methacrylate)-divinylbenzene cross-linked microspheres and functionally modified with quaternary ammonium salt-modified hyperbranched polyethyleneimine.

[0008] It is worth noting that porous organic polymers cannot be used as protein chromatography media due to the strong hydrophobic effect on their surfaces. Therefore, they need to be surface modified to make their surfaces hydrophilic and reduce nonspecific adsorption. Hydrophilic porous polymer microspheres can be used as ionic chromatography media for protein separation by surface functional modification. The functional groups can be combined with hydrophilic carriers through covalent bonds to directly modify the ions on the carrier surface, or they can be polymerized on the matrix surface through chemical reactions through chemical bonds, thereby being grafted onto the carrier surface.

[0009] The invention uses monodisperse porous poly(glycidyl methacrylate)-divinylbenzene crosslinked microspheres as a matrix, the surface of which contains abundant epoxy groups; hyperbranched polyethyleneimine is used to graft the porous microspheres so that the surfaces thereof are rich in amino groups; the grafted microspheres are functionally modified with quaternary ammonium salts containing quaternary ammonium groups, thereby obtaining a strong anion chromatographic filler with a strong anion retention effect.

[0010] Furthermore, the quaternary ammonium salt is betaine.

[0011] A second object of the present invention is to provide a method for preparing a strong anion exchange chromatographic filler, comprising the following steps:

[0012] The porous poly(glycidyl methacrylate)-divinylbenzene (PGMA)-DVB synthesized by seed swelling technology is used as a carrier. After being hydrophilically modified by hyperbranched polyethyleneimine, the quaternary ammonium salt is introduced into the hydrophilic porous microspheres by a "grafting" method to obtain the strong anion exchange chromatography filler, which is then loaded into the column by a homogenization method.

[0013] It is worth noting that, in the present invention, polystyrene seed microspheres are subjected to two-step swelling and then polymerized to form a PGMA-DVB porous microsphere matrix with a surface rich in epoxy groups, and then the epoxy groups on the matrix react with the primary amines in the hyperbranched polyethyleneimine under alkaline conditions to perform a ring-opening reaction, thereby modifying the branched polyethyleneimine on the porous microsphere skeleton to obtain a hydrophilic resin skeleton; the porous PGMA-DVB microspheres modified with polyethyleneimine are subjected to an amidation reaction with the amino groups and the carboxyl groups in the quaternary ammonium salt under the action of an activator EDC, thereby functionalizing the hydrophilic porous PGMA-DVB microspheres to obtain a chromatographic filler with anion retention characteristics.

[0014] Furthermore, the preparation method specifically comprises the following steps:

[0015] Step 1, preparation of porous poly(glycidyl methacrylate)-divinylbenzene PGMA-DVB;

[0016] The non-porous non-cross-linked polystyrene obtained by dispersion polymerization is used as a seed, added to a solvent, and added to a four-necked flask after ultrasonic dispersion, and stirring is started; dibutyl phthalate and toluene, a swelling agent, are added to the solvent for emulsification to form a uniform emulsion, and then dripped into the above flask, and stirring is continued to activate the seeds;

[0017] After activation, monomer glycidyl methacrylate, crosslinking agent divinylbenzene, porogen toluene and initiator dibenzoyl peroxide were added to a solvent containing an emulsifier sodium dodecyl sulfate, and ultrasonic emulsification was performed for 30 minutes to form a uniform emulsion, and the above reaction system was added to continue swelling;

[0018] After the swelling is completed, stabilizer polyvinyl alcohol and inhibitor sodium nitrite are added, stirred for 20 minutes under N2 atmosphere, and the temperature is raised to initiate polymerization. The reaction is continued for 10 to 24 hours to obtain monodisperse porous poly(glycidyl methacrylate)-divinylbenzene PGMA-DVB microspheres;

[0019] Step II, hydrophilic modification of hyperbranched polyethyleneimine;

[0020] Ultrasonic dispersion of the porous poly(glycidyl methacrylate)-divinylbenzene PGMA-DVB microspheres obtained in step I into a dimethyl sulfoxide solution of polyethyleneimine, stirring at a constant temperature for 24 hours, and then washing with pure water and anhydrous ethanol in turn to obtain hydrophilic porous microspheres;

[0021] Step III, functional modification of quaternary ammonium salt;

[0022] The hydrophilic porous microspheres obtained in step II are dispersed in a 2-(N-morpholine)ethanesulfonic acid solution, and a quaternary ammonium salt and a carboxyl activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added, and the reaction is carried out at a constant temperature for 24 hours. After the reaction is completed, the microspheres are washed alternately with pure water and anhydrous ethanol to obtain the strong anion exchange chromatography filler.

[0023] Furthermore, the non-porous non-cross-linked polystyrene seed particles used in step I have a particle size of 3-10 μm and a dispersion coefficient D90 / D10≤1.3; the obtained porous poly(glycidyl methacrylate)-divinylbenzene has a particle size of 10-50 μm and a dispersion coefficient≤1.3.

[0024] Preferably, the non-porous non-cross-linked polystyrene seed particles have a particle size of 5-9 μm, a uniform particle size distribution, and a dispersion coefficient D90 / D10 of 1.2.

[0025] Furthermore, in step I, the seed activation reaction temperature is 25-40°C, and the activation time is 5-12h; the swelling reaction temperature is 25-40°C, and the swelling time is 12-60h; the heating polymerization temperature is 65-80°C, and the heating polymerization time is 12-48h.

[0026] Preferably, the seed activation reaction temperature is 30°C, and the activation time is 6-10h; the swelling reaction temperature is 30°C, and the swelling time is 24-48h; the heating polymerization temperature is 70°C, and the heating polymerization time is 24h.

[0027] Furthermore, in step II, the molecular weight of the hyperbranched polyethyleneimine is 25000-70000, the concentration of the polyethyleneimine in DMSO solution is 15-50%, and the reaction temperature is 30-70°C.

[0028] Preferably, the molecular weight of the hyperbranched polyethyleneimine is 25,000, the concentration of the polyethyleneimine in DMSO solution is 30%, the reaction temperature is 50° C.; and the mass ratio of the monodisperse porous microspheres to the hyperbranched polyethyleneimine is 1:1 to 1:4.5.

[0029] Furthermore, in step III, the quaternary ammonium salt is betaine, the concentration of the 2-(N-morpholine)ethanesulfonic acid solution is 0.05-0.5 mol / L, and the reaction temperature is 25-50°C.

[0030] Preferably, the concentration of the 2-(N-morpholine)ethanesulfonic acid solution is 0.1 mol / L, and the reaction temperature is 37°C.

[0031] It is worth noting that the ionic properties of the strong anion exchange chromatography filler can be represented by the protein loading after column loading, and the hydrophilicity can be represented by the protein recovery rate after column loading. In the present invention, the degree of quaternization of the anion surface can be controlled by the amount of the modified quaternary ammonium salt and the branching degree and dosage of the grafted branched polyethyleneimine. Within a certain range, the more the amount of quaternary ammonium salt, the higher the degree of quaternization; the higher the degree of branching of polyethyleneimine, the higher the final degree of anion quaternization.

[0032] The third object of the present invention is to provide an application of a strong anion exchange chromatography filler, specifically the application of the strong anion exchange chromatography filler in protein separation.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] Compared with the prior art, the PGMA-DVB-based strong anion chromatographic filler disclosed in the present invention introduces a hydrophilic layer on the surface of the microspheres by modifying a layer of branched polyethyleneimine (PEI) on the surface and in the pores of the PGMA-DVB porous microspheres, thereby reducing the nonspecific adsorption of the material and increasing the binding sites of the protein; and the hydrophilicity and surface characteristics of the PGMA-DVB can be adjusted by changing the molecular weight and dosage of the branched polyethyleneimine of the medium. In addition, the hydrophilic PGMA-DVB microspheres are subjected to a quaternization reaction on their surfaces by an amidation reaction, using betaine as a carboxyl quaternary ammonium salt and 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) as a carboxyl activator, and the efficiency of the amidation reaction is regulated by the amount of the activator EDC, thereby adjusting the surface charge density of the hydrophilic PGMA-DVB. The technical route of quaternization by amidation reaction in the present invention has good stability and firm ligand binding, and the anion filler prepared thereby has a high protein loading capacity. At the same time, the technical route is simple to operate and is easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0036] Figure 1 The invention discloses a synthetic route for the branched polyethyleneimine hydrophilic modified PGMA-DVB strong anion chromatographic filler.

[0037] Figure 2 This is an optical microscope image of the strong anion chromatography filler in Example 1.

[0038] Figure 3 This is an optical microscope image of the strong anion chromatography filler in Example 4. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments, and in order to better illustrate the content of the present application, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that without certain specific details, the present application can be implemented equally. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail, so as to highlight the purpose of the present application under the premise of not conflicting, the technical features disclosed in the embodiments of the present application can be arbitrarily combined, and the obtained technical scheme belongs to the content disclosed in the embodiments of the present application.

[0040] Example 1

[0041] 100 g of polystyrene seed microspheres with a particle size of 4.5 μm were added to 3 kg of 0.25% SDS solution, ultrasonically dispersed for 30 min, and transferred to a 20 L reactor, stirred at 30 ° C and 180 rpm; 225 g of dibutyl phthalate (DBP) as a swelling agent and 75 g of toluene were weighed, 1.5 kg of 0.25% SDS solution was added, ultrasonic emulsification was performed for 30 min, and after emulsification, the solution was added to the above reaction system and stirred for 6 h; 600 g of glycidyl methacrylate (GMA), 300 g of divinylbenzene (DVB), 400 g of toluene and 26.25 g of dibenzoyl peroxide were weighed, and 9.5 kg of 0.25% SDS solution was added, ultrasonic emulsification was performed, and the ultrasonic time was 1 h. After emulsification, the solution was added to the above reaction system and stirred for 24 h; 1750 g of 5% PVA solution, and add 150g NaNO2 as an inhibitor, and introduce nitrogen for 30 minutes, then heat up to initiate polymerization, and continue the reaction for 12 hours; after the reaction is completed, filter and wash to obtain monodisperse porous PGMA-DVB microspheres with rich epoxy groups on the surface. The particle size of the porous microspheres is 15μm, and the pore size is

[0042] Take 10g of the porous microspheres drained by the sand core funnel, ultrasonically disperse them into 150g of dimethyl sulfoxide (DMSO), add 25g of hyperbranched polyethyleneimine with a molecular weight of 25,000, and stir at room temperature for 4h; add 5g of 50% NaOH solution, heat to 50°C, and continue stirring for 20h. After the reaction is completed, centrifuge to remove the unreacted polyethyleneimine, filter and wash with water for many times through a G4 sand core funnel to obtain a hydrophilic PGMA-PEI chromatographic medium. The column is loaded by the homogenization method, and bovine serum albumin (BSA) is used as the test protein. The protein recovery rate is 95%.

[0043] The hydrophilic PGMA-PEI separation medium drained from the G4 sand core funnel was ultrasonically dispersed into 0.1 M MES buffer, 5 g of betaine and 4 g of EDC were added, stirred until completely dissolved, reacted at 37°C overnight, and washed with water several times to obtain anion exchange chromatography filler.

[0044] Chromatographic filler protein loading test:

[0045] 10mL of anionic filler was loaded into the chromatographic column by homogenization method and connected to the liquid chromatography protein purification system. Sample concentration was 50mg / mL BSA solution, pump head was loaded, flow rate was 1mL / min, Buffer A pH=7.3, 0.05M Tris-HCl buffer, Buffer B pH=7.3 0.05MTris-HCl+1M NaCl, pump head was loaded until the protein solution 100% flowed through, and then Buffer B was used for elution and collection to measure the loading capacity. The test results showed that the protein loading capacity of this embodiment was 42mg / mL.

[0046] Example 2

[0047] Take 10g of the porous microspheres in Example 1 above, which are drained by a sand core funnel, and ultrasonically disperse them into 150g of dimethyl sulfoxide (DMSO), add 45g of hyperbranched polyethyleneimine with a molecular weight of 25,000, and stir at room temperature for 4h; add 5g of 50% NaOH solution, heat to 50°C, and continue stirring for 20h. After the reaction is completed, centrifuge to remove the unreacted polyethyleneimine, filter and wash with water for several times through a G4 sand core funnel to obtain a hydrophilic PGMA-PEI chromatographic medium. The column is loaded by the homogenization method, and bovine serum albumin (BSA) is used as the test protein, and the protein recovery rate is 99%.

[0048] The hydrophilic PGMA-PEI separation medium drained from the sand core funnel was taken, ultrasonically dispersed into 0.1 M MES buffer, 5 g betaine and 4 g EDC were added, stirred until completely dissolved, reacted at 37° C. overnight, and washed with water several times to obtain anion exchange chromatography filler.

[0049] Chromatographic filler protein loading test:

[0050] 10mL of anionic filler was loaded into the chromatographic column by homogenization method and connected to the liquid chromatography protein purification system. Sample concentration was 50mg / mL BSA solution, pump head was loaded, flow rate was 1mL / min, Buffer A pH=7.3, 0.05M TrisHCl buffer, BufferB pH=7.3 0.05M Tris-HCl+1MNaCl, pump head was loaded until the protein solution 100% flowed through, and then BufferB was used for elution and collection to measure the loading capacity. The test results showed that the protein loading capacity of this embodiment was 47mg / mL.

[0051] Example 3

[0052] 100 g of polystyrene seed microspheres with a particle size of 9 μm were added to 3 kg of 0.25% SDS solution, ultrasonically dispersed for 30 min, and transferred to a 20 L reactor, stirred at 30 ° C and 180 rpm; 225 g of dibutyl phthalate (DBP) as a swelling agent and 75 g of toluene were weighed, 1.5 kg of 0.25% SDS solution was added, ultrasonic emulsification was performed for 30 min, and after emulsification, the solution was added to the above reaction system and stirred for 6 h; 1200 g of glycidyl methacrylate (GMA), 600 g of divinylbenzene (DVB), 900 g of toluene and 26.25 g of dibenzoyl peroxide were weighed, and 9.5 kg of 0.25% SDS solution was added, ultrasonic emulsification was performed, and the ultrasonic time was 1 h. After emulsification, the solution was added to the above reaction system and stirred for 24 h; 1750 g of 5% PVA solution was added, and 150 g of inhibitor was added NaNO2, nitrogen was introduced for 30 minutes, and then the temperature was raised to initiate polymerization, and the reaction was continued for 12 hours; after the reaction, the monodisperse porous PGMA-DVB microspheres with rich epoxy groups on the surface were obtained by filtration and washing. The particle size of the porous microspheres was 30μm and the pore size was

[0053] Take 10g of the porous microspheres drained by the sand core funnel, ultrasonically disperse them into 150g of dimethyl sulfoxide (DMSO), add 45g of hyperbranched polyethyleneimine with a molecular weight of 25,000, and stir at room temperature for 4h; add 5g of 50% NaOH solution, heat to 50°C, and continue stirring for 20h. After the reaction is completed, remove the unreacted polyethyleneimine by centrifugation, filter and wash with water for many times through a G4 sand core funnel to obtain a hydrophilic PGMA-PEI chromatographic medium. The column is loaded by the homogenization method, and bovine serum albumin (BSA) is used as the test protein. The protein recovery rate is 98%.

[0054] The hydrophilic PGMA-PEI separation medium drained from the sand core funnel was taken, ultrasonically dispersed into 0.1 M MES buffer, 5 g betaine and 4 g EDC were added, stirred until completely dissolved, reacted at 37° C. overnight, and washed with water several times to obtain anion exchange chromatography filler.

[0055] Chromatographic filler protein loading test:

[0056] 10mL of anionic filler was loaded into the chromatographic column by homogenization method and connected to the liquid chromatography protein purification system. Sample concentration was 50mg / mL BSA solution, pump head was loaded, flow rate was 1mL / min, Buffer A pH=7.3, 0.05M Tris-HCl buffer, BufferB pH=7.3 0.05M Tris-HCl+1MNaCl, pump head was loaded until the protein solution 100% flowed through, and then BufferB was used for elution and collection to measure the loading capacity. The test results showed that the protein loading capacity of this embodiment was 54mg / mL.

[0057] Example 4

[0058] Take 10g of the porous microspheres of Example 3 drained by the sand core funnel, ultrasonically disperse them into 150g of dimethyl sulfoxide (DMSO), add 45g of hyperbranched polyethyleneimine with a molecular weight of 25000, and stir at room temperature for 4h; add 5g of 50% NaOH solution, heat to 50°C, and continue stirring for 20h. After the reaction is completed, centrifuge to remove the unreacted polyethyleneimine, filter and wash with water for several times through a G4 sand core funnel to obtain a hydrophilic PGMA-PEI chromatographic medium. The column is loaded by the homogenization method, and bovine serum albumin (BSA) is used as the test protein. The protein recovery rate is 98%.

[0059] The hydrophilic PGMA-PEI separation medium drained from the sand core funnel was taken, ultrasonically dispersed into 0.1 M MES buffer, 10 g betaine and 4 g EDC were added, stirred until completely dissolved, reacted at 37° C. overnight, and washed with water several times to obtain anion exchange chromatography filler.

[0060] Chromatographic filler protein loading test:

[0061] 10mL of anionic filler was loaded into the chromatographic column by homogenization method and connected to the liquid chromatography protein purification system. Sample concentration was 50mg / mL BSA solution, pump head was loaded, flow rate was 1mL / min, Buffer A pH=7.3, 0.05M Tris-HCl buffer, Buffer B pH=7.3 0.05MTris-HCl+1M NaCl, pump head was loaded until the protein solution 100% flowed through, and then Buffer B was used for elution and collection to measure the loading capacity. The test results showed that the protein loading capacity of this embodiment was 68mg / mL.

[0062] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following comparative examples are used to further illustrate the technical features of the strong anion exchange chromatography filler and the preparation method thereof disclosed in the present invention, but they should not be understood as limiting the present invention. The parameters and process flow adjustments made by those skilled in the art according to the above invention content or the applications made according to the above properties are also considered to fall within the protection scope of the present invention.

[0063] Comparative Example 1

[0064] Take 10g of the porous microspheres of Example 3 drained by the sand core funnel, ultrasonically disperse them into 150g of dimethyl sulfoxide (DMSO), add 45g of linear polyethyleneimine with a molecular weight of 25000, and stir at room temperature for 4h; add 5g of 50% NaOH solution, heat to 50°C, and continue stirring for 20h. After the reaction is completed, centrifuge to remove the unreacted polyethyleneimine, filter and wash with water for several times through a G4 sand core funnel to obtain a hydrophilic PGMA-PEI chromatographic medium. The column is loaded by the homogenization method, and bovine serum albumin (BSA) is used as the test protein. The protein recovery rate is 85%.

[0065] The hydrophilic PGMA-PEI separation medium drained from the G4 sand core funnel was ultrasonically dispersed into 0.1M MES buffer, 5g betaine and 4g EDC were added, stirred until completely dissolved, reacted at 37°C overnight, and washed with water several times to obtain anion exchange chromatography filler. The chromatographic test results showed that the protein loading of this comparative example was 26mg / mL.

[0066] The comparative example results show that the hydrophilic effect of linear polyethyleneimine as the hydrophilic modification ligand of PGMA porous microspheres is much lower than that of hyperbranched polyethyleneimine. The subsequent grafting of quaternary ammonium salt can provide much fewer grafting active sites than branched polyethyleneimine liquid. Therefore, the use of hyperbranched polyethyleneimine to hydrophilically modify the porous PGMA microspheres in the example is an important technical contribution of the present invention.

[0067] In summary, the present invention provides a strong anion exchange chromatography filler and its preparation method and application. The present invention first uses a two-step seed swelling technology to synthesize porous poly(glycidyl methacrylate)-divinylbenzene (PGMA-DVB) microspheres of a certain size; then uses the abundant epoxy groups on its surface to carry out a ring-opening reaction with branched polyethyleneimine, and modifies the surface and pores of the porous microspheres with amino groups to make them highly hydrophilic; finally, the amino groups are grafted with quaternary ammonium salts to obtain high-density anion groups, thereby synthesizing hydrophilic PGMA-DVB-based strong anion exchange chromatography fillers. The present invention modifies a layer of branched polyethyleneimine on the surface of PGMA-DVB porous microspheres, greatly improving the hydrophilic properties of the material, so that the porous PGMA-DVB has good biocompatibility, avoiding nonspecific adsorption caused by hydrophobic effects during protein separation and purification, and at the same time, the high-density branched polyethyleneimine is convenient for modifying high-density quaternary ammonium groups, thereby greatly improving the surface charge density of the filler and improving the adsorption capacity of the filler on proteins.

[0068] Although the present invention has been described and illustrated with specific embodiments, it will be apparent to those skilled in the art that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, all such changes and modifications within the scope of the present invention are consciously included in the appended claims.

Claims

1. A strong anion exchange chromatography filler, characterized in that: The strong anion exchange chromatography filler uses monodisperse porous poly(glycidyl methacrylate)-divinylbenzene cross-linked microspheres as a matrix and is functionally modified with hyperbranched polyethyleneimine modified with quaternary ammonium salt.

2. The strong anion exchange chromatography filler according to claim 1, characterized in that The quaternary ammonium salt is betaine.

3. A method for preparing a strong anion exchange chromatography filler as claimed in claim 1, characterized in that: The following steps are involved: The porous poly(glycidyl methacrylate)-divinylbenzene synthesized by seed swelling technology is used as a carrier, and after being hydrophilically modified by hyperbranched polyethyleneimine, the quaternary ammonium salt is introduced into the hydrophilic porous microspheres by a grafting method to obtain the strong anion exchange chromatography filler, which is then loaded into the column by a homogenization method.

4. The preparation method according to claim 3, characterized in that: The specific steps of the preparation method are as follows: Step I, using non-porous non-cross-linked polystyrene obtained by dispersion polymerization as seeds, adding it to a solvent, adding it to a four-necked flask after ultrasonic dispersion, and starting stirring; adding swelling agent dibutyl phthalate and toluene to the solvent for emulsification to form a uniform emulsion, and dripping it into the above flask, continuously stirring, and activating the seeds; After activation, monomer glycidyl methacrylate, crosslinking agent divinylbenzene, porogen toluene and initiator dibenzoyl peroxide were added to a solvent containing an emulsifier sodium dodecyl sulfate, and ultrasonic emulsification was performed for 30 minutes to form a uniform emulsion, and the above reaction system was added to continue swelling; After the swelling is completed, stabilizer polyvinyl alcohol and inhibitor sodium nitrite are added, stirred for 20 minutes under N2 atmosphere, and the temperature is raised to initiate polymerization. The reaction is continued for 10 to 24 hours to obtain monodisperse porous poly(glycidyl methacrylate)-divinylbenzene microspheres; Step II, ultrasonically dispersing the porous poly(glycidyl methacrylate)-divinylbenzene microspheres obtained in step I into a dimethyl sulfoxide solution of polyethyleneimine, stirring at a constant temperature for 24 hours, and then washing with pure water and anhydrous ethanol in turn to obtain hydrophilic porous microspheres; Step III, dispersing the hydrophilic porous microspheres obtained in step II into a 2-(N-morpholine)ethanesulfonic acid solution, adding a quaternary ammonium salt and a carboxyl activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and reacting at a constant temperature for 24 hours; after the reaction is completed, washing with pure water and anhydrous ethanol in turn to obtain the strong anion exchange chromatographic filler.

5. The preparation method according to claim 4, characterized in that: The non-porous non-cross-linked polystyrene seed particles used in step I have a particle size of 3-10 μm and a dispersion coefficient D90 / D10≤1.3; the obtained porous poly(glycidyl methacrylate)-divinylbenzene particles have a particle size of 10-50 μm and a dispersion coefficient≤1.

3.

6. The preparation method according to claim 5, characterized in that: In the step I, The seed activation reaction temperature is 25-40°C and the activation time is 5-12h; The swelling reaction temperature is 25-40°C and the swelling time is 12-60h; The temperature of the polymerization is 65-80°C, and the time of the polymerization is 12-48h.

7. The preparation method according to claim 4, characterized in that: In the step II, the molecular weight of the hyperbranched polyethyleneimine is 25000-70000, the concentration of the polyethyleneimine in DMSO solution is 15-50%, and the reaction temperature is 30-70°C.

8. The preparation method according to claim 4, characterized in that: In the step III, the quaternary ammonium salt is betaine.

9. The preparation method according to claim 4 or 8, characterized in that: In the step III, the concentration of the 2-(N-morpholine)ethanesulfonic acid solution is 0.05-0.5 mol / L, and the reaction temperature is 25-50°C.

10. A use of the strong anion exchange chromatography filler as claimed in claim 1, characterized in that: The application of the strong anion exchange chromatography filler in protein separation.

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