Porous high-hydrophilic ion exchange purification filler as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: by taking a seed ball matrix as a base ball, preparing monodisperse porous polymer microspheres through one-step swelling polymerization, then converting residual double bonds on the surfaces of the polymer microspheres into epoxy groups by adopting an oxidation reaction, then grafting a dextran hydrophilic layer to completely cover hydrophobic sites on the surfaces, and then carrying out chemical modification and ligand bonding by utilizing hydroxyl on dextran, so as to obtain the polymer microspheres. The prepared ion exchange polymer filler is porous, high in hydrophilicity and high in exchange capacity and is successfully applied to protein separation and purification. Therefore, the ion exchange purification filler prepared by the method has a good application prospect.

Description

Technical Field

[0001] The invention relates to a purification filler, in particular to a porous high-hydrophilic ion exchange purification filler, a preparation method and application thereof. Background Art

[0002] Ion exchange fillers play an important role in the separation and purification of biomacromolecules due to their good biocompatibility and mild elution pattern. Traditional polysaccharide matrix fillers have the disadvantages of poor mechanical strength and are limited to medium and low pressure separations, while polymer matrices have high mechanical strength and good chemical stability, which can achieve rapid medium and high pressure separations and have broad application prospects in sample separation and purification. Currently, common polymer fillers are mostly based on surface-modified cross-linked polystyrene (PS) microspheres, which have strong surface hydrophobicity and low biocompatibility. They are very likely to cause nonspecific adsorption in the separation of biomacromolecules and affect the recovery rate and biological activity of the product. Although surface chemical modification and modification technology can improve its hydrophobicity, the elimination of nonspecific adsorption is still a problem, resulting in poor subsequent protein separation. Therefore, the preparation of highly hydrophilic, monodisperse polymer fillers with high exchange capacity is an urgent problem to be solved.

[0003] Porous polymer fillers have the advantages of maximum mass transfer and minimum lateral diffusion, which can enable rapid and efficient separation of samples. However, the low specific surface area of ​​porous resins results in low separation capacity, which limits their application in practice. Therefore, the preparation of porous, highly hydrophilic polymer fillers with high ion exchange capacity is still a current hot topic and difficulty. Summary of the invention

[0004] In order to solve the problems existing in the above prior art, the purpose of the present invention is to provide a porous highly hydrophilic ion exchange purification filler, a preparation method and its application. The present invention uses a seed ball matrix as a base ball, prepares monodisperse porous polymer microspheres by one-step swelling polymerization, then uses an oxidation reaction to convert the residual double bonds on the surface of the polymer microspheres into epoxy groups, then grafts a layer of dextran hydrophilic layer, and finally chemically modifies the bonding ligand to prepare a porous highly hydrophilic ion exchange polymer filler, which is successfully applied to protein separation and purification.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a porous highly hydrophilic ion exchange purification filler, the filler comprising a seed ball matrix, the surface of the seed ball matrix being grafted with a polymer, the polymer being polymerized by a monomer of glycidyl methacrylate (GMA) under the action of a crosslinking agent divinylbenzene (DVB); the polymer being modified with dextran, the dextran being coupled with a ligand; the mass of the glycidyl methacrylate (GMA) accounting for 50-80% of the total mass of glycidyl methacrylate (GMA) and divinylbenzene (DVB), the mass of the divinylbenzene (DVB) accounting for 20-50% of the total mass of glycidyl methacrylate (GMA) and divinylbenzene (DVB), and the mass ratio of the seed ball matrix to the glycidyl methacrylate (GMA) is (1-2):(20-40).

[0007] Furthermore, the specific surface area of ​​the seed ball matrix grafted with the polymer is 40-400m 2 / g, pore size is 30-100nm.

[0008] Further, the ligand is a sulfonic acid group, and the sulfonic acid group is Na 2 SO 3 The sulfonic acid group is prepared from raw materials, and a spacer arm is connected to the surface of the filler.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned porous highly hydrophilic ion exchange purification filler, comprising the following steps:

[0010] (1) Preparation of porous polymer microspheres

[0011] emulsifying the oil phase solution in the water phase solution to obtain an emulsion, mixing the seed ball matrix with the emulsion and preparing monodisperse porous polymer microspheres through a one-step swelling polymerization;

[0012] The oil phase solution comprises glycidyl methacrylate (GMA), a cross-linking agent, a porogen and an initiator;

[0013] The aqueous phase solution includes a stabilizer and a surfactant;

[0014] (2) Modification of dextran

[0015] epoxidizing the residual double bonds on the surface of the porous polymer microspheres prepared in step (1) and reacting with dextran to obtain dextran-modified porous polymer microspheres;

[0016] (3) Ligand coupling

[0017] The hydroxyl groups of dextran are activated and then coupled to the ligand.

[0018] Furthermore, the porogen is a compound of no less than two porogens including toluene, and the mass of the porogen is 50-100% of the total mass of glycidyl methacrylate GMA and the crosslinking agent.

[0019] Furthermore, the stabilizer in step (1) is one or more of polyvinyl alcohol (PVOH), polyvinyl pyrrolidone (PVP), and hydroxypropyl methylcellulose (HMPC); and the content of the stabilizer in the aqueous solution is 0.1-1 wt%.

[0020] Furthermore, in step (1), the surfactant is one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and sodium dodecyl sulfonate, and the content of the surfactant in the aqueous phase is 0.01-0.1 wt%.

[0021] Furthermore, in step (2), the molecular weight of dextran is 4000-40000, and the mass ratio of dextran to porous polymer microspheres is (1-3):2.

[0022] Furthermore, the ligand coupled in step (3) is a sulfonic acid group, and the specific steps of the sulfonic acid group coupling are: the porous polymer microspheres modified with dextran are first reacted with allyl glycidyl ether, then reacted with bromine water and alkali solution, and finally reacted with Na 2 SO 3 The aqueous solution reacts to form sulfonic acid groups.

[0023] Furthermore, in step (3), the mass ratio of allyl glycidyl ether to the porous polymer microspheres modified with dextran is (0.5-3):1, and the reaction temperature is 25-55°C;

[0024] The mass ratio of bromine water to dextran-modified porous polymer microspheres is (1-10):20, and the final concentration of the alkali solution is 0.5-5 mol / L;

[0025] The Na 2 SO 3 Na in aqueous solution 2 SO 3 The mass percentage is 1: (5-40), Na 2 SO 3 The mass ratio of the porous polymer microspheres modified with dextran is (1-5):20.

[0026] In a third aspect, the present invention provides use of the porous highly hydrophilic ion exchange purification filler described above or the porous highly hydrophilic ion exchange purification filler prepared by the preparation method described above in the purification of biomacromolecules.

[0027] Compared with the prior art, this application has the following beneficial effects:

[0028] The present invention uses a seed ball matrix as a base ball, prepares monodisperse porous polymer microspheres by one-step swelling polymerization, then uses an oxidation reaction to convert the residual double bonds on the surface of the polymer microspheres into epoxy groups, then grafts a layer of dextran hydrophilic layer to completely cover the surface hydrophobic sites, and then uses the hydroxyl groups on the dextran for chemical modification and bonding ligands to prepare porous, highly hydrophilic, and high exchange capacity ion exchange polymer fillers, which are successfully applied to protein separation and purification. Therefore, the ion exchange purification filler prepared by the present invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Flow chart for the preparation of porous highly hydrophilic sulfonic acid-based ion exchange purification packing.

[0030] Figure 2 This is a transmission electron micrograph of the polystyrene seed ball matrix prepared in Example 1.

[0031] Figure 3 This is a transmission electron micrograph of the porous PGMA-DVB polymer microspheres after swelling and polymerization prepared in Example 1.

[0032] Figure 4 This is a transmission electron micrograph of the sulfonic acid group purification filler prepared in Example 1.

[0033] Figure 5 HPLC chart of the purification filler prepared in Example 1 used to separate three standard proteins: hemolysin, myoglobin and trypsin.

[0034] Figure 6 This is the HPLC chart of the purified filler prepared in Comparative Example 1 for separating three standard proteins: hemolysin, myoglobin, and trypsin.

[0035] Figure 7 This is the HPLC chart of the purified filler prepared in Comparative Example 3 for separating three standard proteins: hemolysin, myoglobin, and trypsin.

[0036] Figure 8 This is the HPLC chart of the purified filler prepared in Comparative Example 4 for separating three standard proteins: hemolysin, myoglobin, and trypsin. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0039] In a first aspect, the present invention provides a porous highly hydrophilic ion exchange purification filler, the filler comprising a seed ball matrix, the surface of the seed ball matrix is ​​grafted with a polymer, the polymer is polymerized by a monomer glycidyl methacrylate GMA under the action of a crosslinking agent; the polymer is modified with dextran, and the dextran is coupled with a ligand;

[0040] Wherein, the crosslinking agent is divinylbenzene DVB.

[0041] In certain embodiments, the mass of the glycidyl methacrylate GMA accounts for 50-80% of the total mass of glycidyl methacrylate GMA and divinylbenzene DVB, the mass of the divinylbenzene DVB accounts for 20-50% of the total mass of glycidyl methacrylate GMA and divinylbenzene DVB, and the mass ratio of the seed ball matrix to glycidyl methacrylate GMA is (1-2):(20-40).

[0042] In certain embodiments, the specific surface area of ​​the seed ball matrix grafted with the polymer is 40-400 m 2 / g, pore size is 30-100nm.

[0043] In certain embodiments, the ligand is a sulfonic acid group, wherein the sulfonic acid group is a Na 2 SO 3 The sulfonic acid group is connected to the surface of the filler by a spacer arm. 2 SO 3 The sulfonic acid group is coupled to the filler surface through the spacer arm, so that there is a certain distance between the sulfonic acid group and the filler surface, and the sulfonic acid group is not directly coupled to the filler surface.

[0044] In certain embodiments, the seed ball matrix is ​​polystyrene microspheres or other polymer microspheres.

[0045] In a second aspect, the present invention provides a method for preparing the above-mentioned porous highly hydrophilic ion exchange purification filler, comprising the following steps:

[0046] (1) Preparation of porous polymer microspheres

[0047] emulsifying the oil phase solution in the water phase solution to obtain an emulsion, mixing the seed ball matrix with the emulsion and preparing monodisperse porous polymer microspheres through a one-step swelling polymerization;

[0048] The oil phase solution comprises glycidyl methacrylate (GMA), a cross-linking agent, a porogen and an initiator;

[0049] The aqueous phase solution includes a stabilizer and a surfactant;

[0050] (2) Modification of dextran

[0051] epoxidizing the residual double bonds on the surface of the porous polymer microspheres prepared in step (1) and reacting with dextran to obtain dextran-modified porous polymer microspheres;

[0052] In certain embodiments, the epoxidation reaction can be carried out by meta-chloroperbenzoic acid, wherein the mass ratio of the meta-chloroperbenzoic acid to the porous polymer microspheres is (0.1-1): 1; the solvent used in the epoxidation reaction is one or more of dichloromethane, dichloroethane, chloroform, and tetrahydrofuran. The catalyst is a basic catalyst or an acidic catalyst, and the mass of the catalyst is 1-10% of the mass of the microspheres, wherein the basic catalyst is one or more of triethylamine, tributylamine, 4-methylaminopyridine, and sodium hydroxide, preferably 4-dimethylaminopyridine; the acidic catalyst is one or more of boron trifluoride etherate, aluminum trichloride, ferric trichloride, and tin tetrachloride, preferably boron trifluoride etherate.

[0053] (3) Ligand coupling

[0054] The hydroxyl groups of dextran are activated and then coupled with a ligand. The coupled ligand can be a cationic ligand or an anionic ligand. A suitable coupling method can be selected according to the reactive groups of the activated hydroxyl groups and the reactive groups of the ionic ligand.

[0055] In certain embodiments, the porogen is one or more of toluene, heptane, dioctyl phthalate DOP or octadecyl alcohol, and the mass of the porogen is 50-100% of the total mass of glycidyl methacrylate GMA and the crosslinking agent;

[0056] In certain embodiments, the initiator is one or more of benzoyl peroxide, lauroyl peroxide, methyl ethyl ketone peroxide, or di-tert-butyl peroxide.

[0057] In certain embodiments, the stabilizer in step (1) is one or more of polyvinyl alcohol (PVOH), polyvinyl pyrrolidone (PVP), and hydroxypropyl methylcellulose (HMPC); the content of the stabilizer in the aqueous solution is 0.1-1 wt%;

[0058] In certain embodiments, the surfactant in step (1) is one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and sodium dodecyl sulfonate, and the content of the surfactant in the aqueous phase is 0.01-0.1 wt %.

[0059] In certain embodiments, the molecular weight of dextran in step (2) is 4000-40000, for example, 4000, 6000, 8000, 10000, and the mass ratio of dextran to porous polymer microspheres is (1-3):2.

[0060] In some embodiments, the ligand coupled in step (3) is a sulfonic acid group, and the specific steps of the sulfonic acid group coupling are: the porous polymer microspheres modified with dextran are first reacted with allyl glycidyl ether, then reacted with bromine water and alkali solution, and finally reacted with Na 2 SO 3 The aqueous solution reacts to form sulfonic acid groups. Wherein, the alkali solution can be selected from NaOH solution.

[0061] In certain embodiments, in step (3), the mass ratio of allyl glycidyl ether to the porous polymer microspheres modified with dextran is (0.5-3):1, and the reaction temperature is 25-55°C;

[0062] The mass ratio of bromine water to dextran-modified porous polymer microspheres is (1-10):20, and the final concentration of the alkali solution is 0.5-5 mol / L;

[0063] The Na 2 SO 3 Na in aqueous solution 2 SO 3 The mass percentage is 1: (5-40), Na 2 SO 3 The mass ratio of the porous polymer microspheres modified with dextran is (1-5):20.

[0064] In a third aspect, the present invention provides use of the porous highly hydrophilic ion exchange purification filler described above or the porous highly hydrophilic ion exchange purification filler prepared by the preparation method described above in the purification of biomacromolecules.

[0065] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0066] The present application is described in detail below in conjunction with specific embodiments. These embodiments and comparative examples are used to understand rather than limit the present application.

[0067] Example 1

[0068] The preparation flow chart of the purified filler in this embodiment is as follows Figure 1 shown.

[0069] 1) Preparation of seed ball matrix

[0070] The reaction medium and the dispersion stabilizer PVP were added in sequence into a jacketed reactor equipped with a magnetic stirring device and a constant temperature water bath, and stirred for 10 minutes to fully dissolve the PVP; after the temperature reached the set temperature, monomer styrene (St) dissolved in azobisisobutyronitrile (AIBN) was added into the jacketed reactor, and the reaction was terminated after 12 hours of reaction; the polystyrene microsphere suspension sample was collected and stored in a plastic bottle, and the particle size measured by transmission electron microscopy was 3-4 μm.

[0071] The mass of the reaction medium ethanol is 30g, the mass of water is 0.5g, the mass of the stabilizer PVP is 0.5g, the mass of the monomer styrene is 10g, and the mass of the initiator azobisisobutyronitrile is 0.11g. The preparation conditions are: temperature 70°C, stirring speed 350r / min.

[0072] 2) Preparation of porous polymer microspheres

[0073] Take 1.3g of the polystyrene microspheres prepared above, add 50g of deionized water, ultrasonically disperse for 10min, pour into a 250mL four-necked bottle, take 20g of glycidyl methacrylate monomer, 7g of divinylbenzene, 8g of porogen toluene, 8g of n-heptane, and 0.7g of initiator benzoyl peroxide as the oil phase solution, ultrasonically dissolve and add to 100g of the aqueous phase solution, wherein the aqueous phase solution consists of 0.5g PVOH, 0.2g PVP, and 0.05g of sodium dodecyl sulfate. The two phases are mixed and ultrasonically crushed for 10min to obtain a uniform emulsion, which is poured into a four-necked bottle and swelled for 24h, then heated to 75°C and reacted for 16h. The porous PGMA-DVB polymer microspheres are collected and repeatedly washed with ethanol and water until there is no odor. The average particle size is measured by transmission electron microscopy at 12μm. The transmission electron microscope picture is as shown Figure 2 The pore size of the porous polymer microspheres is 30 nm and the surface area is 389 m 2 / g.

[0074] 3) Dextran modification

[0075] Take 10g of the porous PGMA-DVB polymer microspheres, disperse them in 130mL of dichloromethane, and stir them at room temperature at a stirring rate of 250rpm for 1h. Add 5g of m-chloroperbenzoic acid (MCPBA) under stirring, react at room temperature for 2h, wash repeatedly with acetone and water, and air-dry. Take 10g of the air-dried microspheres, add 100g of dimethyl sulfoxide, 0.6g of 4-dimethylaminopyridine, and 10g of dextran 6000, and react at 70℃ for 16h by ultrasonic dispersion. Wash and air-dry to obtain dextran-modified hydrophilic PGMA-DVB microspheres. Transmission electron microscopy images are shown in the figure. Figure 3 shown.

[0076] 4) Sulfonic acid modification

[0077] Take 10g of the polymer microspheres modified with dextran, disperse them in 100mL of 40% sodium hydroxide solution by ultrasonic, add 10g of allyl glycidyl ether dropwise at 40℃, wash and air-dry, add Br 2 The mixture was reacted in a sodium hydroxide solution for 6 h. After washing, 200 mL of a 25 wt% sodium sulfite aqueous solution was added and reacted at 75 °C for 16 h. The cation exchange purification filler with sulfonic acid ligands was obtained by washing with pure water. The ligand density was measured to be 100 mmol / L. The transmission electron microscope picture is shown in FIG. Figure 4 shown.

[0078] Example 2

[0079] 1) The seed ball matrix was prepared in the same manner as in Example 1;

[0080] 2) Preparation of porous polymer microspheres

[0081] Take 1.3g of the polystyrene microspheres prepared above, add 50g of deionized water, ultrasonically disperse for 10min, pour into a 250mL four-necked bottle, take 18g of methacrylate glycidyl ester monomer, 18g of divinylbenzene, porogen including 16g of toluene and 10g of DOP, 0.7g of initiator benzoyl peroxide as the oil phase solution, ultrasonically dissolve and add to 100g of aqueous phase solution, wherein the aqueous phase solution consists of 0.5g PVOH, 0.2g PVP, 0.05g of sodium dodecyl sulfate, mix the two phases and ultrasonically crush for 10min to obtain a uniform emulsion, pour into a four-necked bottle and swell for 24h, then heat to 75℃, react for 16h, collect the porous PGMA-DVB polymer microspheres and repeatedly wash with ethanol and water until there is no odor, and the average particle size is measured to be 14μm. The pore size of the porous polymer microspheres is 60nm and the surface area is 210m 2 / g.

[0082] 3) Dextran modification

[0083] Take 10g of the porous PGMA-DVB microspheres, disperse them in 130mL of dichloromethane, and stir them at room temperature at a stirring rate of 250rpm for 1h. Add 10g of MCPBA under stirring, react at room temperature for 2h, wash repeatedly with acetone and water, and air-dry. Take 10g of the air-dried microspheres, add 100g of dimethyl sulfoxide, 1g of boron trifluoride ether, and 5g of dextran 40000, and react at 70℃ for 16h under ultrasonic dispersion. Wash and air-dry to obtain dextran-modified hydrophilic PGMA-DVB microspheres.

[0084] 4) Sulfonic acid modification

[0085] Take 10g of hydrophilically modified microspheres, disperse them in 100mL of 40% sodium hydroxide solution by ultrasonic, add 5g of allyl glycidyl ether at 50℃, wash and air-dry, add Br 2The mixture was reacted in a sodium hydroxide solution for 6 h. After washing, 200 mL of a 15 wt% sodium sulfite aqueous solution was added, and the mixture was reacted at 75° C. for 16 h. The mixture was washed with pure water to obtain a cation exchange filler with a sulfonic acid ligand. The density of the sulfonic acid ligand was measured to be 105 mmol / L.

[0086] Example 3

[0087] 1) Seed ball preparation is the same as in Example 1;

[0088] 2) Preparation of porous polymer microspheres

[0089] Take 1.3g of the polystyrene microspheres prepared above, add 50g of deionized water, ultrasonically disperse for 10min, pour into a 250mL four-necked bottle, take 35g of methacrylate glycidyl ester monomer, 18g of divinylbenzene, porogen including 8g of toluene and 20g of octadecyl alcohol, and 0.7g of initiator benzoyl peroxide as the oil phase solution, ultrasonically dissolve and add to 100g of the aqueous phase solution, wherein the aqueous phase solution consists of 0.5g of PVOH, 0.2g of PVP, and 0.05g of sodium dodecyl sulfate. Mix the two phases and ultrasonically crush for 10min to obtain a uniform emulsion, pour into a four-necked bottle and swell for 24h, then heat to 75°C, react for 16h, collect the porous PGMA-DVB polymer microspheres and repeatedly wash them with ethanol and water until there is no odor, and the average particle size is measured to be 14μm. The pore size of the porous polymer microspheres is 75nm and the surface area is 100m 2 / g.

[0090] 3) Dextran modification

[0091] Take 10g of the porous PGMA-DVB microspheres, disperse them in 130mL of dichloromethane, and stir them at room temperature at a stirring rate of 250rpm for 1h. Add 10g of MCPBA under stirring, react at room temperature for 2h, wash repeatedly with acetone and water, and air-dry. Take 10g of the air-dried microspheres, add 100g of dimethyl sulfoxide, 1g of boron trifluoride ether, and 15g of dextran 10000, and react at 70℃ for 16h under ultrasonic dispersion. Wash and air-dry to obtain dextran-modified hydrophilic PGMA-DVB microspheres.

[0092] 4) Sulfonic acid modification

[0093] Take 10g of hydrophilically modified microspheres, disperse them in 100mL of 40% sodium hydroxide solution by ultrasonic, add 15g of allyl glycidyl ether dropwise at 50℃, wash and air-dry, add Br 2 The mixture was reacted in a sodium hydroxide solution for 6 h. After washing, 200 mL of a 15 wt% sodium sulfite aqueous solution was added, and the mixture was reacted at 75 °C for 16 h. The mixture was washed with pure water to obtain a cation exchange filler with a sulfonic acid ligand. The density of the sulfonic acid ligand was measured to be 155 mmol / L.

[0094] Example 4

[0095] The difference between Example 4 and Example 3 is that the glycidyl methacrylate monomer is 40 g, divinylbenzene is 20 g, and the porogen includes 5 g toluene and 30 g octadecyl alcohol.

[0096] The porous polymer microspheres prepared under the experimental conditions of Example 4 have an average particle size of 14 μm. The pore size of the porous polymer microspheres is 95 nm and the surface area is 50 m 2 / g, and the sulfonic acid ligand density of the purified filler is 169mmol / L.

[0097] The sulfonic acid purification filler prepared in Example 1-4 was filled into a chromatographic column for purification performance testing. The protein sample used was a mixture of three standard proteins: hemolysin, myoglobin, and trypsin. The concentration of each protein was 1 mg / mL.

[0098] The test conditions are: using a stainless steel chromatographic column with an inner diameter of 4.6 mm and a length of 50 mm, the mobile phase is A: 20 mM phosphate solution, pH 6.0; B: A+1.0 M NaCl; the gradient conditions are: 10-100% B within 25 min, flow rate 0.7 mL / min, injection volume 5 μL, column temperature: 25°C, detection wavelength: UV 280 nm.

[0099] The HPLC separation pattern measured in Example 1 is as follows Figure 5 As shown, from Figure 5 It can be seen that the purification filler prepared by using the example can achieve good separation of three standard proteins: hemolysin, myoglobin and trypsin.

[0100] Similarly, Examples 2-4 also achieved the same separation effect as Example 1.

[0101] Comparative Example 1

[0102] The difference between Comparative Example 1 and Example 3 is that the glycidyl methacrylate monomer is 15 g. The measured ligand density is 18 mmol / L. The purified filler prepared in Comparative Example 1 is subjected to purification performance test according to the above test method. The HPLC spectrum shows no separation effect ( Figure 6 ).

[0103] Comparative Example 2

[0104] The difference between Comparative Example 2 and Example 3 is that the porogen toluene is 28 g. The pore size of the porous polymer microspheres is 15 nm and the surface area is 250 m 2 / g, the ligand density is 27mmol / L, and the purification performance test of the purification filler prepared in Comparative Example 2 is carried out according to the above test method.

[0105] The purification performance of the purified filler prepared in Comparative Example 1 was tested according to the above test method. The HPLC spectrum showed no separation effect, and the separation result was the same as that of Comparative Example 1.

[0106] Comparative Example 3

[0107] The difference between Comparative Example 3 and Example 3 is that the crosslinking agent in Comparative Example 3 is ethylene glycol dimethacrylate EGDM, and other process conditions remain unchanged. The density of the sulfonic acid ligand was measured to be 60 mmol / L, and the ligand density was relatively low. The purified filler prepared in Comparative Example 3 was subjected to a purification performance test according to the above test method, and the obtained HPLC spectrum showed a relatively wide peak shape ( Figure 7 ), resulting in poor protein separation.

[0108] Comparative Example 4

[0109] The difference between Comparative Example 4 and Example 3 is that in the process of sulfonic acid group modification in step (4), allyl glycidyl ether was not used for activation, and 1,3-propane sultone was directly used to react with the hydroxyl group on the dextran to obtain the sulfonic acid ligand. The purification filler prepared in Comparative Example 4 was tested for purification performance according to the above test method, and the obtained HPLC spectrum showed that the peak shape became narrower ( Figure 8 ), and it is impossible to achieve complete separation of the three proteins.

[0110] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A porous highly hydrophilic ion exchange purification filler, characterized in that: The filler comprises a seed ball matrix, the surface of which is grafted with a polymer, wherein the polymer is polymerized by a monomer of glycidyl methacrylate (GMA) under the action of a crosslinking agent, divinylbenzene (DVB); the polymer is modified with dextran, and the dextran is coupled with a ligand; The mass of the glycidyl methacrylate GMA accounts for 50-80% of the total mass of glycidyl methacrylate GMA and divinylbenzene DVB, the mass of the divinylbenzene DVB accounts for 20-50% of the total mass of glycidyl methacrylate GMA and divinylbenzene DVB, and the mass ratio of the seed ball matrix to glycidyl methacrylate GMA is (1-2): (20-40).

2. A porous highly hydrophilic ion exchange purification filler according to claim 1, characterized in that: The specific surface area of ​​the seed ball matrix grafted with polymer is 40-400m 2 / g, pore size is 30-100nm.

3. The porous highly hydrophilic ion exchange purification filler according to claim 1, characterized in that: The ligand is a sulfonic acid group, which is prepared using Na2SO3 as a raw material, and the sulfonic acid group is connected to the surface of the filler via a spacer arm.

4. The method for preparing the porous highly hydrophilic ion exchange purification filler according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of porous polymer microspheres emulsifying the oil phase solution in the water phase solution to obtain an emulsion, mixing the seed ball matrix with the emulsion and preparing monodisperse porous polymer microspheres through a one-step swelling polymerization; The oil phase solution comprises glycidyl methacrylate (GMA), a cross-linking agent, a porogen and an initiator; The aqueous phase solution includes a stabilizer and a surfactant; (2) Modification of dextran epoxidizing the residual double bonds on the surface of the porous polymer microspheres prepared in step (1) and reacting with dextran to obtain dextran-modified porous polymer microspheres; (3) Ligand coupling The hydroxyl groups of dextran are activated and then coupled to the ligand.

5. The preparation method according to claim 4, characterized in that: The porogen is a compound of no less than two porogens including toluene, and the total mass of the porogen is 50-100% of the total mass of glycidyl methacrylate GMA and the crosslinking agent.

6. The preparation method according to claim 4, characterized in that: The stabilizer in step (1) is one or more of polyvinyl alcohol (PVOH), polyvinyl pyrrolidone (PVP), and hydroxypropyl methylcellulose (HMPC); the content of the stabilizer in the aqueous solution is 0.1-1 wt%; In the step (1), the surfactant is one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and sodium dodecyl sulfonate, and the content of the surfactant in the aqueous phase solution is 0.01-0.1 wt%.

7. The preparation method according to claim 4, characterized in that: In the step (2), the molecular weight of dextran is 4000-40000, and the mass ratio of dextran to porous polymer microspheres is (1-3):

2.

8. The preparation method according to claim 4, characterized in that: The ligand coupled in step (3) is a sulfonic acid group, and the specific steps of sulfonic acid group coupling are: the porous polymer microspheres modified with dextran are first reacted with allyl glycidyl ether, then reacted under the action of bromine water and alkali solution, and finally reacted with Na2SO3 aqueous solution to form a sulfonic acid group.

9. The preparation method according to claim 8, characterized in that: In the step (3), the mass ratio of allyl glycidyl ether to the porous polymer microspheres modified with dextran is (0.5-3):1, and the reaction temperature is 25-55°C; The mass ratio of bromine water to dextran-modified porous polymer microspheres is (1-10):20, and the final concentration of the alkali solution is 0.5-5 mol / L; The mass ratio of the Na2SO3 to the porous polymer microspheres modified with dextran is (1-5):

20.

10. Use of the porous highly hydrophilic ion exchange purification filler according to any one of claims 1 to 3 or the porous highly hydrophilic ion exchange purification filler prepared by the preparation method according to any one of claims 4 to 9 in the purification of biomacromolecules.

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