A method for preparing a salt-tolerant strong cation exchange chromatography medium

Abstract

The present invention discloses a method for preparing a salt-tolerant strong cation exchange chromatography medium. The method comprises reacting a compound having a sulfate group with a polymerizable monomer having a vinyl group to prepare a polymerizable functional monomer having a sulfate group; and initiating graft polymerization of the obtained functional monomer on the surface of porous microspheres to obtain a salt-tolerant cation exchange chromatography medium. The preparation process of the method is simple and easy to operate, and a wide range of ligands can be selected. The prepared salt-tolerant cation exchange chromatography medium has a wide ionic strength tolerance range, can maintain a high protein adsorption capacity at high salt concentrations, has a wide range of applications, and can improve the separation and purification efficiency of protein drugs.

Description

Technical Field

[0001] The invention relates to the technical field of ion exchange chromatography medium preparation, in particular to a method for preparing a salt-tolerant strong cation exchange chromatography medium. Background Art

[0002] As a mainstream technology for separation and purification, chromatography technology has been widely used in the downstream production of biopharmaceuticals. With the increasing scale of biopharmaceutical production, the requirements for separation and purification efficiency are becoming higher and higher. Ion exchange chromatography is the most widely used of various chromatographic modes. This mode has the characteristics of high loading capacity, large processing volume and wide range of applications. Protein adsorption capacity is an extremely important parameter in the ion exchange chromatography process. Especially with the advancement of upstream genetic engineering and cell engineering technologies, the expression level of target protein molecules and their production scale have increased rapidly, and the requirements for the processing volume and separation flux of a single batch of downstream separation and purification have also increased accordingly. The processing volume mainly depends on the performance of the chromatography seasoning itself. Furthermore, it places higher requirements on the protein adsorption capacity of the chromatography filler. Therefore, the development of high-performance ion exchange chromatography fillers has always been a research hotspot that has attracted much attention.

[0003] In the ion exchange chromatography mode, the adsorption-elution operation mode is a commonly used operation mode. Its operation process usually includes four steps: equilibrium, sample loading, elution, and regeneration. The protein molecules are adsorbed mainly through electrostatic interactions with the chromatographic filler. The ionic strength in the feed solution has a significant effect on the protein adsorption capacity. The higher the ionic strength, the lower the protein adsorption capacity. Therefore, the ionic strength of the feed solution needs to be reduced before loading. For example, desalting or diluting the feed solution usually requires reducing the ionic strength to below 0.05M. The desalting process increases the number of operating steps and equipment, and usually loses some of the yield or activity of the target protein. The dilution of the feed solution increases the feed solution volume, increases the sample loading volume, increases the processing time, etc., which correspondingly increases the separation and purification cost and reduces production efficiency. In order to solve the above problems, developing salt-tolerant ion exchange chromatography media is one of the better solutions. For example, a salt-tolerant cation exchange chromatography medium was prepared by bonding polymer molecules with numerous carboxyl and sulfonic acid groups to the surface of a nylon membrane via a reaction between aldehyde and amine groups. At a concentration of 0.5 M KSCN, the protein adsorption capacity of this cation exchange membrane (using lysozyme as a model protein) remained around 50 mg / mL, demonstrating good salt tolerance (Journal of Chromatography A, 2017, 1521, 19-26). Furthermore, a report has also reported bonding high molecular weight (average molecular weight 900,000) polyallylamine polymer molecules to agarose-based microspheres via an amine-epoxy reaction, resulting in a salt-tolerant weak anion exchange chromatography medium. This medium maintained a bovine serum albumin adsorption capacity of around 200 mg / mL at a concentration of 0.5 M NaCl.

[0004] The above-mentioned preparation methods of the prior art mostly involve chemically bonding high molecular weight polymers to the surface of the material. The functional groups of the cation exchange chromatography medium are carboxyl groups and sulfonic acid groups, while the functional groups of the anion exchange chromatography medium are mainly primary amine groups. The ion tolerance strength of this type of preparation method and its medium type mainly depends on the molecular weight of the bonded polymer. The larger the molecular weight, the higher the ionic strength it can tolerate. However, an increase in the molecular weight of the polymer is likely to cause an increase in the reaction steric effect, thereby affecting the number of bonds, and ultimately leading to a decrease in its ionic strength tolerance, that is, a decrease in salt concentration tolerance. Summary of the Invention

[0005] The present invention aims to provide a method for preparing a salt-tolerant strong cation exchange chromatography medium. The preparation process of the method is simple and easy to operate, a wide range of ligands can be selected, and the prepared salt-tolerant cation exchange chromatography medium has a wide ionic strength tolerance range.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing a salt-tolerant strong cation exchange chromatography medium, comprising:

[0008] Step 1: reacting a compound having a sulfate group with a polymerizable monomer having a vinyl group to prepare a polymerizable functional monomer having a sulfate group;

[0009] Step 2: Initiate graft polymerization of the functional monomer obtained in step 1 on the surface of the porous microspheres to obtain a salt-tolerant cation exchange chromatography medium.

[0010] In step 1, the compound having a sulfate group includes 2-aminoethyl hydrogen sulfate, 2-ethylaminoethyl hydrogen sulfate, 2-methylaminoethyl hydrogen sulfate, 2-benzylaminoethyl hydrogen sulfate or 2-mercaptoethyl hydrogen sulfate.

[0011] In step 1, the polymerizable monomer having a vinyl group includes a vinyl monomer having an epoxy group, an aldehyde group, a carboxyl group, or an acyl chloride group.

[0012] In step 1, the molar ratio of the compound having a sulfate group to the polymerizable monomer having a vinyl group is 1:1 to 2:1.

[0013] In step 1, the temperature range for the reaction of the two compounds is 25 to 70° C.; and the reaction time is 2 to 24 hours.

[0014] In step 2, the porous microspheres include polyacrylate, polystyrene or polysaccharide materials, and their surface groups include amino groups, hydroxyl groups, carboxyl groups, thiol groups or halogen groups. These groups can form a redox reaction system with an initiator or initiate atom transfer radical polymerization.

[0015] In step 2, the initiator for initiating the graft polymerization includes ammonium cerium sulfate, ammonium cerium nitrate, potassium persulfate or ammonium persulfate.

[0016] In step 2, the obtained salt-tolerant cation exchange chromatography medium has a tolerance ionic strength range of 0.1 to 0.7 mol / L NaCl solution; and a protein loading range of 30 to 180 mg lysozyme / mL wet medium.

[0017] As can be seen from the technical solution provided by the present invention, the preparation process of the above method is simple and easy to operate, the range of ligands available for selection is wide, and the prepared salt-tolerant cation exchange chromatography medium has a wide ionic strength tolerance range and can maintain a high protein adsorption capacity at higher salt concentrations. It has a wide range of applications and can improve the separation and purification efficiency of protein drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 A schematic flow chart of a method for preparing a salt-tolerant strong cation exchange chromatography medium provided in an embodiment of the present invention;

[0020] Figure 2 This is a scanning electron microscope photograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 1 of the present invention;

[0021] Figure 3 This is a scanning electron microscope photograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 2 of the present invention;

[0022] Figure 4 This is a scanning electron microscope photograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 3 of the present invention;

[0023] Figure 5 This is a scanning electron microscope photograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 4 of the present invention;

[0024] Figure 6This is a scanning electron microscope photograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and do not constitute a limitation of the present invention. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0026] like Figure 1 FIG. 1 is a flow chart of a method for preparing a salt-tolerant strong cation exchange chromatography medium according to an embodiment of the present invention, wherein the method comprises:

[0027] Step 1: reacting a compound having a sulfate group with a polymerizable monomer having a vinyl group to prepare a polymerizable functional monomer having a sulfate group;

[0028] In this step, the compound having a sulfate group includes 2-aminoethyl hydrogen sulfate, 2-ethylaminoethyl hydrogen sulfate, 2-methylaminoethyl hydrogen sulfate, 2-benzylaminoethyl hydrogen sulfate or 2-mercaptoethyl hydrogen sulfate.

[0029] In a specific implementation, any compound containing both amino or thiol groups and sulfate groups can be used.

[0030] The polymerizable monomers with vinyl groups include vinyl monomers with epoxy groups, aldehyde groups, carboxyl groups, or acyl chlorides, such as glycidyl methacrylate, methacrolein, methacrylic acid, and any vinyl monomer that can react with compounds with sulfate groups.

[0031] In addition, the molar ratio of the compound having a sulfate group to the polymerizable monomer having a vinyl group is 1:1 to 2:1.

[0032] The temperature range for the reaction of the two compounds is 25 to 70° C.; the reaction time is 2 to 24 hours.

[0033] The method described in this embodiment achieves the purpose of expanding functional monomers by selecting different vinyl monomers and compounds with sulfate groups for combination as needed. It is no longer limited to the use of commercial polymer ligands in the prior art, and realizes a wider range of applicable salt-resistant cation exchange chromatography media.

[0034] Step 2: Initiate graft polymerization of the functional monomer obtained in step 1 on the surface of the porous microspheres to obtain a salt-tolerant cation exchange chromatography medium.

[0035] In this step, the porous microspheres include polyacrylate, polystyrene or polysaccharide materials, and their surface groups include amino groups, hydroxyl groups, carboxyl groups, thiol groups or halogen groups. These groups can form a redox reaction system with an initiator or initiate atom transfer radical polymerization.

[0036] The initiator for initiating graft polymerization includes ammonium cerium sulfate, ammonium cerium nitrate, potassium persulfate or ammonium persulfate. In a specific implementation, other initiators that can initiate atom transfer radical polymerization can be used.

[0037] The obtained salt-resistant cation exchange chromatography medium has a tolerance ion strength range of 0.1-0.7 mol / L NaCl solution and a protein loading range of 30-180 mg lysozyme / mL wet medium.

[0038] This embodiment overcomes the steric hindrance effect of the prior art chemical coupling high molecular weight polymer method by initiating graft polymerization of functional monomers on the surface of porous materials. The molecular weight of the functional ligand can be controlled on demand by adjusting the graft polymerization reaction conditions, and can tolerate a wider salt concentration range.

[0039] The following examples illustrate the method and product performance of the embodiments of the present invention:

[0040] Example 1, Experimental Step 1: 2-aminoethyl hydrogen sulfate (AHS) and glycidyl methacrylate (GMA) were mixed in an aqueous solution at a molar ratio of 1:1, stirred, and the reaction temperature was controlled to 70°C for 2 hours. After the reaction was completed, the temperature was lowered to room temperature and the obtained functional monomer was refrigerated and stored at 4°C for later use.

[0041] Experimental step 2: The polysaccharide microspheres with surface hydroxyl groups were mixed with the GMA-AHS solution prepared in step 1) at a mass to volume ratio of 10 (g): 20 (mL), and then solid ammonium cerium nitrate was added (the amount added was 1% of the mass of the microspheres). The mixture was stirred at 40°C for 4 hours. After the reaction was completed, it was washed with deionized water to prepare a salt-resistant cation exchange chromatography medium.

[0042] The salt-tolerant cation exchange chromatography medium has a ligand density of 0.10 mmol / mL and a salt tolerance range of 0.1-0.7 M NaCl aqueous solution. The protein adsorption capacity range is shown in Table 1 below. Within the range of 0.1-0.7 M NaCl solution, the protein adsorption capacity ranges from 180-30 mg / mL. As the salt concentration increases, the protein adsorption capacity decreases. Figure 2The figure shows an electron scanning micrograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 1 of the present invention. A is a full-view image of the microspheres, and B is a magnified local image of the surface of the microspheres. The surface of the microspheres has a through-hole macroporous structure, which provides a guarantee for maintaining a high protein mass transfer rate.

[0043] Table 1 Salt concentration tolerance range of the salt-tolerant cation exchange chromatography medium obtained in Example 1 and its corresponding protein adsorption capacity

[0044]

[0045] Example 2, Experimental Step 1: 2-Mercaptoethyl hydrogen sulfate (SHS) and methacrolein (MAL) were mixed in an aqueous solution at a molar ratio of 2:1, stirred, and the reaction temperature was controlled at 70°C for 24 hours. After the reaction was completed, the temperature was lowered to room temperature and the resulting functional monomer MAL-SHS was refrigerated and stored at 4°C for later use.

[0046] Experimental step 2: The polyacrylate microspheres with surface amino groups were mixed with the MAL-SHS solution prepared in step 1) at a mass to volume ratio of 10 (g): 30 (mL), and then solid ammonium cerium sulfate was added (the amount added was 2% of the mass of the microspheres). The mixture was stirred and reacted at 50°C for 12 hours. After the reaction was completed, the medium was washed with deionized water to prepare a salt-tolerant cation exchange chromatography medium with a ligand density of 0.65 mmol / mL and a salt tolerance range of 0.1-0.5 M NaCl aqueous solution. The protein adsorption capacity range is shown in Table 2 below. Within the range of 0.1-0.5 M NaCl solution, the protein adsorption capacity ranged from 170 to 50 mg / mL, and the protein adsorption capacity decreased with increasing salt concentration.

[0047] like Figure 3 The figure shows an electron scanning micrograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 2 of the present invention. A is a full-view image of the microspheres, and B is a magnified local image of the surface of the microspheres. The surface of the microspheres has a through-going micron-scale macroporous structure, which provides a guarantee for maintaining a high protein mass transfer rate.

[0048] Table 2 Salt concentration tolerance range of the salt-tolerant strong cation exchange chromatography medium obtained in Example 2 and its corresponding protein adsorption capacity

[0049]

[0050]

[0051] Example 3, Experimental Step 1: 2-Methylaminoethyl hydrogen sulfate (MAHS) and methacrylic acid (MAA) were mixed in an aqueous solution at a molar ratio of 1:1 and stirred. The reaction temperature was controlled at 50°C for 12 hours. After the reaction was completed, the temperature was lowered to room temperature and the resulting functional monomer MAA-MAHS was refrigerated and stored at 4°C for later use.

[0052] Experimental step 2: The polystyrene microspheres with carboxyl groups on the surface were mixed with the MAA-MAHS solution prepared in step 1) at a mass to volume ratio of 10 (g): 50 (mL), and then potassium persulfate solid was added (the addition amount was 3% of the mass of the microspheres), and the reaction was stirred at 50°C for 10 hours. After the reaction was completed, the medium was washed with deionized water to prepare a salt-tolerant cation exchange chromatography medium with a ligand density of 0.35 mmol / mL and a salt tolerance range of 0.1-0.7 M NaCl aqueous solution. The protein adsorption capacity range is shown in Table 3. Within the range of 0.1-0.7 M NaCl solution, the protein adsorption capacity ranged from 152 to 31 mg / mL, and the protein adsorption capacity decreased with increasing salt concentration.

[0053] like Figure 4 The figure shows an electron scanning micrograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 3 of the present invention. A is a full-view image of the microspheres, and B is a magnified local image of the surface of the microspheres. The surface of the microspheres has a through-going micron-scale macroporous structure, which provides a guarantee for maintaining a high protein mass transfer rate.

[0054] Table 3 Salt concentration tolerance range of the salt-tolerant cation exchange chromatography medium obtained in Example 3 and its corresponding protein adsorption capacity

[0055]

[0056] Example 4, Experimental Step 1: 2-Ethylaminoethyl hydrogen sulfate (EAHS) and methacryloyl chloride (MAC) were mixed in anhydrous dioxane solvent at a molar ratio of 1:1 and stirred. The reaction temperature was controlled at 30°C for 12 hours. After the reaction was completed, the temperature was lowered to room temperature and the resulting functional monomer MAC-EAHS was refrigerated and stored at 4°C for later use.

[0057] Experimental step 2: The polystyrene microspheres with hydroxyl groups on the surface were mixed with the MAC-EAHS solution prepared in step 1) in a mass to volume ratio of 10 (g): 20 (mL), and then 30 mL of deionized water was added. Then, solid ammonium persulfate was added (the amount added was 2% of the mass of the microspheres), and the reaction was stirred at 50°C for 14 hours. After the reaction was completed, the medium was washed with deionized water to prepare a salt-tolerant cation exchange chromatography medium with a ligand density of 0.25 mmol / mL and a salt tolerance range of 0.1-0.7 M NaCl aqueous solution. The protein adsorption capacity range is shown in Table 4. In the range of 0.1-0.7 M NaCl solution, the protein adsorption capacity ranged from 142 to 35 mg / mL, and the protein adsorption capacity decreased with increasing salt concentration.

[0058] like Figure 5 The figure shows an electron scanning micrograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 4 of the present invention. A is a full-view image of the microspheres, and B is a magnified local image of the surface of the microspheres. The surface of the microspheres has a through-going micron-scale macroporous structure, which provides a guarantee for maintaining a high protein mass transfer rate.

[0059] Table 4 Salt concentration tolerance range of the salt-tolerant cation exchange chromatography medium obtained in Example 4 and its corresponding protein adsorption capacity

[0060]

[0061] Example 5, Experimental Step 1: 2-Benzylaminoethyl hydrogen sulfate (BAHS) and methacryloyl chloride (MAC) were mixed in anhydrous dioxane solvent at a molar ratio of 1:1.1 and stirred. The reaction temperature was controlled at 35°C for 14 hours. After the reaction was completed, the temperature was lowered to room temperature and the resulting functional monomer MAC-BAHS was refrigerated and stored at 4°C for later use.

[0062] Experimental step 2: The polystyrene microspheres with halogen on the surface were mixed with the MAC-BAHS solution prepared in step 1) in a mass to volume ratio of 10 (g): 20 (mL), and then 20 mL of deionized water was added. After nitrogen was added for deoxygenation, a mixture of cuprous chloride and bipyridine (the molar ratio of the two was 1:2) was added. The mixture was stirred at 70°C for 6 hours. After the reaction was completed, it was washed with deionized water to prepare a salt-tolerant cation exchange chromatography medium with a ligand density of 0.35 mmol / mL and a salt tolerance range of 0.1-0.7 M NaCl aqueous solution. The protein adsorption capacity range is shown in Table 5. In the range of 0.1-0.7 M NaCl solution, the protein adsorption capacity ranged from 172 to 35 mg / mL, and the protein adsorption capacity decreased with increasing salt concentration.

[0063] like Figure 6The figure shows an electron scanning micrograph of the salt-tolerant strong cation exchange chromatography medium obtained in Example 5 of the present invention. A is a full-view image of the microspheres, and B is a magnified local image of the surface of the microspheres. The surface of the microspheres has a through-going micron-scale macroporous structure, which provides a guarantee for maintaining a high protein mass transfer rate.

[0064] Table 5 Salt concentration tolerance range of the salt-tolerant cation exchange chromatography medium obtained in Example 5 and its corresponding protein adsorption capacity

[0065]

[0066]

[0067] The density of grafted ligands on the surface of the obtained salt-tolerant cation exchange chromatography medium and the protein adsorption capacity were measured as follows:

[0068] Determination of density of grafted ligands on the surface of media

[0069] Step 1: Hydrogen Transformation

[0070] The medium was transferred to the exchange column and slowly settled until the volume reached 20 mL. 60 mL of 1 mol / L hydrochloric acid solution was passed through the column and the solution was completely drained within 1 h. The column was then washed with deionized water until the outflowing liquid did not change color when exposed to methyl orange indicator.

[0071] Step 2: Acid-base neutralization

[0072] 1) Accurately measure 50 mL of standard hydrochloric acid solution using a basic burette, add it to the above exchange column, allow it to drip slowly, and collect it in a conical flask;

[0073] 2) Add 30 mL of 1 mol / L sodium chloride solution dropwise through the column and collect it in the above-mentioned conical flask.

[0074] Step 3: Back-drip collected sodium hydroxide

[0075] 1) Add 2-3 drops of phenolphthalein indicator to the liquid collected in step 2, and titrate with 0.1 mol / L hydrochloric acid standard solution until the solution becomes colorless and remains colorless for 15 seconds, which is the endpoint. Record the volume of hydrochloric acid consumed.

[0076] 2) The ligand density of the medium sample is calculated according to the following formula.

[0077] E=(C1 V1-C2 V2) / V(mmol / mL)

[0078] Where: C1 is the concentration of sodium hydroxide standard solution (mol / L); V1 is the volume of sodium hydroxide standard solution (mL); C2 is the concentration of hydrochloric acid standard solution (mol / L); V2 is the volume of hydrochloric acid standard solution consumed during titration (mL); and V is the volume of the medium in the column (mL).

[0079] The grafted ligand density of the salt-tolerant cation exchange chromatography media prepared in Examples 1 to 5 above was determined using this method.

[0080] Determination of protein adsorption capacity of salt-tolerant cation exchange chromatography media:

[0081] Using pH = 7.0, 20mM phosphate buffer as the solvent, NaCl solutions of different concentrations were prepared, ranging from 0.1 to 0.7M. Lysozyme protein (abbreviated as Lyz) with a concentration of 3mg / mL was used as the adsorption model protein. A certain volume of salt-tolerant cation exchange chromatography medium was mixed with the protein solution. After adsorption at room temperature for 5h, the mixture was centrifuged at 5000rpm for 10 minutes. The supernatant was taken to measure the ultraviolet absorbance at 280nm, and the protein adsorption capacity per unit volume of the medium was calculated.

[0082] The protein adsorption capacity of the salt-tolerant cation exchange media prepared in Examples 1 to 5 above at different salt concentrations was determined using this method.

[0083] From the above test results, it can be seen that the ligand density of this type of medium is 0.10-0.65 mmol / mL wet medium. Using lysozyme as the test object, the protein loading range of this type of medium is determined to be 30-180 mg / mL, and the protein recovery rate range is above 90%. The loading capacity increases with the increase of the ligand density of the microspheres, and the salt tolerance concentration also increases with the increase of the ligand density. The salt tolerance concentration range is 0.1-0.7 mol / L NaCl solution.

[0084] It should be noted that the contents not described in detail in the embodiments of the present invention belong to the prior art known to those skilled in the art.

[0085] In summary, the method described in the embodiment of the present invention graft-polymerizes a salt-tolerant functional monomer with a sulfate group on the surface of a porous microsphere material to obtain a cation exchange chromatography medium with salt resistance, wherein the salt-tolerant functional monomer with a sulfate group is obtained by chemically connecting a vinyl monomer and a sulfate group compound. Its structure can be designed and regulated over a wide range and can tolerate a wide range of salt concentrations. The developed preparation method is simple and easy to scale up, and the resulting salt-tolerant strong cation exchange chromatography medium can maintain a high protein adsorption capacity in a high salt solution.

[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims. The information disclosed in the background technology section of this article is only intended to deepen the understanding of the overall background technology of the present invention, and should not be regarded as an admission or any form of implication that the information constitutes prior art already known to those skilled in the art.

Claims

1. A method for preparing a salt-tolerant strong cation exchange chromatography medium, characterized in that: The method comprises: Step 1: reacting a compound having a sulfate group with a polymerizable monomer having a vinyl group to prepare a polymerizable functional monomer having a sulfate group; In step 1, the compound having a sulfate group includes 2-aminoethyl hydrogen sulfate, 2-ethylaminoethyl hydrogen sulfate, 2-methylaminoethyl hydrogen sulfate, 2-benzylaminoethyl hydrogen sulfate or 2-mercaptoethyl hydrogen sulfate; The polymerizable monomer with a vinyl group includes glycidyl methacrylate, methacrolein or methacrylic acid; Step 2: Initiate graft polymerization of the functional monomer obtained in step 1 on the surface of the porous microspheres to obtain a salt-tolerant strong cation exchange chromatography medium; The porous microspheres include polyacrylate, polystyrene or polysaccharide materials, and their surface groups include amino groups, hydroxyl groups, carboxyl groups, thiol groups or halogen groups. These groups can form a redox reaction system with an initiator or initiate atom transfer radical polymerization.

2. The method for preparing the salt-tolerant strong cation exchange chromatography medium according to claim 1, wherein In step 1, the molar ratio of the compound having a sulfate group to the polymerizable monomer having a vinyl group is 1:1 to 2:

1.

3. The method for preparing the salt-tolerant strong cation exchange chromatography medium according to claim 1, wherein In step 1, the temperature range of the reaction between the two compounds is 25~70 o C; reaction time is 2~24 h.

4. The method for preparing the salt-tolerant strong cation exchange chromatography medium according to claim 1, wherein In step 2, the initiator for initiating the graft polymerization includes ammonium cerium sulfate, ammonium cerium nitrate, potassium persulfate or ammonium persulfate.

5. The method for preparing the salt-tolerant strong cation exchange chromatography medium according to claim 1, wherein In step 2, the obtained salt-tolerant strong cation exchange chromatography medium has a tolerance ionic strength range of 0.1-0.7 mol / L NaCl solution; The protein loading range was 30-180 mg lysozyme / mL wet medium.

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