Cation exchange chromatography medium and preparation method thereof

Abstract

The invention provides a cation exchange chromatography medium and a preparation method thereof, the cation exchange chromatography medium comprises microspheres and ligand monomers grafted on the surfaces of the microspheres; wherein the microspheres are styrene-divinyl benzene microspheres, and the molecular weight of the microspheres is 100-200; the ligand monomer comprises propene sulfonic acid, 2-acrylamido-2-methyl propane sulfonic acid and a triazole compound. The cation exchange chromatography medium provided by the invention has the advantages of large ion exchange capacity and good salt tolerance, and can maintain high adsorption capacity under high conductivity.

Description

Technical Field

[0001] The present invention relates to the technical field of chromatography separation, in particular to a cation exchange chromatography medium and a preparation method thereof. Background Art

[0002] Cation exchange chromatography media are widely used in the separation and purification of protein drugs, etc. Because the chromatographic media contain exchangeable ions, during the separation of protein molecules, the sample to be purified can be adsorbed or eluted according to the conductivity (i.e., the salt concentration). Specifically, when the conductivity of the mobile phase is low, the salt concentration is low, and the ion exchange charge interaction force is strong. Therefore, the sample to be purified can be adsorbed on the chromatographic media; when the conductivity of the mobile phase is high, the salt concentration is high, and the ion exchange charge interaction between the sample to be purified and the chromatographic media is shielded. Therefore, the sample adsorbed on the chromatographic media will be eluted.

[0003] Cation exchange chromatography media typically consist of polysaccharide and polymer matrices. Polymer matrices offer greater chemical stability and mechanical strength than polysaccharide matrices. However, their strong hydrophobicity limits ion transport and interaction on the media surface, resulting in lower ion exchange adsorption capacity and increased susceptibility to nonspecific adsorption. Furthermore, at high conductivity, adsorption capacity typically decreases dramatically due to reduced electrostatic interactions, and salt tolerance is poor.

[0004] In view of this, it is urgent to propose a cation exchange chromatography medium that can maintain a high adsorption capacity at a higher conductivity. Summary of the Invention

[0005] The object of the present invention is to provide a cation exchange chromatography medium to solve the problems of poor salt tolerance and low adsorption capacity of the cation exchange chromatography medium in the prior art.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] A cation exchange chromatography medium comprising microspheres and ligand monomers grafted onto the surfaces of the microspheres;

[0008] The microspheres are styrene-divinylbenzene microspheres; and the ligand monomers include propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and triazole compounds.

[0009] Preferably, the triazole compound has a structure as shown in formula (I):

[0010]

[0011] Preferably, the particle size of the microspheres is 50-100 μm.

[0012] Preferably, the mass ratio of the propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid and the triazole compound is 1:(20-50):(1-15).

[0013] Preferably, the mass ratio of the microspheres to the ligand monomer is (20-60):1.

[0014] The present invention provides a method for preparing the cation exchange chromatography medium, comprising the following steps:

[0015] The microspheres are taken and added into water, and then the ligand monomer and initiator are added thereto, mixed evenly, and reacted to obtain a cation exchange chromatography medium.

[0016] Preferably, the initiator is one of cerium sulfate and ammonium cerium nitrate.

[0017] Preferably, the mass ratio of the initiator to the ligand monomer is 1:(25-100).

[0018] Preferably, the reaction temperature is 40-60° C. and the reaction time is 3-6 h.

[0019] Preferably, the mass ratio of the microspheres to the water is 1:(2-10).

[0020] The method for preparing the cation exchange chromatography medium of the present invention further comprises the steps of filtering and washing the obtained cation exchange chromatography medium.

[0021] The propylene sulfonic acid has a structure as shown in formula (II); the 2-acrylamido-2-methylpropanesulfonic acid has a structure as shown in formula (III);

[0022]

[0023]

[0024] Preferably, the method for preparing the cation exchange chromatography medium further comprises the step of adding dextrin palmitate and / or cyclodextrin.

[0025] The CAS number of the dextrin palmitate is 83271-10-7; the CAS number of the cyclodextrin is 12619-70-4.

[0026] Preferably, the mass ratio of the dextrin palmitate to the microspheres is (0.05-0.09):1.

[0027] Preferably, the mass ratio of the cyclodextrin to the microspheres is (0.01-0.03):1.

[0028] The above solution of the present invention includes at least the following beneficial effects:

[0029] The cation exchange chromatography medium of the present invention comprises microspheres and a ligand monomer grafted onto the surface of the microspheres; the microspheres are styrene-divinylbenzene microspheres; and the ligand monomer comprises propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and a triazole compound. The cation exchange chromatography medium of the present invention has a large ion exchange capacity, good salt tolerance, and can maintain a high adsorption capacity at high conductivity.

[0030] The present invention adds a triazole compound having a structure as shown in formula (I). The cyclic polynitrogen atom structure can form a higher charge density on the local surface of the microsphere, weakening the shielding effect of high-concentration salt ions on electrostatic interactions, thereby improving the salt tolerance of the medium. At the same time, the triazole compound is polar and has good hydrophilicity, which can reduce nonspecific adsorption to a certain extent. Due to the introduction of the triazole compound, the ligand density of the 2-acrylamide-2-methylpropanesulfonic acid is reduced to a certain extent, and the coordinated use of the propenesulfonic acid and 2-acrylamide-2-methylpropanesulfonic acid allows ligand monomers of different molecular structures and sizes to be arranged and connected to the surface of the microsphere in different ways during the polymerization process, thereby increasing the number of ion exchange groups, achieving a higher ion exchange capacity, and increasing the adsorption load. DETAILED DESCRIPTION

[0031] In the examples of the present invention, if specific conditions are not specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products. Raw materials of different manufacturers and types do not affect the implementation of the technical solutions of the present invention and the achievement of the technical effects.

[0032] The triazole compounds in the following embodiments of the present invention have a structure as shown in formula (I):

[0033]

[0034] The CAS number of the triazole compound is 779354-77-7, which is a product of the prior art and can also be synthesized using the prior art.

[0035] Example 1

[0036] The cation exchange chromatography medium in this embodiment includes microspheres and ligand monomers grafted onto the surface of the microspheres;

[0037] The microspheres are styrene-divinylbenzene microspheres with a particle size of 50 μm. The ligand monomers include propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and a triazole compound. The mass ratio of propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and triazole compound is 1:20:15. The mass ratio of the microspheres to the ligand monomers is 60:1.

[0038] The method for preparing the cation exchange chromatography medium described in this embodiment comprises the following steps:

[0039] The microspheres were taken and added to water, and then the ligand monomer and initiator were added thereto, mixed evenly, and reacted at 40° C. for 4 h. The reaction mixture was filtered and the filtrate was washed with deionized water to obtain a cation exchange chromatography medium.

[0040] The initiator is cerium sulfate, the mass ratio of the initiator to the ligand monomer is 1:25, and the mass ratio of the microspheres to the water is 1:6.

[0041] Example 2

[0042] The cation exchange chromatography medium in this embodiment includes microspheres and ligand monomers grafted onto the surface of the microspheres;

[0043] The microspheres are styrene-divinylbenzene microspheres with a particle size of 100 μm. The ligand monomers include propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and a triazole compound. The mass ratio of propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and triazole compound is 1:50:8. The mass ratio of the microspheres to the ligand monomers is 40:1.

[0044] The method for preparing the cation exchange chromatography medium described in this embodiment comprises the following steps:

[0045] The microspheres were taken and added to water, and then the ligand monomer and initiator were added thereto, mixed evenly, and reacted at 60° C. for 6 h. The reaction mixture was filtered and the filtrate was washed with deionized water to obtain a cation exchange chromatography medium.

[0046] The initiator is ammonium cerium nitrate, the mass ratio of the initiator to the ligand monomer is 1:100, and the mass ratio of the microspheres to the water is 1:10.

[0047] Example 3

[0048] The cation exchange chromatography medium in this embodiment includes microspheres and ligand monomers grafted onto the surface of the microspheres;

[0049] The microspheres are styrene-divinylbenzene microspheres with a particle size of 60 μm. The ligand monomers include propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and a triazole compound. The mass ratio of propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and triazole compound is 1:30:1. The mass ratio of the microspheres to the ligand monomers is 20:1.

[0050] The method for preparing the cation exchange chromatography medium described in this embodiment comprises the following steps:

[0051] The microspheres were taken and added to water, and then the ligand monomer and initiator were added thereto, mixed evenly, and reacted at 50° C. for 3 h. The reaction mixture was filtered and the filtrate was washed with deionized water to obtain a cation exchange chromatography medium.

[0052] The initiator is cerium sulfate, the mass ratio of the initiator to the ligand monomer is 1:60, and the mass ratio of the microspheres to the water is 1:2.

[0053] Example 4

[0054] The cation exchange chromatography medium in this embodiment includes microspheres and ligand monomers grafted onto the surface of the microspheres;

[0055] The microspheres are styrene-divinylbenzene microspheres with a particle size of 80 μm. The ligand monomers include propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and a triazole compound. The mass ratio of propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and triazole compound is 1:40:10. The mass ratio of the microspheres to the ligand monomers is 50:1.

[0056] The method for preparing the cation exchange chromatography medium described in this embodiment comprises the following steps:

[0057] The microspheres were taken and added to water, and then the ligand monomer and initiator were added thereto, mixed evenly, and reacted at 50° C. for 6 h. The reaction mixture was filtered and the filtrate was washed with deionized water to obtain a cation exchange chromatography medium.

[0058] The initiator is cerium nitrate, the mass ratio of the initiator to the ligand monomer is 1:75, and the mass ratio of the microspheres to the water is 1:5.

[0059] Example 5

[0060] The cation exchange chromatography medium in this embodiment is the same as the cation exchange chromatography medium in Example 4, with the only difference being that the preparation method of the cation exchange chromatography medium further includes the step of adding dextrin palmitate; and the mass ratio of dextrin palmitate to the microspheres is 0.05:1.

[0061] The preparation method of the cation exchange chromatography medium of this embodiment comprises the following steps:

[0062] Take the microspheres, add them to water, first add dextrin palmitate thereto, mix evenly, then add the ligand monomer and initiator thereto, mix evenly, react at 50°C for 6 hours, filter the reaction mixture, and wash the filtrate with deionized water to obtain a cation exchange chromatography medium.

[0063] Example 6

[0064] The cation exchange chromatography medium in this embodiment is the same as the cation exchange chromatography medium in Example 4, with the only difference being that the preparation method of the cation exchange chromatography medium further includes the step of adding dextrin palmitate; and the mass ratio of dextrin palmitate to the microspheres is 0.07:1.

[0065] The preparation method of the cation exchange chromatography medium of this embodiment comprises the following steps:

[0066] Take the microspheres, add them to water, first add dextrin palmitate thereto, mix evenly, then add the ligand monomer and initiator thereto, mix evenly, react at 50°C for 6 hours, filter the reaction mixture, and wash the filtrate with deionized water to obtain a cation exchange chromatography medium.

[0067] Example 7

[0068] The cation exchange chromatography medium in this embodiment is the same as the cation exchange chromatography medium in Example 4, with the only difference being that the preparation method of the cation exchange chromatography medium further includes the step of adding dextrin palmitate; and the mass ratio of dextrin palmitate to the microspheres is 0.09:1.

[0069] The preparation method of the cation exchange chromatography medium of this embodiment comprises the following steps:

[0070] Take the microspheres, add them to water, first add dextrin palmitate thereto, mix evenly, then add the ligand monomer and initiator thereto, mix evenly, react at 50°C for 6 hours, filter the reaction mixture, and wash the filtrate with deionized water to obtain a cation exchange chromatography medium.

[0071] Example 8

[0072] The cation exchange chromatography medium in this embodiment is the same as the cation exchange chromatography medium in Example 4, with the only difference being that the preparation method of the cation exchange chromatography medium further includes the step of adding cyclodextrin; and the mass ratio of the cyclodextrin to the microspheres is 0.02:1.

[0073] The preparation method of the cation exchange chromatography medium of this embodiment comprises the following steps:

[0074] Take the microspheres, add them to water, first add cyclodextrin, mix them evenly, then add the ligand monomer and initiator, mix them evenly, react at 50°C for 6 hours, filter the reaction mixture, and wash the filtrate with deionized water to obtain a cation exchange chromatography medium.

[0075] Example 9

[0076] The cation exchange chromatography medium in this embodiment is the same as the cation exchange chromatography medium in Example 4, with the only difference being that the preparation method of the cation exchange chromatography medium further includes the steps of adding dextrin palmitate and cyclodextrin; the mass ratio of dextrin palmitate to the microspheres is 0.05:1; and the mass ratio of cyclodextrin to the microspheres is 0.02:1.

[0077] The preparation method of the cation exchange chromatography medium of this embodiment comprises the following steps:

[0078] Take the microspheres, add them to water, first add dextrin palmitate and cyclodextrin, mix them evenly, then add the ligand monomer and initiator, mix them evenly, react at 50°C for 6 hours, filter the reaction mixture, and wash the filtrate with deionized water to obtain a cation exchange chromatography medium.

[0079] Comparative Example 1

[0080] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, with the only difference being that the ligand monomer does not include the triazole compound.

[0081] Comparative Example 2

[0082] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, with the only difference being that the ligand monomer does not include propylene sulfonic acid.

[0083] Comparative Example 3

[0084] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, with the only difference being that the triazole compound is replaced by 4-allyl-4H-1,2,4-triazole.

[0085] The CAS number of the 4-allyl-4H-1,2,4-triazole is 99091-96-0, and it has a structure as shown in formula (IV):

[0086]

[0087] Comparative Example 4

[0088] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, with the only difference being that the triazole compound is replaced by 1-vinyl-1,2,4-triazole.

[0089] The CAS number of the 1-vinyl-1,2,4-triazole is 2764-83-2, and it has a structure as shown in formula (V):

[0090]

[0091] Comparative Example 5

[0092] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, except that the mass ratio of the propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and the triazole compound is 0.2:40:10.

[0093] Comparative Example 6

[0094] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, except that the mass ratio of the propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and the triazole compound is 4:40:10.

[0095] Comparative Example 7

[0096] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, except that the mass ratio of the propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and the triazole compound is 1:40:0.5.

[0097] Comparative Example 8

[0098] The cation exchange chromatography medium of this comparative example is the same as that of Example 4 and is prepared by the same method, except that the mass ratio of the propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and the triazole compound is 1:40:30.

[0099] Comparative Example 9

[0100] This comparative example is the same as the cation exchange chromatography medium of Example 9 and is prepared by the same method. The only difference is that in the preparation method of the cation exchange chromatography medium, the mass ratio of the dextrin palmitate to the microspheres is 0.12:1; the mass ratio of the cyclodextrin to the microspheres is 0.05:1.

[0101] The preparation method of the cation exchange chromatography medium of this embodiment comprises the following steps:

[0102] Take the microspheres, add them to water, first add dextrin palmitate and cyclodextrin, mix them evenly, then add the ligand monomer and initiator, mix them evenly, react at 50°C for 6 hours, filter the reaction mixture, and wash the filtrate with deionized water to obtain a cation exchange chromatography medium.

[0103] Effect Experiment Example

[0104] To verify the technical effect of the cation exchange chromatography medium of the present invention, the following experiments were performed:

[0105] The cation exchange chromatography medium obtained in Examples 1-9 and Comparative Examples 1-9 was loaded into a glass chromatography column with an inner diameter of 10 mm, washed with 20 mL of a 1.0 mol / L hydrochloric acid aqueous solution, and then washed with deionized water until the outflowing washing liquid was neutral; 50 mL of a 0.1 mol / L sodium hydroxide solution was accurately measured and added to the chromatography column, allowed to drip naturally and the alkali solution was collected, and then 20 mL of a 1.0 mol / L sodium chloride aqueous solution was added, allowed to drip naturally, and combined with the collected alkali solution to obtain a collected liquid; two drops of methyl orange indicator were added to the collected liquid, and the combined collected liquid was titrated with a 0.1 mol / L hydrochloric acid solution to calculate the ion exchange capacity of the medium.

[0106] The cation exchange chromatography medium obtained in Examples 1-4 and Comparative Examples 1-8 was loaded into a chromatographic column (medium volume of 1.0 mL). The loaded chromatographic column was connected to a liquid chromatograph, and the pump head was loaded with a low-salt loading solution (sodium chloride concentration of 35 mM, lysozyme concentration of 2 mg / mL) and a high-salt loading solution (sodium chloride concentration of 100 mM, lysozyme concentration of 2 mg / mL). The chromatographic conditions were as follows: flow rate 1 mL / min; mobile phase A (pH = 7.0, 50 mM phosphate buffer solution); mobile phase B (pH = 7.0, 1 mol / L NaCl, 50 mM phosphate buffer solution); first, the chromatographic column was equilibrated with mobile phase A for 10 column volumes (CV), then the protein solution was loaded onto the pump head until 100% flow-through was achieved, and then the adsorbed protein was eluted with mobile phase B. The corresponding chromatogram was recorded and the 5% flow-through value of the medium was calculated based on the flow-through curve to obtain the dynamic protein adsorption capacity of the medium.

[0107] After experimentation, the results are as follows:

[0108]

[0109]

[0110] According to the results of Examples 1-9 and Comparative Examples 1-9, the cation exchange chromatography medium of the present invention has a large ion exchange capacity, good salt resistance, and can achieve a high adsorption capacity.

[0111] According to the results of Example 4 and Comparative Examples 1, 3, and 4, the cation exchange chromatography medium of the present invention can significantly improve the salt tolerance of the chromatography medium by adding the triazole compound, although the ion exchange capacity will be slightly reduced. The compound in Comparative Example 3, although also having a triazole group, is a non-polar structure with relatively poor hydrophilicity, which has a greater negative impact on the ion exchange capacity and a limited effect on improving salt tolerance. The compound in Comparative Example 4, although polar, does not contain an ester bond, and its effect on the ion exchange capacity is relatively small, but the improvement in salt tolerance is also unsatisfactory.

[0112] The results of Examples 4 and 5-7 indicate that the addition of dextrin palmitate significantly improves the salt tolerance of the chromatography medium. This is likely due to the palmitate chains of dextrin palmitate adsorbing onto the microsphere surface through hydrophobic interactions, forming a steric barrier. This, upon reaction with the ligand monomers, inhibits microsphere aggregation, reduces ligand clustering, and evens out the charge distribution of the chromatography medium, thereby effectively improving its salt tolerance.

[0113] According to the results of Examples 5-7, 8-9 and Comparative Example 9, the addition of the cyclodextrin has a significant effect on the improvement of the ion exchange capacity, but has a relatively small effect on the salt tolerance of the chromatography medium. This may be due to the cyclodextrin having a polyhydroxyl ring structure, which can guide the spatial orderliness of the ligand monomer reaction and improve the ligand density. When the cyclodextrin and the dextrin palmitate are used in appropriate amounts (Example 9), the chromatography medium obtained has the best comprehensive performance. It can be seen that there is a certain synergistic effect between the cyclodextrin and the dextrin palmitate. However, when the cyclodextrin and the dextrin palmitate are used in excess (Comparative Example 9), the various properties of the chromatography medium obtained all decline.

[0114] According to the results of Example 4 and Comparative Examples 7 and 8, if the amount of the triazole compound used is too small, it is difficult to fully exert its effect of improving salt tolerance, and if the amount used is too large, the adsorption capacity will be greatly reduced.

[0115] The results of Example 4 and Comparative Examples 2, 5, and 6 indicate that the addition of propylene sulfonic acid has a minimal effect on the salt tolerance of the chromatography medium, but significantly increases the ion exchange capacity and adsorption loading, while weakening the effect of the triazole compound on adsorption. Using too little propylene sulfonic acid may result in poor adsorption, while using too much may lead to excessive ligand density, increased steric hindrance, and difficulty in achieving effective binding with the target substance.

[0116] It is understood from common knowledge in the art that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, illustrative only and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A cation exchange chromatography medium, characterized in that It includes microspheres and ligand monomers grafted onto the surface of the microspheres; The microspheres are styrene-divinylbenzene microspheres; and the ligand monomers include propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and triazole compounds.

2. The cation exchange chromatography medium according to claim 1, characterized in that The triazole compound has a structure as shown in formula (I):

3. The cation exchange chromatography medium according to claim 1, characterized in that The mass ratio of the propylene sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid and the triazole compound is 1:(20-50):(1-15).

4. The cation exchange chromatography medium according to claim 1, characterized in that The mass ratio of the microspheres to the ligand monomer is (20-60):

1.

5. The method for preparing a cation exchange chromatography medium according to any one of claims 1 to 4, characterized in that: The steps include: The microspheres are taken and added into water, and then the ligand monomer and initiator are added thereto, mixed evenly, and reacted to obtain a cation exchange chromatography medium.

6. The method for preparing a cation exchange chromatography medium according to claim 5, wherein The initiator is one of cerium sulfate and ammonium cerium nitrate.

7. The method for preparing a cation exchange chromatography medium according to claim 5, wherein The mass ratio of the initiator to the ligand monomer is 1:(25-100).

8. The method for preparing a cation exchange chromatography medium according to claim 5, wherein: The reaction temperature is 40-60° C. and the reaction time is 3-6 hours.

9. The method for preparing a cation exchange chromatography medium according to claim 5, wherein The mass ratio of the microspheres to the water is 1:(2-10).

10. The method for preparing a cation exchange chromatography medium according to claim 5, wherein: The method also includes the steps of filtering and washing the obtained cation exchange chromatography medium.

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