Chromatographic medium, preparation method and application thereof

Abstract

The present invention relates to the technical field of recombinant protein and antibody purification, and in particular to a chromatography medium, a preparation method thereof, and an application thereof. The chromatography medium is formed by coupling a matrix and a ligand; the structural formula of the chromatography medium is shown in Formula I: wherein: Q is a matrix; L1 is a spacer arm; R1 is a methyl group or an ethyl group; R2 is an aromatic group; R3 is a linear alkyl group substituted with -OH, a substituted aromatic group, or a substituted cycloalkyl group; L2 is NH, O, S, or a covalent bond; and n=1 or 2. The chromatography medium of the present invention has a functional ligand with a specific structure and has a high adsorption capacity for proteins, so that the chromatography medium has an ionic and hydrophobic composite mode of action, which helps to improve separation efficiency, reduce purification process steps, reduce contaminant levels, and greatly reduce production costs. Furthermore, it is possible to obtain a product that meets quality standards with as few chromatography steps as possible.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant protein and antibody purification, and in particular to a chromatography medium and a preparation method and application thereof. Background Art

[0002] In the production of recombinant proteins and antibodies, purifying the target protein to acceptable quality standards from large quantities of fermentation stock while maintaining overall process economics is a key consideration in large-scale purification. Current trends indicate that the use of chromatographic media for capture and separation purification is the most cost-effective solution.

[0003] Taking monoclonal antibodies as an example, the classic purification method often uses a three-step approach: affinity chromatography to capture the target protein, anionic ion exchange, and cation exchange. Therefore, there is a desire to develop a chromatography medium with a novel structural ligand that exhibits a combined ionic and hydrophobic mode of action, thereby reducing the number of purification steps, lowering contaminant levels, and significantly lowering production costs. Summary of the Invention

[0004] The present invention provides a chromatography medium and a preparation method and application thereof, which are used to solve the problem of complicated purification process in the separation and purification process of existing chromatography media.

[0005] According to a first aspect of the present invention, the present invention provides a chromatography medium, which is formed by coupling a matrix and a ligand;

[0006] The structural formula of the chromatography medium is shown in Formula I:

[0007]

[0008] Wherein: Q is a substrate; L1 is a spacer arm; R1 is a methyl group or an ethyl group; R2 is an aromatic group; R3 is a straight-chain alkyl group substituted with -OH, an aromatic group substituted with -OH, or a cycloalkyl group substituted with -OH; L2 is -NH-, -O-, -S-, or a covalent bond; n=1 or 2.

[0009] In the chromatography medium of the present invention, R1-N + R2 and R3 are positively charged groups that can bind anions, exerting anionic chromatography. The aromatic and cycloalkyl groups in R2 and R3 are hydrophobic groups that can bind hydrophobic substances, exerting hydrophobic chromatography. Therefore, the chromatography medium of the present invention possesses both anionic and hydrophobic chromatography properties. A higher pH and lower conductivity facilitate anionic binding, while a higher conductivity promotes hydrophobic binding. When the appropriate pH and conductivity are selected, antibody monomers can flow through, while impurities such as aggregates bind to the chromatography medium, thereby achieving the desired purity after a single purification step.

[0010] Further, the ligand is selected from 4-(methyl(phenoxymethyl)amine)-2-butanol, 4-(((3,5-dimethoxyphenoxy)methyl(methyl)amine)-2-butanol, 4-(((3,4-dimethoxyphenoxy)methyl(methyl)amine)-2-butanol, 4-(methyl((4-(trifluoromethyl)phenoxy)methyl)amine)-2-butanol, 4-(((4-methoxypyridine)methyl)amine)-2-butanol, 4-(methyl(naphthyloxymethyl)amine)-2-butanol, 4-(((3,5-dimethylphenoxy)methyl)(methyl)amine)-2-butanol, 4-(((3,5-diisopropylphenoxy)methyl) )(methyl)amine)-2-butanol, 4-(methyl((pentafluorophenoxy)methyl)amine)-2-butanol, 1-(methyl(2-methoxythiophene)amine)-2-butanol, 1-(methyl(2-methoxyfuran)amine)-2-butanol, 1-(methyl(phenoxymethyl)amine)-2-butanol, 4-(([1,1'-biphenyl]-4-methoxy)(methyl)amine)-2-butanol, 4-(((3,5-dimethoxyphenethyl)(methyl)amine)methyl)phenol, 4-((3,5-dimethoxyphenethyl)(methyl)amine)-1-cyclohexanol, 3-((3,5-dimethoxyphenethyl)(methyl)amine)-1 -cyclopentanol, 3-((3,5-dimethoxyphenethyl)(methyl)amine)-1-cyclobutanol, 4-(ethyl(phenethyl)amine)-2-butanol, 4-((3,5-difluorophenethyl)(ethyl)amine)-2-butanol, 4-((3,4-difluorophenethyl)(ethyl)amine)-2-butanol, 4-((3,4-dimethylphenethyl)(ethyl)amine)-2-butanol, 4-(((3,4-dimethoxyphenyl)(ethyl)amine)methyl)phenol, 4-((3,5-dimethoxyphenyl)(ethyl)amine)-1-cyclohexanol, 3-((3,5-dimethoxyphenyl)(ethyl)amine)-1-cyclopentanol, 3-(ethyl(4-methoxyphenyl)amine)-1-cyclobutanol, 1-(3,5-dimethylbenzyl)-3-hydroxypyrrolidine, 1-(3,5-dimethylbenzyl)-4-hydroxypiperidine, 1-(3,5-diethylbenzyl)-azetidin-3-ol, 1-(3,5-dimethoxybenzyl)-3-hydroxypyrrolidine, 1-(3,5-dimethoxybenzyl)-4-hydroxypiperidine, 1-(3,5-dimethoxybenzyl)-azetidin-3-ol, 1-([1,1'-biphenyl]-4-methyl)-4-hydroxypiperidine, and one or more of 1-(4-(trifluoromethyl)benzyl)-4-hydroxypiperidine.

[0011] Preferably, the ligand is selected from 4-(methyl(phenoxymethyl)amine)-2-butanol, 4-(((3,5-dimethylphenoxy)methyl)(methyl)amine)-2-butanol, 4-(((3,5-dimethoxyphenethyl)(methyl)amine)methyl)phenol, 4-(ethyl(phenethyl)amine)-2-butanol, 1-([1,1'-biphenyl]-4-methyl)-4-hydroxypiperidine, and 4-((3,4-dimethylphenethyl)(ethyl)amine)-2-butanol.

[0012] It can be understood that by rationally selecting the type of ligand, the chromatography medium of the present invention as shown in Formula I can be effectively prepared.

[0013] Furthermore, the straight-chain alkyl group in R3 is a straight-chain alkyl group of C1 to C4.

[0014] Furthermore, the cycloalkyl group in R3 is a four-membered aliphatic cycloalkyl group, a five-membered aliphatic cycloalkyl group or a six-membered aliphatic cycloalkyl group.

[0015] Furthermore, the spacer arm contains 2-8 carbon atoms and further includes one or more ether groups and / or one or more hydroxyl groups.

[0016] As can be understood, the introduction of a spacer arm on the substrate surface reduces steric hindrance for coupled macromolecules, improves their activity and utilization, and reduces nonspecific interactions between the macromolecule and the substrate surface. Spacers containing ether or hydroxyl groups react with molecules containing sulfhydryl, amino, or carboxyl groups, overcoming the substrate's inherent low reactivity.

[0017] Furthermore, the matrix is ​​agarose microspheres.

[0018] It can be understood that agarose microspheres, as a natural polysaccharide biological filler, are micron-sized spheres with a hollow network structure inside. They have the advantages of good biocompatibility, porosity, hydrophilicity and no charged groups. At the same time, the surface of agarose microspheres contains a large number of hydroxyl groups, which are easy to react chemically with many groups. Therefore, it is easier to modify their surface and bond with groups with different functions, making them suitable for different separation objects and separation requirements.

[0019] Furthermore, the ligand density of the chromatography medium is 20-85 μmol / ml wet gel.

[0020] Understandably, the antibody dynamic binding capacity of the chromatography medium within this density range is higher.

[0021] According to a second aspect of the present invention, the present invention also provides a method for preparing the above-mentioned chromatography medium, comprising the following steps:

[0022] (1) Microsphere activation: The substrate is reacted with an activator and a NaOH solution at 40-45°C for 15-20 hours. The initially activated microspheres obtained after the reaction are again added with water to form a suspension. Saturated bromine water is then added and reacted for 20-40 minutes to obtain an activated microsphere suspension.

[0023] (2) Coupling ligand: Add the ligand to the activated microsphere suspension, adjust the pH to 12-13.5, and then react at 40-45° C. for 15-20 hours.

[0024] It can be understood that the preparation method of the chromatography medium of the present invention first performs an initial activation of the matrix using an activator, then adds bromine to bromine the initially activated microspheres, and then adds a specific ligand to the activated microsphere suspension to form a chromatography medium with a structural formula as shown in Formula I. The preparation process is simple and low-cost. The prepared chromatography medium has an ionic and hydrophobic composite mode effect and can be used for chromatographic separation and purification of biomacromolecules such as recombinant proteins and antibodies, which helps to improve separation efficiency and effectively reduce the purification process steps.

[0025] Furthermore, the weight ratio of the matrix to the activator is (8-12):1, preferably 10:1.

[0026] It can be understood that by limiting the weight ratio of the substrate to the activating agent within a reasonable range, the activation efficiency of the substrate can be improved.

[0027] Furthermore, the activator is selected from one or more of allyl glycidyl ether, allyl bromide, and 4-vinylphenyl glycidyl ether.

[0028] It can be understood that by selecting a suitable type of activating agent, the substrate can be activated more effectively, thereby improving its coupling efficiency with the ligand.

[0029] Furthermore, the concentration of the NaOH solution is 1-3M, preferably 2M.

[0030] It can be understood that by selecting a suitable concentration of NaOH solution, the matrix can be activated more effectively, thereby improving the coupling efficiency between the matrix and the ligand.

[0031] Furthermore, the weight ratio of the initial activated microspheres to the bromine water is (90-120):1, preferably 100:1.

[0032] It can be understood that limiting the weight ratio of the initial activated microspheres to the bromine within a reasonable range of values ​​helps to improve the efficiency of the bromination process.

[0033] According to the third aspect of the present invention, the present invention also provides use of the above-mentioned chromatography medium in separating and purifying antibodies.

[0034] The novel chromatography medium provided by the present invention has a functional ligand with a specific structure, which has both cationic groups and hydrophobic groups. This allows the chromatography medium to have an anionic and hydrophobic composite mode of action, allowing monomers such as recombinant proteins and antibodies to flow through, while aggregates are bound to the chromatography medium, thereby achieving the purpose of effective separation in one-step chromatography. This can reduce the number of purification process steps, obtain products that meet quality standards, reduce the level of pollutants, and greatly reduce production costs. The monoclonal antibody expressed by CHO cells has a molecular weight of 150KD and an isoelectric point of approximately 8.0. After being captured by a protein A affinity chromatography medium (such as MaXtar ARPA from Biolink), the monomer purity is approximately 95%; after purification by the chromatography medium of the present invention in the flow-through mode, the monomer purity is increased to more than 99%, meeting the quality requirements of biopharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are 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.

[0036] Figure 1 is the SEC-HPLC spectrum of the sample after purification by the chromatographic medium of Example 7 of the present invention;

[0037] Figure 2 It is the SEC-HPLC spectrum of the sample after purification by the chromatography medium of the comparative example of the present invention. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0039] The ligands used in preparing the chromatography medium of the present invention are shown in Table 1 below.

[0040] Table 1

[0041]

[0042]

[0043]

[0044]

[0045] The synthesis methods of the ligands in Table 1 include method A, method B and method C, wherein the synthesis of ligand 1, ligand 18 and ligand 27 are used as examples to illustrate these three methods.

[0046] Method A: Taking ligand 1 as an example, the synthesis route is as follows:

[0047]

[0048] The specific steps include:

[0049] (1) Synthesis of intermediate 1:

[0050] To a 500ml three-necked flask, add N-hydroxymethylphthalimide (50g), phenol (26.6g), and THF (200ml). Stir and cool to 0°C to allow thorough mixing. Triphenylphosphine (148g) is then added to the reaction system. Once dissolved, a DEAD / THF solution (98.3g dissolved in 100ml THF) is added dropwise. The reaction mixture is stirred at 0°C for 6 hours.

[0051] The reaction mixture was poured into stirred ice water, followed by extraction with ethyl acetate. The organic phase was dried and the crude product was purified by column chromatography to obtain an off-white intermediate 1 (57 g).

[0052] (2) Synthesis of intermediate 2:

[0053] Add intermediate 1 (57 g) and anhydrous ethanol (500 ml) to a 1000 ml three-necked flask, start stirring, cool to 0°C, and mix thoroughly. Add hydrazine hydrate (140 g) dropwise to the reaction system, and then heat the reaction mixture to reflux for 4 hours.

[0054] After the reaction was completed, the system temperature was lowered to room temperature, and the product was extracted with dichloromethane. The organic phase was dried and spin-dried to obtain intermediate 2 (27 g), which was used directly in the next step without further purification.

[0055] (3) Synthesis of intermediate 3:

[0056] In a 500ml three-necked flask, intermediate 2 (27g), 3-hydroxybutyraldehyde (19.3g), and methanol (200ml) were added, stirred, and cooled to 0°C to allow thorough mixing. NaBH3CN (20.6g) was then added portionwise to the reaction system, and the reaction mixture was warmed to room temperature and stirred for 4 hours.

[0057] The reaction mixture was poured into stirred ice water, and then extracted with ethyl acetate. The organic phase was dried and the crude product was purified by column chromatography to obtain intermediate 3 (35 g) as an oil.

[0058] (4) Synthesis of ligand 1:

[0059] To a 500ml three-necked flask, intermediate 3 (35g), paraformaldehyde (32g), methanol (100ml), and dichloromethane (100ml) were added, stirred, and cooled to 0°C to allow thorough mixing. NaBH(OAc)3 (57g) was then added portionwise to the reaction system, and the reaction mixture was warmed to room temperature and stirred for 4 hours.

[0060] The reaction mixture was poured into stirred ice water, followed by extraction with dichloromethane. The organic phase was dried and the crude product was purified by column chromatography to obtain ligand 1 (34 g).

[0061] Method B: Taking ligand 18 as an example, the synthesis route is as follows:

[0062]

[0063] The specific steps include:

[0064] (1) Synthesis of intermediate 1:

[0065] To a 500ml three-necked flask, add phenylacetaldehyde (50g), 4-amino-2-butanol (37g), methanol (100ml), and dichloromethane (100ml). Stirring was initiated and the temperature was lowered to 0°C to allow thorough mixing. Acetic acid (1ml) was then added to the reaction system, followed by the addition of NaBH(OAc)3 (132g) in portions. The reaction mixture was then warmed to room temperature and stirred for 4 hours.

[0066] After the reaction, the mixture was poured into stirred ice water, and then extracted with dichloromethane. The organic phase was dried and the crude product was purified by column chromatography to obtain intermediate 1 (69 g).

[0067] (2) Synthesis of ligand 18:

[0068] To a 500ml three-necked flask, intermediate 1 (69g), 40% aqueous acetaldehyde solution (58ml), methanol (100ml), and dichloromethane (100ml) were added. Stirring was initiated and the mixture was cooled to 0°C to allow thorough mixing. Acetic acid (1ml) was then added to the reaction system, followed by the addition of NaBH(OAc)3 (113g) in portions. The reaction mixture was then allowed to warm to room temperature and stirred for 4 hours.

[0069] After the reaction, the mixture was poured into stirred ice water, and then extracted with dichloromethane. The organic phase was dried and the crude product was purified by column chromatography to obtain ligand 18 (70 g).

[0070] Method C: Taking ligand 27 as an example, the synthesis route is as follows:

[0071]

[0072] The specific steps include:

[0073] To a 500ml three-necked flask, add 4-hydroxypiperidine hydrochloride (50g), 3,5-dimethylbenzaldehyde (49g), methanol (100ml), and dichloromethane (100ml). Stir and heat to reflux until the solution becomes clear. Then, cool to room temperature, add acetic acid (1ml) and NaBH(OAc)3 (115g) to the reaction system. Heat the reaction mixture to 60°C for 4 hours, then cool to room temperature for 12-16 hours.

[0074] After the reaction, the mixture was poured into stirred ice water, and then extracted with dichloromethane. The organic phase was dried and the crude product was purified by column chromatography to obtain ligand 27 (67 g).

[0075] Examples 1-33

[0076] This embodiment provides a chromatography medium, which is formed by coupling a matrix and ligands 1 to 33 in Table 1. The specific preparation method is as follows:

[0077] (1) Microsphere activation: Agarose microspheres were reacted with allyl glycidyl ether and NaOH solution at 40-45°C for 18 hours, wherein the ratio of agarose microspheres to allyl glycidyl ether was 1 g:0.1 g, and the NaOH solution was 2 M. After the reaction, the microspheres were washed with water, and the resulting microspheres were again suspended in water. Saturated bromine water was added, and the ratio of microspheres to bromine was 1 g:0.01 g. The reaction was continued for 30 minutes, and the microspheres were washed with water to obtain an activated microsphere suspension.

[0078] (2) Coupling ligand: Add the ligand to the activated microsphere suspension, add NaOH solution (2M), adjust the pH to 13, and react the reaction mixture at 40-45°C for 18 hours. After the reaction is complete, rinse with water and set aside.

[0079] The obtained chromatography medium was subjected to a ligand density test, and the test method was as follows:

[0080] (1) Load the chromatography medium into the chromatography column and connect it to the peristaltic pump to perform the following experimental operations.

[0081] (2) Wash the chromatographic medium with 2-3 times 2 mol / L potassium chloride solution and 5 times ultrapure water, then replace it with 2-4 times 2 mol / L potassium nitrate solution. Collect the effluent, add potassium chromate indicator, and titrate with a calibrated 0.1 mol / L silver nitrate standard solution until the solution changes from yellow to brick red. Record the titration volume V of the silver nitrate standard solution consumed. AgNO3 . Each mL of filler

[0082] C AgNO3: concentration of the standard silver nitrate solution obtained by calibration, mol / L;

[0083] V AgNO3 : Volume of silver nitrate consumed in titration, mL;

[0084] V 填料 : Volume of the packing after compaction, mL.

[0085] The test results are shown in Table 1.

[0086] Comparative Example

[0087] Commonly used multi-step chromatography media include ion exchange and hydrophobic chromatography, and their structures are as follows:

[0088]

[0089] Experimental example

[0090] The chromatography medium of the embodiment of the present invention and the chromatography medium of the comparative example were respectively subjected to antibody purification experiments, and the specific steps were as follows:

[0091] Experiment 1: Using the chromatography medium in Example 7 of the present invention

[0092] The supernatant of a monoclonal antibody (molecular weight 150KD, isoelectric point of approximately 8.0) expressed in CHO cells was captured by protein A affinity chromatography medium (such as MaXtar ARPA from Biolink), inactivated at low pH, and captured by protein A affinity chromatography medium. The monomer purity was approximately 95%; the pH and conductivity of the supernatant were adjusted to: 20-50mM Tris-HCl, containing 100-300mM NaCl, pH 7-8. The column height was controlled at approximately 10cm, the retention time was 6min, the sample was loaded, and the flow-through sample was collected, which was the purified monoclonal antibody liquid sample. SEC-HPLC was used for detection, such as Figure 1 The figure shows the SEC-HPLC spectrum of the sample after purification by the chromatography medium of the embodiment of the present invention. It can be seen that the purity of the monoclonal antibody after purification by the chromatography medium of the embodiment of the present invention can reach 99.07%.

[0093] Experiment 2: Chromatographic medium using comparative example

[0094] The supernatant of a monoclonal antibody expressed in CHO cells was captured on a protein A affinity chromatography medium and then inactivated at low pH. The pH and conductivity of the supernatant were adjusted to 20-50 mM Tris-HCl, pH 7-8, with a column height of approximately 10 cm and a retention time of 6 minutes. Samples were then loaded and the flow-through sample was collected. The pH and conductivity of the collected solution were then adjusted to 20-50 mM Tris-HCl, 1 M Na2SO4, pH 7-8, with a column height of approximately 10 cm and a retention time of 6 minutes. Samples were then loaded and eluted with a buffer of 20-50 mM Tris-HCl, 0.2-0.4 M Na2SO4, pH 7-8. The eluted sample was collected to obtain the purified monoclonal antibody feed sample.

[0095] Use SEC-HPLC detection, such as Figure 2 The figure shows the SEC-HPLC spectrum of the sample after purification by the chromatography medium of the comparative example of the present invention. It can be seen that the purity of the monoclonal antibody after purification by the chromatography medium of the comparative example of the present invention can reach 96.02%.

[0096] Comparing Experiment 1 and Experiment 2, it was found that the one-step purification using the chromatography medium of the embodiment of the present invention in Experiment 1 could achieve a purity of 99.07%; this was superior to the purity (96.02%) achieved by the traditional three-step method using anionic and hydrophobic chromatography media in Experiment 2. Furthermore, the one-step purification method of the present invention only requires one chromatography column, the labor and time required for one operation, and has great advantages in terms of economic efficiency.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A chromatography medium, characterized in that It is formed by coupling the matrix and ligand; The structural formula of the chromatography medium is shown in Formula I: Wherein: Q is a substrate; L1 is a spacer arm; R1 is a methyl group or an ethyl group; R2 is an aromatic group; R3 is a straight-chain alkyl group substituted with -OH, an aromatic group substituted with -OH, or a cycloalkyl group substituted with -OH; L2 is -NH-, -O-, or -S-; and n=1 or 2.

2. The chromatography medium according to claim 1, characterized in that The straight-chain alkyl group in R3 is a C1 to C4 straight-chain alkyl group.

3. The chromatography medium according to claim 1, characterized in that The cycloalkyl group in R3 is a four-membered aliphatic cycloalkyl group, a five-membered aliphatic cycloalkyl group or a six-membered aliphatic cycloalkyl group.

4. The chromatography medium according to any one of claims 1 to 3, characterized in that The spacer arm contains 2 to 8 carbon atoms and further comprises one or more ether groups and / or one or more hydroxyl groups.

5. The chromatography medium according to claim 4, characterized in that The matrix is ​​agarose microspheres.

6. The chromatography medium according to claim 1, characterized in that The ligand density of the chromatography medium is 20-85 μmol / ml wet gel.

7. A chromatography medium, characterized in that It is formed by coupling the matrix and ligand; The structural formula of the chromatography medium is shown in Formula I: Wherein: Q is a substrate; L1 is a spacer arm; R1 is a methyl group or an ethyl group; R2 is an aromatic group; R3 is a linear alkyl group substituted with -OH, an aromatic group substituted with -OH, or a cycloalkyl group substituted with -OH; L2 is -NH-, -O-, -S-, or a covalent bond; n = 1 or 2; The ligand is selected from 4-(methyl(phenoxymethyl)amine)-2-butanol, 4-(((3,5-dimethoxyphenoxy)methyl(methyl)amine)-2-butanol, 4-(((3,4-dimethoxyphenoxy)methyl(methyl)amine)-2-butanol, 4-(methyl((4-(trifluoromethyl)phenoxy)methyl)amine)-2-butanol, 4-(((4-methoxypyridine)methyl)amine)-2-butanol, 4-(methyl(naphthyloxymethyl)amine)-2-butanol, 4-(((3,5-dimethylphenoxy)methyl)(methyl)amine)-2-butanol, 4-(((3,5-diisopropylphenoxy)methyl)(methyl)amine)-2-butanol, )amine)-2-butanol, 4-(methyl((pentafluorophenoxy)methyl)amine)-2-butanol, 1-(methyl(2-methoxythiophene)amine)-2-butanol, 1-(methyl(2-methoxyfuran)amine)-2-butanol, 1-(methyl(phenoxymethyl)amine)-2-butanol, 4-(([1,1'-biphenyl]-4-methoxy)(methyl)amine)-2-butanol, 4-(((3,5-dimethoxyphenethyl)(methyl)amine)methyl)phenol, 4-((3,5-dimethoxyphenethyl)(methyl)amine)-1-cyclohexanol, 3-((3,5-dimethoxyphenethyl)(methyl)amine)-1-cyclopentane alcohol, 3-((3,5-dimethoxyphenylethyl)(methyl)amine)-1-cyclobutanol, 4-(ethyl(phenylethyl)amine)-2-butanol, 4-((3,5-difluorophenylethyl)(ethyl)amine)-2-butanol, 4-((3,4-difluorophenylethyl)(ethyl)amine)-2-butanol, 4-((3,4-dimethylphenylethyl)(ethyl)amine)-2-butanol, 4-(((3,4-dimethoxyphenyl)(ethyl)amine)methyl)phenol, 4-((3,5-dimethoxyphenyl)(ethyl)amine)-1-cyclohexanol, 3-((3,5-dimethoxyphenyl)(ethyl)amine)-1-cyclopentanol, 3- One or more of (ethyl(4-methoxyphenyl)amine)-1-cyclobutanol, 1-(3,5-dimethylbenzyl)-3-hydroxypyrrolidine, 1-(3,5-dimethylbenzyl)-4-hydroxypiperidine, 1-(3,5-diethylbenzyl)-azetidin-3-ol, 1-(3,5-dimethoxybenzyl)-3-hydroxypyrrolidine, 1-(3,5-dimethoxybenzyl)-4-hydroxypiperidine, 1-(3,5-dimethoxybenzyl)-azetidin-3-ol, 1-([1,1'-biphenyl]-4-methyl)-4-hydroxypiperidine, and 1-(4-(trifluoromethyl)benzyl)-4-hydroxypiperidine.

8. The method for preparing the chromatography medium according to any one of claims 1 to 7, characterized in that: The steps include: (1) Microsphere activation: The substrate is reacted with an activator and a NaOH solution at 40-45°C for 15-20 hours. The initially activated microspheres obtained after the reaction are again added with water to form a suspension. Saturated bromine water is then added and reacted for 20-40 minutes to obtain an activated microsphere suspension. (2) Coupling ligand: Add the ligand to the activated microsphere suspension, adjust the pH to 12-13.5, and then react at 40-45° C. for 15-20 hours.

9. The preparation method according to claim 8, characterized in that The weight ratio of the matrix to the activator is (8-12):1; And / or, the activator is selected from one or more of allyl glycidyl ether, allyl bromide, and 4-vinylphenyl glycidyl ether; and / or, the concentration of the NaOH solution is 1-3 M; And / or, the weight ratio of the initial activated microspheres to the bromine water is (90-120):

1.

10. Use of the chromatography medium according to any one of claims 1 to 7 in separating and purifying antibodies.

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