Preparation method and application of a high-ligand strong hydrophobic packing

Abstract

The present invention discloses a preparation method and application of a high-ligand strong hydrophobic packing material. By adopting a preparation method of a high-ligand strong hydrophobic packing material, the method comprises the following steps: (1) providing a matrix I; (2) constructing an epoxy spacer arm on the surface of the matrix I to obtain a matrix II with epoxy groups on the surface; (3) constructing a primary hydrophobic group on the surface of the matrix II with epoxy groups to obtain a matrix III with primary hydrophobic groups on the surface; (4) constructing a secondary hydrophobic group on the surface of the matrix III with primary hydrophobic groups to obtain a high-ligand strong hydrophobic packing material. The technical solution of the obtained high-ligand strong hydrophobic packing material preferably solves the problems in the prior art that the packing load is low due to the low ligand density of the hydrophobic group and the binding force of the protein is weak due to the weak hydrophobic interaction force of the packing material when purifying proteins with weak hydrophobicity. Moreover, in the preparation process, high-cost raw materials are avoided, and the process is simple and stable.

Description

Technical Field

[0001] The present invention relates to the field of chromatography packing materials, and particularly to a preparation method and application of a high-ligand strong hydrophobic packing material. Background Art

[0002] A protein composed of a certain amino acid sequence is coiled into a specific spatial structure. Generally, the non-polar amino acid side chains that make up the protein are mostly distributed inside the molecule to form a hydrophobic core, while the polar amino acids are distributed in the hydrophilic environment on the surface. Analysis of the surface properties of some proteins with known structures shows that some hydrophobic groups also appear on the protein surface, making the protein surface have a certain degree of hydrophobicity. The amino acid residues distributed on the protein surface play a very important role in the biological functions and biophysical and biochemical properties of the molecule. The hydrophobicity of the surface of different proteins or the same protein in different solvent environments may be different. Different proteins have different hydrophobicity strengths, resulting in different binding forces with the column packing material.

[0003] Hydrophobic Interaction Chromatography (HIC) is a liquid chromatography method for separating biological macromolecules using a moderately hydrophobic stationary phase and an aqueous salt solution as the mobile phase. It uses the binding between the hydrophobic sites on the protein surface and the hydrophobic groups on the stationary phase to purify proteins, and is one of the commonly used means for protein separation.

[0004] Hydrophobic interaction chromatography has many advantages: (1) High activity recovery rate. Using a neutral or nearly neutral salt solution system as the mobile phase enables proteins that are easily inactivated under acidic and organic solvent conditions to be separated on hydrophobic chromatography in an active state with less inactivation. (2) Since an aqueous salt solution is used as the mobile phase, the physicochemical properties of many proteins that need to maintain biological activity can be studied under similar conditions, such as determining the salting-out concentration of proteins and studying the conformational change behavior of proteins in solution. (3) The cost of the mobile phase is low, greatly reducing the environmental pollution caused by the organic solvents used in reverse-phase chromatography.

[0005] Hydrophobic ligands are the most important part of the stationary phase in hydrophobic chromatography and are the primary consideration when selecting chromatography conditions. Common hydrophobic ligands are mainly alkyl and aromatic groups. For alkyl ligands, the chain length of the alkane determines the strength of the medium's hydrophobicity. Generally speaking, the longer the hydrocarbon chain, the stronger the hydrophobicity and the stronger the adsorption capacity. Aromatic ligands are more complex, exhibiting both hydrophobic interactions and, due to the conjugated structure of the benzene ring, showing mixed-mode separation behavior. Commonly used types of hydrophobic ligands mainly include phenyl, butyl, octyl, ethyl, etc. The strength of hydrophobicity in hydrophobic chromatography is also related to the ligand density, which refers to the degree to which hydrophobic groups occupy the activation sites of the matrix. An increase in ligand density can increase the adsorption capacity of the medium, but after reaching a certain level, due to steric hindrance between biomolecules, the adsorption tends to saturate.

[0006] In many traditional fields such as polyurethane foams, synthetic rubbers, and flame retardants, some functional hydrophobic materials basically choose substances containing triazine rings such as melamine or structures similar to triazine rings like THEIC (tris(2-hydroxyethyl)isocyanurate). Because firstly, these ring structures are all trifunctional compounds, and after modification, they can form relatively stable three-dimensional structures in the material and maintain their original chemical properties without change under extreme temperature or pH conditions. Secondly, the ring structures belong to non-polar groups, while water molecules are polar solvents, so they have a certain degree of hydrophobicity and can be made into various functional hydrophobic materials. Summary of the Invention

[0007] Object of the Invention: Based on this, for some proteins with relatively weak hydrophobicity, it is difficult for the proteins to bind to the packing material during the purification operation, and the purification is difficult. Therefore, it is necessary to prepare a high-ligand strong hydrophobic packing material to solve this problem.

[0008] Technical Solution: A preparation method of a high-ligand strong hydrophobic packing material according to the present invention includes the following steps:

[0009] (1) Provide a matrix I;

[0010] (2) Construct an epoxy spacer arm on the surface of matrix I to obtain matrix II with epoxy groups on its surface;

[0011] (3) Construct primary hydrophobic groups on the surface of matrix II with an epoxy spacer arm to obtain matrix III with primary hydrophobic groups on its surface;

[0012] (4) Construct secondary hydrophobic groups on the surface of matrix III with primary hydrophobic groups to obtain a high-ligand strong hydrophobic packing material.

[0013] In some embodiments, the matrix I in step (1) is a polymer matrix containing hydroxyl groups on its surface.

[0014] In some of these embodiments, in step (1), the polymer matrix I is selected from one of polymethacrylate microspheres and polystyrene-divinylbenzene microspheres. The microspheres can, under acidic conditions, through hydrolysis of the epoxy groups, obtain microspheres with hydroxyl groups on the surface.

[0015] In some of these embodiments, the step of constructing an epoxy spacer arm on the surface of matrix I in step (2) is: mixing the matrix I, an alkali catalyst, and an epoxide, and reacting to obtain matrix II with epoxy groups on the surface.

[0016] In some of these embodiments, the specific steps of constructing an epoxy spacer arm on the surface of matrix I in step (2) are: putting 100 g of the matrix I into a pressure-bearing closed container, then adding 30 - 180 g of an epoxide, mixing evenly, and then adding 5 - 100 mL of a 0.2 - 2 mol / L NaOH solution to the material; setting the temperature in the pressure-bearing closed container to 25 - 90 °C, and the reaction time to 2 - 24 h; after the reaction, washing and drying the solvent by suction to obtain matrix II with an epoxy spacer arm on the surface. Preferably, after the reaction, it is washed with one or several of ethanol, pure water, distilled water, deionized water, or water for injection.

[0017] In some of these embodiments, the epoxide is one or several of epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, bis(2-glycidylpropyl) ether, and dipropylene glycol diglycidyl ether.

[0018] In some of these embodiments, the step of constructing a primary hydrophobic group on the surface of matrix II with an epoxy spacer arm in step (3) is: mixing the matrix II, an alkaline solution, and a primary hydrophobic group compound, and carrying out an epoxy ring-opening reaction at 25 - 90 °C, and then obtaining matrix III with a primary hydrophobic group on the surface through filtration and washing; wherein, the alkaline solution includes sodium hydroxide, sodium carbonate, or sodium bicarbonate solution. The washing is with water, and preferably, pure water is used for washing.

[0019] In some of these embodiments, the specific steps of constructing a primary hydrophobic group on the surface of matrix II with an epoxy spacer arm in step (3) are: putting 100 g of the matrix II into a pressure-bearing closed container, then adding 10 - 40 mL of a 0.1 - 1 mol / L sodium bicarbonate solution and 5 - 40 g of a primary hydrophobic group compound, reacting for 3 - 24 h, with the reaction temperature at 25 - 90 °C, and then filtering and washing the obtained reaction product to remove the unreacted primary hydrophobic group compound, thus obtaining matrix III with a primary hydrophobic group on the surface.

[0020] In some of these embodiments, the primary hydrophobic group compound is a trifunctional compound, selected from one or more of melamine, cyanuric acid, trichlorotriazine, hexamethylol melamine, 1,3,5-tris(2-hydroxyethyl) cyanurate, and 1,3,5-triglycidyl-s-triazine trione.

[0021] In some of these embodiments, the step of constructing a secondary hydrophobic group on the surface of matrix III having a primary hydrophobic group in step (4) is as follows: Mix the matrix III, an alkaline solution, and a secondary hydrophobic group compound evenly, and carry out an epoxy ring-opening reaction at 25-90 °C, and then obtain a high-ligand strong hydrophobic packing through filtration and washing; wherein, the alkaline solution includes sodium hydroxide, sodium carbonate, or sodium bicarbonate solution. The washing is carried out with water, and preferably, pure water is used for washing.

[0022] In some of these embodiments, the specific steps of constructing a secondary hydrophobic group on the surface of matrix III having a primary hydrophobic group in step (4) are as follows: Put 100 g of the matrix III into a pressure-bearing closed container, and then add 10-40 mL of 0.1-1 mol / L sodium bicarbonate solution and 5-50 g of a secondary hydrophobic group compound, react for 3-24 h at a reaction temperature of 25-90 °C, and then filter and wash the obtained reaction product to remove the unreacted secondary hydrophobic group compound, thereby obtaining a high-ligand strong hydrophobic packing.

[0023] In some of these embodiments, the secondary hydrophobic group compound is one of phenyl glycidyl ether and alkyl glycidyl ether, and the chemical structural formula of the alkyl glycidyl ether is:

[0024]

[0025] Wherein R is an alkyl group of C1-C16, and the alkyl group is selected from a straight-chain alkyl group or a branched-chain alkyl group with or without a substituent group; the substituent group is selected from a halogen, a nitro group, a C1-C4 alkyl group, a C1-C4 haloalkyl group, an aromatic group, a silicon group, a fatty acyl group, and a sulfur group.

[0026] Alkyl group: The alkyl group is composed of carbon and hydrogen atoms, and they are usually non-polar, so they are typical hydrophobic groups. For example, methyl (-CH3), ethyl (-C2H5), etc.

[0027] Aromatic group: The aromatic ring can also be hydrophobic, especially when it has no substituent group. Typical aromatic groups include a benzene ring (C6H5), etc.

[0028] Silicon group: The silicon group usually has high hydrophobicity. For example, a methyl silicon group (-Si(CH3)3).

[0029] Halogen groups: Halogens such as chlorine (Cl), bromine (Br), and iodine (I) are generally regarded as hydrophobic groups because their interaction with water is relatively weak.

[0030] Fatty acyl groups: Fatty acyl groups are groups related to fatty acids, such as methyl acyl group (-COCH3).

[0031] Sulfur groups: Sulfur groups in some sulfur compounds, such as alkylthio groups (-SR) or arylthio groups (-SAr), are also regarded as hydrophobic groups.

[0032] Nitro group (-NO2): The nitro group contains nitrogen and oxygen atoms, and the electronegativity of both nitrogen and oxygen is relatively high. Therefore, there is a strong electronegativity difference between the atoms in the nitro group, making the nitro group partially polar and generally tending to exhibit hydrophobic properties.

[0033] Preferably, the alkyl glycidyl ether is selected from butyl glycidyl ether and octyl glycidyl ether.

[0034] Preferably, the pressure-bearing closed container is selected from reaction kettles.

[0035] The present invention provides a high-ligand strongly hydrophobic filler prepared by the above preparation method.

[0036] Application of the high-ligand strongly hydrophobic filler in the separation and purification of proteins.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) A high-ligand strongly hydrophobic filler provided by the present invention has a polymer matrix as the matrix, with a large number of hydroxyl groups on the surface and good hydrophilicity. Therefore, all reaction conditions can be carried out in the aqueous phase, which is more environmentally friendly. (2) The chemical structure of the epoxy spacer arm on the microscopic surface of the high-ligand strongly hydrophobic filler reduces the influence of intermolecular steric hindrance and increases the probability of capturing proteins. The primary hydrophobic groups and secondary hydrophobic groups act together, and the primary hydrophobic groups are all trifunctional compounds, increasing the ligand density of the filler and enhancing the hydrophobic strength of the filler, which is beneficial to the adsorption of proteins with relatively weak hydrophobicity. (3) The high-ligand strongly hydrophobic filler of the present invention has excellent comprehensive performance and is particularly suitable for the separation and purification of proteins with relatively weak hydrophobicity in the feed liquid. The preparation method is simple, the raw materials are widely available, and it has broad application prospects. Description of the Drawings

[0038] Figure 1 Synthesis route of the filler in Example 1;

[0039] Figure 2 Dynamic loading test of the filler in Example 1. Detailed Embodiments

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the related listed items.

[0042] The following are the specific embodiments:

[0043] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available.

[0044] Example 1

[0045] This example provides a high-ligand strong hydrophobic packing material and its preparation method.

[0046] The polymethacrylate microspheres used in this example have the model Monomix MC60 (product number: 283960951, particle size of 60 μm, pore size of 100 nm, a large number of hydroxyl groups on the surface, and white in color), and the manufacturer is Suzhou Sepax Technologies Co., Ltd. The rest of the raw materials are all commercially available.

[0047] Accurately weigh 100 g of polymethacrylate microspheres with a balance, transfer them to a 500 mL reaction kettle, add 85 g of epichlorohydrin, then add 100 mL of 0.75 M NaOH solution, and carry out mechanical stirring reaction at a temperature of 42 °C and 100 rpm for 5 h. After the reaction is completed, the reaction solution is dried by suction with a Buchner funnel, and then the solid product is washed 3 times with absolute ethanol and washed with deionized water until neutral to obtain polymethacrylate microspheres with epoxy group spacer arms on the surface.

[0048] Take the polymethacrylate microspheres with epoxy spacer arms on the surface obtained in the above steps (i.e., Matrix II). Put 100 g of the Matrix II into a reaction kettle, then add 20 mL of 0.25 mol / L sodium bicarbonate solution and 10 g of the primary hydrophobic group compound melamine, and react for 18 h at a reaction temperature of 50 °C. Then filter and wash the reactants clean to remove the unreacted melamine. Next, add the solid obtained from the previous filtration and washing into a 500 mL reaction kettle, add 15 mL of 0.25 mol / L sodium bicarbonate solution and 15 g of the secondary hydrophobic group compound phenyl glycidyl ether, and react for 12 h at a reaction temperature of 45 °C. Then filter and wash the reactants clean to remove the unreacted phenyl glycidyl ether, thus obtaining a high-ligand strongly hydrophobic packing material. The specific synthesis route is as Figure 1 shown.

[0049] Example 2

[0050] This example provides a high-ligand strongly hydrophobic packing material and a preparation method thereof.

[0051] The preparation method is basically the same as that of Example 1, except that the epoxy compound is replaced by 1,4-butanediol diglycidyl ether, the primary hydrophobic group compound is replaced by 1,3,5-tris(2-hydroxyethyl) cyanuric acid, and the secondary hydrophobic group compound is replaced by tetradecyl glycidyl ether, obtaining a high-ligand strongly hydrophobic packing material.

[0052] Example 3

[0053] This example provides a high-ligand strongly hydrophobic packing material and a preparation method thereof.

[0054] The preparation method is basically the same as that of Example 1, except that the polymethacrylate microspheres, model Monomix MC60 (product number: 283960951, particle size 60 μm, pore size 100 nm, manufacturer Suzhou Sepax Technologies Co., Ltd.) are replaced by polymethacrylate microspheres, model Monomix MC30 (product number: 283930951, particle size 30 μm, pore size 100 nm, manufacturer Suzhou Sepax Technologies Co., Ltd.), the primary hydrophobic group compound is replaced by 1,3,5-triglycidyl-S-triazine trione, and the secondary hydrophobic group compound is replaced by butyl glycidyl ether, obtaining a high-ligand strongly hydrophobic packing material.

[0055] Example 4

[0056] This example provides a high-ligand strongly hydrophobic packing material and a preparation method thereof.

[0057] The preparation method is basically the same as that of Example 1, except that the epoxide is replaced by 1,2-cyclohexanediol diglycidyl ether, the primary hydrophobic group compound is replaced by hexahydroxymethyl melamine, and the secondary hydrophobic group compound is replaced by octyl glycidyl ether, to obtain a high-ligand strongly hydrophobic packing material.

[0058] Example 5

[0059] This example provides a high-ligand strongly hydrophobic packing material and a preparation method thereof.

[0060] The preparation method is basically the same as that of Example 1, except that the epoxide is replaced by ethylene glycol diglycidyl ether, and the primary hydrophobic group compound is replaced by trichlorotriazine, to obtain a high-ligand strongly hydrophobic packing material.

[0061] Comparative Example 1

[0062] The difference between this comparative example and Example 1 is that the primary hydrophobic group is not constructed, and the hydrophobic ligand phenyl glycidyl ether is directly coupled. The specific steps are as follows:

[0063] The polymethacrylate microspheres used in this example have the model of Monomix MC60 (product number: 283960951, particle size of 60 μm, pore size of 100 nm, containing a large number of hydroxyl groups on the surface, and white in color); the manufacturer is Suzhou Sepax Technologies Co., Ltd. The rest of the raw materials are commercially available.

[0064] Accurately weigh 100 g of polymethacrylate microspheres with a balance, transfer them to a 500 mL reaction kettle, add 85 g of epichlorohydrin, then add 100 mL of 0.75 M NaOH solution, and carry out mechanical stirring reaction at a temperature of 42 °C and 100 rpm for 5 h. After the reaction is completed, filter the reaction solution with a Buchner funnel, and then wash the solid product 3 times with absolute ethanol and wash it with deionized water until neutral. Polymethacrylate microspheres with epoxy group spacer arms on the surface are obtained.

[0065] Weigh 100 g of the polymethacrylate microspheres with epoxy group spacer arms obtained in the above step and put them into a reaction kettle, then add 80 mL of 0.5 mol / L sulfuric acid solution, react for 6 h at a reaction temperature of 55 °C to hydrolyze the epoxy groups on the sphere surface into hydroxyl groups, and then filter and wash the reactants clean. Then add the obtained solid into a 500 mL reaction kettle, add 15 mL of 0.25 mol / L sodium bicarbonate solution and 15 g of the hydrophobic group compound phenyl glycidyl ether, and react for 12 h at a reaction temperature of 45 °C. Then filter and wash the reactants clean to remove the unreacted phenyl glycidyl ether, and the ordinary hydrophobic packing material of Comparative Example 1 is obtained.

[0066] Comparative Example 2

[0067] Compared with Comparative Example 1, the preparation method of Comparative Example 2 is basically the same, except that the epoxy spacer arm is not attached to the surface of the microspheres, and the hydrophobic compound phenyl glycidyl ether is directly coupled using the hydroxyl groups on the surface of the spheres. The specific steps are as follows:

[0068] The polymethacrylate microspheres used in this example are of the Monomix MC60 type (product number: 283960951, particle size 60 μm, pore size 100 nm, with a large number of hydroxyl groups on the surface, and white in color); the manufacturer is Suzhou Sepax Technologies Co., Ltd. The rest of the raw materials are all commercially available.

[0069] Accurately weigh 100 g of polymethacrylate microspheres with a balance, transfer them to a 500 mL reaction kettle, then add 15 mL of 0.25 mol / L sodium bicarbonate solution and 15 g of the hydrophobic group compound phenyl glycidyl ether, and react for 12 h at a reaction temperature of 45 °C. Then filter and wash the reactants thoroughly to remove the unreacted phenyl glycidyl ether, and the ordinary hydrophobic packing material of Comparative Example 2 is obtained.

[0070] Dynamic loading test

[0071] Ribonuclease A is an enzyme with relatively weak hydrophobicity and it is very difficult to bind to the column when purified with ordinary hydrophobic packing materials, and the loading capacity is relatively low. The dynamic loading test of ribonuclease A was carried out using a high-ligand strong hydrophobic packing material prepared in Example 1 of the present invention. The test method and results are as follows:

[0072] Chromatography column: Pack a 6.6 * 30 mm chromatography column with a high-ligand strong hydrophobic packing material prepared in Example 1

[0073] Instrument: FPLC

[0074] Equilibration buffer A: 100 mM PB + 2 M (NH4)2SO4, pH 7.0

[0075] Elution buffer B: 100 mM PB, pH 7.0

[0076] Sample: 6 mg / mL ribonuclease A (purchased externally)

[0077] Column temperature: Room temperature

[0078] Test conditions: Use 10 column volumes of the mobile phase equilibration buffer A to equilibrate the system and the chromatography column, and load the sample after the baseline returns to zero; then calculate the dynamic loading according to the 10% protein breakthrough dynamic loading calculation formula, and the formula is as follows: Dynamic loading = (the time corresponding to the peak value of the 10% breakthrough signal * flow rate - 0.6) * protein concentration / column volume.

[0079] The test results are as Figure 2As shown in the figure, the dynamic loading capacity of the high-ligand strong hydrophobic packing was calculated according to the formula to be 28.62 mg / mL.

[0080] The same method was used to test the dynamic loading capacities of the high-ligand strong hydrophobic packing in Examples 2 to 5 and the ordinary hydrophobic packing in Comparative Examples 1 to 2. The results are shown in Table 1.

[0081] Table 1

[0082]

[0083] From the performance data in Table 1, it can be seen that for the high-ligand strong hydrophobic packing prepared in Examples 1 to 5 of the present invention application, in terms of the dynamic loading capacity of ribonuclease A, it can reflect the strength of the packing's binding ability. The higher the loading capacity, the stronger the binding ability; the lower the loading capacity, the weaker the binding ability, and the faster the ribonuclease A breaks through. It can be clearly seen from the experimental data that by constructing primary and secondary hydrophobic groups on the surface of the microspheres, the hydrophobicity of the packing becomes stronger and the binding ability with proteins is stronger. While the ordinary hydrophobic packing has only one hydrophobic functional group, with weak hydrophobic strength and is not easy to bind proteins. Therefore, it can be seen that the high-ligand strong hydrophobic packing has obvious advantages in terms of ligand density and hydrophobic strength, can capture proteins with relatively weak hydrophobicity, bringing new opportunities and choices to the biopharmaceutical industry. The preparation method of this packing is simple and has broad application prospects.

Claims

1. A preparation method of a high-ligand and strongly hydrophobic filler, characterized in that, It includes the following steps: (1) Provide a matrix Ⅰ; (2) Construct an epoxy spacer arm on the surface of matrix Ⅰ to obtain matrix Ⅱ with epoxy groups on the surface; (3) Construct a primary hydrophobic group on the surface of matrix Ⅱ with an epoxy spacer arm to obtain matrix Ⅲ with a primary hydrophobic group on the surface; the primary hydrophobic group compound is a trifunctional compound, selected from one or more of melamine, cyanuric acid, trichlorotriazine, hexamethylolmelamine, 1,3,5-tris(2-hydroxyethyl) cyanurate, 1,3,5-triglycidyl-S-triazinetrione; (4) Construct a secondary hydrophobic group on the surface of matrix Ⅲ with a primary hydrophobic group to obtain a high-ligand strongly hydrophobic packing material; the secondary hydrophobic group compound is one of phenyl glycidyl ether and alkyl glycidyl ether, and the chemical structural formula of the alkyl glycidyl ether is: ； where R is an alkyl group of C1-C16, and the alkyl group of C1-C16 is selected from a straight-chain alkyl group or a branched-chain alkyl group with or without a substituent group; the substituent group is selected from a halogen, a nitro group, a C1-C4 alkyl group, a C1-C4 haloalkyl group, an aromatic group, and a fatty acyl group.

2. The preparation method of the high-ligand strong hydrophobic packing according to claim 1, characterized in that, The matrix Ⅰ is a polymer matrix with hydroxyl groups on the surface; the polymer matrix Ⅰ is selected from one of polymethacrylate microspheres and polystyrene-divinylbenzene microspheres.

3. The preparation method of the high-ligand strong hydrophobic packing according to claim 1, characterized in that The step of constructing an epoxy spacer arm on the surface of matrix Ⅰ in step (2) is: mixing the matrix Ⅰ, an alkali catalyst, and an epoxide, and reacting to obtain matrix Ⅱ with epoxy groups on the surface.

4. The preparation method of the high-ligand strong hydrophobic packing according to claim 3, wherein The epoxide is one or more of epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, bis(2-glycidyl) ether, and dipropylene glycol diglycidyl ether.

5. The preparation method of the high-ligand strong hydrophobic packing according to claim 1, wherein, The step of constructing a primary hydrophobic group on the surface of matrix Ⅱ with an epoxy spacer arm in step (3) is: mixing the matrix Ⅱ, an alkaline solution, and a primary hydrophobic group compound, and uniformly mixing to carry out an epoxy ring-opening reaction at 25-90°C, and then obtaining matrix Ⅲ with a primary hydrophobic group on the surface through filtration and washing.

6. The preparation method of the high ligand strong hydrophobic packing according to claim 1, wherein The step of constructing a secondary hydrophobic group on the surface of matrix Ⅲ with a primary hydrophobic group in step (4) is: mixing the matrix Ⅲ, an alkaline solution, and a secondary hydrophobic group compound, and uniformly mixing to carry out an epoxy ring-opening reaction at 25-90°C, and then obtaining a high-ligand strongly hydrophobic packing material through filtration and washing.

7. A packing with high ligand and strong hydrophobicity, characterized in that, It is prepared by the preparation method of the high-ligand strongly hydrophobic packing material according to any one of claims 1-6.

8. Application of the high-ligand strongly hydrophobic packing material according to claim 7 in the separation and purification of proteins.

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