Preparation method and application of high ion exchange capacity and high loading gel filtration medium

Abstract

The application discloses a preparation method and application of a high-ionic exchange capacity and high-loading gel filtration medium. The gel filtration medium is prepared by an allyl activation-bromine activation-dextran grafting-epoxy secondary activation-ion ligand coupling method. The ionic exchange capacity and dynamic loading of the gel filtration medium are significantly greater than those of other similar media. The bromination end point control process is used to ensure that the activation levels of different batches of media are consistent, the batch-to-batch stability of the product is improved, the secondary activation uses epoxy activation to reduce the use of bromine, raw materials are more easily obtained, the operation process is safer, and the generation of waste water and waste gas is reduced.

Description

Technical Field

[0001] This invention belongs to the field of high performance liquid chromatography packing materials technology, specifically relating to a method for preparing and applying a gel filtration medium with high ion exchange capacity and high loading capacity. Background Technology

[0002] Ion exchange chromatography media are core separation materials for the industrial purification of biomacromolecules such as antibodies and recombinant proteins. Polysaccharide-based microspheres, with their excellent hydrophilicity and biocompatibility, are currently the most widely used chromatography matrix. The industry commonly employs an allyl-bromination activation process to modify agarose matrix, thereby binding functional ligands to prepare ion exchange packing materials.

[0003] Existing classic preparation routes are mainly divided into two categories. The first is a one-step activation process: the matrix is ​​modified with allyl glycidyl ether, brominated with bromine, and then directly coupled with ionic ligands. A representative technology is the preparation scheme disclosed in patent CN116262222A. This process is concise, but the activation sites are only distributed on the surface of the microspheres and inside the shallow pores, the number of functional groups that can be bonded is limited, and the dynamic adsorption capacity of the medium is relatively low; at the same time, the ligand distribution is uneven, and the non-specific adsorption is relatively high, making it difficult to meet the requirements of high-load purification.

[0004] The second improvement involves grafting dextran after bromination activation, relying on the long polysaccharide chain to increase reaction sites, a typical example being the high-load agarose chromatography medium disclosed in patent CN105498701B. This patent grafts dextran after matrix bromination, but subsequently uses a second allyl-bromination activation method to prepare the metal chelate medium, without setting an independent epoxy activation step on the dextran backbone. Most existing patents of the same type directly perform ligand coupling after dextran grafting, resulting in the inefficient activation of the large number of hydroxyl groups on the polysaccharide side chains, insufficient release of the site gain brought by dextran grafting, and limited ligand utilization.

[0005] Furthermore, most existing bromination processes only describe the addition of bromine water to initiate the bromination reaction, lacking quantifiable endpoint control methods. The degree of bromination is highly susceptible to fluctuations due to human intervention, directly resulting in inconsistent activation levels between different batches and poor batch-to-batch product stability. None of the aforementioned existing technologies employ a complete tandem process: allyl activation - bromine activation - dextran grafting - epoxy secondary activation - ionic ligand coupling. Either a dextran grafting compatibilization step is missing, or the dextran modification is followed by secondary bromine activation instead of controlled epoxy activation. This fails to construct a large number of uniformly distributed epoxy active sites on the polysaccharide chain, ultimately resulting in insufficient capacity enhancement, mediocre mass transfer performance, and poor batch repeatability.

[0006] As the biopharmaceutical industry continues to expand, industrial purification places higher demands on the protein adsorption capacity, separation precision, and batch stability of chromatographic media. Therefore, developing a method for preparing ion-modified chromatographic matrices that features stepwise multi-stage activation, controllable bromination processes, and full utilization of the advantages of dextran backbone sites has significant engineering application value. Summary of the Invention

[0007] 1. Technical problems to be solved The purpose of this invention is to solve the problems of low protein adsorption capacity, poor separation accuracy, and low batch stability in the protein purification process, and to provide a gel filtration medium with high ion exchange capacity and high loading capacity. It also aims to develop a method for preparing an ion-modified chromatography matrix that features stepwise multi-stage activation, controllable bromination process, and full utilization of the advantages of dextran backbone sites.

[0008] 2. Technical Solution

[0009] To achieve this objective, the present invention adopts the following technical solution: A method for preparing and applying a gel filter medium with high ion exchange capacity and high loading capacity, characterized in that the preparation method includes the following steps: S1. Allyl activation: Add cross-linked agarose matrix to a 20%-60% sodium hydroxide solution with a mass equal to 0.5-2 times the mass of the cross-linked agarose matrix, and react at 40-50℃ for 1-2 hours. Then add anhydrous sodium sulfate with a mass equal to 0.1-0.3 times the mass of the cross-linked agarose matrix and allyl glycidyl ether with a mass equal to that of sodium hydroxide, and continue the reaction at 20-80℃ for 8-28 hours. After the reaction is complete, wash with pure water and dry to obtain the allyl activated matrix. S2. Bromine activation: Add the allyl activated matrix obtained in S1, 0.5-2 times the mass of the allyl activated matrix of pure water, and 0.05-0.1 times the mass of the allyl activated matrix of sodium acetate to a reaction flask. Keep the mixture warm at 25°C and stir for 5-10 min. Then slowly add liquid bromine dropwise until the solution turns pale yellow and does not change color within 5 min. Continue the reaction for 1 h. Add sodium formate until the solution turns milky white. Continue the reaction for 0.5 h. After the reaction is complete, wash with pure water and dry to obtain the bromine activated matrix. S3. Dextran grafting: The bromine-activated matrix obtained in S2, 0.5-2 times the mass of the bromine-activated matrix of dextran, and 2-5 times the mass of the bromine-activated matrix of pure water are thoroughly mixed and reacted at 40-50℃ for 1-2 hours. Then, 1-3 times the mass of the bromine-activated matrix of 1-5M sodium hydroxide solution is added and reacted at 30-60℃ for 8-28 hours. After the reaction is completed, the mixture is washed with pure water and dried to obtain the dextran grafted matrix. S4. Secondary Activation of Epoxy: Take the dextran graft matrix obtained in S3, add pure water of the same mass as the dextran graft matrix, catalyst of 0.0009-0.005 times the mass of the dextran graft matrix, and activator of 0.1-0.3 times the mass of the dextran graft matrix, mix, remove oxygen with nitrogen, react at 30-40℃ for 3-5 hours, wash with pure water after the reaction, and dry to obtain the epoxy activated matrix;

[0010] S5. Ionic ligand coupling: Take the epoxy activated matrix obtained in S4, put it into a large reaction vessel, add 1-5 times the mass of the epoxy activated matrix and a sodium hydroxide solution with a concentration of 5-20M, and slowly add 1-3 times the mass of the epoxy activated matrix and a ligand solution with a concentration of 20-35M. React at 30-40℃ for 15-20h. After the reaction is completed, wash with pure water and dry to obtain the ion-modified matrix.

[0011] In step S4, the catalyst is cerium ammonium sulfate, cerium sulfate, or cerium ammonium nitrate.

[0012] In step S4, the activator is epichlorohydrin, allyl glycidyl ether, 1,4-butanediol diglycidyl ether, glycidyl methacrylate, or ethylene glycol methacrylate.

[0013] In step S5, the ligands are chloroacetic acid, 3-mercaptopropionic acid, bromoacetic acid, sodium metabisulfite, trimethylamine hydrochloride, and diethylaminochloroethane hydrochloride.

[0014] The high ion exchange capacity and high loading capacity gel filter medium obtained by the preparation method described above is used for the purification of vancomycin.

[0015] 3. Beneficial effects

[0016] (1) This invention involves grafting an epoxy structure onto the surface of a glucan medium and then modifying it. After epoxy activation, the activity of the microspheres increases, enhancing the reactivity between the microspheres and the ligands. Simultaneously, the elongated spacer arms reduce the steric hindrance of the agarose microspheres and increase the number of active protein sites, resulting in a gel filtration medium with significantly higher ion exchange capacity and dynamic loading than other similar media.

[0017] (2) The bromination endpoint control process can quantitatively control the reaction endpoint, reduce the fluctuation of the degree of bromination due to human operation, ensure that the activation level of the medium in different batches is consistent, and improve the stability between product batches.

[0018] (3) Secondary activation uses epoxy activation to reduce the use of bromine, the raw materials are more readily available, the operation process is safer, and the generation of wastewater and waste gas is reduced. Attached Figure Description

[0019] Figure 1The particle size distribution diagrams are for the gel filter media in Examples 1, 2, and 3.

[0020] Figure 2 The chromatogram shows the purified vancomycin obtained using Example 5. Detailed Implementation

[0021] To better understand the present invention, embodiments are provided. Example 1

[0022] (1) Allyl activation: 110g of cross-linked agarose matrix was added to 100g of 40% NaOH solution and reacted at 40℃ for 1h. Then, 17g of anhydrous sodium sulfate and 110g of allyl glycidyl ether were added and the reaction was continued for 12h. After the reaction was completed, the mixture was washed with purified water and dried for later use.

[0023] (2) Bromine activation: Add 110g of allyl activation matrix, 100g of pure water and 7g of sodium acetate to a reaction flask, stir at 25°C for 5min, then add liquid bromine in small amounts several times until the solution is pale yellow and does not change color after 5min, continue the reaction for 1h, then add sodium formate until the reaction solution turns milky white, continue the reaction for 0.5h, after the reaction is completed, wash with purified water and dry for later use.

[0024] (3) Grafting of dextran: Mix 110g of bromine-activated matrix, 100g of dextran and 250g of pure water thoroughly, react at 50℃ for 2h, then add 140g of 3M NaOH solution and react at 50℃ for 18h; after the reaction is completed, wash with purified water and dry for later use.

[0025] (4) Secondary activation of epoxy: Take 110g of dried dextran graft matrix, add 110g of pure water, 0.1g of cerium ammonium nitrate and 15g of glycidyl methacrylate, remove oxygen with nitrogen, keep warm at 30℃ for 3h, wash with pure water and dry for later use.

[0026] (5) Ionic ligand coupling: Take 100 mL of epoxy secondary activated matrix, add 300 mL of 19 M sodium hydroxide solution, slowly add 150 mL of 22 M chloroacetic acid solution, react at 35 °C for 18 h, and wash with pure water after the reaction is completed.

[0027] (6) The obtained matrix was tested for ion exchange capacity and dynamic loading. The specific test method was the same as in Example 1. The test results were ion exchange capacity of 701 μmol / mL and dynamic loading of 152 mg / mL.

[0028] The specific testing method is as follows: ① Ion exchange capacity testing method: Take a 12 mL SPE column, accurately pipette 3 mL of deionized water, and mark the 3 mL mark on the SPE column with a marker. Pipette the sample to the mark, pass 30 mL of 1 mol / L HCl through the column, wash with deionized water until neutral, add 30 mL of 1 mol / L KNO3 solution and pass through the column again. Collect the filtrate and titrate with 0.1 mol / L AgNO3 standard solution and 5% potassium chromate indicator until a red precipitate appears. Calculate the exchange capacity. ②Dynamic load test method: Prepare the following solutions: Buffer A (50mM Tris-HCl, pH 7.6±0.05) and Buffer B (50mM Tris-HCl + 1M NaCl, pH 7.6±0.05). For dynamic loading detection of the sample: approximately 3 mg / mL BSA, dissolve and dilute an appropriate amount of BSA to 3 mg / mL with Buffer A. Set the UV detector wavelength to 280 nm, automatically zero the UV, and pump the sample into the pre-packed column at a flow rate of 0.5 mL / min while collecting the flow through. Stop loading when the UV value reaches 10% of the sample's UVmax. Elute the pre-packed column with Buffer B at a flow rate of 1 mL / min. Example 2

[0029] (1) Allyl activation: 110g of cross-linked agarose matrix was added to 80g of 60% NaOH solution and reacted at 50℃ for 1h. Then, 25g of anhydrous sodium sulfate and 110g of allyl glycidyl ether were added and the reaction was continued for 18h. After the reaction was completed, the mixture was washed with purified water and dried for later use.

[0030] (2) Bromine activation: Add 110g of allyl activation matrix, 200g of pure water and 10g of sodium acetate to a reaction flask and stir at 25°C for 10min; then add liquid bromine in small amounts several times until the solution is pale yellow and does not change color after 5min, and continue the reaction for 1h; then add sodium formate until the reaction solution turns milky white, and continue the reaction for 0.5h. After the reaction is completed, wash with purified water and dry for later use.

[0031] (3) Grafting of dextran: Mix 110g of bromine-activated matrix, 70g of dextran and 350g of pure water thoroughly, react at 40℃ for 1h, then add 200g of 5M NaOH solution and react at 60℃ for 10h; after the reaction is completed, wash with purified water and dry for later use.

[0032] (4) Secondary activation of epoxy: Take 110g of dried dextran graft matrix, add 110g of pure water, 0.2g of cerium sulfate and 22g of epichlorohydrin, remove oxygen with nitrogen, keep warm at 40℃ for 5h, wash with pure water and dry for later use.

[0033] (5) Ionic ligand coupling: Take 100 mL of epoxy secondary activated matrix, add 100 mL of 15 M sodium hydroxide solution, slowly add 200 mL of 22 M 3-mercaptopropionic acid, react at 40 °C for 15 h, and wash with pure water after the reaction is completed.

[0034] (6) The obtained matrix was tested for ion exchange capacity and dynamic loading. The specific test method was the same as in Example 1. The test results were ion exchange capacity of 628 μmol / mL and dynamic loading of 138 mg / mL. Example 3

[0035] (1) Allyl activation: 110g of cross-linked agarose matrix was added to 2000g of 20% NaOH solution and reacted at 50℃ for 2h. Then, 17g of anhydrous sodium sulfate and 110g of allyl glycidyl ether were added and the reaction continued for 25h. After the reaction was completed, the mixture was washed with purified water and dried for later use.

[0036] (2) Bromine activation: Add 110g of allyl activation matrix, 100g of pure water and 7g of sodium acetate to a reaction flask, stir at 25°C for 10min, then add liquid bromine in small amounts several times until the solution is pale yellow and does not change color after 5min, and continue the reaction for 1h; then add sodium formate until the reaction solution turns milky white, and continue the reaction for 0.5h. After the reaction is completed, wash with purified water and dry for later use.

[0037] (3) Grafting of dextran: Mix 110g of bromine-activated matrix, 200g of dextran and 500g of pure water thoroughly, react at 40℃ for 2h, then add 300g of 5M NaOH solution and react at 30℃ for 250h; after the reaction is completed, wash with purified water and dry for later use.

[0038] (4) Secondary activation of epoxy: Take 110g of dried dextran graft matrix, add 110g of pure water, 0.4g of cerium ammonium sulfate and 31g of allyl glycidyl ether, remove oxygen with nitrogen, keep warm at 40℃ for 5h, wash with pure water and dry for later use.

[0039] (5) Ionic ligand coupling: Take 100 mL of epoxy secondary activated matrix, add 500 mL of 6 M sodium hydroxide solution, slowly add 300 mL of 20 M bromoacetic acid solution, react at 40 °C for 20 h, and wash with pure water after the reaction is completed.

[0040] (6) The obtained matrix was tested for ion exchange capacity and dynamic loading. The specific test method was the same as in Example 1. The test results were ion exchange capacity of 519 μmol / mL and dynamic loading of 71 mg / mL. Example 4

[0041] (1) Allyl activation: 110g of cross-linked agarose matrix was added to 100g of 40% NaOH solution and reacted at 50℃ for 1h. Then, 17g of anhydrous sodium sulfate and 110g of allyl glycidyl ether were added and the reaction was continued for 18h. After the reaction was completed, the mixture was washed with purified water and dried for later use.

[0042] (2) Bromine activation: Add 110g of allyl activation matrix, 100g of pure water and 7g of sodium acetate to a reaction flask and stir at 25°C for 5-10 min; then add liquid bromine in small amounts several times until the solution is pale yellow and does not change color after 5 min, continue the reaction for 1 h, then add sodium formate until the reaction solution turns milky white, continue the reaction for 0.5 h, after the reaction is completed, wash with purified water and dry for later use.

[0043] (3) Grafting of dextran: Mix 110g of bromine-activated matrix, 100g of dextran and 250g of pure water thoroughly, react at 50℃ for 2h, then add 140g of 3M NaOH solution and react at 50℃ for 18h; after the reaction is completed, wash with purified water and dry for later use.

[0044] (4) Ionic ligand coupling: Take 100 mL of dextran graft matrix, add 300 mL of 19 M sodium hydroxide solution, slowly add 150 mL of 22 M chloroacetic acid solution, react at 35 °C for 18 h, and wash with pure water after the reaction is completed.

[0045] (5) The obtained matrix was tested for ion exchange capacity and dynamic loading. The specific test method was the same as in Example 1. The test results were ion exchange capacity of 230 μmol / mL and dynamic loading of 98 mg / mL. Example 5

[0046] The crude vancomycin was separated and purified using the gel filtration medium prepared in Example 1: Device Model: Bio-Lab100 Flow rate range: 0-100 mL / min Operating temperature: 4-40℃ Loading volume: 5 ml column, load 25 ml of sample at 2 mg / ml Operating procedures: (1) Equilibrium phase: Wash with 50 mM ammonium acetate solution for 5 column volumes. (2) Pre-washing stage: Use 100mM ammonium acetate solution to wash for 10 column volumes. (3) Separation stage: Wash with 250mM ammonium acetate solution for 10 column volumes. (4) Elution phase: Wash with 500mM ammonium acetate solution for 5 column volumes. Results: Purity 93%, yield 86%.

Claims

1. A method for preparing and applying a gel filter medium with high ion exchange capacity and high loading capacity, characterized in that, The preparation method includes the following steps: S1. Allyl activation: Add cross-linked agarose matrix to a 20%-60% sodium hydroxide solution with a mass equal to 0.5-2 times the mass of the cross-linked agarose matrix, and react at 40-50℃ for 1-2 hours. Then add anhydrous sodium sulfate with a mass equal to 0.1-0.3 times the mass of the cross-linked agarose matrix and allyl glycidyl ether with a mass equal to that of sodium hydroxide, and continue the reaction at 20-80℃ for 8-28 hours. After the reaction is complete, wash with pure water and dry to obtain the allyl activated matrix. S2. Bromine activation: Add the allyl activated matrix obtained in S1, 0.5-2 times the mass of the allyl activated matrix of pure water, and 0.05-0.1 times the mass of the allyl activated matrix of sodium acetate to a reaction flask. Keep the mixture warm at 25°C and stir for 5-10 min. Then slowly add liquid bromine dropwise until the solution turns pale yellow and does not change color within 5 min. Continue the reaction for 1 h. Add sodium formate until the solution turns milky white. Continue the reaction for 0.5 h. After the reaction is complete, wash with pure water and dry to obtain the bromine activated matrix. S3. Dextran grafting: The bromine-activated matrix obtained in S2, 0.5-2 times the mass of the bromine-activated matrix of dextran, and 2-5 times the mass of the bromine-activated matrix of pure water are thoroughly mixed and reacted at 40-50℃ for 1-2 hours. Then, 1-3 times the mass of the bromine-activated matrix of 1-5M sodium hydroxide solution is added and reacted at 30-60℃ for 8-28 hours. After the reaction is completed, the mixture is washed with pure water and dried to obtain the dextran grafted matrix. S4. Secondary Activation of Epoxy: Take the dextran graft matrix obtained in S3, add pure water of the same mass as the dextran graft matrix, catalyst of 0.0009-0.005 times the mass of the dextran graft matrix, and activator of 0.1-0.3 times the mass of the dextran graft matrix, mix, remove oxygen with nitrogen, react at 30-40℃ for 3-5 hours, wash with pure water after the reaction, and dry to obtain the epoxy activated matrix; S5. Ionic ligand coupling: Take the epoxy activated matrix obtained in S4, put it into a large reaction vessel, add 1-5 times the mass of the epoxy activated matrix and a sodium hydroxide solution with a concentration of 5-20M, and slowly add 1-3 times the mass of the epoxy activated matrix and a ligand solution with a concentration of 20-35M. React at 30-40℃ for 15-20h. After the reaction is completed, wash with pure water and dry to obtain the ion-modified matrix.

2. The preparation method according to claim 1, characterized in that: In step S4, the catalyst is cerium ammonium sulfate, cerium sulfate, or cerium ammonium nitrate.

3. The preparation method according to claim 1, characterized in that: In step S4, the activator is epichlorohydrin, allyl glycidyl ether, 1,4-butanediol diglycidyl ether, glycidyl methacrylate, or ethylene glycol methacrylate.

4. The preparation method according to claim 1, characterized in that: In step S5, the ligands are chloroacetic acid, 3-mercaptopropionic acid, bromoacetic acid, sodium metabisulfite, trimethylamine hydrochloride, and diethylaminochloroethane hydrochloride.

5. The application of the high ion exchange capacity, high loading capacity gel filter medium obtained by the preparation method according to claim 1 for the purification of vancomycin.

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