An anion exchange chromatography medium, its preparation method and application

Abstract

This invention discloses an anion exchange chromatography medium, its preparation method, and its application. An anion exchange chromatography medium is obtained by coupling quaternary ammonium cationic polyvinyl alcohol to a hydrophilic base medium via a spacer arm reagent. The structural formula is SP-R-Quaternary ammonium cationic polyvinyl alcohol, where SP is the base medium, Quaternary ammonium cationic polyvinyl alcohol is quaternary ammonium cationic polyvinyl alcohol, and R is the spacer arm for coupling the quaternary ammonium cationic polyvinyl alcohol to the base medium. Cationic polyvinyl alcohol contains a large amount of positive charge, which can bind to lipid A. Quaternization introduces positively charged quaternary ammonium salt groups, resulting in stronger anion exchange performance of polyvinyl alcohol in the aqueous phase and improving its stability in the aqueous phase. The anion exchange chromatography medium of this invention exhibits a removal rate greater than 98.1% in endotoxin removal tests.

Description

Technical Field

[0001] This invention relates to the field of chromatography media, specifically to an anion exchange chromatography media, its preparation method, and its applications. Background Technology

[0002] Endotoxins, also known as lipopolysaccharides, are components of the cell walls of Gram-negative bacteria. Located in the outermost layer of the cell wall, they are covered by peptidoglycans. Endotoxins are typically released after bacterial death and lysis or after artificial disruption of bacterial cells. Their chemical composition includes phospholipid polysaccharide-protein complexes, with lipid A being the primary toxic component. Endotoxins are harmful to humans and can cause fever, microcirculatory disturbances, endotoxic shock, and disseminated intravascular coagulation.

[0003] Traditional endotoxin removal methods include heat treatment, acid-base treatment, and adsorbent methods. However, these methods suffer from poor selectivity and low sample recovery rates. For example, heat treatment may cause irreversible damage to biomolecules such as proteins, while acid-base treatment may lead to environmental degradation and affect the sustainability of the process. Therefore, finding an efficient, selective, and controllable endotoxin removal method has become a current research focus.

[0004] Anion exchange chromatography media, as a highly selective separation and purification technique, have shown broad application prospects in the preparation of biopharmaceuticals. Polycations can bind to hydrophobic and electronegative lipid A in endotoxins, and therefore can be used to remove endotoxins. However, currently available anion exchange media still have some problems in removing endotoxins, such as insufficient adsorption capacity, poor stability, and complex processes. Summary of the Invention

[0005] The primary objective of this invention is to provide an anion exchange chromatography medium based on quaternized cationic polyvinyl alcohol and its preparation method.

[0006] Technical solution: An anion exchange chromatography medium, with the following structural formula: SP-R-Quaternary ammonium cationic polyvinyl alcohol, In this system, SP is the base medium, Quaternary ammonium cationic polyvinyl alcohol is a quaternary ammonium cationic polyvinyl alcohol, and R is the spacer arm of the quaternary ammonium cationic polyvinyl alcohol coupled to the base medium.

[0007] Specifically, the base medium is a compound with multiple hydroxyl groups on its surface, selected from one or more of agarose, chitosan, dextran, gelatin, polymethacrylate microspheres, polyacrylamide, and polystyrene-divinylbenzene microspheres.

[0008] Preferably, the base medium is agarose, more preferably Agarosix 65. Agarosix 65 is agarose microspheres produced by Suzhou Saifen Technology Co., Ltd., which have high cross-linking degree, certain mechanical properties, and can be used for industrial chromatography purification. Its surface is unmodified and has a large number of natural hydroxyl groups, exhibiting excellent hydrophilicity and negligible non-specific binding to impurities. The agarose matrix of the present invention preferably has a particle size of 50-65 μm and a pore size of 200 nm.

[0009] Specifically, the spacer arm is an epoxy compound selected from one or more of epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, di(2-epoxypropyl) ether, and dipropylene glycol diglycidyl ether.

[0010] Preferably, the spacer arm is epichlorohydrin.

[0011] This invention also provides a method for preparing anion exchange chromatography media, comprising the following steps: S1. A polyvinyl alcohol solution and an epoxy quaternary ammonium salt are reacted under alkaline conditions to obtain quaternized cationic polyvinyl alcohol; S2. Quaternized cationic polyvinyl alcohol is coupled to the base medium via a spacer arm to obtain anion exchange chromatography medium.

[0012] Specifically, in step S1, several epoxy quaternary ammonium salts can be grafted onto the hydroxyl groups of each monomer of quaternized cationic polyvinyl alcohol. The quaternary ammonium salts grafted multiple times can be the same or different quaternary ammonium salts.

[0013] Specifically, in step S2, the base medium is first mixed and coupled with the spacer arm, and then quaternized cationic polyvinyl alcohol is added and coupled onto the spacer arm.

[0014] The structural formula of the epoxy quaternary ammonium salt is:

[0015] In the formula: R1 is a C1-C5 straight-chain alkyl or branched alkyl, R2, R3, and R4 are C1-C3 alkyl, and M﹣ is a chloride or bromide ion.

[0016] Preferably, the epoxy quaternary ammonium salt is one or more of 2,3-epoxypropyltrimethylammonium chloride, 3,4-epoxybutyltriethylammonium bromide, 4,5-epoxypentyltripropylammonium chloride, and 3,4-epoxybutyltrimethylammonium chloride.

[0017] Specifically, this invention does not impose any particular limitation on the degree of polymerization of polyvinyl alcohol, which can be 500-3500.

[0018] Specifically, the present invention does not impose any particular limitation on the degree of alcoholysis of polyvinyl alcohol, but preferably it is 87.0 to 89.0%.

[0019] The above-mentioned anion exchange chromatography media can be applied to most application areas of anion exchange chromatography media, such as the separation and purification of lipids, proteins, antibodies, plasmids, RNA, DNA, VLPs, antigens, vaccines, viral vectors, viruses, and bacteria, and are particularly suitable for removing endotoxins from biological products.

[0020] Beneficial Effects: Compared with existing technologies, the significant advantages of this invention are: cationic polyvinyl alcohol contains a large amount of positive charge, which can bind to lipid A; quaternization introduces positively charged quaternary ammonium salt groups, making polyvinyl alcohol exhibit stronger anion exchange performance in the aqueous phase, improving its performance stability in the aqueous phase, and simultaneously affecting its water solubility. This helps to regulate the operating conditions of the anion exchange medium, allowing it to exhibit optimal endotoxin removal performance within a specific pH range. The anion exchange chromatography medium of this invention achieved a removal rate of greater than 98.1% in endotoxin removal tests. Detailed Implementation

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0022] The present invention will be further explained below with reference to specific embodiments. Example

[0023] The base medium used in this embodiment is Agarosix 65, which is agarose microspheres produced by Suzhou Saifen Technology Co., Ltd. It has a high degree of cross-linking, its surface is unmodified and has a large number of natural hydroxyl groups, excellent hydrophilicity, and negligible non-specific binding of impurities. The particle size range is 50-65 μm and the pore size is 200 nm.

[0024] 1. Preparation of Quaternized Cationic Polyvinyl Alginate Add 1 L of deionized water to a 2 L four-necked flask, and while stirring, add 100 g of polyvinyl alcohol 1788 (Shanghai Maclean Biochemical Technology Co., Ltd., degree of alcoholysis: 87.0-89.0%). Raise the temperature to about 80 °C to completely dissolve the polyvinyl alcohol, and then lower it to room temperature. Then, at room temperature, slowly add 400 g of 2,3-epoxypropyltrimethylammonium chloride to the four-necked flask, and add dropwise 0.5 mol / L sodium hydroxide aqueous solution to adjust the pH to 10. Gradually raise the temperature to 60 °C and stir at 50 rpm for 4 h. After the reaction is complete, use 0.1 mol / L dilute hydrochloric acid aqueous solution to adjust the pH to neutral to obtain quaternized cationic polyvinyl alcohol ligands.

[0025] 2. Synthesis of anion exchange chromatography media Add 100 g Agarosix 65, 175 g epichlorohydrin, 200 g NaBH4 and 400 mL 1 mol / L NaOH to a 2 L four-necked flask. Then heat to 40 °C and stir at 50 rpm for 4 h. Wash with 5 L of pure water until neutral. Then add 100 mL of the quaternized cationic polyvinyl alcohol ligand prepared in step 1, and then add 50 mL of toluene. Heat to 60 °C and stir at 50 rpm for 1 h. Cool to room temperature to obtain the anion exchange chromatography medium.

[0026] The synthesis steps are shown in the following figure: (1) Preparation of quaternized cationic polyvinyl alcohol ligands

[0027] (2) Coupled spacer arm

[0028] (3) Coupling of quaternized cationic polyvinyl alcohol ligands

[0029] In the picture, The base medium is Agarosix 65.

[0030] Example 2 The base medium used in this embodiment is commercially available polymethyl methacrylate microspheres, which need to be hydrophilically modified before use.

[0031] Hydrophilic modification of polymethyl methacrylate microspheres 100 g of polymethacrylate microspheres (provided by Suzhou Saifen Technology Co., Ltd., with a particle size of 60-80 μm and a pore size of 100-200 nm) and 200 mL of 1 mol / L NaOH solution were added to a 1 L four-necked flask. The mixture was stirred at 100 rpm for 1 h. Then, 100 g of ethylene glycol diglycidyl ether and 100 g of dextran were added. The mixture was heated to 60 °C and stirred at 50 rpm for 24 h. After washing with 5 L of pure water, the mixture was filtered to obtain polymethacrylate-based spheres with a large number of -OH groups on the surface.

[0032] 2. Epoxy Activation Add 100 g of polymethacrylate-based spheres with a large number of -OH groups on the surface and 200 mL of 1 mol / L NaOH solution to a 1 L four-necked flask. Stir at 100 rpm for 1 h, then add 100 g of ethylene glycol diglycidyl ether, heat to 60 °C, stir at 50 rpm for 4 h, wash with 5 L of pure water and filter to obtain the intermediate.

[0033] 3. Synthesis of Chromatographic Media Add 100 g of the intermediate from step 2 and 50 mL of toluene to a 1 L four-necked flask, heat to 60 °C, stir at 50 rpm for 2 h, then add 100 mL of the quaternized cationic polyvinyl alcohol ligand prepared by the method described in step 1 of Example 1, stir at 50 rpm for 1 h, and then cool to room temperature to obtain the anion exchange chromatography medium. Example

[0034] In Example 1, polyvinyl alcohol 1788 was replaced with polyvinyl alcohol 2488 (Shanghai Maclean Biochemical Technology Co., Ltd., degree of alcoholysis: 87.0-89.0%), 2,3-epoxypropyltrimethylammonium chloride was replaced with 4,5-epoxypentyltripropylammonium chloride, and epichlorohydrin was replaced with 1,4-butanediol diglycidyl ether. Anion exchange chromatography media were prepared under the same conditions as in Example 1. Example

[0035] Preparation of Quaternized Cationic Polyvinyl Alginate Add 1 L of deionized water to a 2 L four-necked flask, and while stirring, add 100 g of polyvinyl alcohol 1788 (Shanghai Maclean Biochemical Technology Co., Ltd., degree of alcoholysis: 87.0~89.0%). Raise the temperature to 80℃ to completely dissolve the polyvinyl alcohol, and then cool it to room temperature. At room temperature, 400 g of 2,3-epoxypropyltrimethylammonium chloride was slowly added to a four-necked flask. The pH was adjusted to 10 by adding 0.5 mol / L sodium hydroxide aqueous solution dropwise. The temperature was gradually increased to 60 °C, and the mixture was stirred at 50 rpm for 4 h. After the reaction was completed, the pH was adjusted to neutral using 0.1 mol / L dilute hydrochloric acid aqueous solution. Then, 200 mL of 1 mol / L hydrochloric acid aqueous solution and 200 g of 4,5-epoxypentyltripropylammonium chloride were added. The temperature was slowly increased to 70 °C, and the mixture was stirred at 100 rpm for 2 h. After the reaction was completed, the pH of the aqueous solution was adjusted to neutral using 0.5 mol / L NaOH to obtain the quaternized cationic polyvinyl alcohol ligand.

[0036] This step involves grafting another quaternary ammonium salt onto the quaternary ammonium salt from the initial synthesis of polyvinyl alcohol. The dual quaternary ammonium salt groups enhance ion exchange. The two grafted quaternary ammonium salts can be the same or different. Grafting can be performed under both alkaline and acidic conditions. If necessary, quaternary ammonium salts can be further grafted onto the hydroxyl groups left from the reaction.

[0037] 2. Synthesis of Chromatographic Media 100g Agarosix 65, 200g 1,2-cyclohexanediol diglycidyl ether, 200g NaBH4 and 400mL 1mol / L NaOH were added to a 2 L four-necked flask. The mixture was then heated to 40℃ and stirred at 50 rpm for 4 h. After washing with 5 L of pure water until neutral, 50 mL of toluene was added. The mixture was then heated to 60℃ and stirred at 50 rpm for 2 h. 100 mL of the quaternized cationic polyvinyl alcohol ligand prepared in step 1 was added. The mixture was stirred at 50 rpm for 1 h and then cooled to room temperature to obtain an anion exchange chromatography medium. Example

[0038] Endotoxin removal test: 1. Investigating the factors influencing endotoxin removal rate: (1) Effect of NaCl concentration on endotoxin removal Accurately weigh 20 mg of the chromatography medium prepared in Example 1, add 1 mL of 20 Eu / mL endotoxin standard (provided by Kanglang Biotechnology, catalog number KL-140405-10) and different concentrations of NaCl solution into a shaker. The NaCl concentrations are 0 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, 1 mol / L, 3 mol / L, and 5 mol / L. Shake at 200 r / min for 2 h at an experimental temperature of 25℃. Centrifuge and collect the supernatant to determine the endotoxin content. The endotoxin detection method is the dynamic colorimetric Limulus amebocyte lysate (LAL) reagent kit provided by Beijing Solarbio Science & Technology Co., Ltd. (catalog number: T7570).

[0039] After the test was completed, the chromatography medium was rinsed with 2 mol / L NaOH solution, 0.5 mol / L NaCl solution and pyrogen-free water respectively to fully dissociate the endotoxins on the chromatography medium and clean the chromatography medium.

[0040] The endotoxin removal rate results are shown in Table 1 below: Table 1. Effect of NaCl concentration on endotoxin removal

[0041] As can be seen from the results in Table 1, the endotoxin removal rate first increases and then decreases with the increase of NaCl concentration. The removal rate reaches its highest level of 96.15% when the sodium chloride concentration is 0.1 mol / L.

[0042] (2) Effect of pH on endotoxin removal Accurately weigh 20 mg of the chromatography medium prepared in Example 1, add 1 mL of 20 Eu / mL endotoxin standard (provided by Kanglang Biotechnology, catalog number KL-140405-10) and solutions of different pH values ​​into a shaker. The solutions include 50 mM HAC-NaAC (pH 5.0), 20 mM sodium phosphate buffer (pH 6.0), 20 mM sodium phosphate buffer (pH 7.0), 20 mM Tris-HCl (pH 8.0), and 20 mM Tris-HCl (pH 9.0). Add 0.1 mol / L NaCl solution to each of the five buffer solutions. Shake at 200 r / min for 2 h at an experimental temperature of 25℃. Centrifuge and collect the supernatant to determine the endotoxin content. The endotoxin detection method is the dynamic colorimetric Limulus amebocyte lysate (LAL) assay kit provided by Beijing Solarbio Science & Technology Co., Ltd. (catalog number: T7570).

[0043] Table 2 Effect of pH on endotoxin removal

[0044] The results in the table above show that the endotoxin removal rate is greater than 95% when the pH is between 6.0 and 8.0. Therefore, pH 6.0-8.0 can be selected as the loading condition.

[0045] 2. High-throughput screening of optimal experimental conditions for endotoxin removal In this embodiment, a Freedom EVO 200 fully automated liquid handling workstation was used in flow-through mode. The anion exchange chromatography medium prepared in Example 1 was used for testing. The chromatography steps were as follows: first, 5 column volumes were regenerated with 0.1 mol / L NaOH solution, followed by 10 column volumes of washing with pure water, and then 10 column volumes of equilibration buffer (1% sodium deoxycholate + 50 mM phosphate, pH 6.0-8.0) were used to load rituximab. The sample concentration was 5.30 g / L, PI was 8.9, molecular weight was 150 kDa, and endotoxin content was 500 Eu / mg. The loading flow rate was 0.2 mL / min, and 5 column volumes were loaded. The flow-through was collected, and after 5 cycles of loading, the endotoxin content in the flow-through was detected using an endotoxin kit (Beijing Solarbio, catalog number: T7570). The experimental temperature was 32℃. To determine the optimal sample loading conditions, the samples were adjusted to three different pH values ​​and three different conductivity values. Specific experimental data are shown in Table 3-4 below: Table 3 Test Conditions

[0046] Table 4. Experimental Results of Optimal Experimental Conditions for High-Throughput Screening to Remove Endotoxins

[0047] The above experimental results show that when using the anion exchange chromatography medium prepared in Example 1 to remove endotoxins, the endotoxin content in the monoclonal antibody can be reduced from 500 Eu / mg to below 10 Eu / mg, with a removal rate of more than 98.1%, demonstrating a good effect in removing endotoxins.

[0048] Using the same test conditions, the anion exchange chromatography media prepared in Examples 2-4 were tested, and the optimal test results for each example are shown in Table 5 below.

[0049] Table 5. Effect of the chromatographic media prepared in each example on endotoxin removal.

[0050] As can be seen from the data in the table above, the anion exchange chromatography media prepared in Examples 1-4 all achieved an endotoxin removal rate of greater than 99.9% under optimal test conditions.

Claims

1. An anion exchange chromatography medium, characterized in that, The structural formula is as follows: SP-R-quaternized cationic polyvinyl alcohol, SP is the base medium, the quaternized cationic polyvinyl alcohol is obtained by reacting polyvinyl alcohol solution and epoxy quaternary ammonium salt under alkaline conditions, R is the spacer arm of the base medium coupled with the quaternized cationic polyvinyl alcohol, the spacer arm is an epoxy compound, and the surface of the base medium contains multiple hydroxyl groups.

2. The anion exchange chromatography medium according to claim 1, characterized in that: The base medium includes one or more of agarose, chitosan, dextran, gelatin, polymethacrylate microspheres, polyacrylamide, and polystyrene-divinylbenzene microspheres.

3. The anion exchange chromatography medium according to claim 1, characterized in that: The spacer arm comprises one or more of epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, di(2-epoxypropyl) ether, and dipropylene glycol diglycidyl ether.

4. A method for preparing anion exchange chromatography medium as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. A polyvinyl alcohol solution and an epoxy quaternary ammonium salt are reacted under alkaline conditions to obtain quaternized cationic polyvinyl alcohol; S2. Quaternized cationic polyvinyl alcohol is coupled to the base medium via a spacer arm to obtain anion exchange chromatography medium.

5. The preparation method according to claim 4, characterized in that: In step S2, the base medium is first mixed and coupled with the spacer arm, and then quaternized cationic polyvinyl alcohol is added and coupled to the spacer arm.

6. The preparation method according to claim 4, characterized in that: The structural formula of the epoxy quaternary ammonium salt is: In the formula: R1 is a C1-C5 straight-chain alkyl or branched alkyl, R2, R3, and R4 are C1-C3 alkyl, and M... ﹣ It consists of chloride and bromide ions.

7. The preparation method according to claim 6, characterized in that: The epoxy quaternary ammonium salt is one or more of 2,3-epoxypropyltrimethylammonium chloride, 3,4-epoxybutyltriethylammonium bromide, 4,5-epoxypentyltripropylammonium chloride, and 3,4-epoxybutyltrimethylammonium chloride.

8. The application of the anion exchange chromatography medium according to any one of claims 1-3, or the anion exchange chromatography medium prepared by the preparation method according to any one of claims 4-7, in the separation and purification of lipids, proteins, RNA, DNA, VLPs, antigens, vaccines, viral vectors, viruses, and bacteria, characterized in that: Used to remove endotoxins.

9. The application of the anion exchange chromatography medium according to any one of claims 1-3, or the anion exchange chromatography medium prepared by the preparation method according to any one of claims 4-7, in the removal of endotoxins from biological products.

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