Weak cation chromatographic filler and preparation method thereof
By using porous polystyrene-divinylbenzene microspheres and undergoing hydrophilic modification and copolymerization, a weak cationic chromatography filler with high load is formed, which solves the problems of existing media being prone to deformation under high pressure flow, long production cycle and low load, and achieves stable chemical properties, high strength, high load and low cost filler.
Patent Information
- Application Number
- CN202411899272.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-28
AI Technical Summary
Existing weak cation chromatography media are prone to deformation under high pressure flow, have long production cycles, high costs, and low loads, making it difficult to meet the needs of industrial-scale applications.
Porous polystyrene-divinylbenzene microspheres were used as the matrix, and the copolymerization reaction of copolyenol monomers and weak cationic ligand monomers was formed with high load weak cationic chromatography filler. The filler has a highly branched three-dimensional tentacle-shaped structure that improves mechanical strength and load capacity.
It has achieved weak cationic chromatography fillers with stable chemical properties, high strength, high loading and low non-specific adsorption, with low production cost and short production cycle, and is suitable for industrial-scale applications.
Smart Images
Figure CN119708595A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a filler, in particular to a weak cationic chromatography filler and a preparation method thereof. Background Art
[0002] In the separation and purification process of proteins and biomacromolecules, ion exchange chromatography is one of the most widely used separation chromatography processes and one of the most commonly used methods for the purification of biomacromolecules. According to the type of functional groups of ion exchange, ion chromatography media are divided into two categories: cationic chromatography and weak cationic chromatography. Among them, weak cationic chromatography is particularly effective in removing fragments and aggregates in the purification step of antibody proteins.
[0003] Weakly cationic chromatography media are mainly divided into agarose weakly cationic chromatography media and synthetic resin matrix weakly cationic media. Agarose weakly cationic chromatography media have the following main disadvantages: 1) Low mechanical strength: Although agarose improves diversified adaptability, it is easy to deform under high-pressure flow and is difficult to apply to industrial scale; 2) Long production cycle and high cost. Although synthetic resin matrix weakly cationic media can overcome the above technical problems, their application is limited because their loading capacity is usually lower than that of agarose matrix weakly cationic media. Summary of the invention
[0004] Purpose of the invention: The purpose of the present invention is to provide a weak cationic chromatography filler with a polymer matrix having stable chemical properties, high strength, high loading capacity, and low nonspecific adsorption; another purpose of the present invention is to provide a method for preparing the weak cationic chromatography filler.
[0005] Technical solution: The weak cationic chromatography filler of the present invention has a highly branched three-dimensional tentacle-like structure, and the simplified formula is as follows:
[0006]
[0007] Wherein: A is porous polystyrene-divinylbenzene microspheres; R1 is a hydroxyl group, n is any integer from 1 to 50; R2 is a carboxyl group, m is any integer from 1 to 50.
[0008] The preparation method of the weak cationic chromatography filler of the present invention comprises the following steps:
[0009] (1) hydrophilically modifying uncoated polystyrene-divinylbenzene microspheres to obtain hydrophilic polystyrene-divinylbenzene microspheres;
[0010] (2) Preparation of weak cationic chromatography filler: The hydrophilic polystyrene-divinylbenzene microspheres obtained in step (1) are mixed with an initiator, a copolymerized enol monomer, and a weak cationic ligand monomer, and stirred to react to obtain a weak cationic chromatography filler, i.e., a high-load polystyrene-divinylbenzene microsphere chromatography medium.
[0011] Preferably, the specific preparation method of step (2) is: the hydrophilic polystyrene-divinylbenzene microspheres obtained in step (1) are mixed with an initiator, a copolymerized enol monomer, and a weak cationic ligand monomer, and stirred at 40-45° C. for 4-8 hours, and after the reaction is completed, the mixture is washed with distilled water for more than three times to obtain a weak cationic chromatography filler, i.e., a high-load polystyrene-divinylbenzene microsphere chromatography medium.
[0012] Preferably, the initiator in step (2) is cerium perchlorate.
[0013] Preferably, the concentration of the cerium perchlorate solution is 10 to 20 mmol / L.
[0014] Preferably, the copolymerized enol monomer in step (2) is at least one of 3-butene-1,2-diol, ethylene glycol vinyl ether, and N-(2-hydroxyethyl)acrylamide.
[0015] Preferably, the weak cationic ligand monomer in step (2) is at least one of 6-heptenoic acid, 5-hexenoic acid and 4-pentenoic acid.
[0016] Preferably, in step (2), the concentrations of the copolymerized enol monomer and the weak cationic ligand monomer are consistent, and the concentration range is 50-150 mg / mL.
[0017] Preferably, in step (2), the mass ratio of the hydrophilic polystyrene-divinylbenzene microspheres: initiator: copolymerized enol monomer: weak cationic ligand monomer is 1: 0.03-0.08: 0.5-1.5: 0.5-1.5.
[0018] Preferably, the specific preparation method of step (1) is:
[0019] (1-1) Preparation of polystyrene-divinylbenzene microspheres modified with dextran: uncoated polystyrene-divinylbenzene microspheres are dissolved in water, a bromination reagent is added and stirred for reaction, and then sodium formate is added to neutralize excess bromination reagent; sodium hydroxide solution, dextran solution and sodium borohydride are added to the bromination-treated microspheres, and the mixture is stirred for reaction to obtain polystyrene-divinylbenzene microspheres modified with dextran;
[0020] (1-2) Preparation of hydrophilic polystyrene-divinylbenzene microspheres: Dextran-modified polystyrene-divinylbenzene microspheres are dissolved in a sodium hydroxide solution, and an epoxy crosslinking agent is added. The mixture is stirred and reacted to prepare hydrophilic polystyrene-divinylbenzene microspheres.
[0021] Preferably, the specific preparation method of step (1-1) is: dissolving uncoated polystyrene-divinylbenzene microspheres in water, adding a bromination reagent and stirring the reaction for 1-2 hours, adding sodium formate to neutralize excess bromination reagent after the reaction, and washing with distilled water for more than three times; adding sodium hydroxide solution, dextran solution and sodium borohydride to the microspheres after bromination, mixing and stirring at 40-50° C. for 4-8 hours, and washing with distilled water for more than three times after the reaction to obtain dextran-modified polystyrene-divinylbenzene microspheres.
[0022] Preferably, the particle size of the uncoated polystyrene-divinylbenzene microspheres in step (1-1) is 45-55 μm.
[0023] Preferably, the pore size of the microspheres in step (1-1) is
[0024] Preferably, the pore size of the microspheres in step (1-1) is or
[0025] Preferably, the bromination reagent in step (1-1) is at least one of bromine water and N-bromosuccinimide.
[0026] Preferably, the concentration of the sodium hydroxide solution in step (1-1) is 1 to 3 mol / L.
[0027] Preferably, the concentration of the dextran solution in step (1-1) is 0.1 to 0.3 g / mL.
[0028] Preferably, the molecular weight of the dextran in step (1-1) is 20,000 to 100,000.
[0029] Preferably, the specific preparation method of step (1-2) is: dissolving the polystyrene-divinylbenzene microspheres modified with dextran in a sodium hydroxide solution, adding an epoxy crosslinking agent, mixing and stirring at 40-50° C. for 16-18 hours, and washing with anhydrous ethanol and distilled water for more than three times after the reaction to prepare hydrophilic polystyrene-divinylbenzene microspheres.
[0030] Preferably, the concentration of the sodium hydroxide solution in step (1-2) is 3 to 5 mol / L.
[0031] Preferably, the epoxy crosslinking agent in step (1-2) is at least one of epichlorohydrin, ethylene glycol diglycidyl ether, glycerol diglycidyl ether, and 1,4-butanediol diglycidyl ether.
[0032] The weak cationic chromatography filler of the present invention is applied in the fields of antibodies, vaccines, recombinant proteins and nucleic acids.
[0033] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the high-load weak cations of the polystyrene-divinylbenzene microspheres prepared by the method have a production cost far lower than that of agarose fillers, and the time spent on the synthesis of 50L-scale fillers from base balls to the final finished product is only 4 to 5 days, and the production cycle and cost are far lower than those of agarose-based ball fillers, and the fillers have the characteristics of high rigidity and chemical corrosion resistance; at the same time, the dextran used in the preparation method is used as a hydrophilic modification, so that the filler has low nonspecific adsorption; the hyperbranched tentacle-type structure formed by the copolymerization of enol and weak cationic ligand monomers makes the filler have the advantage of high load. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The peak area of the first injection of the hydrophobic test spectrum of the microspheres of the present invention;
[0035] Figure 2 The peak area of the 16th injection of the hydrophobic test spectrum of the microspheres of the present invention;
[0036] Figure 3 It is the reaction structural formula of the present invention. DETAILED DESCRIPTION
[0037] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.
[0038] Example 1
[0039] Preparation of polystyrene-divinylbenzene microspheres modified with dextran: 100 g dry weight of The uncoated polystyrene-divinylbenzene microspheres were dissolved in 300 mL of distilled water, bromine water was added dropwise until the aqueous solution was bright yellow, and the reaction was stirred for 2 hours. After the reaction was completed, sodium formate was added to neutralize the excess bromine water, so that the color of the system changed from light yellow to white, and the microspheres were washed with distilled water for more than three times; 150 mL of 1 mol / L sodium hydroxide solution, 150 mL of 0.3 g / mL 40000 molecular weight dextran solution and 0.5 g of sodium borohydride were added to the brominated microspheres, and the mixture was stirred at 50° C. for 6 hours. After the reaction was completed, the microspheres were washed with distilled water for more than three times to prepare dextran-modified polystyrene-divinylbenzene microspheres;
[0040] Preparation of hydrophilic polystyrene-divinylbenzene microspheres: dissolve polystyrene-divinylbenzene microspheres modified with dextran in 200 mL of 5 mol / L sodium hydroxide solution, add 50 mL of glycerol diglycidyl ether as a cross-linking agent, stir and react at 50° C. for 18 hours, and wash with anhydrous ethanol and distilled water for more than three times after the reaction to prepare hydrophilic polystyrene-divinylbenzene microspheres;
[0041] Preparation of weak cationic chromatography filler: Dissolve hydrophilic polystyrene-divinylbenzene microspheres in 150 mL of pure water, add 100 mL of 3-butene-1,2-diol and 6-heptenoic acid solutions with a concentration of 100 mg / mL, mix and stir, finally add 50 mL of cerium perchlorate solution with a concentration of 15 mmol / L, react at 45°C for 6 hours, wash with distilled water for more than three times after the reaction, and prepare weak cationic chromatography filler, i.e., high-load polystyrene-divinylbenzene microsphere chromatography medium.
[0042] Example 2
[0043] Preparation of polystyrene-divinylbenzene microspheres modified with dextran: 100 g dry weight of The uncoated polystyrene-divinylbenzene microspheres were dissolved in 300 mL of distilled water, bromine water was added dropwise until the aqueous solution was bright yellow, and the reaction was stirred for 1 hour. After the reaction was completed, sodium formate was added to neutralize the excess bromine water, so that the color of the system changed from light yellow to white, and the microspheres were washed with distilled water for more than three times; 150 mL of 1 mol / L sodium hydroxide solution, 150 mL of 0.3 g / mL 100000 molecular weight dextran solution and 0.5 g of sodium borohydride were added to the brominated microspheres, and the mixture was stirred at 50° C. for 6 hours. After the reaction was completed, the microspheres were washed with distilled water for more than three times to prepare dextran-modified polystyrene-divinylbenzene microspheres;
[0044] Preparation of hydrophilic polystyrene-divinylbenzene microspheres: polystyrene-divinylbenzene microspheres modified with dextran were dissolved in 200 mL of 3 mol / L sodium hydroxide solution, 25 mL of epichlorohydrin and ethylene glycol diglycidyl ether were added as cross-linking agents, mixed and stirred at 50° C. for 18 hours, and washed with anhydrous ethanol and distilled water for more than three times after the reaction to prepare hydrophilic polystyrene-divinylbenzene microspheres;
[0045] Preparation of weak cationic chromatography filler: Dissolve hydrophilic polystyrene-divinylbenzene microspheres in 150 mL of pure water, add 100 mL of ethylene glycol vinyl ether and 5-hexenoic acid solution with a concentration of 50 mg / mL, mix and stir, finally add 50 mL of cerium perchlorate solution with a concentration of 10 mmol / L, react at 45°C for 6 hours, wash with distilled water for more than three times after the reaction, and prepare a weak cationic chromatography filler, i.e., a high-load polystyrene-divinylbenzene microsphere chromatography medium.
[0046] Example 3
[0047] Preparation of polystyrene-divinylbenzene microspheres modified with dextran: 100 g dry weight of The uncoated polystyrene-divinylbenzene microspheres were dissolved in 300 mL of distilled water, and N-bromosuccinimide was added until the aqueous solution was bright yellow, and the reaction was stirred for 1 hour. After the reaction, sodium formate was added to neutralize the excess N-bromosuccinimide, so that the color of the system changed from light yellow to white, and the microspheres were washed with distilled water for more than three times; 150 mL of 1 mol / L sodium hydroxide solution, 150 mL of 0.3 g / mL 20000 molecular weight dextran solution and 0.5 g of sodium borohydride were added to the brominated microspheres, and the mixture was stirred at 50° C. for 6 hours. After the reaction, the microspheres were washed with distilled water for more than three times to prepare dextran-modified polystyrene-divinylbenzene microspheres;
[0048] Preparation of hydrophilic polystyrene-divinylbenzene microspheres: dissolve polystyrene-divinylbenzene microspheres modified with dextran in 200 mL of 4 mol / L sodium hydroxide solution, add 50 mL of 1,4-butanediol diglycidyl ether as a cross-linking agent, stir and react at 50° C. for 18 hours, and wash with anhydrous ethanol and distilled water for more than three times after the reaction to prepare hydrophilic polystyrene-divinylbenzene microspheres;
[0049] Preparation of weak cationic chromatography filler: Dissolve hydrophilic polystyrene-divinylbenzene microspheres in 150 mL of pure water, add 100 mL of N-(2-hydroxyethyl)acrylamide and 4-pentanoic acid solution with a concentration of 150 mg / mL, mix and stir, finally add 50 mL of cerium perchlorate solution with a concentration of 20 mmol / L, react at 45° C. for 6 hours, wash with distilled water for more than three times after the reaction, and prepare a weak cationic chromatography filler, i.e., a high-load polystyrene-divinylbenzene microsphere chromatography medium.
[0050] Comparative Example 1
[0051] The other steps of the preparation method of Comparative Example 1 are basically the same as those of Example 1, with the only difference being that the copolymerized enol monomer is not added.
[0052] HIgG dynamic loading test method:
[0053] The chromatographic medium prepared in the examples and comparative examples was used to prepare a chromatographic column (6.6×50 mm, CV: 1.71 mL);
[0054] Equilibrium mobile phase A: 50 mM NaAC, pH 5.0, 5 CV;
[0055] Elution mobile phase B: 50 mM NaAC + 1 M NaCl, pH 5.0, 100% B elution, 5 CV;
[0056] Loading protein: HIgG (2 mg / mL) in 50 mM NaAC, pH 5.0;
[0057] Flow rate: 0.342 mL / min; Detection instrument: protein purification instrument.
[0058] Nonspecific adsorption test:
[0059] The hydrophilic polystyrene-divinylbenzene microspheres prepared in the examples and comparative examples were packed into a prepacked column (CV: 1.0 mL);
[0060] Washing mobile phase: 150 mM sodium phosphate buffer (pH 7.0); flow rate: 0.25 mL / min; injection volume: 20 μL;
[0061] Loading protein: BSA (1 mg / mL) in 150 mM sodium phosphate buffer (pH 7.0);
[0062] Instrument: Agilent 1260;
[0063] Number of tests: 16 times.
[0064] Standard bovine serum albumin (BSA) was used to detect the nonspecific adsorption of the chromatographic medium. The BSA standard sample was loaded 16 times continuously with a constant injection volume. The peak area of the sample was integrated and the percentage of the peak area of the first injection sample and the last injection sample was calculated.
[0065] The experimental results are shown in Table 1 and Figure 1-2 As shown, the synthesis cases containing copolymerized enol monomers are all samples with highly branched three-dimensional tentacle-like structures, which can achieve high loading characteristics; the samples without copolymerized enol monomers have a final structure of a single long-chain tentacle-like structure with a low loading capacity.
[0066] Table 1
[0067] Serial number <![CDATA[Dynamic loading of IgG antibody [mg·mL -1 > A1 / A16 peak area ratio [%] Comparative Example 1 70 92.16 Example 1 100 94.93 Example 2 90 87.70 Example 3 85 61.74
Claims
1. A weak cationic chromatography filler, characterized in that It has a highly branched three-dimensional tentacle-like structure, and the simplified formula is as follows: Wherein: A is porous polystyrene-divinylbenzene microspheres; R1 is a hydroxyl group, n is any integer from 1 to 50; R2 is a carboxyl group, m is any integer from 1 to 50.
2. A method for preparing the weak cationic chromatography filler according to claim 1, characterized in that: The following steps are involved: (1) hydrophilically modifying uncoated polystyrene-divinylbenzene microspheres to obtain hydrophilic polystyrene-divinylbenzene microspheres; (2) Preparation of weak cationic chromatography filler: The hydrophilic polystyrene-divinylbenzene microspheres obtained in step (1) are mixed with an initiator, a copolymerized enol monomer, and a weak cationic ligand monomer, and stirred to react to obtain a weak cationic chromatography filler, i.e., a high-load polystyrene-divinylbenzene microsphere chromatography medium.
3. The method for preparing the weak cationic chromatography filler according to claim 2, characterized in that: The specific preparation method of step (2) is as follows: the hydrophilic polystyrene-divinylbenzene microspheres obtained in step (1) are mixed with an initiator, a copolymerized enol monomer, and a weak cationic ligand monomer, and stirred at 40-45° C. for 4-8 hours. After the reaction is completed, the mixture is washed with distilled water for more than three times to obtain a weak cationic chromatography filler, i.e., a high-load polystyrene-divinylbenzene microsphere chromatography medium.
4. The method for preparing the weak cationic chromatography filler according to claim 2, characterized in that: The initiator in step (2) is cerium perchlorate.
5. The method for preparing the weak cationic chromatography filler according to claim 2, characterized in that: The copolymerized enol monomer in step (2) is at least one of 3-butene-1,2-diol, ethylene glycol vinyl ether, and N-(2-hydroxyethyl)acrylamide.
6. The method for preparing the weak cationic chromatography filler according to claim 2, characterized in that: In step (2), the weak cationic ligand monomer is at least one of 6-heptenoic acid, 5-hexenoic acid and 4-pentenoic acid.
7. The method for preparing the weak cationic chromatography filler according to claim 2, characterized in that: The mass ratio of the hydrophilic polystyrene-divinylbenzene microspheres: initiator: copolymerized enol monomer: weak cationic ligand monomer in step (2) is 1: 0.03-0.08: 0.5-1.5: 0.5-1.
5.
8. The method for preparing the weak cationic chromatography filler according to claim 2, characterized in that: The specific preparation method of step (1) is: (1-1) Preparation of polystyrene-divinylbenzene microspheres modified with dextran: uncoated polystyrene-divinylbenzene microspheres are dissolved in water, a bromination reagent is added and stirred for reaction, and then sodium formate is added to neutralize excess bromination reagent; sodium hydroxide solution, dextran solution and sodium borohydride are added to the bromination-treated microspheres, and the mixture is stirred for reaction to obtain polystyrene-divinylbenzene microspheres modified with dextran; (1-2) Preparation of hydrophilic polystyrene-divinylbenzene microspheres: Dextran-modified polystyrene-divinylbenzene microspheres are dissolved in a sodium hydroxide solution, and an epoxy crosslinking agent is added. The mixture is stirred and reacted to prepare hydrophilic polystyrene-divinylbenzene microspheres.
9. The method for preparing the weak cationic chromatography filler according to claim 8, characterized in that: The specific preparation method of step (1-1) is as follows: uncoated polystyrene-divinylbenzene microspheres are dissolved in water, a bromination reagent is added, and the reaction is stirred for 1-2 hours. After the reaction is completed, sodium formate is added to neutralize the excess bromination reagent, and the microspheres are washed with distilled water for more than three times; sodium hydroxide solution, dextran solution and sodium borohydride are added to the microspheres after bromination treatment, mixed and stirred at 40-50° C. for 4-8 hours, and then washed with distilled water for more than three times to obtain dextran-modified polystyrene-divinylbenzene microspheres.
10. The method for preparing the weak cationic chromatography filler according to claim 8, characterized in that: The specific preparation method of step (1-2) is: dissolving the polystyrene-divinylbenzene microspheres modified with dextran in a sodium hydroxide solution, adding an epoxy crosslinking agent, mixing and stirring at 40-50° C. for 16-18 hours, and washing with anhydrous ethanol and distilled water for more than three times after the reaction to prepare hydrophilic polystyrene-divinylbenzene microspheres.