Preparation method of carboxymethyl weak cation medium

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of a carboxymethyl weak cation medium, which comprises the following steps: by taking an agarose microsphere as a matrix, introducing a short-chain activating group on a hydroxyl group of the agarose microsphere, opening a ring in acid, and coupling carboxymethyl on an activating site to obtain the carboxymethyl weak cation exchange medium. The weak cation exchange medium prepared by the invention has higher ligand density and better dynamic adsorption loading capacity, can be applied to purification of protein with positive charges, is easy for industrial preparation and production, and has a good application prospect in separation and purification of biomacromolecules.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing a carboxymethyl weak cationic medium. Background Technology

[0002] Currently, the demand for the separation and purification of biological products is large and diverse, especially in the medical field where diagnostic reagents, drugs, and vaccines are most widely used. The structure and properties of the chromatographic medium significantly affect the separation and purification effect. Today, common separation and purification methods include gel chromatography, affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. Among these, ion exchange chromatography is one of the most frequently used methods for the separation and purification of biomolecules. Therefore, chromatography is a crucial tool in the field of biological separation and purification.

[0003] Ion exchange chromatography media are further divided into anion exchange media and cation exchange media. Based on the charge intensity of the functional ligands under the operating conditions, they are classified into strong cation exchange media and weak cation exchange media. Weak cation exchange media are widely used in the separation and purification of biomolecules such as recombinant proteins, natural products, and insulin in biopharmaceuticals and bioengineering.

[0004] For example, the existing technology with authorization announcement number CN101928341B and name "Purification Process for Separating Recombinant Human Interferon α1b Isomers" repeatedly uses weak cation exchange chromatography media to remove related proteins and isomers to obtain high-purity recombinant human interferon α1b.

[0005] For example, in the prior art, which has the announcement number CN115197311A and the name "A Purification Method for Recombinant Human Interleukin-12 Protein", the harvested protein sample is subjected to three purification steps, including complex weak cation exchange chromatography, and finally the purified recombinant human interleukin-12 protein is obtained, which retains the natural activity of rhIL-12 and can ultimately achieve a purity of up to 99% for rhIL-12 protein.

[0006] It is evident that weak cation exchange media have a wide range of applications; however, most of the media currently used in the market are imported brands, which are expensive.

[0007] In addition, there are currently high-capacity macroporous weak cation exchange chromatography media prepared by the "Grafting from" method using macroporous polyacrylate microspheres as the matrix. The resulting media shows good dynamic binding capacity and can be used to remove lysozyme from chicken egg white, achieving the purification effect of chicken egg white. However, the biggest drawback of this type of media is that it has high specific adsorption and most of them cannot be regenerated and recycled.

[0008] In summary, there is a need to provide a method for preparing weak cation exchange media to overcome the shortcomings of high production costs and inability to regenerate and recycle, so as to enable the industrialization of the preparation of weak cation exchange media. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a carboxymethyl cation exchange medium. This method involves introducing an activator onto the surface of agarose gel, characterized by the presence of epoxy groups. The reaction conditions are relatively mild, the preparation is simple, and the medium exhibits high activation efficiency. The activated microspheres possess spacer arms of suitable chain length, reducing steric hindrance for subsequent carboxymethyl coupling and facilitating binding to target biological proteins, thus demonstrating excellent dynamic adsorption capacity. This medium can be used for the adsorption and purification of cationic proteins. The preparation of this medium is simple, suitable for industrial-scale production, and applicable to the separation and purification of biomolecules.

[0010] The technical solution of this invention is achieved as follows: a method for preparing a carboxymethyl cation exchange medium, comprising the following steps: i) Activation (also called epoxidation): A certain amount of agarose microspheres are dispersed in a mixed solvent of purified water and dimethyl sulfoxide, NaOH is added, and the mixture is thoroughly mixed at 25~45℃ to obtain a reaction solution. Then, an epoxy agent is added, and the reaction is continued for 3-10 hours. After washing, epoxidized agarose microspheres are obtained. ii) Hydrolysis: Disperse the agarose microspheres obtained in step i in sulfuric acid aqueous solution, and hydrolyze them fully at 40~60℃ for 18~30h. Adjust the pH to neutral and wash with water to obtain the hydrolyzed agarose microspheres. iii) Coupling with carboxymethyl groups: The agarose microspheres obtained in step ii are dispersed in a mixed solution of purified water and dimethyl sulfoxide, a ligand compound is added, the pH of the reaction solution is adjusted to between 9 and 13 with NaOH, and the reaction is stirred at 30-50℃ for 6-12 h. After washing, a weak cation exchange medium is obtained.

[0011] Furthermore, the particle size of the agarose microspheres in step i is 45~300μm, with 45~165μm being optimal.

[0012] Furthermore, the agarose microspheres in step i can be any one of agarose microsphere 4B, agarose microsphere 6B, agarose microsphere 4FF, or agarose microsphere 6FF, with agarose microsphere 6FF being the optimal choice.

[0013] Furthermore, the epoxy agent in step i is any one of epichlorohydrin, epibromopropane, 1,4-butanediol glycidyl ether, and 2-(4-bromophenyl)ethylene oxide, with epibromopropane being the most preferred.

[0014] Furthermore, in step i, the mass ratio of epoxy agent to agarose microspheres is 1:0.5~4, with 1:1~2 being optimal.

[0015] Furthermore, the concentration of NaOH in the reaction solution in step i is 0.5~4 mol / L, with 0.5~2 mol / L being optimal.

[0016] Furthermore, the concentration of the sulfuric acid aqueous solution in step ii is 0.8~2 mol / L, with 1 mol / L being optimal.

[0017] Furthermore, the ligand compound in step iii is any one of bromoacetic acid, chloroacetic acid, and glycidyl oxypropionic acid, and when the ligand compound is chloroacetic acid, the mass ratio of chloroacetic acid to agarose microspheres is 1:1 to 5, with 1:1 to 2 being optimal.

[0018] Furthermore, the optimal pH range for the reaction solution in step iii is 11-12.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: 1. During the preparation process, epoxy groups are introduced onto the surface of agarose microspheres and then acidified to reduce steric hindrance for subsequent coupling group incorporation, thereby increasing the amount of subsequent coupling group incorporation. The final prepared medium has a dynamic adsorption capacity of 40.2 mg / mL·gel for BSA protein, which is far higher than that of the prior art (see the specific embodiments of the present invention for details).

[0020] 2. The carboxymethyl group is coupled to the surface of agarose microspheres. This reaction is simple, economical and efficient, and produces little industrial pollution. The cation exchange medium prepared can be eluted, regenerated and reused repeatedly. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the carboxymethyl cationic medium in a specific embodiment of the present invention under an optical microscope. Figure 2 The dynamic adsorption loading test spectrum of the medium prepared in specific embodiment 2 of the present invention; Figure 3 The image shows the dynamic adsorption loading test spectrum of the medium prepared in Comparative Example 3 in a specific embodiment of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: 5 g of agarose microspheres 6FF (particle size 45-165 μm, the same below) were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 0.72 g of NaOH and 5 g of epichlorohydrin were added, and the mixture was placed in a shaker at 30 °C for 3 h. The microspheres were washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 21.78 μmol / g·gel by titration.

[0025] The agarose microspheres were redispersed in a 1 mol / L sulfuric acid aqueous solution, shaken at 170 r / min for 24 h at 55 °C, and then washed with water until neutral to obtain hydrolyzed agarose microspheres.

[0026] The hydrolyzed agarose microspheres were dispersed in 20 mL of a 1:3 mixture of water and dimethyl sulfoxide. 2.5 g of chloroacetic acid was added to adjust the pH to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity for BSA (bovine serum albumin) was determined to be 32.0 mg / mL·gel.

[0027] Example 2: 5 g of agarose microspheres 6FF were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 0.48 g of NaOH and 5 g of epichlorohydrin were added, and the mixture was reacted in a shaker at 30 °C for 3 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 19.94 μmol / g·gel by titration.

[0028] The microspheres were redispersed in a 1 mol / L sulfuric acid aqueous solution and shaken at 150 r / min for 24 h at 55 °C. After washing with water until neutral, hydrolyzed agarose microspheres were obtained.

[0029] The hydrolyzed agarose microspheres were dispersed in 20 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 2.5 g of chloroacetic acid was added, and the pH was adjusted to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 40.2 mg / mL·gel.

[0030] Example 3: 5 g of agarose microspheres 6B were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 0.38 g of NaOH and 2.5 g of epichlorohydrin were added, and the mixture was reacted in a shaker at 30 °C for 6 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 17.32 μmol / g·gel by titration.

[0031] The microspheres were redispersed in a 0.8 mol / L sulfuric acid aqueous solution and shaken at 150 r / min for 24 h at 50 °C. After washing with water until neutral, hydrolyzed agarose microspheres were obtained.

[0032] The hydrolyzed agarose microspheres were dispersed in 20 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 2.5 g of chloroacetic acid was added, and the pH was adjusted to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 23.6 mg / mL·gel.

[0033] Example 4: 5 g of agarose microspheres 4FF were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 0.96 g of NaOH and 5 g of epichlorohydrin were added, and the mixture was reacted in a shaker at 30 °C for 6 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 24.93 μmol / g·gel by titration.

[0034] The microspheres were redispersed in a 1 mol / L sulfuric acid aqueous solution and shaken at 150 r / min for 24 h at 55 °C. After washing with water until neutral, hydrolyzed agarose microspheres were obtained.

[0035] The hydrolyzed agarose microspheres were dispersed in 20 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 5 g of chloroacetic acid was added to adjust the pH to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 30.6 mg / mL·gel.

[0036] Example 5: 5 g of agarose microspheres 6FF were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 0.72 g of NaOH and 5 g of 2-(4-bromophenyl)ethylene oxide were added, and the mixture was reacted in a shaker at 30 °C for 9 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 18.94 μmol / g·gel by titration.

[0037] The microspheres were redispersed in a 1 mol / L sulfuric acid aqueous solution and shaken at 150 r / min for 24 h at 55 °C. After washing with water until neutral, hydrolyzed agarose microspheres were obtained.

[0038] The hydrolyzed agarose microspheres were dispersed in 20 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 2.5 g of chloroacetic acid was added, and the pH was adjusted to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 26.8 mg / mL·gel.

[0039] The following are comparative examples provided by this invention: Comparative Example 1: 5 g of agarose microspheres 4FF were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 1.44 g of NaOH and 5 g of epichlorohydrin were added, and the mixture was reacted in a shaker at 30 °C for 10 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 30.17 μmol / g·gel by titration.

[0040] The microspheres were redispersed in a 1 mol / L sulfuric acid aqueous solution and shaken at 150 r / min for 24 h at 55 °C. After washing with water until neutral, hydrolyzed agarose microspheres were obtained.

[0041] The hydrolyzed agarose microspheres were dispersed in 20 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 2.5 g of chloroacetic acid was added to adjust the pH to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 19.4 mg / mL·gel.

[0042] Comparative Example 2: 5 g of agarose microspheres 6FF were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a 1:3 mixture of water and dimethyl sulfoxide. 0.48 g of NaOH and 5 g of epichlorohydrin were added, and the mixture was reacted in a shaker at 30 °C for 3 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The epoxy group density was determined to be 18.62 μmol / g·gel by titration.

[0043] The microspheres were redispersed in a 1 mol / L sulfuric acid aqueous solution and shaken at 150 r / min for 24 h at 55 °C. After washing with water until neutral, hydrolyzed agarose microspheres were obtained.

[0044] The hydrolyzed agarose microspheres were dispersed in 20 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 5 g of chloroacetic acid was added to adjust the pH to 9. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 16.2 mg / mL·gel.

[0045] Comparative Example 3: 5 g of agarose microspheres 6FF were added to a 50 mL Erlenmeyer flask and dispersed in 12 mL of a mixed solvent of water and dimethyl sulfoxide in a ratio of 1:3. 2.5 g of chloroacetic acid was added to adjust the pH to 12. The mixture was reacted in a shaker at 45 °C and 150 r / min for 8 h. The microspheres were then washed with deionized water and an appropriate amount of anhydrous ethanol. The dynamic adsorption capacity of the microspheres for BSA was determined to be 15.4 mg / mL·gel.

[0046] As can be seen from the above examples and comparative examples, the adsorption capacity of the weak cation exchange medium obtained without the epoxidation acidification step is only 15.4 mg / mL·gel, while the adsorption capacity of the weak cation exchange medium obtained by the preparation method of the present invention can reach 40.2 mg / mL·gel. The latter is far superior to the former.

[0047] In addition, the present invention also provides the following methods: 1. Method for measuring epoxy group density (i.e., the titration method in this invention): Step 1) Accurately weigh approximately 1 g (W) 样品 The epoxy-activated microsphere sample was placed in a 100 mL iodine flask. 5 mL of Na2S2O3 standard solution (1M) was added to the flask by pipetting. The flask was then placed in a shaker at 30 °C and shaken thoroughly for 3 h. Step 2) Add 10 mL of deionized water; Step 3) Titrate with 0.05 M hydrochloric acid standard solution, using methyl red-methylene blue as an indicator. The endpoint is reached when the titration solution changes from green to red. Record the volume V of hydrochloric acid standard solution consumed. 样品 .

[0048] C 环氧基 The formula for calculating (μmol / g.gel) is as follows: C 环氧基 =C HCL *V 样品 / W 样品 2. Dynamic adsorption loading measurement method: Equilibrium: The prepared weak cation exchange medium is packed into the chromatography column (V B=1 mL), connect to the protein purification system, purge 10 column volumes of deionized water, and after the bed stabilizes, equilibrate 20 column volumes with buffer (20 mM citrate, pH 3.5); Sample loading: Prepare a 2 mg / mL (c0) BSA protein solution and flow it through the protein purification system at a flow rate of 0.5 mL / min. After obtaining the UV detection signal value (100%) of the BSA solution at the outlet, let the protein flow into the equilibrated chromatography column and record the volume V0 (mL). Stop the sample loading when the UV detection signal of the flow-through curve reaches 10% and record the sample loading volume V1 (mL). Elution: After equilibrating the column with 20 column volumes of buffer (20 mM PB, pH 3.5), proteins were eluted with high-salt buffer (20 mM PB + 1.0 M NaCl, pH 3.5). The dynamic loading capacity of the protein adsorption medium, Q, was measured. 10% The formula for calculating (mg / mL) is as follows: 3. Recycling method: Flow 0.8M NaOH through the eluted chromatography column (the elution requirements can be referred to the elution steps in the dynamic adsorption loading measurement method), wash for 20 column volumes, then equilibrate 20 column volumes with buffer (20 mM citrate, pH 3.5), and finally store the chromatography column in 20% ethanol solution for reuse next time.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a carboxymethyl weak cationic medium, characterized in that: Includes the following steps: i) Activation: A certain amount of agarose microspheres are dispersed in a mixed solvent of purified water and dimethyl sulfoxide, NaOH is added, and the mixture is thoroughly mixed at 25~45℃ to obtain a reaction solution. Then, an epoxy agent is added, and the reaction is continued for 3-10 hours. After washing, epoxidized agarose microspheres are obtained. ii) Hydrolysis: Disperse the agarose microspheres obtained in step i in sulfuric acid aqueous solution, and hydrolyze them fully at 40~60℃ for 18~30h. Adjust the pH to neutral and wash with water to obtain the hydrolyzed agarose microspheres. iii) Coupling with carboxymethyl groups: The agarose microspheres obtained in step ii are dispersed in a mixed solution of purified water and dimethyl sulfoxide, a ligand compound is added, the pH of the reaction solution is adjusted to between 9 and 13 with NaOH, and the reaction is stirred at 30-50℃ for 6-12 h. After washing, a weak cation exchange medium is obtained.

2. The method for preparing a carboxymethyl weak cationic medium according to claim 1, characterized in that: In step i, the particle size of the agarose microspheres is 45~300μm.

3. The method for preparing a carboxymethyl weak cationic medium according to claim 1 or 2, characterized in that: The agarose microspheres in step i are any one of agarose microspheres 4B, agarose microspheres 6B, agarose microspheres 4FF, and agarose microspheres 6FF.

4. The method for preparing a carboxymethyl weak cationic medium according to claim 1, characterized in that: The epoxy agent in step i is any one of epichlorohydrin, epibromopropane, 1,4-butanediol glycidyl ether, and 2-(4-bromophenyl)ethylene oxide.

5. The method for preparing a carboxymethyl weak cationic medium according to claim 1 or 4, characterized in that: In step i, the mass ratio of epoxy agent to agarose microspheres is 1:0.5~4.

6. The method for preparing a carboxymethyl weak cationic medium according to claim 1, characterized in that: In step i, the concentration of NaOH in the reaction solution is 0.5~4 mol / L.

7. The method for preparing a carboxymethyl weak cationic medium according to claim 1, characterized in that: The concentration of the sulfuric acid aqueous solution in step ii is 0.8~2 mol / L.

8. The method for preparing a carboxymethyl weak cationic medium according to claim 1, characterized in that: The ligand compound in step iii is any one of bromoacetic acid, chloroacetic acid, and glycidyl oxypropionic acid.

9. The method for preparing a carboxymethyl weak cationic medium according to claim 8, characterized in that: The ligand compound in step iii is chloroacetic acid, and the mass ratio of chloroacetic acid to agarose microspheres is 1:1~5.

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