Ligands-optimized ion exchange chromatographic filler as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of ion exchange chromatographic packing, and particularly relates to ligand-optimized ion exchange chromatographic packing as well as a preparation method and application thereof. The optimized ion exchange chromatography packing comprises a chromatography packing matrix and an ion exchange ligand. The ion exchange ligand comprises a functional group, a connecting arm and an auxiliary group. The auxiliary group is an alkyl chain segment or an alkoxy chain segment or an aryl chain segment with hydrophobic property. The ligand-optimized ion exchange chromatographic filler provided by the invention is used for adjusting the acting force of the ion exchange chromatographic filler, and selective adsorption and elution of different proteins on a chromatographic column are realized by utilizing various synergistic action mechanisms such as electrostatic interaction, hydrophobic interaction and the like between the optimized ligand and the proteins; therefore, the purpose of efficiently separating and purifying a complex protein mixed system is achieved, and the protein separation performance is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of ion exchange chromatography packing materials, and particularly relates to an ion exchange chromatography packing material with optimized ligand, a preparation method thereof, and an application thereof. Background Art

[0002] Due to the characteristics of high separation precision, mild separation conditions, simple operation and high repeatability, chromatography technology has become one of the most commonly used separation and purification means in the production process of large-scale biological products. As an important separation and analysis technology, ion exchange chromatography plays a key role in the separation and purification field of proteins. Although the existing ion exchange chromatography packing materials can achieve a certain degree of separation of proteins, there are still many limitations.

[0003] On the one hand, the ligand structure adopted by traditional ion exchange chromatography packing materials is relatively single. When interacting with complex and diverse proteins, it is difficult to accurately achieve efficient and highly selective separation based on the different physical and chemical properties of proteins. For example, for some proteins with similar isoelectric points, small molecular weight differences but subtle structural differences, conventional packing materials often cannot achieve ideal separation effects. On the other hand, in the actual biological samples or the production process of biopharmaceuticals, it is often necessary to process complex mixed systems containing multiple proteins, and the performance indicators such as separation degree, sample loading capacity and separation speed of the existing packing materials still need to be improved when dealing with such complex situations. This greatly limits the efficiency of protein separation and purification work and the quality of the final products, and increases the production cost and time cost.

[0004] The existing multi-mode packing materials related to ion exchange mostly focus on the modification of the ion exchange ligand itself, which can effectively increase the retention time of proteins on the multi-mode chromatography packing materials related to ion exchange. However, in the separation process of different proteins, due to the enhancement of the interaction between the packing material and proteins after the modification of the ion exchange ligand itself, while the selectivity has not been significantly improved, problems such as low chromatogram resolution and low column efficiency are often encountered. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides an ion exchange chromatography packing material with optimized ligand, a preparation method thereof, and an application thereof, so as to significantly improve its separation performance of proteins and better meet the requirements of high-efficiency separation and purification of proteins in the fields of biochemical research, biopharmaceuticals, etc.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first object of the present invention is to provide a ligand-optimized ion exchange chromatography packing material. The ion exchange chromatography packing material includes a packing matrix and an optimized ligand. The packing matrix is connected to the optimized ligand. The optimized ligand includes a functional group, a linker, and an auxiliary group. The functional group is a group having an ion exchange effect. The linker is a segment connecting the packing matrix and the functional group. The auxiliary group is an aryl segment, an alkyl segment, or an alkoxy segment having hydrophobic properties located on the linker.

[0008] Furthermore, the group having an ion exchange effect is an amino group with a charge, a quaternary ammonium group with a charge, a carboxyl group with a charge, or a sulfonic acid group with a charge.

[0009] Furthermore, the auxiliary group is a benzyl group, a phenyl group, or an alkyl group having hydrophobic properties.

[0010] Furthermore, the chromatography packing matrix is a silica gel matrix, polymer microspheres, or a polysaccharide gel matrix.

[0011] The second object of the present invention is to provide a preparation method of the above ligand-optimized ion exchange chromatography packing material, including the following steps: S1. Introduce an optimized ligand onto the chromatography packing matrix, and the linker on the optimized ligand has an active site; or react a multi-active site compound containing an epoxy group with the chromatography packing matrix to introduce a multi-active site segment, and then introduce an ion exchange group at one of the active sites to form a linker with an active site.

[0012] S2. Modify the linker by reacting the active site on the linker with a compound containing a hydrophobic group to obtain an auxiliary ligand, and further obtain a ligand-optimized ion exchange chromatography packing material.

[0013] Furthermore, the method for introducing a linker with an active group is as follows: React a compound with an epoxy group at one end and an ion exchange group at the other end with a hydroxyl group or an amino group of the chromatography packing matrix to form a linker with an active hydroxyl group; or react a multi-active site compound containing an epoxy group with a hydroxyl group or an amino group of the chromatography packing matrix to introduce a multi-active site segment, and then introduce an ion exchange group at one of the active sites to form a linker with an active site.

[0014] Furthermore, the ion exchange group is introduced by a compound containing an amino group with a charge, a compound containing a quaternary ammonium group with a charge, a compound containing a carboxyl group with a charge, or a compound containing a sulfonic acid group with a charge; or introduced by a reaction for generating a charged group through an amination reaction, a quaternization reaction, a carboxylation reaction, or a sulfonation reaction.

[0015] Further, the compound containing a hydrophobic group is a compound with an aryl segment, an alkyl segment, or an alkoxy segment at one end.

[0016] Further, the multi-active-site compound containing an epoxy group is a compound with an epoxy group at one end and a modifiable group at the other end, and the modifiable group is an alkenyl group, an alkynyl group, a hydroxyl group, a carboxyl group, an aldehyde group, a mercapto group, an epoxy group, or a halogen.

[0017] The third object of the present invention is to provide the application of the above ligand-optimized ion exchange chromatography packing material in protein separation and purification.

[0018] The present invention has the following beneficial effects compared with the prior art: The ligand-optimized ion exchange chromatography packing material provided by the present invention comprises a packing matrix and an optimized ligand. The optimized ligand includes a functional group, a linker, and an auxiliary group. The functional group is the group that plays the role of ion exchange, the linker is the segment connecting the packing matrix and the functional group, and the auxiliary group is an aryl segment, an alkyl segment, or an alkoxy segment with hydrophobic properties located on the linker. The functional group can have electrostatic interaction with the amino acid residues with opposite charges on the protein surface, providing the main driving force for the adsorption of the protein on the packing surface. The auxiliary group can finely adjust the hydrophilicity and hydrophobicity of the packing surface, contributing to the hydrophobic interaction with the hydrophobic region in the protein, and obviously generating hydrophobic forces. By using various synergistic action mechanisms such as electrostatic interaction and hydrophobic interaction between the optimized ligand and the protein, selective adsorption and elution of different proteins on the chromatographic column are realized, so as to achieve the purpose of efficient separation and purification of a complex protein mixture system, and significantly improve its separation performance for proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic structural diagram of the ligand-optimized ion exchange chromatography packing material of the present invention.

[0020] Figure 2 It is a schematic structural diagram of the ligand-optimized quaternary ammonium type anion exchange chromatography packing material of the present invention.

[0021] Figure 3 It is a schematic structural diagram of the ligand-optimized sulfonic acid type cation exchange chromatography packing material of the present invention.

[0022] Figure 4 It is the separation effect diagram of different proteins by the ligand-optimized ion exchange chromatography packing material prepared in Example 1 of the present invention and the packing structure diagram.

[0023] Figure 5 It is the separation effect diagram of different proteins by the ion exchange packing material prepared in Comparative Example 1 of the present invention and the packing structure diagram.

[0024] Figure 6 Separation effect diagrams and packing structure diagrams of ion exchange chromatography packings prepared in Comparative Example 2 and Comparative Example 3 of the present invention for different proteins.

[0025] Figure 7 Separation effect diagrams and packing structure diagrams of the ion exchange chromatography packing prepared in Comparative Example 4 of the present invention for different proteins.

[0026] Figure 8 Separation effect diagrams and packing structure diagrams of ion exchange chromatography packings prepared in Example 2 and Comparative Example 5 of the present invention for different proteins.

[0027] Figure 9 Separation effect diagrams and packing structure diagrams of the ligand-optimized ion exchange chromatography packing prepared in Example 1 of the present invention for hydrophobic mode.

[0028] Figure 10 Separation effect diagrams and packing structure diagrams of the ligand-optimized ion exchange chromatography packing prepared in Example 2 of the present invention for hydrophobic mode.

[0029] Figure 11 Separation effect diagrams and corresponding polyacrylamide gel electrophoresis diagrams of the ligand-optimized ion exchange chromatography packings prepared in Example 1 and Example 2 of the present invention for egg white samples, Figure 11 wherein Figure a is the separation effect diagram and the corresponding polyacrylamide gel electrophoresis diagram of Example 1, and Figure b is the separation effect diagram and the corresponding polyacrylamide gel electrophoresis diagram of Example 2. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0031] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention. In the present invention, certain terms are used to refer to specific components. Those skilled in the art should understand that technicians will use different nouns to refer to the same component. The present invention does not distinguish components by the difference in nouns, but by the difference in the functions of components. As mentioned throughout the specification and claims, "including" is an open-ended term, so it should be understood as "including but not limited to".

[0032] Existing multimodal packing materials related to ion exchange mainly focus on the modification of the ion exchange ligand itself, which can effectively increase the retention time of proteins on multimodal chromatographic packing materials related to ion exchange. However, during the separation of different proteins, after the modification of the ion exchange ligand itself, the interaction force between the packing material and the protein increases, but the selectivity does not increase significantly. Problems such as low chromatogram resolution and low column efficiency are often encountered.

[0033] Based on the above problems, the present invention provides an ion exchange chromatographic packing material with ligand optimization. The ion exchange chromatographic packing material includes a packing matrix and an optimized ligand. The packing matrix is connected to the optimized ligand. The optimized ligand includes a functional group, a linker, and an auxiliary group. The functional group is a group with ion exchange function. The linker is a segment connecting the packing matrix and the functional group. The auxiliary group is an aryl segment, an alkyl segment, or an alkoxy segment with hydrophobic properties located on the linker.

[0034] In the present invention, the ion exchange group can have an electrostatic interaction with the amino acid residues with opposite charges on the protein surface, providing the main force for the adsorption of the protein on the packing material surface. The auxiliary group can finely adjust the hydrophilicity and hydrophobicity of the packing material surface, contributing to the generation of hydrophobic interaction with the hydrophobic region in the protein, obviously generating hydrophobic force and adjusting the adsorption capacity of the packing material.

[0035] In some embodiments, the group with ion exchange function is a group containing a charged amino group, a charged quaternary ammonium group, a charged carboxyl group, or a charged sulfonic acid group. It should be noted that as a preferred scheme of the present invention, the group with ion exchange function is a charged amino group, quaternary ammonium group, carboxyl group, or sulfonic acid group. Among them, the amino group can be a primary amine, secondary amine, or tertiary amine. The charged group can have an electrostatic interaction with the amino acid residues with opposite charges on the protein surface, providing the main force for the adsorption of the protein on the packing material surface. As the most preferred scheme of the present invention, the group with ion exchange function is a group containing a charged quaternary ammonium group or a charged sulfonic acid group.

[0036] In some embodiments, the auxiliary group is a hydrophobic compound containing a benzyl group, a phenyl group, or an alkyl group with hydrophobic properties. It should be noted that the alkyl group with hydrophobic properties is an alkyl segment or an alkoxy segment. The alkyl segment or alkoxy segment is a C3-C18 alkyl segment or alkoxy segment. As a preferred scheme of the present invention, the auxiliary group is a benzyl group, a phenyl group, or an alkyl group with hydrophobic properties. The hydrophilicity and hydrophobicity of the packing material surface can be finely adjusted by changing the length of the hydrophobic chain, contributing to the generation of hydrophobic interaction with the hydrophobic region of the protein, generating hydrophobic force, and adjusting the adsorption capacity of the packing material. As the most preferred scheme of the present invention, the auxiliary group is a benzyl group or a phenyl group with hydrophobic properties.

[0037] It should be noted that the present invention can also adjust the hydrophilic-hydrophobic property of the filler surface by changing the hydrophobic strength of the hydrophobic compound containing benzyl or phenyl and the bonding degree of the alkyl chain, generate hydrophobic interaction with the hydrophobic region of the protein, produce hydrophobic force, and adjust the adsorption capacity of the filler. In addition, based on the different charges of different proteins, the difference in hydrophobic interaction is increased, so the adsorption selectivity is also enhanced, which is beneficial to controlling the elution behavior of proteins under different elution conditions. In particular, the auxiliary ligand can endow the chromatographic filler matrix with bifunctionality. For example, the benzyl-assisted quaternary ammonium modified filler has both optimized strong anion exchange ability and can be used for hydrophobic chromatography.

[0038] In some embodiments, the chromatographic filler matrix is a silica gel matrix, polymer microspheres or polysaccharide gel matrix. It should be noted that the chromatographic filler matrix used in the present invention is a matrix commonly used by those skilled in the art for conventional protein separation. The purpose of the present invention is to improve the separation performance of the chromatographic filler matrix for proteins by modifying it with multifunctional ion exchange ligands. Therefore, the chromatographic filler matrix is not specifically limited. After the chromatographic filler matrix is selected, the selected matrix needs to be pretreated. For example, for a silica gel matrix, it is first soaked in a strong acid solution for a certain time (such as 1 h to 3 h) to remove surface impurities and increase the activity of silanol groups; then it is thoroughly rinsed with deionized water until neutral, and then dried for standby by high-temperature drying or vacuum drying. For polymer microspheres or polysaccharide gel matrices, appropriate organic solvents are used for cleaning, activation and other operations to ensure that their surfaces have good reaction activity, which is beneficial to the subsequent immobilization of ligands. For example, for cross-linked cellulose microspheres, they can be washed with 1 mol / L sodium hydroxide to improve the activity of cellulose hydroxyl groups. In a preferred embodiment, the preferred chromatographic filler matrix of the present invention is cross-linked cellulose microspheres, the particle size of the cross-linked cellulose microspheres is 10 μm to 25 μm, and the cross-linked cellulose microspheres are cellulose microspheres modified with epichlorohydrin. Among them, the cellulose microspheres are prepared according to the method disclosed in CN 101274985 A, except that magnetic substances are not added. After obtaining the cellulose microspheres, the cellulose microspheres are cross-linked with epichlorohydrin to obtain cross-linked cellulose microspheres, and the cross-linked cellulose microspheres with a particle size distribution of 10 μm to 25 μm are screened out.

[0039] The present invention also provides a preparation method of the above-mentioned ligand-optimized ion exchange chromatographic filler, including the following steps: S1. Introduce an optimized ligand on the chromatographic filler matrix, and the linker on the optimized ligand has active sites; or react a multi-active-site compound containing an epoxy group with the chromatographic filler matrix to introduce a segment with multi-active sites, and then introduce an ion exchange group on one of the active sites to form a linker with active sites.

[0040] S2. React the active sites on the linking arm with a compound containing a hydrophobic group to modify the linking arm, obtaining an auxiliary ligand, and further obtaining an ion exchange chromatography packing with optimized ligand.

[0041] It should be noted that introducing an ion exchange ligand onto the chromatography packing matrix adopts the conventional methods of those skilled in the art. The difference lies in that after the reaction between the ion exchange ligand and the chromatography packing matrix, its linking arm has active sites (such as hydroxyl groups). The linking arm with active sites and the ion exchange ligand can be introduced step by step or simultaneously, and by reacting the active sites with a compound containing a hydrophobic group, a hydrophobic group branch chain is formed on the linking arm, which can finely adjust the hydrophilicity and hydrophobicity of the packing surface, contributing to the generation of hydrophobic interaction with the hydrophobic region in proteins. Obviously, hydrophobic force will be generated to adjust the adsorption capacity of the packing.

[0042] In some embodiments, as a preferred solution of the present invention, the method of introducing a linking arm with an active group is as follows: React a compound with an epoxy group at one end and an ion exchange group at the other end with the hydroxyl group or amino group of the chromatography packing matrix to form a linking arm with an active hydroxyl group; or react a multi-active-site compound containing an epoxy group with the hydroxyl group or amino group of the chromatography packing matrix to introduce a segment with multi-active sites, and then introduce an ion exchange group at one of the active sites to form a linking arm with active sites.

[0043] In some embodiments, as a preferred solution of the present invention, the ion exchange group is introduced by a compound containing a charged amino group, a compound containing a charged quaternary ammonium group, a compound containing a charged carboxyl group, or a compound containing a charged sulfonic acid group; or introduced by a reaction of generating a charged group through an amination reaction, a quaternization reaction, a carboxylation reaction, or a sulfonation reaction.

[0044] It should be noted that as the most preferred solution, the present invention ensures that while connecting an ion exchange functional group (such as a quaternary ammonium group) to the chromatography packing matrix, a linking arm containing active sites is introduced. One way is to use a compound containing a quaternary ammonium group, and the compound containing a quaternary ammonium group is a quaternary ammonium salt containing glycidyl. In a most preferred embodiment, the quaternary ammonium salt containing glycidyl is 2,3-epoxypropyltrimethylammonium chloride, and cross-linked cellulose microspheres are modified with 2,3-epoxypropyltrimethylammonium chloride. While connecting the quaternary ammonium group to the chromatography packing matrix, a linking arm containing active hydroxyl groups (the epoxy reacts with the cellulose hydroxyl group to generate new hydroxyl groups) is introduced to form a quaternary ammonium type anion exchange chromatography packing, and the structural schematic diagram is as Figure 2 shown;

[0045] Another way is to react a multi-active-site compound containing epoxy groups with a chromatographic packing matrix to introduce a segment with multi-active sites, and then introduce an ion-exchange group at one of the active sites, substantially forming a linking arm with multi-active sites. In a most preferred embodiment, the multi-active-site compound containing epoxy groups is allyl glycidyl ether. The epoxy group in allyl glycidyl ether reacts with the hydroxyl or amino group of the chromatographic packing matrix to form a segment with multi-active groups (allyl, hydroxyl), and the allyl group can react with other reagents to generate an ion-exchange group (such as reacting with sodium metabisulfite to generate a sulfonic group), forming a sulfonic acid-based cation exchange chromatographic packing. The structural schematic diagram is as Figure 3 shown.

[0046] In some embodiments, as a preferred solution of the present invention, the compound containing a hydrophobic group is a compound with an aryl segment at one end, a compound with an alkyl segment, or a compound with an alkoxy segment. In a most preferred embodiment, the compound with a hydrophobic group can be benzyl chloride or phenyl glycidyl ether. After the substitution reaction of benzyl chloride and hydroxyl, the benzyl group is introduced onto the linking arm.

[0047] In some embodiments, as a preferred solution of the present invention, the multi-active-site compound containing epoxy groups is a compound with an epoxy group at one end and a modifiable group at the other end, and the modifiable group is an alkenyl group, an alkynyl group, a hydroxyl group, a carboxyl group, an aldehyde group, a mercapto group, an epoxy group, or a halogen. In a most preferred embodiment, the multi-active-site compound containing epoxy groups is allyl glycidyl ether, ethylene glycol diglycidyl ether, epichlorohydrin. In a more preferred embodiment, the multi-active-site compound containing epoxy groups is allyl glycidyl ether.

[0048] In addition, the present invention also provides the application of the above ligand-optimized ion exchange chromatography packing material in protein separation and purification. The chromatography packing material provided by the present invention is used to adjust the interaction force of the ion exchange chromatography packing material and has remarkable protein separation performance. In actual operation, a mixed solution to be separated containing multiple proteins is injected into an ion exchange chromatography column filled with the packing material of the present invention. By adjusting the composition of the mobile phase (for example, adjusting the type, concentration, pH value of the buffer solution, adding an appropriate amount of salts or organic solvents, etc.), and utilizing various synergistic action mechanisms such as the electrostatic interaction and hydrophobic interaction between the optimized ligand and the protein, the selective adsorption and elution of different proteins on the chromatography column are realized, so as to achieve the purpose of highly efficient separation and purification of a complex protein mixture system. Specific application scenarios include but are not limited to: a. The biopharmaceutical industry: In the production process of recombinant protein drugs, it is used to separate and purify the target recombinant protein from the fermentation broth or cell lysate, improve the product purity, remove impurity proteins, and ensure the quality and safety of drugs. b. The field of biochemical research: It can be applied to proteomics research to efficiently separate proteins in biological samples (such as cells, tissue extracts, etc.), facilitating subsequent identification, quantitative analysis and other research work, and helping to deeply explore the expression, function and interaction mechanism of proteins in vivo. c. The biotechnology industry: In enzyme engineering, it is used for the separation and purification of enzyme proteins, improving the activity recovery rate of enzymes, providing a high-quality product basis for the production of industrial enzymes, and can also be used in the quality control link of protein-based biological products (such as antibodies, vaccines, etc.) to ensure that the products meet relevant standards and requirements.

[0049] The following is further illustrated by specific examples.

[0050] Example 1 A preparation method of a ligand-optimized ion exchange chromatography packing material includes the following steps: S1. Take 10 mL of cross-linked cellulose microspheres, disperse them into 5 mL of pure water, add 0.5 mL of a sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir and dissolve and mix for 0.5 h, add 1 g of 2,3-epoxypropyltrimethylammonium chloride every 0.5 h, add a total of 7 times, stir and react for 8 h. After the reaction is completed, wash with pure water until neutral to prepare a conventional quaternary ammonium-based anion exchange resin with an active hydroxyl group on the linker arm.

[0051] S2. Take 10 mL of the quaternary ammonium anion exchange resin with an active hydroxyl group on the linker prepared above, disperse it in 5 mL of pure water, add 1 mL of sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h. At the same time, slowly dropwise add 1.5 mL of benzyl chloride and 2 mL of sodium hydroxide solution with a mass fraction of 40%, stir and react for 8 h. After the reaction is completed, wash with ethanol and pure water in turn until neutral. The quaternary ammonium anion exchange resin with optimized ligand is prepared.

[0052] Example 2 A preparation method of an ion exchange chromatography packing with optimized ligand, comprising the following steps: S1. Take 10 mL of cross-linked cellulose microspheres, disperse them in 5 mL of pure water, add 0.5 mL of sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h. Add 1 mL of allyl glycidyl ether every 0.5 h for a total of 5 times, stir and react for 8 h. After the reaction is completed, wash with pure water until neutral to obtain allyl-modified cellulose microspheres.

[0053] S2. Take 10 mL of the above-mentioned allyl-modified cellulose microspheres, disperse them in 5 mL of sodium acetate buffer solution (concentration: 4 M, pH = 5), stir and heat to 30 °C, add 1 g of sodium metabisulfite every 0.5 h for a total of 5 times, stir and react for 8 h. After the reaction is completed, wash with pure water until neutral to prepare a conventional sulfonic acid group cation exchange resin with an active hydroxyl group on the linker.

[0054] S3. Take 10 mL of the sulfonic acid group cation exchange resin with an active hydroxyl group on the linker prepared by the method of Example 3, disperse it in 5 mL of pure water, add 1 mL of sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h. Add 0.5 mL of phenyl glycidyl ether every 0.5 h for a total of 5 times, stir and react for 8 h. After the reaction is completed, wash with ethanol and pure water in turn until neutral to prepare a sulfonic acid group cation exchange resin with optimized ligand.

[0055] Comparative Example 1 A preparation method of an ion exchange chromatography packing, comprising the following steps: Take 10 mL of cross-linked cellulose microspheres, disperse them into 5 mL of pure water, add 0.5 mL of sodium hydroxide solution with a mass fraction of 40% and 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h, add 1 g of 2,3-epoxypropyltrimethylammonium chloride every 0.5 h, add a total of 7 times, stir and react for 8 h, and after the reaction, wash with pure water until neutral. A conventional quaternary ammonium anion exchange resin with an active hydroxyl group on the linker arm is prepared.

[0056] Comparative Example 2 A preparation method of an ion exchange chromatography packing, comprising the following steps: Take 10 mL of cross-linked cellulose microspheres, disperse them into 5 mL of pure water, add 5 mL of sodium hydroxide solution with a mass fraction of 40% and 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h, add 1 g of 6-bromohexyltrimethylammonium bromide every 0.5 h, add a total of 7 times, stir and react for 8 h, and after the reaction, wash with pure water until neutral. A conventional quaternary ammonium anion exchange resin with an inactive group on the linker arm is prepared.

[0057] Comparative Example 3 A preparation method of an ion exchange chromatography packing, comprising the following steps: S1. Take 10 mL of cross-linked cellulose microspheres, disperse them into 5 mL of pure water, add 5 mL of sodium hydroxide solution with a mass fraction of 40% and 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h, add 1 g of 6-bromohexyltrimethylammonium bromide every 0.5 h, add a total of 7 times, stir and react for 8 h, and after the reaction, wash with pure water until neutral. A conventional quaternary ammonium anion exchange resin with an inactive group on the linker arm is prepared.

[0058] S2. Take 10 mL of the above-prepared conventional quaternary ammonium anion exchange resin with an inactive group on the linker arm, disperse it into 5 mL of pure water, add 1 mL of sodium hydroxide solution with a mass fraction of 40% and 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h, while slowly dropping 1.5 mL of benzyl chloride and 2 mL of sodium hydroxide solution with a mass fraction of 40%, stir and react for 8 h, and after the reaction, wash with ethanol and pure water in sequence until neutral. A hydrophobically modified quaternary ammonium anion exchange resin is prepared.

[0059] Comparative Example 4 A preparation method of an ion exchange chromatography packing, comprising the following steps: S1. Take 10 mL of cross-linked cellulose microspheres, disperse them into 5 mL of pure water, add 0.5 mL of sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h. Add 1 mL of allyl glycidyl ether every 0.5 h, for a total of 5 times, stir and react for 8 h. After the reaction, wash with pure water until neutral to obtain allyl-modified cellulose microspheres.

[0060] S2. Take 10 mL of the above-mentioned allyl-modified cellulose microspheres, add 5 mL of pure water and 3.0 g of sodium acetate, stir for 15 min, dropwise add 3% bromine water until the yellow color does not disappear for 1 min, then stir for another 15 min, and add 60 mg of sodium formate to remove the remaining bromine water. After the reaction, wash with pure water until neutral to obtain brominated modified cellulose microspheres.

[0061] S3. Take 10 mL of the above-mentioned brominated modified cellulose microspheres. Disperse them into 5 mL of pure water, add 2 mL of sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h. Add 1 mL of N,N-dimethylbenzylamine every 0.5 h, for a total of 5 times, stir and react for 8 h. After the reaction, wash with pure water until neutral to obtain a quaternary ammonium anion exchange resin with a reactive hydroxyl group on the linker arm, and a benzyl group is introduced on the quaternary ammonium group to obtain a conventional multi-mode packing material.

[0062] Comparative Example 5 A preparation method of an ion exchange chromatography packing material includes the following steps: S1. Take 10 mL of cross-linked cellulose microspheres, disperse them into 5 mL of pure water, add 0.5 mL of sodium hydroxide solution with a mass fraction of 40%, 0.02 g of sodium borohydride, heat to 60 °C, add 4 g of anhydrous sodium sulfate, stir to dissolve and mix for 0.5 h. Add 1 mL of allyl glycidyl ether every 0.5 h, for a total of 5 times, stir and react for 8 h. After the reaction, wash with pure water until neutral to obtain allyl-modified cellulose microspheres.

[0063] S2. Take 10 mL of the above-mentioned allyl-modified cellulose microspheres, disperse them in 5 mL of sodium acetate buffer solution (concentration: 4 M, pH = 5), stir and heat to 30 °C, add 1 g of sodium metabisulfite every 0.5 h, for a total of 5 times, stir and react for 8 h. After the reaction, wash with pure water until neutral to prepare a conventional sulfonic acid group cation exchange resin with a reactive hydroxyl group on the linker arm.

[0064] Use the ion exchange chromatography packing materials prepared in Examples 1 - 2 and Comparative Examples 1 - 5 for protein separation tests.

[0065] Instruments used: AKTA protein purification system.

[0066] Chromatography column: 5 mL (16 mm × 25 mm).

[0067] Column packing and testing: Take 5.5 mL of ion exchange chromatography packing material and pack it into the above-mentioned chromatography column. Equilibrate with buffer A for 3 CV (column volume), load with the mixed protein solution, set a gradient elution of buffer B from 0 to 40 mL (gradient from 0 to 60%), and finally rinse with buffer B (100%) for 10 mL. The flow rate throughout the process is 1 mL / min. The gradient elution mode used for the ion exchange chromatography packing material prepared in Comparative Example 5 is: buffer B from 0 to 65 mL (gradient from 0 to 100%).

[0068] Anion chromatography: Buffer A consists of 50 mmol / L Tris, pH = 7.4.

[0069] Buffer B consists of 50 mmol / L Tris, 1 mol / L sodium chloride, pH = 7.4.

[0070] Mixed protein solution: A mixed solution of myoglobin, carbonic anhydrase, α - amylase, transferrin, α - lactalbumin, bovine serum albumin (BSA), and soy trypsin inhibitor.

[0071] Cation chromatography: Buffer A consists of 20 mmol / L phosphate buffer solution, pH = 7.4.

[0072] Buffer B consists of 20 mmol / L phosphate buffer solution, 1 mol / L sodium chloride, pH = 7.4.

[0073] Mixed protein solution: A mixed solution of myoglobin, lysozyme, cytochrome C, and ribonuclease A.

[0074] Figure 4 It is the test structure diagram of protein chromatographic separation for the ligand - optimized ion exchange chromatography packing material prepared in Example 1 of the present invention. Figure 4 In it, 1 is myoglobin, 2 is carbonic anhydrase, 3 is α - amylase, 4 is transferrin, 5 is α - lactalbumin, 6 is BSA, and 7 is soy trypsin inhibitor. AsFigure 4 As shown, the ion exchange chromatography packing prepared in Example 1 has excellent protein separation effect and successfully separates 7 different proteins.

[0075] Figure 5 This is the structural diagram of the protein chromatography separation test of the ion exchange chromatography packing prepared in Comparative Example 1 of the present invention. Figure 5 In it, 1 is myoglobin, 2 is carbonic anhydrase, 3 is α-amylase, 4 is transferrin, 5 is α-lactalbumin, 6 is BSA, and 7 is soy trypsin inhibitor. As Figure 5 shown, although the ion exchange chromatography packing prepared in Comparative Example 1 has good protein separation effect, there are still proteins that cannot be separated.

[0076] Figure 6 This is the structural diagram of the protein chromatography separation test of the ion exchange chromatography packings prepared in Comparative Example 2 and Comparative Example 3 of the present invention. Figure 6 In it, 1 is myoglobin, 2 is carbonic anhydrase, 3 is α-amylase, 4 is transferrin, 5 is α-lactalbumin, 6 is BSA, and 7 is soy trypsin inhibitor. As Figure 6 shown, the effect of the ion exchange chromatography packing prepared in Comparative Example 2 is equivalent to that of the packing prepared in Example 1. Although it has good protein separation effect, there are still proteins that cannot be separated. Although the effect of Comparative Example 3 is better than that of the ion exchange chromatography packing prepared in Comparative Example 2, the improvement is limited and it is significantly worse than the ion exchange chromatography packing prepared in Example 1.

[0077] In summary, excellent separation effect can be obtained only when the auxiliary ligand is located on the linker arm.

[0078] Figure 7 This is the structural diagram of the protein chromatography separation test of the ion exchange chromatography packing prepared in Comparative Example 4 of the present invention. Figure 7 In it, 1 is myoglobin, 2 is carbonic anhydrase, 3 is α-amylase, 4 is transferrin, 5 is α-lactalbumin, 6 is BSA, and 7 is soy trypsin inhibitor. As Figure 7 shown, the retention times of different proteins of the ion exchange chromatography packing prepared in Comparative Example 4 are all greatly increased, but its separation effect is not good and the column efficiency is not high. This result once again proves that excellent separation effect can be obtained only when the auxiliary ligand is located on the linker arm.

[0079] Figure 8 This is the structural diagram for the chromatographic separation test of proteins using the ion exchange chromatography packing materials prepared in Example 2 of the present invention and Comparative Example 5. Figure 8 In it, 1 is myoglobin, 2 is ribonuclease A, 3 is cytochrome C, and 4 is lysozyme. As Figure 8 shown, the ion exchange chromatography packing material prepared in Example 2 has excellent protein separation effect. Although the ion exchange chromatography packing material prepared in Comparative Example 5 has good protein separation effect, there are still proteins that cannot be separated. Thus, it can be seen that only when the auxiliary ligand is located on the linker arm can excellent separation effect be obtained.

[0080] The ligand-optimized ion exchange chromatography packing materials prepared in Examples 1 to 2 of the present invention were used for the hydrophobic mode protein separation test.

[0081] Instrument: AKTA protein purification system.

[0082] Chromatographic column: 5 mL (16 mm×25 mm).

[0083] Column packing and test: Take 5.5 mL of the ion exchange chromatography packing material and pack it into the above-mentioned chromatographic column. Equilibrate with buffer A for 3 CV (column volume), load the mixed protein solution (1 mL / min), and set a gradient elution of buffer B from 0 mL to 30 mL (gradient from 0% to 100%).

[0084] The composition of buffer A is 3 mol / L ammonium sulfate and 20 mmol / L phosphate, pH = 7.

[0085] The composition of buffer B is 20 mmol / L phosphate, pH = 7.

[0086] Mixed protein solution: A mixed solution of myoglobin, lysozyme, α-amylase, α-chymotrypsin, and insulin.

[0087] Figure 9 This is the separation effect diagram of the ligand-optimized ion exchange chromatography packing material prepared in Example 1 of the present invention for the hydrophobic mode. Figure 9 In it, 1 is myoglobin, 2 is lysozyme, 3 is α-amylase, 4 is α-chymotrypsin, and 5 is insulin. As Figure 9As shown, the packing can be used in the hydrophobic mode. This indicates that the successful introduction of hydrophobic groups has indeed changed the hydrophobicity of the ligand, enabling the separation of five proteins in the hydrophobic chromatography mode. This also proves that the introduction of hydrophobic ligands can strengthen the synergistic effect between the electrostatic and hydrophobic forces of the ligand, achieve precise regulation of the protein binding site, and thus improve the selectivity and resolution of the ligand for protein separation. At the same time, it also shows that the packing has a certain bifunctional effect, with excellent ion exchange chromatography performance and can also be used for separation in the hydrophobic mode.

[0088] Figure 10 Separation effect diagram of the ligand-optimized ion exchange chromatography packing prepared in Example 2 of the present invention for the hydrophobic mode Figure 10 In it, 1 is myoglobin, 2 is lysozyme, 3 is α-amylase, 4 is α-chymotrypsin, and 5 is insulin. As Figure 10 shown, the packing can be used to separate proteins in the hydrophobic mode. Although the five proteins were not completely separated in the hydrophobic mode, this result also proves that the hydrophobicity of the ligand can be effectively improved by introducing hydrophobic groups, strengthening the synergistic effect between the electrostatic and hydrophobic forces of the ligand, achieving precise regulation of the protein binding site, and improving the selectivity and resolution of the ligand for protein separation.

[0089] The ligand-optimized ion exchange chromatography packing prepared in Examples 1 to 2 was used for the separation of egg white proteins and the corresponding SDS-polyacrylamide gel electrophoresis test.

[0090] Instrument: AKTA protein purification system.

[0091] Chromatographic column: 5 mL (16 mm×25 mm).

[0092] Treatment of egg white sample: Take 20 mL of egg white, add 80 mL of 20 mmol / L phosphate buffer solution with pH = 7.4, centrifuge, and filter through a 0.45 μm filter membrane to obtain the original egg white solution.

[0093] Column packing and testing: Take 5.5 mL of the packing and install it in the above chromatographic column. Equilibrate with buffer A for 3 CV (column volume), load 1 mL of the original egg white solution, and then rinse with buffer A for another 3 CV (column volume). Then, set a gradient elution of buffer B from 0 mL to 40 mL (gradient from 0% to 60%), and finally rinse with buffer B (100%) for 10 mL. The flow rate throughout the process is 1 mL / min.

[0094] Anion chromatography: The composition of buffer A is 50 mmol / L Tris, pH = 7.4.

[0095] Buffer B is composed of 50 mmol / L Tris, 1 mol / L sodium chloride, and pH = 7.4.

[0096] Protein solution: Raw egg white solution.

[0097] Cation exchange chromatography: Buffer A is composed of 20 mmol / L phosphate buffer solution, and pH = 7.4.

[0098] Buffer B is composed of 20 mmol / L phosphate buffer solution, 1 mol / L sodium chloride, and pH = 7.4.

[0099] Protein solution: Raw egg white solution.

[0100] Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS - PAGE): Using 5% stacking gel and 12% separating gel, controlling the voltage at 150V. After electrophoresis, Coomassie Brilliant Blue R - 250 staining is carried out, and then decolorization and photography are performed.

[0101] Figure 11 The separation effect diagrams of the ligand - optimized ion - exchange chromatography packing materials prepared in Example 1 and Example 2 of the present invention for the egg white sample and the corresponding polyacrylamide gel electrophoresis diagrams Figure 11 In the figure, Figure a is the separation effect diagram of Example 1, Figure b is the corresponding polyacrylamide gel electrophoresis diagram of Example 1, Figure c is the separation effect diagram of Example 2, and Figure d is the corresponding polyacrylamide gel electrophoresis diagram of Example 2. Figure 11 In the figure, lane M is commercially available ovalbumin and lysozyme, E is the raw egg white solution, and the other lane labels correspond one - to - one with the chromatographic peak labels. As Figure 11 As shown in (a), the ligand - optimized quaternary ammonium - type strong anion packing material successfully purified ovotransferrin (chromatographic peaks 2, 3, lanes 2, 3) and ovalbumin (chromatographic peak 4, lane 4) from egg white. As Figure 11 As shown in (b), the ligand - optimized sulfonic acid - type strong cation packing material successfully purified avidin (chromatographic peak 2, lane 2) and lysozyme (chromatographic peak 3, lane 3) from egg white. Both of them have excellently completed the separation and purification of proteins in egg white, proving that the ligand - optimized ion - exchange packing materials have excellent practical application performance.

[0102] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the adopted step methods are the same as those in the embodiments, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0103] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A ligand-optimized ion exchange chromatography filler, characterized in that: The ion exchange chromatography filler comprises a filler matrix and an optimized ligand, the filler matrix and the optimized ligand are connected, and the optimized ligand comprises a functional group, a connecting arm and an auxiliary group; the functional group is a group having an ion exchange effect, the connecting arm is a segment connecting the filler matrix and the functional group, and the auxiliary group is an aromatic segment, an alkyl segment or an alkoxy segment containing hydrophobic properties and located on the connecting arm.

2. The ligand-optimized ion exchange chromatography filler according to claim 1, characterized in that: The group having an ion exchange function is a group containing a charged amine group, a charged quaternary ammonium group, a charged carboxyl group or a charged sulfonic acid group.

3. The ligand-optimized ion exchange chromatography filler according to claim 1, characterized in that: The auxiliary group is a benzyl, phenyl or alkyl group having hydrophobic properties.

4. The ligand-optimized ion exchange chromatography filler according to claim 1, characterized in that: The chromatographic filler matrix is ​​a silica gel matrix, polymer microspheres or a polysaccharide gel matrix.

5. A method for preparing the ligand-optimized ion exchange chromatography filler according to any one of claims 1 to 4, characterized in that: The following steps are involved: An optimized ligand is introduced into the chromatographic packing matrix, wherein the connecting arm of the optimized ligand has an active site; Alternatively, a multi-active site compound containing an epoxy group is reacted with a chromatographic filler matrix to introduce a segment with multiple active sites, and then an ion exchange group is introduced into one of the active sites to form a connecting arm with an active site; The connecting arm is modified by reacting the active site on the connecting arm with a compound containing a hydrophobic group to obtain an auxiliary ligand, thereby obtaining a ligand-optimized ion exchange chromatography filler.

6. The method for preparing the ligand-optimized ion exchange chromatography filler according to claim 5, characterized in that: The method of introducing a linker with an active site is as follows: a compound having an epoxy group at one end and an ion exchange group at the other end is reacted with the hydroxyl group or amino group of the chromatographic filler matrix to form a linker with an active hydroxyl group; or a chain segment with multiple active sites is introduced by reacting a multi-active site compound containing an epoxy group with the hydroxyl group or amino group of the chromatographic filler matrix, and then an ion exchange group is introduced into one of the active sites to form a linker with an active site.

7. The method for preparing the ligand-optimized ion exchange chromatography filler according to claim 5, characterized in that: The ion exchange groups are introduced through compounds containing charged amine groups, compounds containing charged quaternary ammonium groups, compounds containing charged carboxyl groups or compounds containing charged sulfonic acid groups; or introduced through reactions such as amination, quaternization, carboxylation or sulfonation to generate charged groups.

8. The method for preparing the ligand-optimized ion exchange chromatography filler according to claim 5, characterized in that: The compound containing a hydrophobic group is a compound containing an aromatic segment, an alkyl segment or an alkoxy segment at one end.

9. The method for preparing the ligand-optimized ion exchange chromatography filler according to claim 5, characterized in that: The multi-active site compound containing epoxy groups is a compound having an epoxy group at one end and a modifiable group at the other end, wherein the modifiable group is an olefin group, an alkynyl group, a hydroxyl group, a carboxyl group, an aldehyde group, a thiol group, an epoxy group or a halogen.

10. Use of the ligand-optimized ion exchange chromatography filler according to any one of claims 1 to 4 in protein separation and purification.

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