Chromatographic medium, and preparation method therefor and use thereof

Abstract

The present invention relates to a chromatographic medium, and a preparation method therefor and the use thereof, and specifically to a chromatographic medium. The chromatographic medium uses a polysaccharide microsphere as a matrix, wherein the polysaccharide microsphere is bonded to an epoxy compound, the epoxy compound is coupled with a polysaccharide, and the polysaccharide is coupled with a ligand. The raw material of the polysaccharide microsphere comprises 1-20 wt% agarose. The chromatographic medium provided by the present invention has a high protein loading capacity, and exhibits good resolution with high resolving power when applied to the separation of biomacromolecules.

Description

Chromatographic medium, preparation method and application thereof

[0001] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on March 21, 2024, entitled “A Chromatographic Medium, Preparation Method and Application Thereof” and application number 202410328433.3. The entire contents of the above application are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of separation and purification, and in particular to a chromatography medium and a preparation method and application thereof. Background Art

[0003] Ion exchange chromatography purifies biomacromolecules such as target proteins by exploiting the charge differences between the ligands on the medium and the target molecules. Ion exchange chromatography is one of the most effective and widely used methods for separating and purifying biomacromolecules such as monoclonal antibodies and nucleic acids. Ion exchange chromatography media are the core building blocks of bioseparations. Currently, ion exchange chromatography media are categorized by charge as cation exchange and anion exchange, and further subdivided by charge level into strong cationic, weak cationic, strong anionic, and weak anionic exchange. Strong cationic and strong anionic exchange are the most widely used, accounting for approximately 60%. Currently, ion exchange chromatography media on the market can be divided into two types, polymers and polysaccharides, based on the matrix. For antibody purification, Thermo Fisher's POROS HQ 50 is the most popular anionic media, followed by Cytiva's Capto Q. Other media, such as Purolite's Praesto series and TOSOH's TOYOPEARL series, are less commonly used.

[0004] There are numerous established anionic and cationic fillers on the market, each with varying applications across different sample types. These differences are reflected in microsphere materials, such as polysaccharides and polymers, particle size distributions ranging from microns to millimeters (resins), and pore sizes from nanometers to millimeters. However, these established anionic and cationic fillers all have limitations, preventing them from achieving high-capacity and high-resolution separations simultaneously.

[0005] The present invention hopes to separate monomeric and polymeric proteins, and distinguish two proteins with similar properties through a high-load, high-resolution ion filler.

[0006] Summary of the Invention

[0007] In view of the defects of the prior art, the present invention provides a high-performance chromatography medium with good mechanical properties, large protein loading capacity and good resolution, as well as a preparation method and application thereof.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a chromatography medium, wherein the chromatography medium comprises polysaccharide microspheres as a matrix, the polysaccharide microspheres are bonded to an epoxy compound, the epoxy compound is coupled to a polysaccharide, and the polysaccharide is coupled to a ligand;

[0010] The raw materials of the polysaccharide microspheres include 1-20 wt% agarose, preferably 2-20 wt%.

[0011] Preferably, the raw material of the polysaccharide microspheres further includes cellulose, and the amount of the cellulose is 0.1 to 10 wt%, preferably 0.5 to 10 wt%, and more preferably 5 to 10 wt%.

[0012] Preferably, the polysaccharide coupled with the epoxy compound is selected from one or more of dextran, chitosan, gelatin and sodium alginate, preferably dextran.

[0013] Preferably, the mass ratio of the polysaccharide coupled with the epoxy compound to the polysaccharide microspheres coupled with the epoxy compound is 0.1 to 1:1, preferably 0.2 to 1:1.

[0014] Preferably, the epoxy compound is selected from epichlorohydrin and / or glycidyl ether.

[0015] Further preferably, the glycidyl ether is selected from one or more of 1,4-butanediol diglycidyl ether, allyl glycidyl ether, 1,2-ethylene glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether, preferably 1,4-butanediol diglycidyl ether.

[0016] Preferably, the ligand is an anion exchange ligand or a cation exchange ligand.

[0017] More preferably, the anion exchange ligand is selected from a quaternary ammonium ligand or a diethylaminoethyl ligand, and the cation exchange ligand is selected from a sulfonic acid ligand or a carboxymethyl ligand. Even more preferably, the quaternary ammonium ligand is selected from 2,3-epoxypropyltrimethylammonium chloride.

[0018] More preferably, the diethylaminoethyl type ligand is selected from diethylaminoethyl chloride hydrochloride.

[0019] More preferably, the sulfonic acid type ligand is selected from sulfonic acid esters.

[0020] More preferably, the carboxymethyl type ligand is selected from sodium chloroacetate.

[0021] Preferably, the particle size of the polysaccharide microspheres is 45 to 120 μm.

[0022] Preferably, the polysaccharide microspheres have a porous structure.

[0023] Preferably, the polysaccharide microspheres are bonded with epichlorohydrin, the epichlorohydrin is coupled with dextran, and the dextran is coupled with 2,3-epoxypropyltrimethylammonium chloride, diethylaminoethyl chloride hydrochloride, sulfonic acid ester or sodium chloroacetate.

[0024] In a second aspect, the present invention provides a method for preparing the chromatography medium as described above, comprising the following steps:

[0025] (1) Activated polysaccharide microspheres;

[0026] (2) reacting the activated polysaccharide microspheres obtained in step (1) with polysaccharide to obtain polysaccharide-coupled polysaccharide microspheres;

[0027] (3) coupling the polysaccharide-coupled polysaccharide microspheres obtained in step (2) with a ligand to obtain the chromatography medium.

[0028] Preferably, the step (1) comprises: mixing polysaccharide microspheres, a base, sodium borohydride and an activation reagent to react.

[0029] Further preferably, the mass ratio of the polysaccharide microspheres, base, sodium borohydride and activation reagent is 1:(0.05-0.5):(0.001-0.005):(0.1-0.5), preferably 1:(0.05-0.2):(0.001-0.005):(0.1-0.5).

[0030] Further preferably, the reaction temperature in step (1) is controlled to be 25 to 60° C.; and / or the reaction time in step (1) is controlled to be 2 to 20 h.

[0031] Preferably, the step (2) comprises: mixing the activated polysaccharide microspheres with the polysaccharide, and then adding alkali and sodium borohydride to react.

[0032] Further preferably, the mass ratio of the activated polysaccharide microspheres, polysaccharide, alkali and sodium borohydride is 1:(0.1-1):(0.05-0.5):(0.0005-0.005), preferably 1:(0.1-1):(0.05-0.2):(0.0005-0.002).

[0033] Further preferably, the reaction temperature of step (2) is controlled to be 25 to 60° C.; and / or the reaction time of step (2) is controlled to be 6 to 24 hours.

[0034] Preferably, the step (3) comprises: mixing the polysaccharide microspheres coupled with polysaccharide obtained in step (2) with water, a ligand, a base, and sodium borohydride to react; or,

[0035] The step (3) comprises the following steps: mixing the polysaccharide microspheres coupled with polysaccharides obtained in step (2) with water, a ligand and a base to react.

[0036] Further preferably, the feeding mass ratio of the polysaccharide microspheres, water, ligand, base and sodium borohydride for the coupled polysaccharide is 1:(0.1-0.5):(0.1-1.5):(0.05-0.5):(0.0005-0.005), preferably 1:(0.1-0.5):(0.2-1.2):(0.05-0.2):(0.0005-0.002); or, the feeding mass ratio of the polysaccharide microspheres, water, ligand and base for the coupled polysaccharide is 1:(0.1-1):(0.1-1):(0.1-0.5); preferably 1:(0.2-0.8):(0.3-1):(0.1-0.3).

[0037] Further preferably, the reaction temperature of step (3) is controlled to be 25 to 50° C.; and / or the reaction time of step (3) is controlled to be 6 to 24 hours.

[0038] Preferably, the reactions of step (1), step (2) and step (3) are all carried out under stirring.

[0039] Preferably, the base is sodium hydroxide.

[0040] Preferably, the activation reagent is selected from epichlorohydrin and / or glycidyl ether, and the glycidyl ether is selected from one or more of 1,4-butanediol diglycidyl ether, allyl glycidyl ether, 1,2-ethylene glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether, preferably 1,4-butanediol diglycidyl ether.

[0041] Preferably, the ligand is an anion exchange ligand or a cation exchange ligand.

[0042] Further preferably, the anion exchange ligand is selected from a quaternary ammonium type ligand or a diethylaminoethyl type ligand, and the cation exchange ligand is selected from a sulfonic acid type ligand or a carboxymethyl type ligand.

[0043] More preferably, the quaternary ammonium ligand is selected from 2,3-epoxypropyltrimethylammonium chloride.

[0044] More preferably, the diethylaminoethyl type ligand is selected from diethylaminoethyl chloride hydrochloride.

[0045] More preferably, the sulfonic acid type ligand is selected from sulfonic acid esters.

[0046] More preferably, the carboxymethyl type ligand is selected from sodium chloroacetate.

[0047] In a third aspect, the present invention provides a use of the chromatography medium described in any one of the above or the chromatography medium obtained by the preparation method described in any one of the above in the separation and purification of biomacromolecules.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention uses polysaccharide microspheres as the matrix. After cross-linking modification, the polysaccharide has an excellent rigid structure, which can improve the mechanical properties of the chromatography medium. At the same time, through the ratio between the various components, more epoxy groups are coupled to the surface of the chromatography medium. The epoxy groups are then grafted with polysaccharides and ligands in turn, thereby reducing the steric hindrance of the polysaccharide microspheres. When the chromatography medium provided by the present invention is applied to the purification of macromolecules, it has the advantages of high protein loading capacity, high resolution, and high chromatography efficiency. At the same time, the preparation process provided by the present invention is simple and the reaction conditions are easy to control, which is conducive to large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 shows the separation and analysis results of bovine serum albumin (BSA) and bovine hemoglobin (Hemo) using the chromatography medium prepared in Example 1 of the present invention. Figure 1(A) is a chromatogram; Figure 1(B) is a gel electrophoresis diagram, where 1: Marker, 2: Original, 3: E3, 4: E4, 5: E5, and 7: Marker.

[0051] Figure 2 shows the separation and analysis results of bovine serum albumin (BSA) and bovine hemoglobin (Hemo) using the chromatography medium prepared in Example 2 of the present invention. Figure 2(A) is a chromatogram; Figure 2(B) is a gel electrophoresis diagram, where 1: original sample, 2: E2, 3: E3, 4: E4, 5: E5, 6: E6, 7: E7, and 8: marker.

[0052] Figure 3 shows the separation and analysis results of bovine serum albumin (BSA) and bovine hemoglobin (Hemo) using the chromatography medium prepared in Example 3 of the present invention. Figure 3(A) is a chromatogram; Figure 3(B) is a gel electrophoresis diagram, where 7: Marker; 8: Original; 9: E9; 10: E10; 11: E11.

[0053] Figure 4 shows the separation and analysis results of bovine serum albumin (BSA) and bovine hemoglobin (Hemo) using the chromatography medium prepared in Example 6 of the present invention. Figure 4(A) is a chromatogram; Figure 4(B) is a gel electrophoresis diagram. Here, 1: BSA, 2: Hemo, 3: Original, 4: E1, 5: E2, 6: E3, 7: E4, and Mr: Marker.

[0054] Figure 5 shows the separation and analysis results of bovine serum albumin (BSA) and bovine hemoglobin (Hemo) using the chromatography medium prepared in Comparative Example 1 of the present invention. Figure 5(A) is a chromatogram; Figure 5(B) is a gel electrophoresis diagram. Here, 1: BSA, 2: Hemo, 3: Original, 4: E1, 5: E2, 6: E3, 7: E4, and Mr: Marker.

[0055] FIG6 is a graph showing the separation and analysis results of bovine serum albumin (BSA) and bovine hemoglobin (Hemo) using the chromatography medium prepared in Comparative Example 3 of the present invention.

[0056] Figure 7 shows the results of separation and analysis of cytochrome C and lysozyme using the chromatography medium prepared in Example 8 of the present invention. Figure 7(A) is a chromatogram; Figure 7(B) is a gel electrophoresis diagram, where Mr: Marker, 1: Original, 2: Flowthrough, 3: E1, 4: E2, 5: E3, 6: E4, 7: Cytochrome C standard, 8: Lysozyme standard.

[0057] Figure 8 shows the separation and analysis results of cytochrome C and lysozyme using the chromatography medium prepared in Comparative Example 4 of the present invention. Figure 8(A) is a chromatogram; Figure 8(B) is a gel electrophoresis diagram, where Mr: Marker, 1: Original, 2: Flowthrough, 3: E1, 4: E2, 5: E3, 6: E4, 7: E5, 8: E6, 9: E7, and 10: E8. DETAILED DESCRIPTION

[0058] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which the present invention belongs.

[0059] As used herein, “comprises,” “comprising,” “containing,” “having,” and the like may have the meanings described to them in U.S. patent law, and may mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially of” also have the meanings described in U.S. patent law, and the term is open-ended, allowing for the presence of more than the stated items as long as the basic or novel characteristics of the stated items are not changed by the presence of the more than stated items, but does not include prior art embodiments.

[0060] As used herein, the terms "mass %", "wt %", "mass percent" or "mass percent" are used interchangeably.

[0061] As used herein, the term "polysaccharide" as used herein includes natural polysaccharides, synthetic polysaccharides, polysaccharide derivatives, modified polysaccharides, and any mixtures thereof.

[0062] As used herein, the term "biomolecule" refers to a molecule that occurs naturally in living organisms. Biomolecules include macromolecules (such as viruses, proteins, carbohydrates, lipids, and nucleic acids) as well as small molecules (such as primary and secondary metabolites and natural products).

[0063] As used herein, the term "ligand" is used herein in its conventional sense in chromatography, i.e., for chemical entities that are capable of interacting with a target compound, such as a charged group that is capable of interacting with a target compound of opposite charge in an ion exchange process. Examples of ligand groups are positively charged or positively chargeable groups (anion exchange ligands); negatively charged or negatively chargeable groups (cation exchange ligands).

[0064] As used herein, the terms "separation matrix" and "matrix" are used interchangeably herein to refer to an insoluble carrier to which polysaccharides and / or ligands can be attached, and in the field of chromatography, matrices are sometimes referred to as resins or media.

[0065] In the present invention, microspheres formed by agarose or a blend of agarose and cellulose are selected as the matrix.

[0066] In the present invention, polysaccharide microspheres can be synthesized by any known method, for example, by emulsifying an aqueous solution containing agarose and cellulose in an oil phase (such as toluene or heptane) to form microspheres.

[0067] The polysaccharide microspheres used in the examples of the present invention were commissioned to Wuhan Huiyan Biotechnology Co., Ltd. for production, wherein the agarose-cellulose microspheres are of the Focurose HR series.

[0068] As used herein, the term "ionic capacity" refers to the capacity of a ligand-bearing matrix to bind ionic species. Ionic capacity is typically determined by titration methods known in the art and expressed as micromoles or millimoles per mL of precipitated matrix.

[0069] As used herein, the term "binding capacity" refers to the ability of a matrix to bind particulate species (eg, viral particles).

[0070] As used herein, the term "dynamic binding capacity" refers to the amount of a test species, such as a protein, that a separation matrix can bind in a penetration assay. In the present invention, the terms "capacity" and "dynamic binding capacity" are used interchangeably. Dynamic binding capacity is a good indicator of the suitability of a separation matrix for large-scale operation, as increased binding capacity significantly improves process economics.

[0071] In a first aspect, the present invention provides a chromatography medium, wherein the chromatography medium comprises polysaccharide microspheres as a matrix, the polysaccharide microspheres are bonded to an epoxy compound, the epoxy compound is coupled to a polysaccharide, and the polysaccharide is coupled to a ligand;

[0072] Among them, the raw material of the polysaccharide microspheres includes 1 to 20 wt% of agarose, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, etc.

[0073] In certain embodiments, the mass percentage of the agarose is 2-20%.

[0074] In some embodiments, the mass percentage of the agarose is 2 to 15%. In some embodiments, the raw material of the polysaccharide microspheres further includes cellulose, and the amount of the cellulose is 0.1 to 10 wt%, for example, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, etc. By controlling the ratio of agarose and cellulose in the matrix, the polysaccharide microspheres can have excellent rigidity, which can improve the mechanical properties, protein loading capacity and resolution of the chromatography medium.

[0075] In certain embodiments, the mass percentage of the cellulose is 0.5-10%.

[0076] In certain embodiments, the mass percentage of the cellulose is 5-10%.

[0077] In certain embodiments, the mass percentage of the cellulose is 8-10%.

[0078] In certain embodiments, the polysaccharide coupled with the epoxy compound is selected from one or more of dextran, chitosan, gelatin and sodium alginate.

[0079] In certain embodiments, the polysaccharide coupled with the epoxy compound is selected from dextran.

[0080] In certain embodiments, the dextran may be a type of dextran commonly used in the art.

[0081] In certain embodiments, the mass ratio of the epoxy compound coupled polysaccharide to the polysaccharide microspheres coupled with the epoxy compound is 0.1 to 1:1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, etc.

[0082] In certain embodiments, the mass ratio of the polysaccharide to the polysaccharide microspheres coupled with the epoxy compound is 0.2 to 1:1.

[0083] In certain embodiments, the epoxy compound is selected from epichlorohydrin and / or glycidyl ether.

[0084] In certain embodiments, the glycidyl ether is selected from one or more of 1,4-butanediol diglycidyl ether, allyl glycidyl ether, 1,2-ethylene glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether.

[0085] In certain embodiments, the glycidyl ether is 1,4-butanediol diglycidyl ether.

[0086] In certain embodiments, the ligand is an anion exchange ligand.

[0087] In certain embodiments, the anion exchange ligand is selected from quaternary ammonium ligands.

[0088] In certain embodiments, the quaternary ammonium ligand is selected from 2,3-epoxypropyltrimethylammonium chloride.

[0089] In certain embodiments, the anion exchange ligand is selected from diethylaminoethyl type ligands.

[0090] In certain embodiments, the diethylaminoethyl type ligand is selected from diethylaminoethyl chloride. In certain embodiments, the ligand is a cation exchange ligand.

[0091] In certain embodiments, the cation exchange ligand is selected from sulfonic acid-type ligands.

[0092] In certain embodiments, the sulfonic acid-type ligand is selected from sulfonic acid esters.

[0093] In certain embodiments, the cation exchange ligand is selected from carboxymethyl-type ligands.

[0094] In certain embodiments, the carboxymethyl-type ligand is selected from sodium chloroacetate.

[0095] In some embodiments, the mass ratio of the ligand to the polysaccharide microspheres coupled with the polysaccharide is 0.1 to 1.5:1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, etc.

[0096] In certain embodiments, the mass ratio of the ligand to the polysaccharide-coupled polysaccharide microspheres is 0.5-1.5:1.

[0097] In certain embodiments, the particle size of the polysaccharide microspheres is 45 to 120 μm.

[0098] In certain embodiments, the polysaccharide microspheres have a porous structure.

[0099] In certain embodiments, the polysaccharide microspheres are bonded with epichlorohydrin, the epichlorohydrin is coupled to dextran, and the dextran is coupled to 2,3-epoxypropyltrimethylammonium chloride and / or sulfonate.

[0100] In a second aspect, the present invention provides a method for preparing the chromatography medium as described above, the method comprising the following steps:

[0101] (1) Activated polysaccharide microspheres;

[0102] (2) reacting the activated polysaccharide microspheres obtained in step (1) with polysaccharide to obtain polysaccharide-coupled polysaccharide microspheres;

[0103] (3) coupling the polysaccharide-coupled polysaccharide microspheres obtained in step (2) with a ligand to obtain the chromatography medium.

[0104] In certain embodiments, the step (1) comprises: mixing polysaccharide microspheres, a base, sodium borohydride and an activation reagent to react.

[0105] In certain embodiments, the mass ratio of the polysaccharide microspheres, base, sodium borohydride and activation reagent is 1:(0.05-0.5):(0.001-0.005):(0.1-0.5), for example, 1:0.07:0.001:0.1, 1:0.1:0.001:0.1, 1:0.15:0.001:0.1, 1:0.2:0.001: 0.1, 1:0.07:0.002:0.1, 1:0.1:0.003:0.1, 1:0.15:0.004:0.1, 1:0.2:0.005:0.1, 1:0.2:0.002:0.2, 1:0.2:0.002:0.3, 1:0.2:0.002:0.4, 1:0.2:0.002:0.5, etc.

[0106] In certain embodiments, the mass ratio of the polysaccharide microspheres, base, sodium borohydride and activation reagent is 1:(0.05-0.2):(0.001-0.005):(0.1-0.5).

[0107] In certain embodiments, the mass ratio of the polysaccharide microspheres, base, sodium borohydride and activation reagent is 1:(0.05-0.2):(0.001-0.003):(0.1-0.5).

[0108] In certain embodiments, the reaction temperature in step (1) is controlled to be 25-60°C, for example, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, etc.

[0109] In certain embodiments, the reaction temperature in step (1) is controlled to be 25-40°C.

[0110] In certain embodiments, the reaction time in step (1) is controlled to be 2 to 20 h, for example, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, etc.

[0111] In certain embodiments, the reaction time in step (1) is controlled to be 2 to 10 hours.

[0112] In certain embodiments, the step (2) comprises: mixing the activated polysaccharide microspheres with the polysaccharide, and then adding alkali and sodium borohydride to react.

[0113] In certain embodiments, the mass ratio of the activated polysaccharide microspheres, polysaccharide, base and sodium borohydride is 1:(0.1-1):(0.05-0.5):(0.0005-0.005), for example, 1:0.2:0.1:0.001, 1:0.3:0.1:0.001, 1:0.4:0.1:0.001, 1:0.5:0.1:0.001, 1:0.6:0.1:0.001, 1:0.7:0.1:0.001, 1:0.8:0.1:0.00 1. 1:0.9:0.1:0.001, 1:1:0.1:0.001, 1:0.2:0.05:0.001, 1:0.3:0.05:0.001, 1:0.4:0.05:0.001, 1:0.5:0.05:0.001, 1:0.6:0.05:0.001, 1:0.7:0.05:0.001, 1:0.8:0.05:0.001, 1:0.9:0.05:0.001, 1:1:0.05:0.001, etc.

[0114] In certain embodiments, the mass ratio of the activated polysaccharide microspheres, polysaccharide, base and sodium borohydride is 1:(0.1-1):(0.05-0.2):(0.0005-0.002).

[0115] In certain embodiments, the mass ratio of the activated polysaccharide microspheres, polysaccharide, base and sodium borohydride is 1:(0.5-1):(0.05-0.15):(0.001-0.002).

[0116] In certain embodiments, the reaction temperature of step (2) is controlled to be 25-60°C, for example, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, etc.

[0117] In certain embodiments, the reaction temperature of step (2) is controlled to be 40-60°C.

[0118] In certain embodiments, the reaction time of step (2) is controlled to be 6 to 24 hours, for example, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc.

[0119] In certain embodiments, the reaction time of step (2) is controlled to be 15 to 24 hours.

[0120] In certain embodiments, step (3) is to prepare an anionic chromatography medium, and the step (3) comprises: mixing the polysaccharide microspheres coupled with polysaccharide obtained in step (2) with water, a ligand, a base, and sodium borohydride to react.

[0121] In certain embodiments, the mass ratio of the polysaccharide microspheres, water, ligand, base, and sodium borohydride coupled with the polysaccharide is 1:(0.1-0.5):(0.1-1.5):(0.05-0.5):(0.0005-0.005), for example, 1:0.3:0.1:0.05:0.0015, 1:0.3:0.2:0.05:0.0015, 1:0.3:0.3:0.05:0.0015, 1:0.3:0.4: 0.05:0.0015, 1:0.3:0.5:0.05:0.0015, 1:0.3:0.6:0.05:0.0015, 1:0.3:0.7:0.05:0.0015, 1:0.3:0.8:0.05:0.0015, 1:0.3:0.9:0.05:0.0015, 1:0.3:1.2:0.05:0.0015, 1:0.3:0.6:0.1:0.0015, etc.

[0122] In certain embodiments, the mass ratio of the polysaccharide microspheres, water, ligand, base, and sodium borohydride for the coupled polysaccharide is 1:(0.1-0.5):(0.2-1.2):(0.05-0.2):(0.0005-0.002).

[0123] In certain embodiments, the mass ratio of the polysaccharide microspheres, water, ligand, base, and sodium borohydride for the coupled polysaccharide is 1:(0.2-0.5):(0.2-1.2):(0.05-0.15):(0.0005-0.002).

[0124] In certain embodiments, step (3) is to prepare a cationic chromatography medium, and the step (3) comprises: mixing the polysaccharide microspheres coupled with polysaccharide obtained in step (2) with water, a ligand and a base to react.

[0125] In certain embodiments, when preparing a cationic chromatography medium, the mass ratio of the polysaccharide microspheres coupled with the polysaccharide, water, ligand, and base is 1:(0.1-1):(0.1-1):(0.01-1), for example, 1:0.2:0.2:0.2, 1:0.3:0.2:0.2, 1:0.4:0.2:0.2, 1:0.5:0.2:0.2, 1:0.6:0.2:0.2, 1:0.7:0.2:0.2, 1:0.5:0.3:0.2, 1:0.5:0.5:0.2, 1:0.5:0.6:0.2, 1:0.5:0.7:0.2, 1:0.5:0.5:0.5, 1:0.5:0.7:0.7, etc.

[0126] In certain embodiments, when preparing a cationic chromatography medium, the mass ratio of the polysaccharide microspheres coupled with the polysaccharide, water, ligand, and base is 1:(0.2-0.8):(0.3-1):(0.1-0.3).

[0127] In certain embodiments, when preparing a cationic chromatography medium, the mass ratio of the polysaccharide microspheres coupled with the polysaccharide, water, ligand, and base is 1:(0.2-0.8):(0.3-0.8):(0.1-0.3).

[0128] In certain embodiments, the reaction temperature of step (3) is controlled to be 25-50°C, for example, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, etc.

[0129] In certain embodiments, the reaction temperature of step (3) is controlled to be 25-40°C.

[0130] In certain embodiments, the reaction time of step (3) is controlled to be 6 to 24 hours, for example, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc.

[0131] In certain embodiments, the reaction time of step (3) is controlled to be 15 to 24 hours.

[0132] In certain embodiments, the reactions of step (1), step (2) and step (3) are all carried out under stirring conditions.

[0133] In certain embodiments, the base is sodium hydroxide.

[0134] In the present application, the base is prepared into an aqueous solution with a concentration of 0.5 to 18 mol / L to participate in the reaction, for example, 1 mol / L, 2 mol / L, 4 mol / L, 6 mol / L, 8 mol / L, 10 mol / L, 12 mol / L, 14 mol / L, 16 mol / L, 18 mol / L, etc.

[0135] In certain embodiments, the preparation method comprises the following steps:

[0136] (1) Mix the polysaccharide microspheres with sodium hydroxide, add sodium borohydride, stir, and add an activation reagent to mix and react;

[0137] (2) mixing the activated polysaccharide microspheres obtained in step (1) with polysaccharide, stirring, adding sodium hydroxide and sodium borohydride to react, and filtering and washing after the reaction to obtain polysaccharide-coupled polysaccharide microspheres;

[0138] (3) mixing the polysaccharide microspheres coupled with polysaccharide obtained in step (2) with water, adding a ligand, stirring, adding sodium hydroxide and sodium borohydride, heating, stirring to react, and filtering and washing after the reaction to obtain the chromatography medium; or

[0139] (3) Mixing the polysaccharide-coupled polysaccharide microspheres obtained in step (2) with water, adding a ligand, stirring, adding sodium hydroxide, heating, stirring to react, and filtering and washing after the reaction to obtain the chromatography medium.

[0140] In certain embodiments, the concentration of sodium hydroxide is 1 to 15 mol / L.

[0141] In certain embodiments, the activating agent is selected from epichlorohydrin and / or glycidyl ether.

[0142] In certain embodiments, the glycidyl ether is selected from one or more of 1,4-butanediol diglycidyl ether, allyl glycidyl ether, 1,2-ethylene glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether.

[0143] In certain embodiments, the glycidyl ether is 1,4-butanediol diglycidyl ether.

[0144] In certain embodiments, the ligand is an anion exchange ligand.

[0145] In certain embodiments, the anion exchange ligand is selected from quaternary ammonium ligands.

[0146] In certain embodiments, the quaternary ammonium ligand is selected from 2,3-epoxypropyltrimethylammonium chloride (GTA).

[0147] In certain embodiments, the anion exchange ligand is selected from diethylaminoethyl type ligands.

[0148] In certain embodiments, the diethylaminoethyl-type ligand is selected from diethylaminoethyl chloride hydrochloride.

[0149] In certain embodiments, the ligand is a cation exchange ligand.

[0150] In certain embodiments, the cation exchange ligand is selected from sulfonic acid-type ligands.

[0151] In certain embodiments, the sulfonic acid-type ligand is selected from sulfonic acid esters.

[0152] In certain embodiments, the cation exchange ligand is selected from carboxymethyl-type ligands.

[0153] In certain embodiments, the carboxymethyl-type ligand is selected from sodium chloroacetate.

[0154] The preparation process provided by the invention is simple, the reaction conditions are easy to control, and it is conducive to large-scale promotion and application.

[0155] In a third aspect, the present invention provides a use of the chromatography medium described in any one of the above or the chromatography medium obtained by the preparation method described in any one of the above in the separation and purification of biomacromolecules.

[0156] The present invention is further illustrated below by means of specific examples. Unless otherwise specified herein, "%" represents mass percentage. The materials and reagents used in the following examples, unless otherwise specified, are commonly used in the art and can be obtained from commercial sources or synthesized by known methods. Experimental methods in the following examples where no conditions are specified were generally performed according to conventional experimental conditions or the conditions recommended by the manufacturers of the relevant reagents (kits).

[0157] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0158] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0159] The following Examples 1-7 and Comparative Examples 1-3 relate to anionic chromatography media.

[0160] Example 1

[0161] (1) 100 g of agarose microspheres (2 wt% agarose) were washed with deionized water and added to a 250 mL reaction flask. 20 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 20 g of epichlorohydrin was added and the temperature was raised to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0162] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, 20 g of the polysaccharide (dextran) solution was added, and the mixture was stirred at 50°C for 1 hour. 20 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0163] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 30 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 20 g of sodium hydroxide solution (10 mol / L) and 0.15 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0164] Example 2

[0165] (1) 100 g of agarose microspheres (8 wt% agarose) were washed with deionized water and added to a 250 mL reaction flask. 30 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.3 g of sodium borohydride was then added and stirred for 10 minutes. 30 g of epichlorohydrin was added and the temperature was raised to 35°C and stirred for 5 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0166] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, 60 g of the polysaccharide (dextran) solution was added, and the mixture was stirred at 50°C for 1 hour. 30 g of sodium hydroxide solution (10 mol / L) and 0.2 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0167] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 80 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 20 g of sodium hydroxide solution (10 mol / L) and 0.2 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0168] Example 3

[0169] (1) 100 g of agarose microspheres (15 wt% agarose) were washed with deionized water and added to a 250 mL reaction flask. 20 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 30 g of epichlorohydrin was added and the temperature was raised to 40°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0170] (2) Add 100 g of the intermediate microspheres to a 250 mL reaction flask, add 90 g of the polysaccharide (dextran) solution, mix thoroughly, and stir at 50°C for 1 hour. Then, add 20 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride, and stir at 50°C for 20 hours. Filter and rinse with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0171] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 100 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 30 g of sodium hydroxide solution (10 mol / L) and 0.15 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0172] Example 4

[0173] (1) 100 g of agarose cellulose microspheres (1 wt% agarose and 1 wt% cellulose) (Focurose HR) were washed with deionized water and added to a 250 mL reaction flask. 20 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 20 g of epichlorohydrin was then added and the mixture was heated to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0174] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, 10 g of the polysaccharide (dextran) solution was added, and the mixture was stirred at 50°C for 1 hour. 20 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0175] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 20 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 20 g of sodium hydroxide solution (10 mol / L) and 0.15 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0176] Example 5

[0177] (1) 100 g of agarose cellulose microspheres (5 wt% agarose and 5 wt% cellulose) (Focurose HR) were washed with deionized water and added to a 250 mL reaction flask. 30 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 30 g of epichlorohydrin was then added and the mixture was heated to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0178] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, 50 g of polysaccharide (dextran) was added, and the mixture was stirred at 50°C for 1 hour. 30 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0179] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 60 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 20 g of sodium hydroxide solution (10 mol / L) and 0.15 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0180] Example 6

[0181] (1) 100 g of agarose cellulose microspheres (10 wt% agarose and 8 wt% cellulose) (Focurose HR) were washed with deionized water and added to a 250 mL reaction flask. 40 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 40 g of epichlorohydrin was then added and the mixture was heated to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0182] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, and 80 g of polysaccharide (dextran) was added and mixed. The mixture was stirred at 50°C for 1 hour. 30 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0183] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 90 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 20 g of sodium hydroxide solution (10 mol / L) and 0.15 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0184] Example 7

[0185] (1) 100 g of agarose cellulose microspheres (20 wt% agarose and 10 wt% cellulose) (Focurose HR) were washed with deionized water and added to a 250 mL reaction flask. 50 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 50 g of epichlorohydrin was then added and the mixture was heated to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0186] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, and 100 g of polysaccharide (dextran) was added and mixed. The mixture was stirred at 50°C for 1 hour. 30 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0187] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 30 g of deionized water, and stir to mix. Add 120 g of ligand (2,3-epoxypropyltrimethylammonium chloride) and stir for 1 h. Add 20 g of sodium hydroxide solution (10 mol / L) and 0.15 g of sodium borohydride, heat to 35°C, and stir for 20 h. Filter and rinse with deionized water to obtain an anionic chromatography medium.

[0188] Comparative Example 1

[0189] The preparation method of this embodiment is basically the same as that of Example 6, except that the amount of glucan added in step (2) is 130 g.

[0190] Comparative Example 2

[0191] The preparation method of this embodiment is basically the same as that of Example 6, except that the amount of ligand added is 180 g.

[0192] Comparative Example 3

[0193] The preparation method of this embodiment is basically the same as that of Example 3, except that the agarose microspheres contain 25 wt% agarose and no cellulose.

[0194] The ion exchange capacity and protein binding capacity of the chromatography media prepared in Examples 1-7 and Comparative Examples 1-3 were measured. The results are shown in Table 1.

[0195] Determination of ion exchange capacity:

[0196] S1. Load 5 mL of the prepared exchange chromatography medium into the gravity column.

[0197] S2, wash with 100mL water, add 50mL 0.5M HCl to wash, wash with deionized water until neutral, then add 50mL 1M KNO3 to rinse, collect the washing liquid,

[0198] S3. Add 3 drops of potassium chromate to the collected washing solution, titrate with 0.1MAgNO3 standard solution, and calculate the exchange capacity.

[0199] Determination of protein binding capacity:

[0200] S1. Load 5 mL of anion exchange medium into a 5 mL prepacked column and connect the prepacked column to the AKTA protein purification system.

[0201] S2. Sample concentration: 5 mg / mL BSA solution, load on the pump head, flow rate 2.5 mL / min, Buffer A (0.02 M Tris HCl, pH = 8.5), Buffer B (containing 0.02 M Tris HCl and 1 M NaCl, pH = 8.5), load on the pump head until 50% of the protein solution flows through, then use Buffer B to elute and collect to measure the loading capacity.

[0202] Table 1

[0203] Drawing on the preparation conditions of the above anionic chromatography media that can achieve good protein loading effects, they were further applied to cationic chromatography media for verification, as shown in Examples 8-9.

[0204] Example 8

[0205] (1) 100 g of agarose cellulose microspheres (10 wt% agarose and 8 wt% cellulose) (Focurose HR) were washed with deionized water and added to a 250 mL reaction flask. 30 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 30 g of epichlorohydrin was then added and the mixture was heated to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0206] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, and 80 g of the polysaccharide (dextran) solution was added and mixed. The mixture was stirred at 50°C for 1 hour. 20 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0207] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 50 g of deionized water, and stir to mix. Then add 90 g of sulfonate ligand and 50 g of sodium hydroxide solution (10 mol / L). Heat to 35°C and stir for 5 h. Filter and rinse with deionized water to obtain a cationic chromatography medium.

[0208] Example 9

[0209] (1) 100 g of agarose cellulose microspheres (10 wt% agarose and 8 wt% cellulose) (Focurose HR) were washed with deionized water and added to a 250 mL reaction flask. 30 g of sodium hydroxide solution (10 mol / L) was added and stirred to mix thoroughly. 0.2 g of sodium borohydride was then added and stirred for 10 minutes. 30 g of epichlorohydrin was then added and the mixture was heated to 35°C and stirred for 4 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0210] (2) 100 g of the intermediate microspheres were added to a 250 mL reaction flask, and 100 g of the polysaccharide (dextran) solution was added and mixed. The mixture was stirred at 50°C for 1 hour. 20 g of sodium hydroxide solution (10 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was filtered and washed with deionized water to obtain intermediate microspheres modified with a large amount of polysaccharide (dextran).

[0211] (3) Add 100 g of the microspheres to a 250 mL reaction flask, add 50 g of deionized water, and stir to mix. Then add 100 g of sulfonate ligand and 70 g of sodium hydroxide solution (10 mol / L). Heat to 35°C and stir for 5 h. Filter and rinse with deionized water to obtain a cationic chromatography medium.

[0212] Comparative Example 4

[0213] The difference between Comparative Example 4 and Example 8 is that the amount of glucan is 100 g and the amount of ligand is 180 g.

[0214] The ion exchange capacity and protein binding capacity of the cationic chromatography media prepared in Examples 8-9 and Comparative Example 4 were measured. The results are shown in Table 2.

[0215] Determination of ion exchange capacity:

[0216] S1. Load 5 mL of the prepared exchange chromatography medium into the gravity column.

[0217] S2, wash with 100mL water, add 50mL 0.5M HCl to wash, wash with deionized water until neutral, then add 50mL 1M KNO3 to rinse, collect the washing liquid,

[0218] S3. Add 3 drops of cresol red to the collected washing solution, titrate with 0.05M NaOH standard solution, and calculate the exchange capacity.

[0219] Determination of protein binding capacity:

[0220] S1. Load 5 mL of cation exchange medium into a 5 mL prepacked column and connect the prepacked column to the AKTA protein purification system.

[0221] S2, sample concentration 5mg / mL IgG solution, pump head load, flow rate 2.5mL / min, Buffer A (containing 0.02MNaAC and 40mM NaCl, pH = 4.5), Buffer B (containing 0.02M NaAC and 1M NaCl, pH = 4.5), pump head load until 50% of the protein solution flows through, then use Buffer B to elute and collect to measure the loading capacity.

[0222] Table 2

[0223] Test Example 1 Determination of the resolution of anion chromatography media

[0224] The chromatographic media prepared in Examples 1-3, Example 6, Comparative Example 1 and Comparative Example 3 were respectively loaded into chromatographic columns to perform purification, separation, and analysis tests on bovine serum albumin (BSA) and bovine hemoglobin (Hemo).

[0225] The conditions for purification, separation and analysis tests are as follows:

[0226] (1) Chromatography column specifications: Chromatography medium is packed into 1 ml pre-packed column.

[0227] (2) Sample pretreatment

[0228] Take 50 mg of bovine serum albumin (BSA) and 7 mg of bovine hemoglobin (Hemo), add equilibrium solution A to make the volume to 20 ml, and then prepare for sample loading.

[0229] (3) Buffer

[0230] Solution A: 0.02M Tris, pH 8.5; Solution B: 0.02M Tris, 1M NaCl, pH 8.5; the above solutions were filtered through a 0.45μm membrane before use.

[0231] (4) Purification process

[0232] The purification patterns of Example 1-3 and Comparative Example 3 and the corresponding gel electrophoresis patterns are shown in Figures 1-3 and 6, respectively. As can be seen from Figures 1-3, a very high peak appears at 400nm (red) when loading, and a very high peak appears at 280nm (blue) during elution, indicating that Hemo and BSA are separated during the loading process, Hemo peaks out in advance (red), and BSA is eluted and enriched later, achieving the separation of BSA and Hemo. As can be seen from Figure 6, BSA (280nm, blue) and Hemo (400nm, red) flow through simultaneously during the loading process, and peak out simultaneously during elution and enrichment, without separating the two, so there is no separation effect. Therefore, a higher agarose content cannot achieve a better separation effect.

[0233] The purification patterns and corresponding gel electrophoresis patterns of Example 6 and Comparative Example 1 are shown in Figures 4 and 5, respectively. Comparing the electrophoresis patterns and chromatograms, Figure 4(A) shows that after the highest peak in the blue elution peak, a small peak (E4) appears, representing the peak of its multimer. The gel image in Figure 4(B) also shows that E3 is a BSA monomer (65 kDa), and E4 has peaks near 65 kDa and 135 kDa, representing its dimer peak. However, in Figure 5, there are no obvious peaks of the polymer in the chromatogram. Furthermore, in the gel image, both the E3 and E4 lanes contain bands of the polymer, thus failing to better separate the polymers. Therefore, a higher dextran content does not achieve a better separation effect.

[0234] At the same time, the separation effects of Example 4, Example 5, Example 7 and Comparative Example 2 were determined using the same method as in Experimental Example 1. The separation effects of Example 4, Example 5, and Example 7 were equivalent to those of Example 6, and all were able to achieve the separation of aggregates; whereas Comparative Example 2 still could not achieve the effect of separating aggregates.

[0235] Test Example 2 Determination of the Resolution of Cationic Chromatography Media

[0236] The chromatographic media prepared in Example 8 and Comparative Example 4 were respectively loaded into chromatographic columns to perform purification, separation, analysis and testing of cytochrome C and lysozyme.

[0237] (1) Chromatography column specifications: Chromatography medium is packed into 1 ml pre-packed column.

[0238] (2) Sample pretreatment

[0239] Take 1 ml of cytochrome C solution (4 mg / ml) and 8 mg of lysozyme, dilute to 20 ml with solution A, filter through 0.45 μm and prepare for sample loading.

[0240] (3) Buffer

[0241] Solution A: 20 mM PB, pH 7.5; Solution B: 20 ​​mM PB, 1 M NaCl, pH 7.5; all solutions were freshly prepared and filtered through a 0.45 μm aqueous filter membrane.

[0242] (4) Purification process

[0243] The molecular weights of cytochrome C and lysozyme are similar, but cytochrome C has a specific band between 63-75KD, and the 63-75KD band can be used to distinguish cytochrome C and lysozyme on gel electrophoresis.

[0244] During the elution process, the pigment slowly migrated downward. Based on the elution peak colors and the chromatogram from Example 8, it was determined that the main components of elution peaks E1-E2 in Figure 7 were cytochrome C, while the main components of E3-E4 were lysozyme. The gel image indicated that E3 contained some cytochrome C, while E4 was relatively pure lysozyme.

[0245] Based on the elution peak colors and chromatogram of Comparative Example 4, it was determined that the main components of elution peaks E1-E6 in Figure 8 were cytochrome C, while the main components of E7-E8 were lysozyme. Based on the unique bands between 63 and 75 kD of cytochrome C on the electrophoretogram, the lysozyme in E7-E8 contained some cytochrome C. Therefore, Comparative Example 4 did not achieve a better separation of cytochrome C and lysozyme than Example 8.

[0246] At the same time, the separation effect of Example 9 was measured using the same method as in Experimental Example 2. The separation effect of Example 9 was equivalent to that of Example 8.

[0247] Based on the above results, it can be concluded that the chromatography medium obtained by optimizing the conditions such as the matrix, dextran, and ligand in the present invention not only has high resolution in the separation of similar substances, but also has the advantage of high protein loading capacity.

[0248] Comparison of Test Example 3 with Competitors

[0249] The dynamic binding capacity (DBC) of commercially available chromatography media for bovine serum albumin (BSA) and the separation performance of BSA and bovine hemoglobin (Hemo) were verified. The results are shown in Table 3.

[0250] Table 3

[0251] As shown in Table 3, the four commercially available competing products lack both high binding capacity and effective separation of bovine serum albumin and bovine hemoglobin. Compared to commercially available products, the chromatography medium provided by the present invention offers significant advantages in both protein binding capacity and resolution, addressing the current lack of high-capacity, high-resolution chromatography media on the market.

[0252] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of the present invention. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A chromatography medium, characterized in that The chromatography medium uses polysaccharide microspheres as a matrix, the polysaccharide microspheres are bonded with epoxy compounds, the epoxy compounds are coupled with polysaccharides, and the polysaccharides are coupled with ligands; The raw materials of the polysaccharide microspheres include 1-20 wt% agarose, preferably 2-20 wt%.

2. The chromatography medium according to claim 1, characterized in that The raw material of the polysaccharide microspheres also includes cellulose, and the amount of the cellulose is 0.1 to 10 wt%, preferably 0.5 to 10 wt%; more preferably 5 to 10 wt%; Preferably, the polysaccharide coupled with the epoxy compound is selected from one or more of dextran, chitosan, gelatin and sodium alginate, preferably dextran; Preferably, the mass ratio of the epoxy compound coupled polysaccharide to the epoxy compound coupled polysaccharide microspheres is 0.1 to 1:1, preferably 0.2 to 1:1; Preferably, the epoxy compound is selected from epichlorohydrin and / or glycidyl ether; The glycidyl ether is selected from one or more of 1,4-butanediol diglycidyl ether, allyl glycidyl ether, 1,2-ethylene glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether, preferably 1,4-butanediol diglycidyl ether.

3. The chromatography medium according to claim 1, characterized in that The ligand is an anion exchange ligand or a cation exchange ligand; Preferably, the anion exchange ligand is selected from a quaternary ammonium type ligand or a diethylaminoethyl type ligand, and the cation exchange ligand is selected from a sulfonic acid type ligand or a carboxymethyl type ligand; Preferably, the quaternary ammonium ligand is selected from 2,3-epoxypropyltrimethylammonium chloride; Preferably, the diethylaminoethyl type ligand is selected from diethylaminoethyl chloride hydrochloride; Preferably, the sulfonic acid type ligand is selected from sulfonic acid esters; Preferably, the carboxymethyl type ligand is selected from sodium chloroacetate; Preferably, the particle size of the polysaccharide microspheres is 45 to 120 μm; Preferably, the polysaccharide microspheres have a porous structure.

4. The chromatography medium according to any one of claims 1 to 3, characterized in that The polysaccharide microspheres are bonded with epichlorohydrin, the epichlorohydrin is coupled with dextran, and the dextran is coupled with 2,3-epoxypropyltrimethylammonium chloride, diethylaminochloroethane hydrochloride, sulfonic acid ester or sodium chloroacetate.

5. A method for preparing a chromatography medium according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) Activated polysaccharide microspheres; (2) reacting the activated polysaccharide microspheres obtained in step (1) with polysaccharide to obtain polysaccharide-coupled polysaccharide microspheres; (3) coupling the polysaccharide-coupled polysaccharide microspheres obtained in step (2) with a ligand to obtain the chromatography medium.

6. The preparation method according to claim 5, characterized in that The step (1) comprises: mixing polysaccharide microspheres, a base, sodium borohydride and an activation reagent to react; Preferably, the mass ratio of the polysaccharide microspheres, base, sodium borohydride and activation reagent is 1:(0.05-0.5):(0.001-0.005):(0.1-0.5), preferably 1:(0.05-0.2):(0.001-0.005):(0.1-0.5); Preferably, the reaction temperature in step (1) is controlled to be 25 to 60° C.; and / or the reaction time in step (1) is controlled to be 2 to 20 h.

7. The preparation method according to claim 5, characterized in that The step (2) comprises: mixing the activated polysaccharide microspheres with the polysaccharide, and then adding alkali and sodium borohydride to react; Preferably, the mass ratio of the activated polysaccharide microspheres, polysaccharide, alkali and sodium borohydride is 1:(0.1-1):(0.05-0.5):(0.0005-0.005), preferably 1:(0.1-0.8):(0.05-0.2):(0.0005-0.002); Preferably, the reaction temperature of step (2) is controlled to be 25 to 60° C.; and / or the reaction time of step (2) is controlled to be 6 to 24 hours.

8. The preparation method according to claim 5, characterized in that The step (3) comprises: mixing the polysaccharide microspheres coupled with polysaccharide obtained in step (2) with water, ligand, base and sodium borohydride to react; or, The step (3) comprises: mixing the polysaccharide microspheres coupled with polysaccharide obtained in step (2) with water, a ligand and a base to react; Preferably, the mass ratio of the polysaccharide microspheres, water, ligand, base, and sodium borohydride for the coupled polysaccharide is 1:(0.1-0.5):(0.1-1.5):(0.05-0.5):(0.0005-0.005), preferably 1:(0.1-0.5):(0.2-1.2):(0.05-0.2):(0.0005-0.002); or, the mass ratio of the polysaccharide microspheres, water, ligand, and base for the coupled polysaccharide is 1:(0.1-1):(0.1-1):(0.1-0.5); preferably 1:(0.2-0.8):(0.3-1):(0.1-0.3); Preferably, the reaction temperature of step (3) is controlled to be 25 to 50° C.; and / or the reaction time of step (3) is controlled to be 6 to 24 hours.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The reactions of step (1), step (2) and step (3) are all carried out under stirring; Preferably, the base is sodium hydroxide; Preferably, the activation agent is selected from epichlorohydrin and / or glycidyl ether, and the glycidyl ether is selected from one or more of 1,4-butanediol diglycidyl ether, allyl glycidyl ether, 1,2-ethylene glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether, preferably 1,4-butanediol diglycidyl ether; Preferably, the ligand is an anion exchange ligand or a cation exchange ligand; Preferably, the anion exchange ligand is selected from a quaternary ammonium type ligand or a diethylaminoethyl type ligand, and the cation exchange ligand is selected from a sulfonic acid type ligand or a carboxymethyl type ligand; Preferably, the quaternary ammonium ligand is selected from 2,3-epoxypropyltrimethylammonium chloride; Preferably, the diethylaminoethyl type ligand is selected from diethylaminoethyl chloride hydrochloride; Preferably, the sulfonic acid type ligand is selected from sulfonic acid esters; Preferably, the carboxymethyl-type ligand is selected from sodium chloroacetate.

10. Use of the chromatography medium according to any one of claims 1 to 4 or the chromatography medium obtained by the preparation method according to any one of claims 5 to 9 in the separation and purification of biomacromolecules.

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