Heparin affinity chromatography medium and preparation method thereof

Abstract

The present invention provides a heparin affinity chromatography medium and a preparation method thereof. The heparin affinity chromatography medium comprises a porous polymer microsphere matrix modified with a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain the heparin affinity chromatography medium. The heparin affinity chromatography medium of the present invention has a high adsorption capacity for target biomolecules, significantly improving separation efficiency; and also exhibits high mechanical strength.

Description

Technical Field

[0001] The present invention relates to chromatography separation technology, in particular to a heparin affinity chromatography medium and a preparation method thereof. Background Art

[0002] Heparin is a naturally occurring mucopolysaccharide that acts as an affinity ligand on a wide range of biomolecules, including coagulation factors, plasma proteins, and lipoproteins. Currently, heparin affinity chromatography media on the market primarily use agarose gel as a matrix. However, agarose has a low ligand density and a low adsorption capacity for target biomolecules, resulting in low separation efficiency. Furthermore, the mechanical strength of the resulting chromatography media is also low when agarose is used as a matrix. This low pressure tolerance further limits the separation efficiency when filling chromatography columns in industrial production. Summary of the Invention

[0003] The object of the present invention is to provide a heparin affinity chromatography medium and a preparation method thereof, so as to solve the problem of low analysis efficiency of the heparin affinity chromatography medium in the prior art.

[0004] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0005] A heparin affinity chromatography medium is provided. The porous polymer microspheres are used as a matrix, the matrix is modified by using a polyamide-amine dendrimer to obtain a modified matrix, and heparin is coupled to the modified matrix to obtain the heparin affinity chromatography medium.

[0006] Preferably, the porous polymer microspheres are polyglycidyl methacrylate microspheres.

[0007] Preferably, dextran is attached to the surface of the porous polymer microspheres and is obtained through a cross-linking reaction.

[0008] Preferably, the cross-linking agent used in the cross-linking reaction is glutaraldehyde.

[0009] Preferably, the mass ratio of the porous polymer microspheres to the polyamidoamine dendritic polymer is 1:(2.0-2.5).

[0010] The present invention also provides a method for preparing a heparin affinity chromatography medium, comprising the following steps:

[0011] (1) taking porous polymer microspheres and modifying them with polyamide-amine dendrimers to obtain a modified matrix;

[0012] (2) Taking the modified matrix obtained in step (1), coupling heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0013] Preferably, step (1) further includes the step of treating the porous polymer microspheres: taking the porous polymer microspheres, placing them in a dextran solution, and stirring at 40-60° C. until the volatile components evaporate to obtain microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, carrying out a cross-linking reaction at 30-40° C. for 5-10 hours, and filtering to obtain the microspheres.

[0014] Preferably, step (1) further comprises the step of activating the porous polymer microspheres obtained by the cross-linking reaction.

[0015] Preferably, the activation specifically comprises: taking the porous polymer microspheres obtained by the cross-linking reaction, adding allyl glycidyl ether, a sodium hydroxide solution with a concentration of 0.4-0.6 mol / L, and dimethyl sulfoxide thereto, stirring and reacting at 30-50° C. for 2-5 hours to obtain activated porous polymer microspheres.

[0016] Preferably, the amount of the porous polymer microspheres obtained by the cross-linking reaction and the allyl glycidyl ether is as follows: 10-30 ml of allyl glycidyl ether is added to every 10 g of the porous polymer microspheres obtained by the cross-linking reaction; and the volume ratio of the allyl glycidyl ether, the sodium hydroxide solution with a concentration of 0.4-0.6 mol / L, and the dimethyl sulfoxide is 1:1:2.

[0017] Preferably, the mass fraction of the dextran solution is 1.2-2.5 wt %; the mass ratio of the porous polymer microspheres to the dextran solution is 100:(10-15);

[0018] The mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is (1.0-1.2):10;

[0019] The concentration of the potassium hydroxide solution is 3-5 mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is (0.5-0.8):10.

[0020] Preferably, step (1) specifically comprises: taking porous polymer microspheres, adding polyamide-amine dendritic polymer and sodium hydroxide solution thereto, stirring and reacting at 40-55° C. for 3-8 hours to obtain a modified matrix.

[0021] Preferably, the concentration of the sodium hydroxide solution is 0.3-0.8 mol / L, the polyamidoamine dendrimer is PAMAM, and the mass ratio of the polyamidoamine dendrimer to the sodium hydroxide solution is 1:(20-30).

[0022] Preferably, step (2) specifically comprises: taking the modified matrix obtained in step (1), adding heparin sodium and ethanol, and stirring the reaction under nitrogen protection for 50-60 hours to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0023] Preferably, the mass ratio of the modified matrix to heparin sodium is (4-10):1.

[0024] Preferably, the amount of ethanol used is 50-80 ml of ethanol per 10 g of the modified substrate.

[0025] The above solution of the present invention includes at least the following beneficial effects:

[0026] (1) The heparin affinity chromatography medium of the present invention uses porous polymer microspheres as a matrix, and uses a polyamide-amine dendritic polymer to modify the matrix to obtain a modified matrix; heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium. In the present invention, the matrix is modified using a polyamide-amine dendritic polymer. The main chain of the polyamide-amine dendritic polymer contains a large number of hydrophilic groups, which can effectively avoid nonspecific adsorption. In particular, it has a spherical three-dimensional structure and a large number of amino groups distributed in the three-dimensional space, which can be connected with the aldehyde group in the heparin ligand, thereby increasing the binding sites of the heparin ligand, thereby increasing the adsorption capacity of the target biomolecule and improving the separation efficiency. At the same time, the spherical three-dimensional structure also improves the mechanical strength of the matrix to a certain extent.

[0027] (2) The heparin affinity chromatography medium of the present invention, wherein the porous polymer microspheres are poly(glycidyl methacrylate) microspheres, and dextran is attached to the surface and obtained by a cross-linking reaction. Poly(glycidyl methacrylate) microspheres have good mechanical strength, but poor hydrophilicity. By attaching dextran, good hydrophilicity can be obtained. More importantly, dextran is attached to the poly(glycidyl methacrylate) microspheres through a cross-linking reaction. After activation, the hydroxyl groups of dextran provide cross-linking points for the modification of the polyamide-amine dendrimer. The cross-linking points between chains can inhibit the sliding between polymer chains, so that the heparin affinity chromatography medium exhibits better mechanical strength. DETAILED DESCRIPTION

[0028] In the examples of the present invention, if specific conditions are not specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products. Raw materials of different manufacturers and types do not affect the implementation of the technical solutions of the present invention and the achievement of the technical effects.

[0029] Example 1

[0030] The heparin affinity chromatography medium in this embodiment uses porous polymer microspheres as a matrix, and the matrix is modified using a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium.

[0031] The porous polymer microspheres are poly(glycidyl methacrylate) microspheres with dextran attached to their surfaces, obtained by cross-linking reaction, wherein the cross-linking agent used in the cross-linking reaction is glutaraldehyde.

[0032] The mass ratio of the porous polymer microspheres to the polyamide-amine dendrimer is 1:2.0.

[0033] The method for preparing the heparin affinity chromatography medium described in this embodiment comprises the following steps:

[0034] (1) taking porous polymer microspheres, placing them in a dextran solution, and stirring at 60° C. until the volatile components evaporate to obtain microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, and cross-linking reaction at 40° C. for 8 hours, filtering, and obtaining porous polymer microspheres obtained by cross-linking reaction;

[0035] Among them, the mass fraction of the dextran solution is 1.2wt%; the mass ratio of the porous polymer microspheres to the dextran solution is 100:13; the mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is 1.0:10; the concentration of the potassium hydroxide solution is 4mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is 0.5:10.

[0036] Taking the porous polymer microspheres obtained by the cross-linking reaction, allyl glycidyl ether, a 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide were added thereto, and the mixture was stirred and reacted at 30°C for 3 hours to obtain activated porous polymer microspheres;

[0037] Among them, the dosage relationship between the porous polymer microspheres obtained by the cross-linking reaction and the allyl glycidyl ether is: 10 ml of allyl glycidyl ether is added to every 10 g of the porous polymer microspheres obtained by the cross-linking reaction; the volume ratio of the allyl glycidyl ether, the sodium hydroxide solution with a concentration of 0.5 mol / L, and the dimethyl sulfoxide is 1:1:2.

[0038] The activated porous polymer microspheres were taken, and polyamide-amine dendrimer and sodium hydroxide solution were added thereto. The mixture was stirred and reacted at 40° C. for 5 hours to obtain a modified matrix.

[0039] It should be noted that polyamidoamine dendrimers are known products in the prior art. For example, the polyamidoamine dendrimer nanomaterial disclosed in Chinese patent document CN1631936A can be used. Those skilled in the art may also select polyamidoamine dendrimers (PAMAM) obtained by other methods according to actual circumstances. The concentration of the sodium hydroxide solution is 0.3 mol / L; the mass ratio of the polyamidoamine dendrimer to the sodium hydroxide solution is 1:30.

[0040] (2) Adding heparin sodium and ethanol to the modified matrix obtained in step (1) and stirring the mixture for 50 hours under nitrogen protection to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0041] The mass ratio of the modified matrix to heparin sodium is 7: 1. The amount of ethanol used is 50 ml of ethanol per 10 g of the modified matrix.

[0042] Example 2

[0043] The heparin affinity chromatography medium in this embodiment uses porous polymer microspheres as a matrix, and the matrix is modified using a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium.

[0044] The porous polymer microspheres are poly(glycidyl methacrylate) microspheres with dextran attached to their surfaces, obtained by cross-linking reaction, wherein the cross-linking agent used in the cross-linking reaction is glutaraldehyde.

[0045] The mass ratio of the porous polymer microspheres to the polyamide-amine dendrimer is 1:2.5.

[0046] The method for preparing the heparin affinity chromatography medium described in this embodiment comprises the following steps:

[0047] (1) taking porous polymer microspheres, placing them in a dextran solution, and stirring at 50° C. until the volatile components evaporate to obtain microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, and performing a cross-linking reaction at 40° C. for 10 hours, filtering, and obtaining porous polymer microspheres obtained by the cross-linking reaction;

[0048] Among them, the mass fraction of the dextran solution is 2.5wt%; the mass ratio of the porous polymer microspheres to the dextran solution is 100:15; the mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is 1.2:10; the concentration of the potassium hydroxide solution is 5mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is 0.8:10.

[0049] Taking the porous polymer microspheres obtained by the cross-linking reaction, allyl glycidyl ether, a 0.6 mol / L sodium hydroxide solution, and dimethyl sulfoxide were added thereto, and the mixture was stirred and reacted at 50° C. for 5 hours to obtain activated porous polymer microspheres;

[0050] Among them, the usage relationship of the porous polymer microspheres obtained by the cross-linking reaction and the allyl glycidyl ether is: 30 ml of allyl glycidyl ether is added to every 10 g of the porous polymer microspheres obtained by the cross-linking reaction; the volume ratio of the allyl glycidyl ether, the sodium hydroxide solution with a concentration of 0.6 mol / L, and the dimethyl sulfoxide is 1:1:2.

[0051] The activated porous polymer microspheres were taken, and polyamide-amine dendrimer and sodium hydroxide solution were added thereto. The mixture was stirred and reacted at 55° C. for 8 hours to obtain a modified matrix.

[0052] It should be noted that polyamidoamine dendrimers are known products in the prior art. For example, the polyamidoamine dendrimer nanomaterial disclosed in Chinese patent document CN1631936A can be used. Those skilled in the art may also select polyamidoamine dendrimers (PAMAM) obtained by other methods according to actual circumstances. The concentration of the sodium hydroxide solution is 0.8 mol / L; the mass ratio of the polyamidoamine dendrimer to the sodium hydroxide solution is 1:25.

[0053] (2) Adding heparin sodium and ethanol to the modified matrix obtained in step (1) and stirring the mixture under nitrogen for 60 hours to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0054] The mass ratio of the modified matrix to heparin sodium is 10: 1. The amount of ethanol used is 80 ml of ethanol per 10 g of the modified matrix.

[0055] Example 3

[0056] The heparin affinity chromatography medium in this embodiment uses porous polymer microspheres as a matrix, and the matrix is modified using a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium.

[0057] The porous polymer microspheres are poly(glycidyl methacrylate) microspheres with dextran attached to their surfaces, obtained by cross-linking reaction, wherein the cross-linking agent used in the cross-linking reaction is glutaraldehyde.

[0058] The mass ratio of the porous polymer microspheres to the polyamide-amine dendrimer is 1:2.2.

[0059] The method for preparing the heparin affinity chromatography medium described in this embodiment comprises the following steps:

[0060] (1) taking porous polymer microspheres, placing them in a dextran solution, and stirring at 40° C. until the volatile components evaporate to obtain microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, and performing a cross-linking reaction at 35° C. for 5 hours, filtering, and obtaining porous polymer microspheres obtained by the cross-linking reaction;

[0061] Among them, the mass fraction of the dextran solution is 1.8wt%; the mass ratio of the porous polymer microspheres to the dextran solution is 100:10; the mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is 1.1:10; the concentration of the potassium hydroxide solution is 3mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is 0.6:10.

[0062] Taking the porous polymer microspheres obtained by the cross-linking reaction, allyl glycidyl ether, a 0.4 mol / L sodium hydroxide solution, and dimethyl sulfoxide were added thereto, and the mixture was stirred and reacted at 40°C for 2 hours to obtain activated porous polymer microspheres;

[0063] Among them, the usage relationship of the porous polymer microspheres obtained by the cross-linking reaction and the allyl glycidyl ether is as follows: 20 ml of allyl glycidyl ether is added to every 10 g of the porous polymer microspheres obtained by the cross-linking reaction; the volume ratio of the allyl glycidyl ether, the sodium hydroxide solution with a concentration of 0.4 mol / L, and the dimethyl sulfoxide is 1:1:2.

[0064] The activated porous polymer microspheres were taken, and polyamide-amine dendrimer and sodium hydroxide solution were added thereto. The mixture was stirred and reacted at 45° C. for 3 hours to obtain a modified matrix.

[0065] It should be noted that polyamidoamine dendrimers are known products in the prior art. For example, the polyamidoamine dendrimer nanomaterial disclosed in Chinese patent document CN1631936A can be used. Those skilled in the art may also select polyamidoamine dendrimers (PAMAM) obtained by other methods according to actual circumstances. The concentration of the sodium hydroxide solution is 0.5 mol / L, and the mass ratio of the polyamidoamine dendrimer to the sodium hydroxide solution is 1:20.

[0066] (2) Adding heparin sodium and ethanol to the modified matrix obtained in step (1), stirring and reacting for 55 hours under nitrogen protection to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0067] The mass ratio of the modified matrix to heparin sodium is 4: 1. The amount of ethanol used is 60 ml of ethanol per 10 g of the modified matrix.

[0068] Example 4

[0069] The heparin affinity chromatography medium in this embodiment uses porous polymer microspheres as a matrix, and the matrix is modified using a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium.

[0070] The porous polymer microspheres are poly(glycidyl methacrylate) microspheres with dextran attached to their surfaces, obtained by cross-linking reaction, wherein the cross-linking agent used in the cross-linking reaction is glutaraldehyde.

[0071] The mass ratio of the porous polymer microspheres to the polyamide-amine dendrimer is 1:2.4.

[0072] The method for preparing the heparin affinity chromatography medium described in this embodiment comprises the following steps:

[0073] (1) taking porous polymer microspheres, placing them in a dextran solution, and stirring at 50° C. until the volatile components evaporate to obtain microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, and cross-linking reaction at 40° C. for 8 hours, filtering, and obtaining porous polymer microspheres obtained by cross-linking reaction;

[0074] Among them, the mass fraction of the dextran solution is 1.6wt%; the mass ratio of the porous polymer microspheres to the dextran solution is 100:12; the mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is 1.2:10; the concentration of the potassium hydroxide solution is 3mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is 0.6:10.

[0075] Taking the porous polymer microspheres obtained by the cross-linking reaction, allyl glycidyl ether, a 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide were added thereto, and the mixture was stirred and reacted at 45°C for 2 hours to obtain activated porous polymer microspheres;

[0076] Among them, the usage relationship of the porous polymer microspheres obtained by the cross-linking reaction and the allyl glycidyl ether is as follows: 30 ml of allyl glycidyl ether is added to every 10 g of the porous polymer microspheres obtained by the cross-linking reaction; the volume ratio of the allyl glycidyl ether, the sodium hydroxide solution with a concentration of 0.5 mol / L, and the dimethyl sulfoxide is 1:1:2.

[0077] The activated porous polymer microspheres were taken, and polyamide-amine dendrimer and sodium hydroxide solution were added thereto. The mixture was stirred and reacted at 45° C. for 8 hours to obtain a modified matrix.

[0078] It should be noted that polyamidoamine dendrimers are known products in the prior art. For example, the polyamidoamine dendrimer nanomaterial disclosed in Chinese patent document CN1631936A can be used. Those skilled in the art may also select polyamidoamine dendrimers (PAMAM) obtained by other methods according to actual circumstances. The concentration of the sodium hydroxide solution is 0.6 mol / L; the mass ratio of the polyamidoamine dendrimer to the sodium hydroxide solution is 1:28.

[0079] (2) Adding heparin sodium and ethanol to the modified matrix obtained in step (1) and stirring the mixture under nitrogen for 60 hours to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0080] The mass ratio of the modified matrix to heparin sodium is 8: 1. The amount of ethanol used is 60 ml of ethanol per 10 g of the modified matrix.

[0081] Comparative Example 1

[0082] In this comparative example, the heparin affinity chromatography medium was prepared using the same method as in Example 4, except that the porous polymer microspheres used were poly(glycidyl methacrylate) microspheres, dextran was not attached to the surface, and no cross-linking reaction was performed.

[0083] That is, the preparation method of the heparin affinity chromatography medium of this comparative example comprises the following steps:

[0084] (1) taking porous polymer microspheres, adding allyl glycidyl ether, a 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide thereto, stirring and reacting at 45°C for 2 h to obtain activated porous polymer microspheres;

[0085] The usage ratio of the porous polymer microspheres and the allyl glycidyl ether is as follows: 30 ml of allyl glycidyl ether is added to every 10 g of porous polymer microspheres; and the volume ratio of the allyl glycidyl ether, 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide is 1:1:2.

[0086] The activated porous polymer microspheres were taken, and polyamide-amine dendrimer and sodium hydroxide solution were added thereto. The mixture was stirred and reacted at 45° C. for 8 hours to obtain a modified matrix.

[0087] The concentration of the sodium hydroxide solution is 0.6 mol / L; and the mass ratio of the polyamide-amine dendritic polymer to the sodium hydroxide solution is 1:28.

[0088] (2) Adding heparin sodium and ethanol to the modified matrix obtained in step (1) and stirring the mixture under nitrogen for 60 hours to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0089] The mass ratio of the modified matrix to heparin sodium is 8: 1. The amount of ethanol used is 60 ml of ethanol per 10 g of the modified matrix.

[0090] Comparative Example 2

[0091] In this comparative example, the heparin affinity chromatography medium was prepared using the same method as in Example 4, except that the porous polymer microspheres used were poly(glycidyl methacrylate) microspheres, and dextran was attached to the surface without undergoing a cross-linking reaction.

[0092] That is, the preparation method of the heparin affinity chromatography medium of this comparative example comprises the following steps:

[0093] (1) taking porous polymer microspheres, placing them in a dextran solution, and stirring at 50°C until the volatile components evaporate, thereby obtaining microspheres with dextran attached to the surface;

[0094] The mass fraction of the dextran solution is 1.6 wt %; the mass ratio of the porous polymer microspheres to the dextran solution is 100:12.

[0095] Taking the microspheres with dextran attached to the surface, adding allyl glycidyl ether, a 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide thereto, stirring and reacting at 45° C. for 2 h to obtain activated porous polymer microspheres;

[0096] The dosage of the microspheres with glucan attached to the surface and the allyl glycidyl ether is as follows: 30 ml of allyl glycidyl ether is added to every 10 g of microspheres with glucan attached to the surface; and the volume ratio of the allyl glycidyl ether, 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide is 1:1:2.

[0097] The activated porous polymer microspheres were taken, and polyamide-amine dendrimer and sodium hydroxide solution were added thereto. The mixture was stirred and reacted at 45° C. for 8 hours to obtain a modified matrix.

[0098] The concentration of the sodium hydroxide solution is 0.6 mol / L; and the mass ratio of the polyamide-amine dendritic polymer to the sodium hydroxide solution is 1:28.

[0099] (2) Adding heparin sodium and ethanol to the modified matrix obtained in step (1) and stirring the mixture under nitrogen for 60 hours to couple heparin to the modified matrix to obtain a heparin affinity chromatography medium.

[0100] The mass ratio of the modified matrix to heparin sodium is 8: 1. The amount of ethanol used is 60 ml of ethanol per 10 g of the modified matrix.

[0101] Comparative Example 3

[0102] In this comparative example, a heparin affinity chromatography medium was prepared using the same method as in Example 4, except that the matrix was not modified with a polyamidoamine dendrimer.

[0103] That is, the preparation method of the heparin affinity chromatography medium of this comparative example comprises the following steps:

[0104] (1) taking porous polymer microspheres, placing them in a dextran solution, and stirring at 50° C. until the volatile components evaporate to obtain microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, and cross-linking reaction at 40° C. for 8 hours, filtering, and obtaining porous polymer microspheres obtained by cross-linking reaction;

[0105] Among them, the mass fraction of the dextran solution is 1.6wt%; the mass ratio of the porous polymer microspheres to the dextran solution is 100:12; the mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is 1.2:10; the concentration of the potassium hydroxide solution is 3mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is 0.6:10.

[0106] Taking the porous polymer microspheres obtained by the cross-linking reaction, allyl glycidyl ether, a 0.5 mol / L sodium hydroxide solution, and dimethyl sulfoxide were added thereto, and the mixture was stirred and reacted at 45°C for 2 hours to obtain activated porous polymer microspheres;

[0107] Among them, the usage relationship of the porous polymer microspheres obtained by the cross-linking reaction and the allyl glycidyl ether is as follows: 30 ml of allyl glycidyl ether is added to every 10 g of the porous polymer microspheres obtained by the cross-linking reaction; the volume ratio of the allyl glycidyl ether, the sodium hydroxide solution with a concentration of 0.5 mol / L, and the dimethyl sulfoxide is 1:1:2.

[0108] (2) The porous polymer microspheres obtained by cross-linking reaction in step (1) were added with heparin sodium and ethanol, and stirred for 60 hours under nitrogen protection to obtain a heparin affinity chromatography medium.

[0109] The mass ratio of the modified matrix to heparin sodium is 8: 1. The amount of ethanol used is 60 ml of ethanol per 10 g of the modified matrix.

[0110] Comparative Example 4

[0111] In this comparative example, the heparin affinity chromatography medium was prepared by the same method as in Example 4, except that the mass ratio of the porous polymer microspheres to the polyamidoamine dendrimer was 1:3.

[0112] Comparative Example 5

[0113] In this comparative example, the heparin affinity chromatography medium was prepared by the same method as in Example 4, except that the mass ratio of the porous polymer microspheres to the polyamidoamine dendrimer was 1:1.

[0114] Effect Experiment Example

[0115] To verify the technical effect of the heparin affinity chromatography medium of the present invention, the following experiments were performed:

[0116] Effect Experiment Example 1: Pressure Resistance Test

[0117] Take 20-25 mL of the heparin affinity chromatography medium prepared in Examples 1-4 and Comparative Examples 1-5, respectively, and place it in a 50 mL G3 sand core funnel extraction and washing system. Pour 20 mL of deionized water and turn on the circulating water vacuum pump for extraction and washing until no liquid drips from the sand core funnel within 1 minute. Repeat the extraction and washing 5 times to treat each heparin affinity chromatography medium.

[0118] Each heparin affinity chromatography medium was loaded into the column according to the standard operating procedures for pre-packed columns, and the packed bed height was controlled to be 10±0.2 cm. The loaded chromatography column (Φ1.60 cm×20.00 cm) was connected to the chromatography equipment.

[0119] Set a certain flow rate Vx (mL / min) starting from 0 (V1 is 1mL / min, Vx = x mL / min), maintain the flow rate for 5 minutes, and record the system display pressure Px (MPa) at this time. Continue to increase the flow rate with a gradient of 1mL / min, repeat the above process, and record Px at the corresponding Vx. Until the system pressure exceeds 1.2Mpa, record the corresponding flow rate at this time as V (mL / min), that is, the maximum flow rate. Or when the flow rate is increased at a certain moment, the system pressure increases sharply, indicating that the filler has reached the pressure tolerance limit. Record the flow rate at the previous moment as the maximum flow rate V, and the corresponding pressure as the maximum pressure P. Bypass the chromatography column and use the above method to record the background pressure Py of the instrument at the same flow rate Vx (mL / min). Calculate Vx and the corresponding column pressure Pc according to the formula; among which, the calculation formula for Vx corresponding to column pressure Pc is as follows:

[0120] P c =P x -P y

[0121] Where Pc is the column pressure (MPa) corresponding to the flow rate Vx; Px is the system pressure (MPa) corresponding to the flow rate Vx; and Py is the background pressure (MPa) corresponding to the instrument at the flow rate Vx.

[0122] Effect Experiment Example 2: Adsorption Capacity Test of Target Biomolecules

[0123] The heparin affinity chromatography media prepared in Examples 1-4 and Comparative Examples 1-5 were respectively taken to measure their ligand densities, and the static adsorption capacity of each heparin affinity chromatography media for lysozyme protein was determined by a static adsorption method.

[0124] Effect experiment results:

[0125] After experimentation, the results are as follows:

[0126]

[0127] According to the comparison of Examples 1-4 and Comparative Examples 1-2, the heparin affinity chromatography medium of the present invention can significantly improve the mechanical strength of the chromatography medium by attaching dextran and performing a cross-linking reaction. It has no effect on the increase in ligand density, but has a greater impact on the static adsorption capacity. In theory, the higher the ligand density of the medium, the higher its loading capacity. However, in reality, the loading capacity is not only related to the ligand density of the medium, but also to the cross-linking mode of the ligand. Dextran is attached to poly(glycidyl methacrylate) microspheres through a cross-linking reaction. After activation, the hydroxyl groups of dextran provide cross-linking points for the modification of polyamide-amine dendrimers, which also affects the three-dimensional structure of the ligand coupling, thereby causing its static adsorption capacity to change. It can be seen that the heparin affinity chromatography medium of the present invention, by attaching dextran and performing a cross-linking reaction, has a higher adsorption capacity for the chromatography medium.

[0128] Comparison of Examples 1-4 with Comparative Example 3 shows that the present invention uses polyamide-amine dendrimers to modify the matrix, significantly increasing the ligand density and static adsorption capacity of target biomolecules, while also achieving higher mechanical strength. Comparison of Examples 1-4 with Comparative Examples 4-5 shows that when the dosage of polyamide-amine dendrimer is too low, it cannot fully exert its effect; while when the dosage of polyamide-amine dendrimer is too high, although it can slightly improve the mechanical strength of the chromatography medium and increase the ligand density, it does not increase the static adsorption capacity of target biomolecules, but instead causes a certain degree of decrease in the static adsorption capacity. When the mass ratio of the porous polymer microspheres to the polyamide-amine dendrimer is 1:(2.0-2.5), the resulting chromatography medium exhibits optimal overall performance.

[0129] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A heparin affinity chromatography medium, characterized in that The porous polymer microspheres are used as a matrix, and the matrix is modified with a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium; The porous polymer microspheres are polyglycidyl methacrylate microspheres; The porous polymer microspheres are attached with dextran on their surfaces and are obtained through a cross-linking reaction; The mass ratio of the porous polymer microspheres to the polyamide-amine dendritic polymer is 1:(2.0-2.5).

2. The heparin affinity chromatography medium according to claim 1, characterized in that The cross-linking agent used in the cross-linking reaction is glutaraldehyde.

3. A method for preparing the heparin affinity chromatography medium according to any one of claims 1 to 2, characterized in that: The steps include: (1) taking porous polymer microspheres and modifying them with polyamide-amine dendrimers to obtain a modified matrix; (2) taking the modified matrix obtained in step (1), coupling heparin to the modified matrix to obtain a heparin affinity chromatography medium; Step (1) also includes the step of treating the porous polymer microspheres: taking the porous polymer microspheres, placing them in a dextran solution, stirring at 40-60° C. until the volatile components evaporate, thereby obtaining microspheres with dextran attached to the surface; adding glutaraldehyde and potassium hydroxide solution to the microspheres with dextran attached to the surface, carrying out a cross-linking reaction at 30-40° C. for 5-10 hours, and filtering to obtain the microspheres.

4. The method for preparing a heparin affinity chromatography medium according to claim 3, wherein The mass fraction of the dextran solution is 1.2-2.5 wt %; the mass ratio of the porous polymer microspheres to the dextran solution is 100:(10-15); The mass ratio of the glutaraldehyde to the microspheres with dextran attached to the surface is (1.0-1.2):10; The concentration of the potassium hydroxide solution is 3-5 mol / L; the mass ratio of the potassium hydroxide solution to the microspheres with dextran attached to the surface is (0.5-0.8):

10.

5. The method for preparing the heparin affinity chromatography medium according to claim 3, wherein Step (1) specifically includes: taking porous polymer microspheres, adding polyamide-amine dendritic polymer and sodium hydroxide solution thereto, stirring and reacting at 40-55° C. for 3-8 hours to obtain a modified matrix.

6. The method for preparing the heparin affinity chromatography medium according to claim 3, wherein Step (2) specifically comprises: taking the modified matrix obtained in step (1), adding heparin sodium and ethanol, stirring and reacting for 50-60 hours under nitrogen protection, so that heparin is coupled to the modified matrix to obtain a heparin affinity chromatography medium.

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