CN114272913A

CN114272913A - Immunoadsorption blood purification material and preparation method thereof

Info

Publication number: CN114272913A
Application number: CN202111645315.8A
Authority: CN
Inventors: 杨正根; 李永桂; 林振南; 邓杰文; 陈校园
Original assignee: Guangzhou Kangsheng Biotechnology Co ltd
Current assignee: Guangzhou Kangsheng Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-05

Abstract

The invention discloses an immunoadsorption blood purification material and a preparation method thereof. The immunoadsorbent material is obtained by activating the solid support with CDI, followed by further covalent coupling of ligand molecules with amino groups. The method has simple process, and compared with the immunoadsorption carrier prepared by using the same raw materials in the prior art, the immunoadsorption carrier has higher pathogenic substance clearance rate, ligand molecules are less prone to fall off, and the immunoadsorption carrier can be used for adsorbing blood plasma, can also be directly used for adsorbing whole blood, and can also be used for removing small-molecule pathogenic substances such as urea, creatinine and the like through hemodialysis or hemodiafiltration. Through introducing the anticoagulant containing amino into the solid phase carrier, the prepared immunoadsorption material has self-anticoagulation property, and in the process of blood purification, the anticoagulant can be reduced or even not used completely, so that the risk of bleeding of patients is greatly reduced.

Description

Immunoadsorption blood purification material and preparation method thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to an immunoadsorption blood purification material and a preparation method thereof.

Background

Many diseases occur and develop as a result of accumulation of pathogenic agents in the body. The specific and effective method for cleaning these pathogenic factors from the body by purifying blood without causing damage to the body is a problem that has been continuously explored in clinical medicine in recent years. One of the important methods is adsorption therapy for blood purification, which is based on the principle that a ligand is firmly bound on a fixed carrier to construct an adsorption column, and pathogen-related molecules in the blood of a patient are specifically adsorbed and removed by an extracorporeal circulation method to purify the blood, thereby achieving the purpose of treating diseases. Blood immunoadsorption therapy is a new clinical medical technology in recent 30 years, and difficult diseases with no obvious curative effect of some medicines, such as atopic dermatitis, autoimmune system diseases, nephrosis syndrome, severe hepatitis, malignant tumors and the like, can often obtain better clinical curative effect by carrying out extracorporeal immunoadsorption blood purification. The blood immunoadsorption treatment technology is widely applied to a plurality of clinical fields of kidney diseases, neurosurgery, blood diseases, respiratory diseases, liver diseases and the like. Therefore, the development and production of immunoadsorbents have become an important high-tech industry.

Staphylococcal Protein A (SPA) is a protein on the cell wall of some Staphylococcus aureus, has a molecular weight of about 42000, and has 4-5 highly similar Fc binding domains of immunoglobulin at its amino terminal end, which can specifically bind to the Fc domain of antibodies (such as IgG) and their immune complexes in human plasma. SPA is fixed on a carrier to prepare a carrier-SPA compound immunoadsorption column, which can specifically adsorb antibodies and immune compounds thereof in blood plasma, and diseases and symptoms caused by the antibodies and the immune compounds, such as autoimmune diseases, organ transplantation, malignant tumors and the like, can be prevented, treated and relieved. The clinical common allergic asthma, allergic urticaria, allergic dermatitis, allergic rhinitis and anaphylactic shock caused by penicillin lead the IgE concentration in a patient body to reach more than 10 times of the normal value, and particularly the concentration of allergen specific IgE can reach about 1000 times of the normal value. At present, immunosuppressant medicines are mainly used for clinical treatment, and the medicine therapy has good treatment effect on accidental anaphylactic reaction, but has great side effect on the treatment of chronic and intractable allergic diseases such as allergic asthma and the like which need to be taken for a long time, not only can the chronic and intractable allergic diseases not be cured, but also can generate medicine dependence. In recent years, the blood purification and adsorption treatment of autoimmune diseases caused by over-high IgG and the treatment of anaphylactic reaction caused by over-high IgE become a new trend, so the synthesis of the immunoadsorbent material is very important.

The existing immunoadsorption products such as protein A, IgE on the market are plasma adsorption products, and need to be matched with a plasma separator for use, so that the treatment cost of a patient is increased, and the risk of membrane rupture of the plasma separator causes blockage of a plasma adsorption column and loss of plasma outside the patient, so that a whole blood adsorption product is urgently needed. In the traditional blood purification treatment process, heparin is required to be injected to prevent blood anticoagulation, so that the treatment cost is increased, and more importantly, the risk of serious hemorrhage of a patient caused by systemic anticoagulation is increased; considering the special clinical problem that the blood coagulation dysfunction is frequently complicated in the course of the disease of critical patients such as autoimmune disease, the blood purification material with the self-anticoagulation property has better blood compatibility, can influence the function of the blood coagulation system of the patient to a smaller extent, and better prevents the serious hemorrhage in the blood purification process or after the blood purification.

The traditional hemodialysis or hemodiafiltration can only remove medium and small molecules, cannot effectively remove macromolecular pathogenic substances, and needs to perform hemoperfusion. And the coupling reagents used in the traditional blood perfusion technical research are cyanogen bromide, trichlorotriazine, Carbonyl Diimidazole (CDI), sodium periodate, epichlorohydrin and the like generally, wherein the cyanogen bromide is a highly toxic substance, the synthetic process has great harm to human bodies and environment, and the cyanogen bromide method is used for coupling ligands which are easy to fall off and enter the human bodies, so that great side effects are generated on patients. In addition, epichlorohydrin and carbonyldiimidazole are used as coupling reagents to activate carriers to couple PMB in the traditional method, although the use of virulent cyanogen bromide is avoided, the reaction steps in the preparation process are more, the adsorption material can be synthesized only by five chemical reactions, and the method is complex, so that the obtained adsorption material product has large batch difference and unstable performance.

Carbonyl diimidazole is a reagent with high reactivity, can rapidly react with hollow fibers with hydroxyl groups to form imidazole carbamate, and the imidazole carbamate can react with ligands with the immunoadsorption function under mild conditions to obtain the stable immunoadsorption material.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides a preparation method for an immunoadsorption blood purification material.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a preparation method of an immunoadsorption material comprises the following steps:

dissolving carbonyl diimidazole CDI with an organic solvent to obtain CDI activating solution;

fully contacting and reacting a solid phase carrier containing hydroxyl with a CDI activation solution to activate the solid phase carrier;

mixing the activated solid phase carrier with a ligand solution, carrying out covalent coupling reaction, and carrying out post-treatment after the reaction is finished to obtain an immunoadsorption material;

the ligand is an amino compound.

In a specific example, the volume mass ratio of the organic solvent to the carbonyldiimidazole is (5-10) mL/(0.5-1) g.

In a specific example, the mass ratio of the solid phase carrier to the carbonyldiimidazole is (5-10): (0.5 to 1).

In a specific example, the volume mass ratio of the solid phase carrier organic solvent to the carbonyldiimidazole is (5-10) g: (5-10) mL: (0.5-1) g.

In a specific example, the temperature of the activation reaction is 4 to 25 ℃.

In a specific example, the time of the activation reaction is 1h to 3 h.

In a specific example, the temperature of the activation reaction is 4-25 ℃, and the time of the activation reaction is 1-3 h.

In one specific example, the temperature of the covalent coupling reaction is between 25 ℃ and 40 ℃.

In a specific example, the time of the covalent coupling reaction is 8-24 h.

In a specific example, the pH of the covalent coupling reaction is 7.0 to 10.0.

In a specific example, the temperature of the covalent coupling reaction is 25-40 ℃, and the time of the covalent coupling reaction is 8-24 h.

In a specific example, the temperature of the covalent coupling reaction is 25 ℃ to 40 ℃, and the pH of the covalent coupling reaction is 7.0 to 10.0.

In a specific example, the temperature of the covalent coupling reaction is 25-40 ℃, the time of the covalent coupling reaction is 8-24 h, and the pH of the covalent coupling reaction is 7.0-10.0.

In a specific example, the post-treatment comprises end-capping the solid support after the covalent coupling reaction.

In a specific example, the capping treatment comprises reacting the solid phase carrier after the covalent coupling reaction with an ethanolamine solution to block excessive active groups.

In a specific example, the organic solvent is acetone, tetrahydrofuran, an aromatic solvent.

In a specific example, the solid support is polysulfone, polyethersulfone, polyaryl (ether) sulfone, or polyvinyl alcohol EVOH.

In a specific example, the ligand is a combination of an amino compound having an immunoadsorption function and an amino-containing anticoagulant.

In a specific example, the amino compound having immunoadsorption function is at least one of protein a, a protein a-like polypeptide, goat anti-human Ig polyclonal antibody, tryptophan, phenylalanine, murine anti-human IgE monoclonal antibody/IgE receptor, beta adrenergic receptor fragment, acetylcholine receptor fragment, and calf thymus DNA.

In a specific example, the protein a is selected from at least one of recombinant protein a, native protein a, and biomimetic protein a.

In a specific example, the recombinant protein A is used at a concentration of 10-100 mg/mL.

In a specific example, the amino-containing anticoagulant is selected from at least one of nafamostat mesylate, heparin, and heparan.

In a specific example, the amino-containing anticoagulant is nafamostat mesylate.

In a specific example, the nafamostat mesylate is used at a concentration of 50-100 mg/mL.

In one particular example, the solid support is a hydroxyl-containing hollow fiber solid support. The hollow fiber solid phase carrier can be better used for blood purification and is a better choice.

In a specific example, the hydroxyl group-containing hollow fiber solid phase carrier is a hollow fiber woven tube, a hollow fiber woven mesh, or a hollow fiber tubular nonwoven material.

In a specific example, the hollow fiber has an inner diameter of 100 to 500 μm.

In a specific example, the wall thickness of the hollow fiber is 20-150 μm.

In a specific example, the hollow fibers are made of polysulfone, polyethersulfone, polyaryl (ether) sulfone, or polyvinyl alcohol EVOH.

In one particular example, the solid support is a hollow fiber made of polyvinyl alcohol EVOH.

In a second aspect of the present invention, there is provided:

an immunoadsorbent material prepared according to the protocol of the first aspect of the invention.

The invention has the beneficial effects that:

the research of the invention finds that after the solid phase carrier containing hydroxyl is activated by CDI, a functional group imidazolyl formate group can be introduced on the surface of the solid phase carrier, and the imidazolyl formate group can be coupled with a ligand containing amino, in particular to stably couple the ligand recombinant protein A and nafamostat mesilate. Compared with the immunoadsorption material prepared by the conventional method, the immunoadsorption material prepared by the method has the advantages that the adsorption effect on pathogenic components is obviously enhanced, the immunoadsorption material has good blood compatibility, not only can be used for adsorbing blood plasma, but also can be directly used for adsorbing whole blood, and meanwhile, small molecular pathogenic substances such as urea, creatinine and the like can be removed through hemodialysis or hemodiafiltration.

According to some preparation method examples, the anticoagulant containing amino is introduced into the solid phase carrier, so that the prepared immunoadsorption material has self-anticoagulation, and the anticoagulant can be reduced or even not used at all in the blood purification process, so that the risk of bleeding of a patient is greatly reduced.

In some embodiments of the preparation method, nafamostat mesilate is introduced as an anticoagulant, so that the immunoadsorption material has better safety, and the risk of bleeding of patients is further reduced.

Some preparation methods of the invention are safe and nontoxic, have simple preparation process, do not need to use strong irritant chemical substances or highly toxic substances, and are suitable for industrial production.

Drawings

FIG. 1 is a schematic representation of the reaction principle of some examples of the invention.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the accompanying examples. The preferred embodiments of the present invention are given in the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

s1) dissolving carbonyl diimidazole CDI with an organic solvent to obtain a CDI activating solution;

s2) fully contacting and reacting the hollow fiber solid phase carrier containing hydroxyl with CDI activating solution to activate the solid phase carrier;

s3) mixing the activated solid phase carrier with a ligand solution, carrying out covalent coupling reaction, and carrying out post-treatment after the reaction is finished to obtain an immunoadsorption material;

the ligand is an amino compound.

If the solid support itself does not contain hydroxyl groups, the hydroxyl groups are first introduced by known techniques, such as by chemical methods.

In one specific example, the temperature of the activation reaction is from 4 ℃ to 25 ℃. It is understood that, in the present invention, the temperature of the activation reaction includes, but is not limited to, 4 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C. At these temperatures, it is sufficient to obtain a satisfactory activation effect, which is sufficient for the subsequent coupling reaction. The experimental data show that in the reaction temperature range, the reaction result is not affected basically.

The time of the activation reaction can be adjusted accordingly according to the activation effect. In a specific example, the time of the activation reaction is 1 to 3 hours. It is understood that in the present invention, the time of the activation reaction includes, but is not limited to, 1h, 1.5 h, 2h, 2.5 h, 3 h.

In a specific example, the volume mass ratio of the organic solvent to the carbonyldiimidazole is (5-10) mL/(0.5-1) g. Different concentrations of CDI have different reaction speeds, and can be applied to the modification of different solid phase carriers. The solid phase carrier is easy to modify, and a CDI solution with lower concentration can be used; for solid supports that are difficult to modify, higher concentrations of CDI solution can be used.

In a specific example, the mass ratio of the solid phase carrier to the carbonyldiimidazole is (5-10): (0.5 to 1). The mixing ratio can be adjusted according to the grafting amount of the ligand.

In one particular example, EVOH hollow fiber matrix is mixed with CDI and then mixed with pyridine; the mass ratio of the mass of the EVOH hollow fiber matrix to the volume of the acetone to the mass of the CDI is (10-5): 1-0.5).

It is understood that in the present invention, the mass ratio of EVOH hollow fiber matrix mass, acetone volume to CDI includes, but is not limited to: 5:5:1, 5:10:1, 10:5:0.5, 10:5:1, 10:10:0.6, 10:10:0.7, 10:10:0.8, 10:10:0.9, 10:10: 1.

In a specific example, the amino compound is added in the form of a solution, and the concentration of the nafamostat mesylate solution is 50mg/mL to 100 mg/mL. It is understood that in the present invention, the concentration of the nafamostat mesylate solution includes, but is not limited to, 50mg/mL, 60mg/mL, 70mg/mL, 80 mg/mL, 90mg/mL, 100 mg/mL. The concentration of the recombinant protein A is 30-100 mg/mL. It is understood that, in the present invention, the concentration of the recombinant protein A solution includes, but is not limited to, 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80 mg/mL, 90mg/mL, 100 mg/mL.

The temperature of the covalent coupling reaction is only required to ensure the covalent coupling reaction. In one specific example, the temperature of the covalent coupling reaction is between 25 ℃ and 40 ℃. It is understood that in the present invention, the temperature of the covalent coupling reaction includes, but is not limited to, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C.

The time of the covalent coupling reaction can be adjusted accordingly as required to ensure that the covalent coupling reaction is to the desired extent. In a specific example, the time of the covalent coupling reaction is 8h to 24 h. It is understood that in the present invention, the time of the covalent coupling reaction includes, but is not limited to, 8h, 10h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h.

The pH of the covalent coupling reaction is such that the covalent coupling reaction proceeds without destroying the activity of the amino compound. In a specific example, the pH of the covalent coupling reaction is 7.0 to 10.0. It is understood that in the present invention, the pH of the covalent coupling reaction includes, but is not limited to, 7.0, 8.0, 9.0, 10.0.

In one particular example, the capping operation comprises a soaking reaction of the covalently coupled substrate with ethanolamine to block excess reactive groups on the substrate.

In one specific example, the temperature of the soaking reaction is 20 ℃ to 30 ℃. It is understood that, in the present invention, the temperature of the soaking reaction includes, but is not limited to, 20 deg.C, 25 deg.C, 30 deg.C.

In a specific example, the soaking reaction time is 6h to 10 h. It is understood that in the present invention, the soaking reaction time includes, but is not limited to, 6h, 7h, 8h, 9 h, 10 h.

The organic solvent is only required to be capable of well dissolving the CDI, not influencing the activation reaction and not destroying the original structure of the solid phase carrier. In a specific example, the organic solvent is acetone, tetrahydrofuran, an aromatic solvent. The better choice is acetone which is safer and less costly. Studies have shown that the kind of organic solvent has substantially no influence on the reaction as long as the activation reaction is ensured to be carried out under anhydrous conditions.

According to different specific applications, in a specific example, the amino compound with immunoadsorption function is at least one of protein A, protein A-like polypeptide, goat anti-human Ig polyclonal antibody, tryptophan, phenylalanine, mouse anti-human IgE monoclonal antibody/IgE receptor, beta-adrenergic receptor fragment, acetylcholine receptor fragment and calf thymus DNA.

In a specific example, the concentration of the nafamostat mesylate solution is 50-100 mg/mL.

In one particular example, the solid support is a hydroxyl-containing hollow fiber solid support.

In a specific example, the wall thickness of the hollow fiber is 20-150 μm.

In one particular example, the polyvinyl alcohol-based (EVOH) solid support is preferably a hollow fiber, including but not limited to a hollow fiber braided tube, a hollow fiber braided mesh, or a hollow fiber tubular nonwoven material.

The hollow fiber braided tube is a tubular material braided by hollow fibers, the hollow fiber braided net is a reticular material braided by the hollow fibers, the hollow fiber tubular non-woven material can be a short hollow fiber tubular non-woven material or a long hollow fiber tubular non-woven material, and the short hollow fiber tubular non-woven material is a tubular material which is cut by the long hollow fibers into the short hollow fibers and is not braided.

In a specific example, the EVOH hollow fiber matrix has an inner diameter of 100 to 500 μm. It is understood that, in the present invention, the inner diameter of the EVOH hollow fiber matrix includes, but is not limited to, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm.

The ligand molecule can bind at least to at least one pathogenic component of blood or plasma. More specifically, the pathogenic component includes IgG, IgM, IgA, IgD, IgE.

In a specific example, the amino compound is selected from recombinant protein a and nafamostat mesylate.

In a more specific example, the method for preparing the immunoadsorbent material comprises the following steps:

s1) mixing the mass ratio of the mass of the EVOH hollow fiber substrate to the volume of acetone to the mass of CDI (10-5) to (1-0.5), and carrying out an activation reaction at the temperature of 4-25 ℃ for 1-3 h;

s2), then adding 50-100 mg/mL of naphthalene methane sulfonate mostat solution and 30-100 mg/mL of recombinant protein A solution, adjusting the pH to 7.0-10.0, and soaking at the temperature of 25-40 ℃ for covalent coupling reaction for 8-24 h;

s3) soaking and reacting the product of the covalent coupling reaction with ethanolamine solution at 20-30 ℃ for 6-10 h, and sealing redundant active groups on the substrate.

Example 1

The embodiment provides a protein a immunoadsorption material using recombinant protein a and nafamostat mesylate as ligands, which comprises the following specific steps:

s1) placing 100g of dry EVOH hollow fiber (tubular shape, the inner diameter is 200 mu m), 10g of Carbonyl Diimidazole (CDI) and 10mL of dry acetone into a container, soaking at 25 ℃ for reaction for 1h, taking out, washing with 70% acetone and dry acetone in sequence to remove imidazole to obtain an activated hollow fiber matrix containing imidazolyl formate, and storing the matrix in the dry acetone for later use and marked as A0;

s2) taking 100g of the A0 prepared above, adding 150mL of 0.1mol/L borate buffer solution, controlling the pH value of the system to be 7.5-8.5, adding 14g of recombinant protein A and 7.5g of nafamostat mesilate, soaking at 37 ℃ for reaction for 24h, stopping the reaction, and then washing with 20 times of volume of injection water;

s3), adding 200mL of 0.2mol/L ethanolamine solution to seal unreacted imidazolyl formate, soaking at 25 ℃ for reaction for 6h, and sealing redundant active group imidazolyl formate on the substrate;

s4), washing with a large amount of water for injection, and preserving in preservative solution to obtain protein A immunoadsorbent material labeled A1.

Example 2

s1) placing 100g of dry EVOH hollow fiber (tubular shape, the inner diameter is 200 mu m), 10g of Carbonyl Diimidazole (CDI) and 10mL of dry acetone into a container, soaking at 25 ℃ for reaction for 1h, taking out, washing with 70% acetone and dry acetone in sequence to remove imidazole to obtain an activated hollow fiber matrix containing imidazolyl formate, and storing the matrix in the dry acetone for later use, wherein the matrix is marked as A0';

s2) taking 100g of the A0' prepared above, adding 150mL of 0.1mol/L borate buffer solution, controlling the pH value of the system to be 7.5-8.5, adding 7.5g of recombinant protein A and 7.5g of nafamostat mesilate, soaking at 37 ℃ for reaction for 24h, stopping the reaction, and then washing with 20 times of volume of injection water;

s4), washing with a large amount of water for injection after the reaction is finished, and storing in a preservative solution containing preservative to obtain the protein A immunoadsorbent material, which is marked as A1'.

Comparative example 1

S1) placing 100g of agarose, 10g of Carbonyl Diimidazole (CDI) and 10mL of dry acetone into a container, stirring and reacting for 1h at 25 ℃, taking out, washing with 70% acetone and dry acetone in sequence to remove imidazole to obtain activated imidazole formate-containing agarose, and storing the matrix in the dry acetone for later use, wherein the matrix is marked as B0;

s2) taking 100g of the prepared B0, adding 150mL of 0.1mol/L borate buffer solution, controlling the pH value of the system to be 7.5-8.5, adding 14g of recombinant protein A and 7.5g of nafamostat mesilate, stirring and reacting at 37 ℃ for 24h, stopping the reaction, and then washing with 20 times of volume of injection water;

s3), adding 200mL of 0.2mol/L ethanolamine solution to seal unreacted imidazolyl formate, stirring at 25 ℃ for reaction for 6h, and sealing redundant active group imidazolyl formate on the substrate;

s4), washing with a large amount of water for injection, and preserving in preservative solution to obtain protein A immunoadsorbent material labeled as B1.

Comparative example 2

S1) adding 80mL of injection water into 100g of dry EVOH hollow fiber (tubular shape, the inner diameter is 200 mu m) for full dispersion, adding 20mL of 1mol/L periodate for soaking and reacting at 25 ℃ for 2h, taking out, washing residual periodate by the injection water to obtain activated aldehyde group-containing hollow fiber, and storing the matrix in the injection water at 4 ℃ for later use and marking as C0;

s2) taking 100g of the prepared C0, adding 150mL of 0.1mol/L borate buffer solution, controlling the pH value of the system to be 7.5-8.5, adding 14g of recombinant protein A and 7.5g of nafamostat mesilate, soaking at 37 ℃ for reaction for 24h, stopping the reaction, and then washing with 20 times of volume of injection water;

s3), washing, adding 200mL of 0.2mol/L sodium borohydride solution, stirring at 25 ℃ for reaction for 6 hours, and reducing Schiff base to generate stable secondary amine;

s4), washing with a large amount of water for injection, and preserving in preservative solution to obtain protein A immunoadsorbent material labeled as C1.

Comparative example 3

S1) placing 100g of dry EVOH hollow fiber (tubular shape, the inner diameter is 200 mu m), 10g of Carbonyl Diimidazole (CDI) and 10mL of dry acetone into a container, soaking at 25 ℃ for reaction for 1h, taking out, washing with 70% acetone and dry acetone in sequence to remove imidazole to obtain an activated hollow fiber matrix containing imidazolyl formate, and storing the matrix in the dry acetone for later use and labeled as D0;

s2) taking 100g of the D0 prepared above, adding 150mL of 0.1mol/L borate buffer solution, controlling the pH value of the system to be 7.5-8.5, adding 14g of recombinant protein A, soaking at 37 ℃ for reaction for 24h, stopping the reaction, and then washing with 20 times of volume of injection water;

s4), washing with a large amount of water for injection, and preserving in preservative solution to obtain protein A immunoadsorbent material labeled D1.

Effect test-adsorption Performance test

The protein a adsorbing materials prepared in the above example 1 and comparative examples 1-2 were tested for adsorption performance, and the specific operations were as follows:

1g each of the protein A adsorbing materials A1, A1', B1 (comparative example 1) and C1 (comparative example 2) synthesized in examples and comparative examples was loaded on a column of Φ 0.8X 5cm, and 10mL of plasma was passed through the column at a flow rate of 1mL/min, after washing the column sufficiently with physiological saline, so that antibody IgG was adsorbed on the adsorbing material. Then, the plasma was washed with 30mL of physiological saline, human immunoglobulin IgG adsorbed on the adsorbent was eluted using 0.01M citric acid-disodium hydrogen phosphate buffer solution having a pH of 2.8 as an eluent, the eluent was collected and fixed to 100mL, an absorption peak absorption value of the eluent at around 280nm was measured using an ultraviolet-visible spectrophotometer, and the absorption peak absorption value of IgG at around 280nm in the eluent was in a proportional relationship, and the measurement results are shown in table 1.

TABLE 1 absorption peaks of immunoadsorbent materials

Immunoadsorption material	A₂₈₀
		Example 1 synthetic immunoadsorbent material A1	0.530
EXAMPLE 2 synthetic immunoadsorbent material A1'	0.350
		Comparative example 1 synthetic immunoadsorbent material B1	0.433
Comparative example 2 synthetic immunoadsorbent material C1	0.469

As is clear from the data in table 1, the adsorption amount of the protein a immunoadsorbent material a1 synthesized by the production method of the present invention > the adsorption amount of the immunoadsorbent material C1 synthesized in comparative example 2 > the adsorption amount of the immunoadsorbent material B1 synthesized in comparative example 1 > the adsorption amount of the immunoadsorbent material a 1' synthesized in example 2.

Effect verification test two-protein A shedding amount contrast test

50mg of each of the synthesized protein A adsorbing materials A1, B1 and C1 are taken and loaded in a chromatography column with the diameter of 4 multiplied by 10cm, 3L of normal saline is used for washing the column, the last 100mL is taken as a test solution, the test solution is taken and detected by a protein A ELISA kit and an enzyme linked immunosorbent assay detector, and the detection results are shown in the following table 2.

TABLE 2 protein A shedding amount on immunoadsorbent materials

Immunoadsorption material	ProteinAmount of A falling off
		Example 1 synthetic adsorbent material A1	0.151μg/mL
Comparative example 1 synthetic adsorbent material B1	0.310μg/mL
		Comparative example 2 synthetic adsorbent material C1	0.201μg/mL

As can be seen from the data in table 2, the protein a immunoadsorbent material a1 synthesized by the preparation method of the present invention has a protein a shedding amount < the adsorbent material C1 synthesized in comparative example 2 has a protein a shedding amount < the adsorbent material B1 synthesized in comparative example 1.

Effect test three blood compatibility test

The blood compatibility test was performed on the adsorbing materials prepared in example 1 and comparative examples 1 to 2, and the following specific operations were performed:

hemolysis experiment: hemolysis experiments are carried out according to the experimental selection of interaction with blood in GB/T16886.4-2003 section 4 of medical device biology evaluation and the experimental method of GB/T16175-2008 of biological evaluation of medical organosilicon materials.

S1) adding 1g of adsorbing material prepared in the example 1 and the comparative examples 1-2 into each tube of the sample group, and then adding 10ml of sodium chloride injection; adding 10ml of sodium chloride injection into each tube of the negative control group; 10ml of distilled water was added to each tube of the positive control group. Each set of 3 tubes was run in parallel;

s2) putting all test tubes into a constant temperature water bath (37 +/-1) DEG C, preserving the temperature for 30min, adding 0.2ml of diluted rabbit blood into each test tube, gently mixing the diluted rabbit blood and the diluted rabbit blood, and continuously preserving the temperature for 60 min in the water bath (37 +/-1) DEG C;

s3), the liquid in the pouring tube is centrifuged at 800g for 5 min, the supernatant is aspirated and transferred into a cuvette, and the absorbance is measured with a spectrophotometer at 545 nm.

The absorbance of the sample combination control group was averaged over 3 tubes. The absorbance of the negative control tube should not be greater than 0.03, the absorbance of the positive control tube should be 0.8 + -0.3, otherwise, the test should be repeated.

Hemolysis rate = (a-B)/(C-B) × 100%, where a is the sample set absorbance; b is the absorbance of the negative control group; and C is the absorbance of the positive control group.

Blood compatibility test:

s1) taking 1g of each adsorbing material prepared in the example 1 and the comparative examples 1-3, soaking the adsorbing materials in normal saline for 10 hours, and then putting the adsorbing materials into a column;

s2) was injected into 10mL of rabbit whole blood without anticoagulation with heparin sodium using a syringe, and adsorbed at a flow rate of 20mL/min for 2 hours, while a control experiment was performed using an empty column adsorbed with rabbit whole blood anticoagulated with heparin for 2 hours.

The change in each component of blood before and after adsorption was measured using a Beckman LH750 blood cell analyzer.

The results show that:

(1) the hemolysis rate of the adsorbing materials prepared in the example 1 and the comparative examples 1-2 is less than 2 percent and less than 5 percent which is required by national standard.

(2) The change of each main component in blood before and after adsorption of the adsorbing materials prepared in the example 1 and the comparative examples 1-2 is not large, and the reduction percentage is within 5 percent, but the adsorbing material prepared in the comparative example 3 generates coagulation, because the adsorbing material of the comparative example 3 does not have self-anticoagulation, rabbit whole blood which is not anticoagulated generates coagulation when passing.

The results show that the self-anticoagulation adsorption material prepared by the invention has good blood compatibility.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.

Claims

1. A preparation method of an immunoadsorption material is characterized by comprising the following steps: the preparation method comprises the following steps:

the ligand is an amino compound.

2. The method of claim 1, wherein: the volume mass ratio of the organic solvent to the carbonyldiimidazole is (5-10) mL/(0.5-1) g; and/or

The mass ratio of the solid phase carrier to the carbonyldiimidazole is (5-10): (0.5 to 1).

3. The method of claim 1, wherein: the temperature of the activation reaction is 4-25 ℃; and/or

The time of the activation reaction is 1-3 h.

4. The method of claim 1, wherein: the temperature of the covalent coupling reaction is 25-40 ℃; and/or

The time of the covalent coupling reaction is 8-24 h; and/or

The pH value of the covalent coupling reaction is 7.0-10.0.

5. The production method according to any one of claims 1 to 4, characterized in that: the post-treatment comprises the end-capping treatment of the solid phase carrier after the covalent coupling reaction.

6. The method of claim 5, wherein: the operation of the end capping treatment comprises the steps of fully reacting the solid phase carrier after the covalent coupling reaction with an ethanolamine solution to block redundant active groups.

7. The production method according to any one of claims 1 to 4, characterized in that: the organic solvent is acetone, tetrahydrofuran or aromatic solvent; and/or

The solid phase carrier is polysulfone, polyethersulfone, polyaryl (ether) sulfone or polyvinyl alcohol EVOH; and/or

The ligand is the combination of an amino compound with an immunoadsorption function and an amino-containing anticoagulant.

8. The method of claim 7, wherein:

1) the amino compound with the immunoadsorption function is at least one of protein A, proteinoid A polypeptide, goat anti-human Ig polyclonal antibody, tryptophan, phenylalanine, mouse anti-human IgE monoclonal antibody/IgE receptor, beta-adrenergic receptor segment, acetylcholine receptor segment and calf thymus DNA;

preferably, the protein A is at least one selected from recombinant protein A, natural protein A and biomimetic protein A; further, the use concentration of the recombinant protein A is 10-100 mg/mL;

2) the amino-containing anticoagulant is at least one of nafamostat mesylate, heparin and heparan;

preferably, the amino-containing anticoagulant is nafamostat mesylate; further, the using concentration of the nafamostat mesylate is 50-100 mg/mL.

9. The production method according to any one of claims 1 to 4, characterized in that: the hydroxyl-containing solid phase carrier is at least one of hollow fiber, a hollow fiber braided tube, a hollow fiber braided net or a hollow fiber tubular non-woven material;

preferably, the inner diameter of the hollow fiber is 100-500 μm; and/or

The wall thickness of the hollow fiber is 20-150 mu m.

10. An immunoadsorbent material prepared by the method of any one of claims 1 to 9.