CN103933947B - For blood purification material removing rheumatoid factor and preparation method thereof - Google Patents

Abstract

The invention provides a blood purification material for removing rheumatoid factor and a preparation method thereof, belonging to the technical field of biomedicine. The blood purification material is composed of a solid-phase carrier and ligands fixed on the solid-phase carrier through chemical coupling; wherein, the solid-phase carrier is a polysaccharide natural polymer material, and the ligand is 1-amino-3-(2 -(4-pyridyl)-ethylmercapto)-2-propanol, the ligand density fixed on the solid phase support by chemical coupling is 1.4-2.8mmol/g dry solid phase support. The blood purification material can selectively adsorb rheumatoid factors in blood, has limited non-specific adsorption to other plasma components such as human serum albumin and IgG, and has low preparation cost and stable physical and chemical properties, so it can be used as an adsorption agent for blood purification devices. The filler is used for the removal of rheumatoid factor in the blood of rheumatoid patients.

Description

Blood purification material for removing rheumatoid factor and preparation method thereof

technical field

The invention relates to a blood purification material for removing rheumatoid factor and its preparation method and application, belonging to the technical field of biomedicine.

Background technique

Rheumatoid factors (RF) is an IgM anti-denatured IgG antibody, which has been proven to be closely related to the occurrence and development of a series of rheumatoid diseases, such as rheumatoid arthritis and systemic lupus erythematosus. For these diseases, reducing the concentration of rheumatoid factors in the patient's blood through in vitro clearance can significantly improve the symptoms, and play a role in relieving and treating the disease (B.C.McLeod, Introduction to the Third Special Issue: Clinical Applications of Therapeutic Apheresis. J Clin Apheresis ,2000,15(1-2):1-5), so the blood purification technology used to remove rheumatoid factor has been widely valued in clinical practice.

Blood purification technology is a treatment method that artificially regulates blood-related components. Since it was developed and applied in the 1980s, it has gradually developed into an important clinical treatment method. The patient's blood is taken out of the body and returned to the body after passing through the purification device. During this process, the purification device is used to remove metabolic waste, pathogenic substances, excess water, etc. in the blood, so as to correct and regulate the imbalance of the internal environment of the body, thereby To achieve the purpose of treating diseases.

Adsorbents used in blood purification generally consist of two parts: a polymer carrier material as the adsorbent matrix and ligand molecules immobilized on the carrier surface by chemical cross-linking. The adsorbent matrix generally adopts porous materials with good hydrophilicity. A good adsorbent matrix also needs to have stable physical and chemical properties, small non-specific adsorption, and good blood compatibility. At present, there are many different types of polymer materials that can be used as carrier substrates for blood purification adsorbents, including polysaccharide natural polymers, such as agarose, cellulose, dextran, chitosan, etc.; synthetic polymer materials, Such as polyacrylamide, polyimide, polyvinyl alcohol, etc.; inorganic materials, such as silica, porous glass beads, etc. Ligand molecules are bound to the carrier material through covalent bonds, and their structure and properties determine the effect of the adsorbent. At present, the adsorbents used for the adsorption and removal of antibody molecules mainly use two functional ligands: biological type and small molecule compound type.

Protein A adsorbent is a typical biological blood purification adsorbent, which uses protein A as the adsorption functional group. Protein A is a staphylococcal cell wall protein, its full name is Staphylococcal Protein A (SPA), it is a single-chain polypeptide with a molecular weight of 4.2kDa. It has reversible affinity for various immunoglobulins of humans and other mammals, especially for the Fc region of IgG. The binding of protein A to rheumatoid factor is mainly based on its ability to bind a broad spectrum of immunoglobulins. At present, protein A, as a typical affinity ligand, has been used for the clearance of autoantibodies and immune complexes in patients with autoimmune diseases in clinic. Prosorba and Immunosorba (Fresenius, Germany) are the two most widely used protein A adsorbents. They use silica gel and agarose gel as the carrier matrix respectively. Among them, Prosorba has obtained the certification of the US Food and Drug Administration (FDA) , for the treatment of idiopathic thrombocytopenic purpura and rheumatoid arthritis. Domestic also has similar product to come out (CN1367181A). For this type of adsorbent that uses protein A as a ligand, its advantage is that it has a high selective recognition ability for antibody molecules by utilizing the natural affinity between biomolecules, and can realize the removal of a large class of autoantibodies. Therefore, it can be widely used in the treatment of a series of autoantibody-related diseases. However, its disadvantage is that the binding ability of protein A to IgM antibodies is significantly weaker than that of IgG antibodies. Protein A mainly acts on the Fc fragment of IgG, but the Fc fragment of IgM is concentrated in the center of the molecule, with a low degree of exposure, and the steric hindrance of binding protein A is relatively large. Therefore, the removal effect of protein A on rheumatoid factor is lower than that on other IgG autoantibodies. To obtain a better therapeutic effect, a high treatment intensity is required, which will cause a large loss of normal IgG. In addition, as a biologically active protein molecule, it needs to be obtained from Staphylococcus aureus or genetically engineered bacteria, and its production cost is very high. At present, the market price of Prosorba adsorption column is about 1,000 Euros, while the market price of a pair of switchable Immunosorba adsorption columns is about 10,000 Euros. At the same time, protein ligands are unstable and easily inactivated during immobilization and storage. Although it can be regenerated and reused, it also has the problem of being difficult to clean and sterilize in-line and the hidden danger of ligand molecule falling off. These shortcomings limit the clinical use of protein A adsorbents in the treatment of rheumatoid diseases.

In addition to protein A, it has also been reported that thermopolymerized IgG was used as the affinity functional group for the adsorption of rheumatoid factor. The application of thermally polymerized IgG is mainly based on the fact that IgM type rheumatoid factor has the biological activity of binding denatured IgG. Therefore, thermally polymerized IgG prepared by artificial thermal denaturation is immobilized on the surface of the matrix material, and it also has the ability to selectively remove rheumatoid factor. . However, due to the high cost of ligand production and possible safety hazards, this type of adsorbent is only found in laboratory research and has not yet been applied industrially.

Compared with biological macromolecules, chemically synthesized small molecule compounds have significant advantages in production cost and physical and chemical stability, so many studies have also focused on screening and designing small molecule ligands that can be applied to blood purification.

Hydrophobic amino acid is currently the most reported and applied small molecule group for antibody adsorption. Japan's Asahi Medical Company has developed two kinds of adsorbent materials with tryptophan and phenylalanine as ligands respectively, and the trade names are Immusorba TR (IM-TR) and Immusorba PH (IM-PH). These two adsorbents both use polyvinyl alcohol microspheres as carrier substrates, and tryptophan or phenylalanine molecules are coupled on the surface of the substrates through amino groups. The force characteristics of these two adsorbents are mainly based on aromatic rings and carboxyl groups, so it is inferred that their interaction with antibody molecules combines hydrophobic and electrostatic interactions. Adsorption experiments showed that IM-TR and IM-PH did not have considerable non-specific adsorption on albumin and fibrinogen in plasma, but had a general removal effect on autoantibodies such as anti-DNA antibody, anti-AchR antibody, and anti-C1q antibody. It is used in the treatment of autoimmune diseases such as Guillain-Barré syndrome, systemic lupus erythematosus, and myasthenia gravis. Chinese patent document CN1666784A provides an adsorbent material using histidine as an adsorbent group. In addition, there are also reports on the use of polyamino acids as adsorption groups in this field (Chinese patent document CN1476908A). In terms of the application of adsorbents, a small number of studies have confirmed that this kind of adsorbent based on hydrophobic amino acids also has a certain degree of adsorption and removal effect on rheumatoid factor. For example, it was reported that phenylalanine was used as a ligand to couple to polyvinyl alcohol microsphere matrix for the adsorption and removal of rheumatoid factor (Wang Weichao et al., Chinese Journal of Biomedical Engineering, 2009, 28 (4): 561- 566).

In addition, the inventor's team also developed two small molecule ligands for the adsorption and removal of autoantibodies in patients with autoimmune diseases, including benzoic acid (Chinese patent document CN101185880A) and 4-mercaptoethylpyridine (J.Ren, et al., J Biomed Mater Res Part A, 2011:98A:589–595). For the adsorption and removal of rheumatoid factor, although the existing small molecule ligand adsorbents show a certain degree of binding ability under laboratory evaluation conditions, the effect is often not satisfactory in the further experimental evaluation of simulated clinical treatment. . The main reason is that the adsorbents based on small molecule ligands represented by aromatic amino acids and 4-mercaptoethylpyridine are broad-spectrum antibody adsorbents developed and designed for the molecular structure characteristics of IgG antibodies, while IgM antibodies It is significantly different from IgG antibodies in terms of molecular scale and structural characteristics: ①The molecular weight of IgM is six times that of IgG; ②Although the two have partially identical conserved sequences, there are still large differences in molecular structural characteristics. Therefore, the adsorbent designed for IgG is not suitable for IgM-based rheumatoid factor. Taking 4-mercaptoethylpyridine as an example, although we found that this molecule has varying degrees of adsorption capacity for various antibody components in human blood, it is not compatible with non-IgG antibodies (such as IgM, IgA, and IgE) and their complexes. The binding ability of the protein is far less than the adsorption effect on IgG. If it is used for the adsorption treatment of IgM type rheumatoid factor, it will face similar problems as protein A, that is, the adsorption capacity of rheumatoid factor is weak. If the removal rate of rheumatoid factor is increased by increasing the treatment intensity, it will inevitably lead to a large number of Loss of normal IgG carries the risk of immunocompromise (J.Ren, et al., J Biomed Mater Res Part A, 2011:98A:589–595).

In essence, 4-mercaptoethylpyridine is an adsorption functional group specially developed for IgG antibodies, and its interaction sites with IgG are mainly concentrated on the Fc fragment of IgG, in which hydrophobic interaction and π-π conjugation play an important role. effect. For IgM whose Fc fragment is wrapped inside the molecule, it is difficult to effectively reflect the ability of 4-mercaptoethylpyridine. Therefore, in order to improve the adsorption selectivity of IgM antibodies and realize the selective adsorption and removal of rheumatoid factors, it is necessary to carry out a new design and structural optimization of the ligand molecule specifically for the molecular structure characteristics of IgM, so that its spatial structure and force It is more suitable for IgM.

Contents of the invention

The object of the present invention is to provide a blood purification material for removing rheumatoid factor and a preparation method thereof. The blood purification material should have good selectivity and high adsorption capacity for rheumatoid factor in human blood, stable physical and chemical properties and relatively low production cost.

The inventors of the present invention designed ligand molecules based on the structural characteristics of IgM, and finally determined a brand new long-chain multifunctional compound: 1-amino-3-(2-(4- Pyridinyl)-ethylthio)-2-propanol (1-amino-3-(2-(pyridin-4-yl)ethylthio)propan-2-ol) as a selective binding ligand for IgM. 1-Amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol molecule with ligands used in prior art (benzoic acid, aromatic amino acid, 4-mercaptoethylpyridine ) compared with the introduction of multiple electron-rich groups (hydroxyl, amine) on the basis of hydrophobic heterocyclic groups. Molecular docking results show that its binding ability to IgM antibodies is significantly higher than that of IgG antibodies; and the analysis of the site of action shows that these electron-rich groups are more inclined to bind to the Fab fragment of IgM, and there are more on the outer surface of IgM. effective binding site. Since the molecular size of IgM is about 6 times that of IgG, and multiple Fab fragments are exposed on the molecular surface, strengthening the binding of the ligand molecule to the Fab will significantly enhance the ability of the ligand to interact with IgM and rheumatoid factor. At the same time, IgM has a larger surface area, and compared with the adsorption of IgG molecules, the ligand density required for effective adsorption of IgM and rheumatoid factor will be lower. By properly controlling the density of the coupling group, the adsorbent can distinguish between IgM and IgG. Based on the above two factors, and on the basis of ligand selection, by further optimizing the reaction conditions of ligand immobilization, a synthetic scheme of adsorption material suitable for selective removal of rheumatoid factor in human blood was determined.

The structural formula of the above-mentioned ligand molecule 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol is as follows:

Specifically, the blood purification material for removing rheumatoid factor of the present invention is composed of a solid phase carrier and a ligand fixed on the solid phase carrier through chemical coupling. The solid phase carrier is a cross-linked polysaccharide, and the ligand The base is 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol, and the ligand density fixed on the solid phase carrier by chemical coupling is 1.4～2.8mmol/g dry solid carrier.

Ligand density affects the adsorption effect of blood purification materials on rheumatoid factors: as the density of ligands increases, the binding capacity of rheumatoid factors per unit volume of blood purification materials increases; but when the density reaches a certain level, it will also bring more Much non-specific adsorption, especially for IgG binding. The inventors of the present invention have found through research that the suitable ligand density is 1.4-2.8 mmol/g dry solid phase carrier. Wherein, the dry solid-phase carrier refers to a solid-phase carrier that does not contain moisture after drying.

The solid phase carrier used for blood purification materials should have good hydrophilicity and should not adsorb proteins by itself, and should have good blood compatibility without causing adverse reactions such as activation of blood coagulation mechanism. As such a solid phase carrier material, for example, can enumerate: polysaccharide natural polymers, such as agar, agarose, cellulose, dextran, chitosan, etc.; synthetic polymer materials, such as substituted or unsubstituted Polyacrylamide, polyimide, polyvinyl alcohol, etc.; inorganic materials, such as silica, etc. Considering the good biocompatibility of polysaccharide natural polymers and their successful application in the field of blood purification materials, the present invention chooses cross-linked polysaccharide materials as carriers, more preferably agarose among them.

The solid phase support is preferably in the form of porous microspheres. In addition, the solid phase carrier needs to have sufficient permeability and a relatively stable spatial structure, and the permeation molecular weight of its gel pores is preferably 150,000 to 5,000,000Da. Because the molecular weight of the IgM antibody and its immune complex among the target molecules to be adsorbed by the blood purification material is often above 900,000Da.

The method for preparing the above-mentioned blood purification material of the present invention includes the following steps: firstly, introducing a ligand 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2 -Active groups that react with the amine groups on propanol, and then 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol is co- bonded to the surface of the solid support.

The immobilization of the ligand molecules on the solid phase support relies on the covalent connection between the amine groups on the ligand molecules and the reactive groups on the solid phase support through chemical reactions. Groups that can react with the amine groups on the above-mentioned ligand molecules include imidazole carbamate groups, sulfonyl chloride groups, carboxyl groups, epoxy groups, haloalkyl groups, succinimide groups, trifluoroethanesulfonic acid monomethyl Oxygen etc. If the solid phase support does not contain active groups that can react with amine groups, some activating reagents are required to generate amine reactive groups. Such activating reagents often have dual functional groups, such as carbonyldiimidazole, epichlorohydrin, epibromohydrin, dichloro(bromo)propanol, dibromobutane, diglycidyl ether, divinyl sulfone, bromide Cyanide etc. In addition, standard immobilization methods for any amine group can be used, and those skilled in the art can refer to relevant books or handbooks, such as Immobilized Affinity Ligand Techniques (Hermanson et al., Academic Press, 1992) and Bioconjugate Techniques (Greg T. Hermanson, Academic Press, Inc, 1996). For solid-phase supports with hydroxyl groups, such as agarose, cellulose, dextran and other cross-linked polysaccharide supports, the surface of the support can be activated by carbonyldiimidazole, epichlorohydrin, epibromohydrin, cyanogen bromide, etc. The hydroxy groups generate active groups, and then use these groups that can react with amine groups to couple ligand molecules. Since cyanogen bromide is a highly toxic substance, it is harmful to the human body and the environment when the carrier is activated, so try to avoid using it. In the present invention, carbonyldiimidazole, epichlorohydrin, and epibromohydrin are preferably used as the activating agent, and carbonyldiimidazole, which is easy to control the degree of activation of the carrier, is more preferably used as the activating agent.

When using carbonyldiimidazole as an activating reagent to prepare the above-mentioned blood purification material, the active group introduced on the solid phase carrier is an imidazole carbamate active group, which specifically includes the following steps:

(1) Activation of solid phase carrier

Activated with carbonyldiimidazole, the amount added is 0.9~1.5g/10mL wet solid phase carrier; the reaction is carried out in acetone, 15~30℃ for 1~2h, the product is washed with acetone; the carrier can be controlled by adjusting carbonyldiimidazole The degree of activation can be adjusted to obtain a blood purification material with a suitable ligand density (1.4-2.8mmol/g dry solid-phase carrier). Wherein, the wet solid phase carrier refers to a solid phase carrier from which unbound water is removed by vacuum filtration, and the volume ratio of the wet solid phase carrier to be activated to acetone is preferably 1:1˜1:2.

(2) Coupling ligand

1-Amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol is covalently bonded to the surface of the solid phase support, and the reaction conditions are as follows: Add 5-10 times the volume of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol to the suspension of solid phase carrier and acetone, and react at 20-35°C for 2 -3h, and then washed repeatedly with acetone, 0.1M HCl solution, 0.1M NaOH solution, and deionized water.

Through this step, the amine group in the ligand molecule 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol can be covalently bonded to the carrier made of carbonyldiimidazole On the active group generated on the surface, the chemical coupling of the ligand molecule and the carrier is realized.

(3) Post-processing

Using a NaOH solution with a pH of 10-13, post-treat the ligand-coupled solid-phase support obtained in step (2), and hydrolyze the active groups derived from carbonyldiimidazole on the solid-phase support that have not reacted with the ligand.

There may also be active groups derived from carbonyldiimidazole (ie, imidazole carbamate active groups) that have not reacted with ligands in the solid phase support obtained in step (2). These active groups are potential factors for non-specific adsorption, so it is necessary to remove them by hydrolysis through the above-mentioned post-treatment.

The application of the present invention refers to the application of the above-mentioned blood purification material in the preparation of a blood purification device for removing rheumatoid factors in the blood of rheumatoid patients. Specifically, this material can be used as an adsorption filler of a blood purification device for rheumatoid patients Clearance of rheumatoid factor and other IgM autoantibodies and circulating immune complexes in the blood.

The beneficial effect of the present invention is that the blood purification material uses a small molecular compound as a ligand, has better stability relative to the protein ligand, and does not have the hidden danger of being degraded by proteolytic enzymes. At the same time, the ligand used is simple in structure and convenient in synthesis, and its production cost is much lower than that of protein. Moreover, the blood purification material of the present invention can withstand harsh treatment conditions such as strong acid, strong alkali, organic solvent, heating, etc., and the adsorption performance will not be affected by it. In addition, compared with other currently used antibody adsorption materials based on small molecular compound ligands, the blood purification material of the present invention can exhibit higher IgM adsorption selectivity, and has a non-specific effect on other plasma components such as human serum albumin and IgG. Heteroadsorption is limited, and the loss of IgG antibodies can be minimized while removing rheumatoid factors.

Detailed ways

The present invention will be described in detail below in conjunction with the examples, but the following examples are only preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto, and any person familiar with the technical field of the present invention is within the technical scope disclosed in the present invention Within the technical scheme of the present invention and its inventive concept, any equivalent replacement or change shall be covered within the protection scope of the present invention.

1. Synthesis of ligand molecule 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol

It is prepared by ring-opening ammonia water after a substitution reaction between 4-mercaptoethylpyridine (CAS: 2127-05-1) and epichlorohydrin (CAS: 106-89-8). The specific method is as follows:

Add equimolar amounts of 4-mercaptoethylpyridine (5g) and epichlorohydrin (3.33g) to 25ml dimethyl sulfoxide, mix well, then add 1ml triethylamine to the reaction system, and react at 25°C for 6 hours . After the reaction was completed, the reaction solution was added dropwise into 100 ml of ammonia solution (10%) under stirring conditions, and the reaction was continued for 12 hours. After the reaction, it was left to stand for 1 hour. After the solution was phase-separated, the organic phase was collected, washed repeatedly with 0.1M sodium hydroxide solution, and the product was separated with a silica gel column. After analysis, the purity of the product was 95%, and the recovery rate was 65%.

2. Preparation of blood purification materials

Comparative Example 1: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 0.75mmol/g dry gel ( activated by carbonyldiimidazole)

1) Take 10 mL of cross-linked agarose gel Sepharose CL-6B (gel pore penetration molecular weight: 10,000-4,000,000 Da) after sufficient sedimentation (overnight), wash with 200 mL of anhydrous acetone several times to remove the water contained in the gel , and then uniformly disperse the gel in an equal volume of acetone, and ensure that the entire system is anhydrous. Weigh 0.5g carbonyldiimidazole (CDI) and mix it with the above gel suspension (the amount of CDI is 0.5g/10mL wet gel), and stir the resulting mixture at 15°C for 1h. After the reaction, the gel was washed several times with 200 mL of anhydrous acetone to obtain a cross-linked agarose gel with imidazole carbamate active groups.

2) Evenly disperse 10mL of CDI-activated gel in an equal volume of acetone, add 1-amino-3-(2-(4-pyridyl)-ethyl mercapto)-2-propanol, and the resulting mixture was stirred at 20°C for 2 h. After the reaction, wash with 100mL of acetone, 50mL of 0.1M HCl solution, 50mL of 0.1M NaOH solution, and 100mL of deionized water for multiple times to obtain agarose gel coupled with ligands.

3) Add the cleaned gel wet cake to 50mL NaOH solution with pH 12, and place it in a shaker at 30°C at a speed of 130rmp for 10h to block the remaining active groups on the gel. After the reaction mixture was suction filtered, the filter cake was taken, washed with 50mL deionized water, 50mL 1M NaCl solution, and 50mL deionized water successively. Elemental analysis (nitrogen content) showed that the ligand coupling density was 0.75mmol/g dry gel.

Comparative Example 2: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 1.12mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that the amount of CDI is increased to 0.8g/10mL wet glue, and the activation reaction is carried out at 30°C for 1h; when coupling the ligand, 1-amino-3-(2-(4-pyridyl )-Ethylmercapto)-2-propanol was used in an amount of 10 times the volume, and the reaction was carried out at 35°C for 2 hours; when the remaining imidazole activation groups were hydrolyzed, a NaOH solution with a pH of 13 was used. Elemental analysis showed that the ligand coupling density was 1.12mmol/g dry glue.

Example 1: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 1.4 mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that: the amount of CDI is increased to 0.9g/10mL wet glue, and the activation reaction is carried out at 20°C for 2h; when the ligand is coupled, the reaction is carried out at 25°C for 2h; when the remaining imidazole activation group is hydrolyzed, NaOH solution at pH 10 was used. Elemental analysis showed that the ligand coupling density was 1.4mmol/g dry glue.

Example 2: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 2.03 mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that the amount of CDI is increased to 1.1g/10mL wet glue, and the activation reaction is carried out at 25°C for 1h; when coupling ligands, 1-amino-3-(2-(4-pyridyl )-Ethylmercapto)-2-propanol is used in an amount of 10 times the volume, and the reaction is carried out at 25°C for 3 hours; when hydrolyzing the remaining imidazole activation groups, a NaOH solution with a pH of 10 is used. Elemental analysis showed that the ligand coupling density was 2.03mmol/g dry glue.

Example 3: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 2.61 mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that the amount of CDI is increased to 1.2g/10mL wet glue, and the activation reaction is carried out at 25°C for 1h; when coupling ligands, 1-amino-3-(2-(4-pyridyl )-Ethylmercapto)-2-propanol is used in an amount of 10 times the volume, and the reaction is carried out at 25°C for 3 hours; when hydrolyzing the remaining imidazole activating groups, a NaOH solution with a pH of 13 is used. Elemental analysis showed that the ligand coupling density was 2.61mmol/g dry glue.

Example 4: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 2.82 mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that the amount of CDI is increased to 1.5g/10mL wet glue, and the activation reaction is carried out at 25°C for 1h; when coupling the ligand, 1-amino-3-(2-(4-pyridyl )-Ethylmercapto)-2-propanol is used in an amount of 10 times the volume, and the reaction is carried out at 25°C for 3 hours; when hydrolyzing the remaining imidazole activation groups, a NaOH solution with a pH of 10 is used. Elemental analysis showed that the ligand coupling density was 2.82mmol/g dry glue.

Comparative Example 3: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 3.06mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that the amount of CDI is increased to 1.6g/10mL wet glue, and the activation reaction is carried out at 30°C for 2h; )-Ethylmercapto)-2-propanol is used in an amount of 10 times the volume, and the reaction is carried out at 25°C for 3 hours; when hydrolyzing the remaining imidazole activation groups, a NaOH solution with a pH of 10 is used. Elemental analysis showed that the ligand coupling density was 3.06mmol/g dry glue.

Comparative Example 4: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cross-linked agarose blood purification material with a coupling density of 3.15mmol/g dry gel ( activated by carbonyldiimidazole)

The specific steps are the same as in Example 1, the difference is that: the amount of CDI is increased to 1.8g/10mL wet glue, and the activation reaction is carried out at 30°C for 1h; when the ligand is coupled, the reaction is carried out at 30°C for 2h; when the remaining imidazole activation group is hydrolyzed, NaOH solution at pH 10 was used. Elemental analysis showed that the ligand coupling density was 3.15mmol/g dry glue.

Example 5: Preparation of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol cellulose blood purification material with a coupling density of 2.45mmol/g dry gel (sulfonated acid chloride group coupling)

Weigh 1g of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol and dissolve it in 10mL of carbonate buffer (0.5M, pH10), and add 5mL of acetone to the solution. Weigh 10g of cellulose microsphere wet glue with sulfonyl chloride groups (the density of sulfonyl chloride groups is greater than 80μmol/mL wet glue, and the gel pore molecular weight is 100,000-4,000,000Da) and disperse it in the prepared solution. ℃ suspension paddle stirring for 6h. After the reaction was completed, the obtained gel was washed with 100 mL of acetone, 100 mL of deionized water, and 100 mL of 0.1M hydrochloric acid solution several times. The cellulose microspheres coupled with 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol were dried and dispersed in 60mL of 1M ethanolamine aqueous solution (pH10), at 30℃ Suspension paddle stirring for 3h. After the reaction was completed, the obtained cellulose microsphere gel was washed with 200 mL of deionized water several times. Elemental analysis showed that the ligand coupling density was 2.45mmol/g dry glue.

Example 6: Preparation of chitosan blood purification material with 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol coupling density of 1.67mmol/g dry gel (via Carboxyl group coupling)

Weigh 1g of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol and dissolve it in 30mL of MES buffer (0.1M, pH4.6). Weigh 10g of chitosan microsphere wet glue with carboxyl groups (carboxyl group density greater than 80μmol/mL wet glue, gel pore molecular weight of 15,000-4,000,000Da) and disperse it in the prepared solution, add 2.5g1 -Ethyl-(3-dimethylaminopropyl)carbodiamine hydrochloride (EDC·HCl), adjust the pH to 4.6, and stir at 30°C for 6h. After the reaction, the obtained gel was washed with 100 mL deionized water, 100 mL 0.1M hydrochloric acid, and 200 mL deionized water for several times. Elemental analysis showed that the ligand coupling density was 1.67mmol/g dry glue.

Example 7: Preparation of dextran blood purification material with 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol coupling density of 1.92mmol/g dry gel (via Epichlorohydrin activation)

1) Weigh 10g of dextran gel (gel pore penetration molecular weight is 15,000~4,000,000Da) and evenly disperse it in 30mL of deionized water to obtain a gel suspension, and add 5mL of epoxy chloride to the above gel suspension in turn Propane, 10mL of 1M NaOH solution, 0.1g of sodium borohydride, stirred at 30°C for 16h. After the reaction, the obtained gel was washed with 200 mL of deionized water several times to obtain a dextran gel with epoxy groups.

2) Take 10 g of epoxy-activated dextran gel and evenly disperse in 50 mL of a mixed solution composed of carbonate buffer (0.05M, pH9) and ethanol (the volume ratio of ethanol to carbonate buffer is 65/ 35) in. Dissolve 1g of 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol in 1mL of deionized water, adjust the pH to 9, add it to the mixed solution, and suspend it at 45°C Stir for 16h. After the reaction, the gel was fully washed with ethanol solution.

3) Disperse the gel in 50mL of 1M ethanolamine solution (pH10.5), and stir at 45°C for 2.5h. After the reaction, the gel was washed several times with 200 mL of deionized water. Elemental analysis showed that the ligand coupling density was 1.92mmol/g dry glue.

Comparative Example 5: Preparation of 4-Mercaptoethylpyridine (4-Mercaptoethylpyridine, 4-MEP) agarose blood purification material (activated by allyl bromide) with a coupling density of 1.97mmol/g dry gel

1) Weigh 10g of Sepharose CL-6B agarose gel (with alkenyl active groups), wash with 200mL of deionized water several times, and evenly disperse in 5mL of 3M NaOH solution to form a gel suspension, take 2mL of bromine Propylene was mixed with the prepared gel suspension, and the resulting mixture was suspended and stirred at 30° C. for 18 hours. After the reaction, the gel was washed several times with 100 mL of anhydrous acetone and 100 mL of deionized water respectively to obtain an agarose gel with alkenyl active groups activated by allyl bromide.

2) Take 1g of 4-MEP·HCl (hydrochloride salt form), dissolve it in 1mL of deionized water, adjust the pH value of the prepared solution to 7 with 10M NaOH solution, and then add 6mL of phosphate buffer (0.1 M, pH7) and 3 mL of acetonitrile. The activated agarose gel was added to the prepared solution to form a gel suspension, which was suspended and stirred at 30° C. for 12 hours. After the reaction, wash the gel with 100mL of acetonitrile, 100mL of deionized water, and 100mL of 0.1M hydrochloric acid for several times to obtain an agarose gel coupled with 4-MEP. The agarose gel is dried and dispersed In 60mL of 1M mercaptoethanol aqueous solution (pH10), stir with a paddle at 30°C for 3 hours. After the reaction, the gel was washed several times with 200 mL of deionized water. Elemental analysis showed that the ligand coupling density was 1.97mmol/g dry glue.

3. Evaluation of the adsorption effect of blood purification materials on rheumatoid factor and related antibody components in human serum

Human serum samples positive for rheumatoid factor were collected and mixed for performance evaluation of blood purification materials. After testing, the IgG concentration in the mixed serum sample was 12.4mg/mL, the IgM concentration was 2.1mg/mL, the human serum albumin (HSA) concentration was 42.6mg/mL, and the rheumatoid factor (RF) content was 948IU/mL.

Take the blood purification materials synthesized in Examples 1-11, wash them three times with normal saline, weigh 0.2 g of the blood purification materials and add them to a sample bottle containing 1 mL of serum (the volume ratio of material to serum is 1:5), 37 Incubate at ℃ for 2h. The concentration of IgG, IgM, HSA and RF in serum samples after adsorption was detected, and the removal rate was calculated. The specific results are shown in Table 1.

Table 1

The evaluation results show that the blood purification material with 1-amino-3-(2-(4-pyridyl)-ethylmercapto)-2-propanol as ligand has a certain effect on IgM and IgG antibodies in human serum. Significant discrimination effect; showed strong adsorption and removal ability for RF; in contrast, less non-specific adsorption for HSA. It can also be seen from the results in Table 1 that the adsorption effect of blood purification materials is greatly affected by the ligand coupling density. With the increase of the ligand density, the removal rates of IgG and IgM both increased, but With a further increase in base density (for example, more than 3.06mmol/g dry gel), the adsorption of IgG increases significantly, while the removal rate of IgM and RF tends to decline, indicating that the molecular weight of blood purification materials can be reduced after the ligand density reaches a certain level. Smaller IgG with higher serum concentration has a stronger effect, and more binding sites will be occupied by it, thus affecting the adsorption and binding of IgM antibodies and RF. Considering the removal effect of blood purification materials on RF, 1.4-2.8mmol/g dry glue is selected as the ideal ligand coupling density. At the same time, from the evaluation results, in addition to cross-linked agarose gel, other polysaccharide carrier materials (Example 9-11) coupled with 1-amino-3-(2-(4-pyridyl)-ethyl After mercapto)-2-propanol, if the ligand density is within the range selected above, the expected and ideal RF removal effect can also be obtained.

In addition, compared with the blood purification material of the present invention, the adsorbent with 4-mercaptoethylpyridine as a ligand has a removal rate of 56.7% for RF under the same experimental conditions, but at the same time, the adsorbent has a negative effect on IgG. Adsorption removal is also very impressive, reaching 84.4%. It shows that although 4-mercaptoethylpyridinium has excellent IgG binding ability, it will cause significant loss of IgG antibodies when applied to RF clearance, thus limiting its application in the removal of rheumatoid factor.

Claims (4)

1. for removing a blood purification material for rheumatoid factor, it is characterized in that, this blood purification material forms by solid phase carrier with by aglucon two parts that chemical coupling is fixed on solid phase carrier; Solid phase carrier is cross-linked polysaccharides, and aglucon is 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol, be fixed on solid phase carrier by chemical coupling, ligand density is the dry solid phase carrier of 1.4 ~ 2.8mmol/g; Moisture free solid phase carrier after described dry solid phase carrier refers to drying process.

2. blood purification material according to claim 1, is characterized in that, described solid phase carrier adopts the form of porous microsphere, and its glue hole is 150000 ~ 5000000Da through molecular weight.

3. a preparation method for the blood purification material described in claim 1 or 2, is characterized in that:

(1) activated solid support: with carbonyl dimidazoles activation, its addition is that 0.9 ~ 1.5g/10mL wets solid phase carrier, and reaction is carried out in acetone, 15 ~ 30 DEG C of reaction 1 ~ 2h, product acetone washs; Described wet solid phase carrier refers to the solid phase carrier removing non-binding state water through vacuum filtration;

(2) coupling aglucon: the 1-amido-3-(2-(4-pyridine radicals adding 5-10 times of suspension volume in the suspension of the solid phase carrier activated containing equal-volume carbonyl dimidazoles and acetone)-ehtylmercapto)-2-propyl alcohol, 20-35 DEG C of reaction 2-3h, then uses acetone, 0.1M HCl solution, 0.1M NaOH solution, washed with de-ionized water respectively;

(3) post processing: use the NaOH solution of pH10 ~ 13, to the coupling that step (2) obtains, the solid phase carrier of aglucon processes, the active group being derived from carbonyl dimidazoles that hydrolysis solid phase carrier does not react with aglucon.

4. the application of the blood purification material described in a claim 1 or 2, it is characterized in that, described blood purification material is used for the removing of rheumatoid factor and other IgM type autoantibody and CIC ELISA in rheumatoid disease human blood as the adsorption stuffing of apparatus for purifying blood.