CN104525150A - IgG1 adsorbent, and preparation method and application thereof - Google Patents

IgG1 adsorbent, and preparation method and application thereof Download PDF

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CN104525150A
CN104525150A CN201410698607.1A CN201410698607A CN104525150A CN 104525150 A CN104525150 A CN 104525150A CN 201410698607 A CN201410698607 A CN 201410698607A CN 104525150 A CN104525150 A CN 104525150A
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cellulose
agarose
microspheres
igg1
carrier
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CN104525150B (en
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董凡
郭延河
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Jafron Biomedical Co Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/3085—Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
    • B01J2220/48—Sorbents characterised by the starting material used for their preparation
    • B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character

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Abstract

An IgG1 adsorbent is prepared through the following steps: cross-linking and activating agarose or cellulose by ethylenediamine tetraacetic anhydride, 1,4-diphenylenediamine tetraacetic anhydride or 1,4-dibenzyldiamine tetraacetic anhydride, and reacting with a tyrosine or tryptophan solution. A carrier has good blood compatibility and mechanical performances, the above cross-linking and activating step is simple, the preparation process is safe, and the above cross-linking and activating agent simultaneously has the dual uses of spacer arm and functional group adsorption; and the adsorbent realizes the relative specific adsorption to immunoglobulin IgG1 through spatial selection, charge effect and hydrophobic effect, has low adsorption rate on IgM and IgA, and an adsorption rate ratio of IgG1/IgM(IgA) is 2-4, so the adsorbent can be used to adsorb IgG1 in blood and clinically remove IgG1 related panel reactive antibodies (PRAs) in the organ transplanting field.

Description

IgG1 adsorbent and preparation method and application thereof
Technical Field
The invention relates to the field of medicine, and particularly relates to an IgG1 adsorbent for blood perfusion and a preparation method and application thereof.
Background
The existence of the colony reactive antibody (PRA) in the kidney transplant recipient and the sensitization degree of the PRA are very close to the acute rejection after the transplantation, and have obvious influence on the survival rate of the transplanted organ. The kidney transplantation effect of patients with high immunity allergy (PRA > 80%), namely patients with high PRA level is poor, and the patients are easy to generate hyperacute, accelerated and acute rejection reactions, which directly cause graft failure or shorten the kidney survival time after transplantation. There are studies that only IgG class anti-HLA antibodies are the ones that actually affect organ transplant survival.
Currently, pre-treatment of patients with high levels of PRA to render them acceptable for renal transplant surgery is a global challenge. The means commonly used in clinic and their limitations are as follows: (1) good HLA matching may stop or reduce the incidence of acute rejection, but waiting for proper matching may result in delayed surgical time. (2) Desensitization therapy by intravenous injection of small doses of immunoglobulin (1vIG) or combined plasmapheresis (PP) is also commonly used, but the incidence of postoperative acute rejection (AHR) is high. (3) Plasmapheresis is able to eliminate part of the PRA antibodies, but loss of beneficial agent is itself a means of inducing sensitization in humans, and is limited in its use. (4) The anti-rejection function of the novel immunosuppressant such as Neoral, MMF, FK506 and the like is well known, but the toxic and side effects on human bodies are not ignored except that the medicament is expensive and needs to be taken for a long time.
Currently, the mode with more clinical application and better effect is Immunoadsorption (IA), and the most representative mode is the first application of the immunoadsorption agent for resisting the transplant rejection protein A in 1986. The effectiveness of protein a adsorbent materials for the treatment of various immune diseases has been demonstrated and approved by the FDA in the united states. However, protein a adsorption materials also have some drawbacks, which limit their spread. Firstly, the price of the protein A is high, and heavy economic burden is brought to common patients; secondly, the protein A is easy to denature as a protein aglucon, and the production, transportation, storage and use of the adsorbing material bring inconvenience; thirdly, protein A is a biological macromolecule, and if the protein A falls off, the protein A is easy to bring risks to patients.
In addition, the carrier activation method adopted by immunoadsorbents such as protein A adsorption columns and the like generally adopts an epichlorohydrin method, arm molecules only have four atoms, chain links are short, and the requirements on spatial positions of adsorption of IgG macromolecules are difficult to match. If the arm molecule is to be extended, the most common method is to connect the hydroxyl group of the polysaccharide carrier with the diamine spacer arm by using epoxy chloropropane, then to generate Schiff base by coupling with the ligand after hydroformylation (such as glutaraldehyde), and finally to reduce the double bond of the Schiff base to obtain the adsorbing material. In the preparation method, the activation steps are complicated, and after multi-step reaction, the number of active functional groups used for immobilizing the ligand on the carrier is continuously reduced. Therefore, the preparation cost of the immunoadsorbent is increased and the adsorption performance thereof is affected.
Therefore, how to overcome the defects of the protein A adsorption material, design and construct a novel immunoadsorbent which has the equivalent IgG adsorption capacity, stable physical and chemical properties and relatively low cost, and has important research value and practical significance.
Disclosure of Invention
The first purpose of the invention is to overcome the defects of the adsorption material and provide an IgG1 adsorbent which is safe, effective, low in cost, stable and reliable in performance. The second purpose of the invention is to provide a preparation method of the IgG1 adsorbent. The third purpose of the invention is to provide the application of the IgG1 adsorbent, namely the application of adsorbing IgG1 in blood and clinically removing a mass-reactive antibody (PRA) in the field of organ transplantation related to IgG 1.
The first aspect of the invention provides an IgG1 adsorbent, wherein, agarose microspheres or cellulose microspheres crosslinked by anhydride are used as a carrier, the anhydride is ethylenediamine tetraacetic anhydride, 1, 4-diphenylenediamine tetraacetic anhydride or 1, 4-dibenzylenediamine tetraacetic anhydride, and the ligand is tyrosine or tryptophan;
the chemical structural formula of the agarose microspheres or cellulose microspheres after anhydride crosslinking is as follows:
the chemical structural formula of the IgG1 adsorbent is shown as follows
Wherein,
w is agarose microsphere or cellulose microsphere,
x is agarose microsphere or cellulose microsphere after acid anhydride crosslinking,
y is ethylene, p-phenylene or p-xylylene,
r is p-hydroxyphenyl or beta-indolyl.
Preferably, the effective amount of anhydride to activate the support is 110-180. mu. mol/ml.
Preferably, the amount of carrier-supported ligand is 70-130. mu. mol/ml.
In a second aspect of the invention, there is provided a method for preparing an IgG1 adsorbent according to the first aspect of the invention, comprising the steps of:
(a) crosslinking of agarose microspheres or cellulose microspheres: agarose microspheres or cellulose microspheres, acid anhydride and liquid organic alkali react for 4 to 10 hours at the temperature of between 30 and 40 ℃, and are filtered, and products are washed by water, wherein the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic alkali is 1 to (0.01 to 0.2) to (20 to 50); the reaction formula is as follows:
wherein, W is agarose microsphere or cellulose microsphere, Y is ethylidene, p-phenylene or p-xylylene;
(b) activation of the carrier: reacting the carrier obtained after crosslinking with acid anhydride and liquid organic base at the temperature of 20-30 ℃ for 4-12h, filtering, and washing a product with water, wherein the volume ratio of the carrier obtained after crosslinking to the acid anhydride to the liquid organic base is 1: 0.01-0.2: 20-50; the reaction formula is as follows:
wherein, X is agarose microspheres or cellulose microspheres after acid anhydride crosslinking;
(c) immobilization of the ligand: reacting the activated carrier with the ligand and the liquid organic alkali at 0-40 ℃ for 4-10h, and washing the product with water, wherein the dosage ratio of the activated carrier, the ligand and the liquid organic alkali is 1mL to (0.05-0.2g) to (40-60 mL); the reaction formula is as follows:
wherein R is p-hydroxyphenyl or beta-indolyl.
Preferably, in step a, the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic base is 1 to (0.05-0.1) to 30.
Preferably, in step b, the volume ratio of the carrier, the acid anhydride and the liquid organic base obtained after crosslinking is 1 to (0.05-0.1) to 30.
Preferably, in step c, the ratio of the carrier, ligand and liquid organic base after activation is 1 mL: 0.1 g: 50 mL.
Preferably, the liquid organic base is one or more selected from pyridine, triethylamine, trimethylamine, triethanolamine, diethanolamine, N-diisopropylethylamine, diisopropylamine, and quinoline.
The agarose microspheres can be prepared by methods known in the art, for example, by reverse phase suspension embedding or membrane emulsification.
The cellulose microspheres may be prepared by a method known in the art, for example, an emulsion-solidification method, a spray-drying method, or an agglomeration method.
Preferably, the agarose microspheres are prepared by the following steps: dissolving the agarose powder in water to prepare 4-15% agarose solution; adding the dissolved agarose solution into the oil phase containing the dispersant, stirring and dispersing at 45-65 ℃ for about 0.5-1.5h to make the agar ball, cooling, stopping stirring, discharging and washing to obtain the agarose microspheres.
Preferably, the mass percentage concentration of the dispersant in the oil phase is 0.5-5.0%; the volume ratio of the agarose solution to the oil phase is 1: 1-5.
The oil phase can adopt the oil phase commonly used in the reversed-phase suspension embedding method, and can be one or more of salad oil, epoxidized soybean oil, aromatic oil, castor oil, 200# solvent oil, liquid paraffin and n-hexane.
Preferably, the oil phase is one or more of salad oil, 200# solvent oil and n-hexane.
The dispersant can be dispersant commonly used in reversed phase suspension embedding method, such as span, tween and the like.
Preferably, the dispersant is one or more of span 85, span 80, tween 80 and tween 20.
Preferably, the cellulose microspheres are prepared by the following steps: dissolving cellulose resin in an organic solvent to prepare a cellulose solution with the mass percentage concentration of 6-20%, then adding a pore-forming agent, stirring uniformly, adding the mixture into a water phase containing a dispersing agent, stirring for 4-10 hours at 25-35 ℃, stopping stirring, discharging and washing to obtain the cellulose microspheres.
Preferably, the volume ratio of the cellulose solution to the pore-foaming agent is (100-; the volume ratio of the total volume of the cellulose solution and the pore-forming agent to the water phase is (1-5) to (1-5).
The cellulose resin of the present invention is one or more of natural cellulose and various esterified and etherified derivatives thereof, and may be one or more of cellulose, cellulose nitrate, cellulose acetate butyrate, and cellulose xanthate, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, cyanoethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose, for example.
Preferably, the cellulose resin is cellulose diacetate.
The organic solvent may be an organic solvent commonly used in the preparation of cellulose microspheres, such as halogenated hydrocarbon (dichloromethane, etc.), edible vegetable oil, silicone oil, etc.
The pore-forming agent can be one or more of pore-forming agents commonly used in the existing preparation of cellulose microspheres, such as methanol, ethanol, 1, 4-butanediol, dimethyl phthalate, dipropyl phthalate, ethyl acetate, polyvinyl alcohol and the like.
Preferably, the pore-foaming agent consists of dodecanol, ethanol and dimethyl phthalate in a volume ratio of (50-85) to (15-35) to (70-120).
The dispersing agent can be a dispersing agent commonly used for preparing cellulose microspheres, such as gelatin, polyvinyl alcohol, polyglycerol monostearate, polyvinylpyrrolidone and the like.
Preferably, the dispersing agent is a mixture of gelatin and polyvinyl alcohol, and the mass ratio of the gelatin to the polyvinyl alcohol is (7-11) to 1.
The third aspect of the invention provides the application of the IgG1 adsorbent of the first aspect in preparing an IgG1 adsorbent product in blood and/or a antibody product for eliminating mass reaction in the field of organ transplantation related to IgG 1.
The invention takes agarose or cellulose gel which contains abundant hydroxyl, has good blood compatibility and mechanical property as a carrier, takes safe, cheap, stable and easily preserved and disinfected amino acid as a ligand, and adopts an efficient and simple way to prepare the IgG1 adsorbent which has relative specific adsorption performance to immunoglobulin IgG1, can effectively eliminate colony reactive antibody (PRA) and has low cost.
Compared with protein A biological macromolecules, the amino acid micromolecule compound has remarkable advantages in cost and physicochemical stability. The tyrosine or tryptophan adopted by the invention is the ligand which is essential amino acid for human body, has no toxicity and good safety, has wide source of amino acid and low price, and can realize high selective adsorption of IgG1 by the hydrophobic effect of the aromatic side chain and the negative charge of carboxyl.
The carrier crosslinking and activating reagent used in the invention adopts ethylenediamine tetraacetic anhydride, 1, 4-diphenyldiamine tetraacetic anhydride and 1, 4-dibenzyldiamine tetraacetic anhydride, and the molecules have moderate length and 13-17 atomic number, and can meet the space requirement of adsorbing IgG protein molecules. Meanwhile, the amino acetic acid fragment is arranged on the arm molecule, the carboxyl of the amino acetic acid is negatively charged under the neutral pH environment of a human body, the adsorption of the IgG antibody with positive charge can be realized through the additional charge effect, in addition, the acid radical ion on the amino acetic acid fragment can play an anticoagulation role, and when the amino acetic acid fragment is applied to blood perfusion, the use of anticoagulants such as EDTA and sodium citrate can be avoided.
Therefore, the adsorbent can realize the adsorption of positively charged IgG protein macromolecules through the space selection effect of long-chain arms and ligands, the charge adsorption of negative charges of carboxyl groups and the hydrophobic effect of aromatic groups of the ligands.
The primary causes of increased levels of PRA are Human Leukocyte Antigen (HLA) class i and class ii IgG antibodies, which are most strongly associated with IgG1, which accounts for 60-70% of the total IgG. The IgG1 adsorbent can effectively reduce the PRA level of a hypersensitive organ transplant patient while adsorbing the immunoglobulin antibody. Therefore, the IgG1 adsorbent has wide adaptation diseases, can be applied to autoimmune diseases such as myasthenia gravis, Guillain-Barre syndrome, systemic lupus erythematosus and rheumatoid arthritis, and can also be applied to the field of organ transplantation such as hypersensitive organ transplant patients and rejection patients after organ transplantation.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the amino acid micromolecules which are cheap, safe, stable and modifiable are used as the ligand to replace the commonly used protein A to prepare the immunoadsorbent, so that the defects of high price, easy shedding, low safety and the like of the protein A immunoadsorbent can be avoided.
(2) The research idea of preparing the immunoadsorbent by activating the carrier by using the ethylenediamine tetraacetic anhydride, the 1, 4-diphenylenediamine tetraacetic anhydride or the 1, 4-dibenzylenediamine tetraacetic anhydride and then directly immobilizing the amino acid ligand is provided, so that the preparation process of the adsorbent can be greatly simplified, the higher ligand immobilization amount is realized, and the adsorption of IgG1 is realized through the synergistic process of space selection, charge action and hydrophobic action.
(3) In-vitro plasma adsorption test data show that the IgG1 adsorbent has higher specific adsorption to IgG1 in human plasma, and can be used for separation of the antibody or treatment of autoimmune diseases.
(4) The bionic immunoadsorbent has good effect of removing the in-vivo colony reactive antibodies (PRA) of the patients with hyperallergenic organ transplantation, and is expected to be applied to the field of organ transplantation such as the patients with hyperallergenic organ transplantation and the patients with rejection after organ transplantation.
Drawings
Fig. 1 is a summary diagram of the detection results of the adsorption performance of the IgG1 adsorbent provided by the present invention.
Detailed Description
The IgG1 adsorbent provided by the invention adopts agarose microspheres or cellulose microspheres crosslinked by anhydride as a carrier, wherein the anhydride is ethylenediamine tetraacetic anhydride, 1, 4-diphenyldiamine tetraacetic anhydride or 1, 4-dibenzyldiamine tetraacetic anhydride, and the ligand is tyrosine or tryptophan;
the chemical structural formula of the agarose microspheres or cellulose microspheres after anhydride crosslinking is as follows:
the chemical structural formula of the IgG1 adsorbent is shown as follows
Wherein,
w is agarose microsphere or cellulose microsphere,
x is agarose microsphere or cellulose microsphere after acid anhydride crosslinking,
y is ethylene, p-phenylene or p-xylylene,
r is p-hydroxyphenyl or beta-indolyl.
Preferably, the effective amount of anhydride to activate the support is 110-180. mu. mol/ml.
Preferably, the amount of carrier-supported ligand is 70-130. mu. mol/ml.
The IgG1 adsorbent provided by the invention can be prepared by the following steps:
(a) crosslinking of agarose microspheres or cellulose microspheres: agarose microspheres or cellulose microspheres, acid anhydride and liquid organic alkali react for 4 to 10 hours at the temperature of between 30 and 40 ℃, and are filtered, and products are washed by water, wherein the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic alkali is 1 to (0.01 to 0.2) to (20 to 50), and preferably 1 to (0.05 to 0.1) to 30;
(b) activation of the carrier: reacting the carrier obtained after crosslinking with acid anhydride and liquid organic alkali at the temperature of 20-30 ℃ for 4-12h, filtering, and washing the product with water, wherein the volume ratio of the carrier obtained after crosslinking to the acid anhydride to the liquid organic alkali is 1: 0.01-0.2: 20-50, preferably 1: 0.05-0.1: 30;
(c) immobilization of the ligand: reacting the activated carrier, the ligand and the liquid organic alkali at 0-40 ℃ for 4-10h, and washing the product with water, wherein the dosage ratio of the activated carrier, the ligand and the liquid organic alkali is 1mL to (0.05-0.2g) to (40-60mL), preferably 1mL to 0.1g to 50 mL.
Wherein the liquid organic base participates in the reaction on the one hand and acts as a solvent on the other hand, preferably selected from one or more of pyridine, triethylamine, trimethylamine, triethanolamine, diethanolamine, N-diisopropylethylamine, diisopropylamine and quinoline.
The agarose microspheres can be prepared by methods known in the art, for example, by reverse phase suspension embedding or membrane emulsification. The cellulose microspheres may be prepared by methods known in the art, for example, by emulsion-solidification, spray-drying, or coacervation.
For example, the agarose microspheres are specifically prepared according to the following steps: dissolving the agarose powder in water to prepare 4-15% agarose solution; adding the dissolved agarose solution into the oil phase containing the dispersant, stirring and dispersing at 45-65 ℃ for about 0.5-1.5h to make the agar ball, cooling, stopping stirring, discharging and washing to obtain the agarose microspheres. Preferably, the mass percentage concentration of the dispersant in the oil phase is 0.5-5.0%; the volume ratio of the agarose solution to the oil phase is 1: 1-5. The oil phase can adopt the oil phase commonly used in the reversed-phase suspension embedding method, and can be one or more of salad oil, epoxidized soybean oil, aromatic oil, castor oil, 200# solvent oil, liquid paraffin and n-hexane. Preferably, the oil phase is one or more of salad oil, 200# solvent oil and n-hexane. The dispersant can be dispersant commonly used in reversed phase suspension embedding method, such as span, tween and the like. Preferably, the dispersant is one or more of span 85, span 80, tween 80 and tween 20. It should be understood that this is only a preferred example of the method for preparing agarose microspheres, and those skilled in the art can also prepare agarose microspheres by other methods known in the art.
For example, the cellulose microspheres are specifically prepared according to the following steps: dissolving cellulose resin in an organic solvent to prepare a cellulose solution with the mass percentage concentration of 6-20%, then adding a pore-forming agent, stirring uniformly, adding the mixture into a water phase containing a dispersing agent, stirring for 4-10 hours at 25-35 ℃, stopping stirring, discharging and washing to obtain the cellulose microspheres. Preferably, the volume ratio of the cellulose solution to the pore-foaming agent is (100-; the volume ratio of the total volume of the cellulose solution and the pore-forming agent to the water phase is (1-5) to (1-5). The organic solvent may be an organic solvent commonly used in the preparation of cellulose microspheres, such as halogenated hydrocarbon (dichloromethane, etc.), edible vegetable oil, silicone oil, etc. The pore-forming agent can be one or more of pore-forming agents commonly used in the existing preparation of cellulose microspheres, such as methanol, ethanol, 1, 4-butanediol, dimethyl phthalate, dipropyl phthalate, ethyl acetate, polyvinyl alcohol and the like. Preferably, the pore-foaming agent consists of dodecanol, ethanol and dimethyl phthalate in a volume ratio of (50-85) to (15-35) to (70-120). The dispersing agent can be a dispersing agent commonly used for preparing cellulose microspheres, such as gelatin, polyvinyl alcohol, polyglycerol monostearate, polyvinylpyrrolidone and the like. Preferably, the dispersing agent is a mixture of gelatin and polyvinyl alcohol, and the mass ratio of the gelatin to the polyvinyl alcohol is (7-11) to 1. It should be understood that this is merely a preferred example of a method for preparing cellulose microspheres, and that other methods known in the art may be used by those skilled in the art to prepare cellulose microspheres.
The cellulose resin of the present invention is one or more of natural cellulose and various esterified and etherified derivatives thereof, and may be one or more of cellulose, cellulose nitrate, cellulose acetate butyrate, and cellulose xanthate, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, cyanoethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose, for example, and preferably cellulose diacetate.
The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings.
Example 1
(a) Preparation of agarose gel
Agar powder was dissolved in water to make a 5.5% agarose solution. The dissolved agarose solution was added to 2 volumes of 200# solvent oil containing 2% Tween 20 as a dispersant, and dispersed at 55 ℃ with stirring for about 1 hour to make the agarose spherical. And stopping stirring when the temperature is reduced to room temperature or below, discharging and washing to obtain the agarose microspheres.
(b) Cross-linking of agarose microspheres
Soaking the wet agarose microspheres in ethanol, drying, taking 2mL agarose microspheres, soaking and washing the agarose microspheres with pyridine for several times, and then adding 0.1mL ethylenediamine tetraacetic anhydride and 50mL pyridine into a three-necked bottle to react for 6h at 35 ℃. Filtered and the product washed with water until no pyridine taste.
(c) Activation of agarose Carrier
Soaking the crosslinked carrier in ethanol, drying, taking 2mL of carrier, soaking and washing the carrier for several times by using pyridine, adding 0.2mL of ethylenediamine tetraacetic dianhydride and 60mL of pyridine, adding the mixture into a three-necked bottle, and reacting for 10h at the temperature of 20 ℃. The product was filtered, washed with water until no pyridine smell was observed, and then washed with ethanol until use.
(d) Preparation of the adsorbent
2mL of the activated carrier was soaked in anhydrous tetrahydrofuran for 3 to 5 times, and then 0.2g of tyrosine and 100mL of anhydrous pyridine were added to react at 30 ℃ for 10 hours. The adsorbent was washed with water and stored at 4 ℃. And testing the content of tyrosine (absorbance at 275 nm) in the tyrosine solution by using an ultraviolet spectrophotometer, and calculating the solid loading amount of the ligand on the adsorbent according to the change of the content of the tyrosine before and after the reaction. The test result shows that the tyrosine immobilization amount on the adsorbent is 97.0 mu mol/mL.
(e) Adsorption Performance test
The adsorption performance of the adsorbent on human immunoglobulin IgG, IgG1, IgA and IgM is tested by an in-vitro static adsorption method. Specific results are shown in table 1 and fig. 1.
The adsorption performance of the adsorbent on Population Reactive Antibodies (PRA) in plasma of a hypersensitive kidney transplant patient is tested by an in vitro static adsorption method:
that is, 1mL of the adsorbent was added to thawed plasma at a ratio of 1:4(V/V) adsorbent to plasma. The sample was placed in a 37 ℃ water bath constant temperature shaker and shaken at 60rpm for 2 h. And after adsorption, taking out the upper layer plasma, and carrying out sample detection. The detection method of the immunoglobulin is an immunoturbidimetry method, a Roche full-automatic biochemical analyzer and an immunoglobulin detection kit are used, and the operation method is carried out according to the kit specification. Specific results are shown in table 1; PRA was detected using a Mixed antigen plate (LATM) from Lyme Deg, USA, and the specific procedures were performed according to the kit's instructions, and the plasma used was highly sensitized renal transplant patient plasma. The specific results are shown in Table 2.
Example 2
(a) Preparation of agarose gel
Agar powder is dissolved in water to prepare an agarose solution with the concentration of 5.0%. Adding the dissolved agarose solution into 2.5 times of n-hexane containing 3% span-80 dispersant, and stirring and dispersing at 65 ℃ for about 0.5h to make the agarose spherical. And stopping stirring when the temperature is reduced to room temperature or below, discharging and washing to obtain the agarose microspheres.
(b) Cross-linking of agarose microspheres
Soaking the wet agarose carrier in ethanol, drying, taking 2mL of the carrier, soaking and washing the carrier for several times by using pyridine, and then adding 0.2mL of 1, 4-dibenzyldiamine tetraacetic anhydride and 50mL of pyridine into a three-necked bottle to react for 4 hours at 40 ℃. Filtered and the product washed with water until no pyridine taste.
(c) Activation of agarose Carrier
Soaking the crosslinked carrier in ethanol, drying, taking 2mL of the carrier, soaking and washing the carrier for several times by using pyridine, adding 0.15mL of dibenzyldiamine tetraacetic anhydride and 60mL of pyridine into a three-necked bottle, and reacting for 10h at 20 ℃. The product was filtered, washed with water until no pyridine smell was observed, and then washed with ethanol until use.
(d) Preparation of the adsorbent
2mL of the activated carrier was soaked with anhydrous tetrahydrofuran 3-5 times, and then 0.2g of tryptophan and 100mL of anhydrous pyridine were added to react at 25 ℃ for 10 hours. The adsorbent was washed with water and stored at 4 ℃. And (3) testing the content of tryptophan (absorbance at 275 nm) in the tryptophan solution by using an ultraviolet spectrophotometer, and calculating the solid loading amount of the ligand on the adsorbent according to the change of the content of the tryptophan before and after the reaction. The test result shows that the solid content of the tryptophan on the adsorbent is 105.3 mu mol/mL.
(e) Adsorption Performance test
The adsorbent was tested for adsorption to human immunoglobulins IgG, IgG1, IgA, IgM and for adsorption to Population Reactive Antibodies (PRA) as in example 1. Specific results are shown in table 1, fig. 1 and table 2, respectively.
Example 3
(a) Preparation of cellulose gel
Dissolving 15g of cellulose diacetate in 120mL of dichloromethane, adding a pore-foaming agent solution containing 58.5mL of dodecanol, 19mL of ethanol and 82.5mL of dimethyl phthalate, uniformly stirring, adding the mixture into 1L of water phase containing 2% of dispersing agent (gelatin: PVA: 9:1W/W), stirring for 6 hours at 35 ℃, stopping stirring and discharging, and washing with 75% alcohol and water in sequence to obtain the cellulose microspheres.
Taking 2mL of the vector according to VWet cellulose:V75% alcohol:V10％NaOHTo an erlenmeyer flask, a volume of 75% alcohol and 10 wt% NaOH solution was added at a ratio of 1:2:3 and saponified for 10 hours at room temperature. After saponification, the carrier is screened, washed with water and stored in a wet state at normal temperature.
(b) Crosslinking of cellulose microspheres
Soaking a wet fiber carrier in ethanol, drying, taking 2mL of the carrier, soaking and washing the carrier with pyridine for several times, and then adding 0.1mL of ethylenediamine tetraacetic dianhydride and 50mL of pyridine into a three-necked bottle to react for 6h at 35 ℃. Filtered and the product washed with water until no pyridine taste.
(c) Activation of cellulose support
Soaking the crosslinked carrier in ethanol, drying, taking 2mL of the carrier, soaking and washing the carrier with pyridine for several times, adding 0.2mL of ethylenediamine tetraacetic dianhydride and 60mL of pyridine, adding the mixture into a three-necked bottle, and reacting for 6h at 25 ℃. The product was filtered, washed with water until no pyridine smell was observed, and then washed with ethanol until use.
(d) Preparation of the adsorbent
2mL of the activated carrier was soaked in anhydrous tetrahydrofuran for 3 to 5 times, and then 0.2g of tyrosine and 100mL of anhydrous pyridine were added to react at 30 ℃ for 10 hours. The adsorbent was washed with water and stored at 4 ℃. And testing the content of tyrosine (absorbance at 275 nm) in the tyrosine solution by using an ultraviolet spectrophotometer, and calculating the solid loading amount of the ligand on the adsorbent according to the change of the content of the tyrosine before and after the reaction. The test results showed that the tyrosine immobilization on the adsorbent was 114.8. mu. mol/mL.
(e) Adsorption Performance test
The adsorbent was tested for adsorption to human immunoglobulins IgG, IgG1, IgA, IgM and for adsorption to Population Reactive Antibodies (PRA) as in example 1. Specific results are shown in table 1, fig. 1 and table 2, respectively.
Example 4
(a) Preparation of cellulose gel
Dissolving 15g of cellulose diacetate in 200mL of dichloromethane, adding a pore-foaming agent solution containing 55.0mL of dodecanol, 20.0mL of ethanol and 90.0mL of dimethyl phthalate, uniformly stirring, adding the mixture into 1L of water phase containing 2% of dispersing agent (gelatin: PVA: 9:1W/W), stirring for 10 hours at 32 ℃, stopping stirring, discharging, and sequentially washing with 75% alcohol and water to obtain the cellulose spheres. Taking 2mL of the vector according to VWet cellulose:V75% alcohol:V10％NaOHTo an erlenmeyer flask, a volume of 75% alcohol and 10 wt% NaOH solution was added at a ratio of 1:2:3 and saponified for 10 hours at room temperature. After saponification, the carrier is screened, washed with water and stored in a wet state at normal temperature.
(b) Crosslinking of cellulose microspheres
Soaking the wet agarose carrier in ethanol, drying, taking 2mL of the carrier, soaking and washing the carrier for several times by using pyridine, and then adding 0.1mL of 1, 4-diphenylenediamine tetraacetic anhydride and 50mL of pyridine into a three-necked bottle to react for 10 hours at 30 ℃. Filtered and the product washed with water until no pyridine taste.
(c) Activation of cellulose support
Soaking the crosslinked carrier in ethanol, drying, taking 2mL of the carrier, soaking and washing the carrier for several times by using pyridine, adding 0.2mL of 1, 4-diphenyldiamine tetraacetic anhydride and 60mL of pyridine into a three-necked bottle, and reacting for 6h at 25 ℃. The product was filtered, washed with water until no pyridine smell was observed, and then washed with ethanol until use.
(d) Preparation of the adsorbent
2mL of the activated carrier was soaked with anhydrous tetrahydrofuran 3-5 times, and then 0.2g of tryptophan and 100mL of anhydrous pyridine were added to react at 30 ℃ for 10 hours. The adsorbent was washed with water and stored at 4 ℃. And (3) testing the content of tryptophan (absorbance at 275 nm) in the tryptophan solution by using an ultraviolet spectrophotometer, and calculating the solid loading amount of the ligand on the adsorbent according to the change of the content of the tryptophan before and after the reaction. The test result shows that the solid content of the tryptophan on the adsorbent is 88.2 mu mol/mL.
(e) Adsorption Performance test
The adsorbent was tested for adsorption to human immunoglobulins IgG, IgG1, IgA, IgM and for adsorption to Population Reactive Antibodies (PRA) as in example 1. Specific results are shown in table 1, fig. 1 and table 2, respectively.
TABLE 1 adsorption Performance of adsorbents for immunoglobulins
As can be seen from table 1 and fig. 1, the adsorbent for blood purification according to the present invention has excellent adsorption performance for immunoglobulin IgG and IgG1, and also has high adsorption capacity for IgA and IgM. The comparison adsorption quantity of IgG1/IgG is in the range of 0.76-0.80, while the normal IgG1 accounts for 60-70% of the IgG, so the adsorbent has certain specificity to the adsorption of IgG1 in the IgG class protein. Comparing the adsorption rates IgG1/IgA (IgM), in the range of 2.0-3.7, it can be concluded that adsorption to IgG1 is relatively specific for IgA and IgM.
TABLE 2 adsorption Performance of adsorbents for Population Reactive Antibodies (PRA)
As can be seen from the test results of table 2, the IgG1 adsorbent for blood purification of the present invention also has a high adsorption capacity for Population Reactive Antibodies (PRAs), and examples 1 and 2 are capable of directly turning off even PRA-positive plasma. The PRA antibody causing the hyperacute rejection reaction is mainly an IgG1 antibody, and the data in the table 1 show that the adsorbent has relative specific adsorption capacity to IgG1, and the PRA value is obviously reduced after IgG1 is eliminated; while IgM has no direct evidence to indicate that the IgM has an influence on causing PRA rejection, IgA also has a benefit on organ transplantation, so that the IgG1 is relatively specifically removed, and the purposes of purifying the in-vivo environment of a patient waiting for organ transplantation and improving the survival rate of the transplanted organ can be achieved.
From the above examples, the present invention provides IgG1 adsorbents with different polycarboxyl amino acids as spacer arms and different grafted amino acids as ligands, and tests on their adsorption performance show that they can effectively reduce PRA levels in highly sensitized organ transplant patients while adsorbing immunoglobulin antibodies. Therefore, the IgG1 immunoadsorbent has wide adaptation diseases, can be applied to autoimmune diseases, and can also be applied to the field of organ transplantation.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (10)

1. The IgG1 adsorbent is characterized in that an anhydride crosslinked agarose microsphere or cellulose microsphere is used as a carrier, the anhydride is ethylenediamine tetraacetic anhydride, 1, 4-diphenyldiamine tetraacetic anhydride or 1, 4-dibenzyldiamine tetraacetic anhydride, and the ligand is tyrosine or tryptophan;
the chemical structural formula of the agarose microspheres or cellulose microspheres after anhydride crosslinking is as follows:
the chemical structural formula of the IgG1 adsorbent is shown as follows
Wherein,
w is agarose microsphere or cellulose microsphere,
x is agarose microsphere or cellulose microsphere after acid anhydride crosslinking,
y is ethylene, p-phenylene or p-xylylene,
r is p-hydroxyphenyl or beta-indolyl.
2. A method for preparing IgG1 adsorbent according to claim 1, wherein the effective amount of acid anhydride for activating the carrier is 110-180 μmol/ml and the amount of the ligand carried on the carrier is 70-130 μmol/ml.
3. A method of making the IgG1 adsorbent of claim 1, comprising the steps of:
(a) crosslinking of agarose microspheres or cellulose microspheres: agarose microspheres or cellulose microspheres, acid anhydride and liquid organic alkali react for 4 to 10 hours at the temperature of between 30 and 40 ℃, and are filtered, and products are washed by water, wherein the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic alkali is 1 to (0.01 to 0.2) to (20 to 50);
(b) activation of the carrier: reacting the carrier obtained after crosslinking with acid anhydride and liquid organic base at the temperature of 20-30 ℃ for 4-12h, filtering, and washing a product with water, wherein the volume ratio of the carrier obtained after crosslinking to the acid anhydride to the liquid organic base is 1: 0.01-0.2: 20-50;
(c) immobilization of the ligand: reacting the activated carrier with the ligand and the liquid organic alkali at 0-40 ℃ for 4-10h, and washing the product with water, wherein the dosage ratio of the activated carrier, the ligand and the liquid organic alkali is 1mL to (0.05-0.2g) to (40-60 mL).
4. The preparation method of claim 3, wherein in the step a, the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic base is 1: 0.05-0.1: 30; in the step b, the volume ratio of the carrier, the acid anhydride and the liquid organic alkali obtained after cross-linking is 1 to (0.05-0.1) to 30; in the step c, the dosage ratio of the activated carrier, the ligand and the liquid organic base is 1 mL: 0.1 g: 50 mL.
5. The method of claim 3, wherein the liquid organic base is selected from one or more of pyridine, triethylamine, trimethylamine, triethanolamine, diethanolamine, N-diisopropylethylamine, diisopropylamine, and quinoline.
6. The preparation method according to claim 3, wherein the agarose microspheres are prepared by an inverse suspension embedding method or a membrane emulsification method; the cellulose microsphere is prepared by an emulsification-solidification method, a spray drying method or an agglomeration method.
7. The preparation method according to claim 6, wherein the agarose microspheres are prepared by the following steps:
dissolving the agarose powder in water to prepare 4-15% agarose solution; adding the dissolved agarose solution into an oil phase containing a dispersing agent, stirring and dispersing at 45-65 ℃ for about 0.5-1.5h to make the agar ball, cooling, stopping stirring, discharging and washing to obtain the agarose microspheres;
wherein the oil phase is one or more of salad oil, 200# solvent oil and n-hexane; the dispersant is one or more of span 85, span 80, tween 80 and tween 20, and the mass percentage concentration of the dispersant in the oil phase is 0.5-5.0%; the volume ratio of the agarose solution to the oil phase is 1: 1-5.
8. The preparation method according to claim 6, wherein the cellulose microspheres are prepared by the following steps:
dissolving cellulose resin in an organic solvent to prepare a cellulose solution with the mass percentage concentration of 6-20%, then adding a pore-foaming agent, uniformly stirring, adding the mixture into a water phase containing a dispersing agent, stirring for 4-10 hours at 25-35 ℃, stopping stirring, discharging, washing, and then performing saponification treatment to obtain cellulose microspheres;
wherein the volume ratio of the cellulose solution to the pore-foaming agent is (100-200) to (120-250); the volume ratio of the total volume of the cellulose solution and the pore-forming agent to the water phase is (1-5) to (1-5).
9. The method of claim 8, wherein the porogen consists of dodecanol, ethanol, and dimethyl phthalate in a volume ratio of (50-85) to (15-35) to (70-120); the dispersing agent is a mixture of gelatin and polyvinyl alcohol, and the mass ratio of the gelatin to the polyvinyl alcohol is (7-11) to 1.
10. The production method according to claim 3 or 8, wherein the cellulose resin from which the cellulose microspheres are produced is cellulose diacetate.
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