High-performance low-risk protein A immunoadsorbent and application thereof

Abstract

The invention provides a high-performance low-risk protein A immunoadsorbent and application thereof. The immunoadsorbent comprises an agarose gel porous microsphere carrier and an immunoglobulin binding protein coupled with the agarose gel porous microsphere carrier. The immunoadsorbent can greatly improve the adsorption effect and shorten the treatment time, particularly has good blood compatibility and biocompatibility, is simple in preparation method and low in cost, and has huge market prospects and economic values.

Description

Technical Field

[0001] The present invention relates to the technical field of medical device blood purification, and particularly to a high-performance and low-risk protein A immunosorbent and its application. Background Art

[0002] The process of removing pathogenic substances from a patient's blood by drawing the blood out of the body and passing it through a purification device to achieve the purpose of treating diseases is called blood purification. Current blood purification techniques include: hemodialysis, hemofiltration, hemodiafiltration, hemoperfusion, plasma exchange, and immunoadsorption, etc. Among them, hemoperfusion and immunoadsorption rely on materials with adsorption properties to achieve the purification process. The adsorption material uses the affinity, electrostatic, hydrophobic, van der Waals forces, etc. of the material molecules themselves or the ligand molecules immobilized on the surface of the material molecules to "adsorb" harmful substances in the blood, and the process is specific or relatively specific.

[0003] Immunosorption therapy uses highly specific antibodies, antigens, or ligands with specific physicochemical affinity properties to combine with a carrier (adsorption material) to form an adsorbent, specifically removing pathogenic substances in the blood, thereby treating some diseases that cannot be cured by traditional therapies. Immunoadsorption is developed on the basis of plasma exchange, and its advantages are that it can efficiently remove various pathogenic factors through specific adsorption without affecting other important components of the plasma, and it does not require plasma replacement, thus avoiding the spread of diseases. Due to the high specificity and reversibility of adsorption, the immunosorbent can elute the adsorbed pathogenic factors with an eluent and be reused, greatly reducing the production cost and the treatment cost of patients. So far, immunosorption therapy has been widely used in various diseases.

[0004] Currently, the commercially available plasma adsorption immunosorption column in the German market is priced at 10,000 euros, and most patients cannot afford it. For the commercially available protein A immunosorption column in the domestic market, the cost for patients to purchase from the hospital is 40,000 - 50,000 yuan, and an additional 3,000 yuan for treatment is required each time. On the other hand, the adsorption performance of this adsorbent is not ideal. During treatment, it needs to be eluted and recycled 10 times to achieve the therapeutic effect. Not only does it require a large amount of eluent, balance solution, and physiological saline, but it also greatly increases the treatment duration, increases the pain of patients, and adds the operation burden on medical staff (as shown in b in Figure 1 For protein A immunosorbents, excellent adsorption performance can ensure that the volume of the adsorption column can be made smaller, without the need for multiple cycles during use, reducing the treatment time, lowering the treatment risk, better improving the product safety and the product application scope, and at the same time reducing the production cost of the product.

[0005] Moreover, in the existing preparation process of immunoadsorption columns, unbound immunoglobulin-binding proteins cannot be effectively and fully eluted and removed after the coupling reaction, resulting in a safety hazard that the proteins may fall off into the plasma during subsequent treatment and cause severe allergic reactions.

[0006] In view of this, it is necessary to provide an improved protein A immunoadsorbent with high performance, low risk and high safety performance to solve the above problems. Summary of the Invention

[0007] The object of the present invention is to provide a protein A immunoadsorbent with high performance and low risk and its application.

[0008] To achieve the above object of the invention, the present invention provides a protein A immunoadsorbent with high performance and low risk, which comprises an agarose gel porous microsphere carrier and an immunoglobulin-binding protein coupled to the agarose gel porous microsphere carrier; the average particle size of the agarose gel porous microsphere carrier is 30-200 μm, and the average pore size is 40-100 nm.

[0009] As a further improvement of the present invention, the immunoglobulin-binding protein is one or a mixture of several of recombinant protein A, recombinant protein G, and recombinant protein L by genetic engineering.

[0010] As a further improvement of the present invention, the agarose gel porous microsphere carrier is modified by the epoxy method to couple the immunoglobulin-binding protein;

[0011] The epoxy reagent used in the epoxy modification is one or a mixture of epibromohydrin, epichlorohydrin, and diepoxy reagents.

[0012] To achieve the above object of the invention, the present invention also provides a preparation method of the above protein A immunoadsorbent with high performance and low risk, which comprises the following steps:

[0013] P1, preparing and screening an agarose gel porous microsphere carrier with a particle size range of 45-165 μm;

[0014] P2, modifying the agarose gel porous microsphere carrier by the epoxy method to obtain a modified carrier;

[0015] Mixing the modified carrier with an immunoglobulin-binding protein solution to couple the protein to the agarose gel, and performing post-treatment to obtain a protein A immunoadsorbent with high performance and low risk.

[0016] As a further improvement of the present invention, the screening method for the agarose gel porous microsphere carrier with a particle size of 45 - 165 μm in step P1 is as follows: screening is carried out by using a rotary vibrating screen process, and the specific process settings for screening are: the mixing ratio of microspheres to water is 1:(10 - 20); the volume of the flushing water is set to be 15 - 50 times the volume of the microspheres.

[0017] As a further improvement of the present invention, when preparing the mixed solution of microspheres and water, a surfactant (with a concentration of 0.1% - 0.5%) is added.

[0018] As a further improvement of the present invention, the surfactant is a quaternary ammonium salt cationic monomer, preferably (3 - acrylamidopropyl) trimethyl ammonium chloride.

[0019] As a further improvement of the present invention, the mixed solution of microspheres and water with the added surfactant is pretreated by ultrasonic waves before entering the rotary vibrating screen.

[0020] As a further improvement of the present invention, before screening, the multi - stage screen is pretreated by spraying a super - hydrophilic self - cleaning coating.

[0021] As a further improvement of the present invention, the specific process of the epoxy method in step P2 is as follows:

[0022] P2 - 1, mix the agarose gel porous microsphere carrier, epoxy reagent and NaOH solution to modify the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain an activated carrier;

[0023] P2 - 2, mix the activated carrier with the immunoglobulin - binding protein solution, adjust the pH value of the mixed solution to 6 - 11, and add solid salt to couple the protein to the carrier to obtain a coupling product;

[0024] P2 - 3, wash the coupling product, then wash it with physiological saline to remove acid and alkali, and after drying by suction, obtain a high - performance and low - risk immunoadsorbent prepared by the epoxy method, and store it with a storage solution.

[0025] As a further improvement of the present invention, in step P2 - 1, the concentration of the NaOH solution is 0.1 - 1.5 mol / L, and the epoxy reagent is one or a mixture of several of epibromohydrin, epichlorohydrin, and bis - epoxy reagent; the ratio of the NaOH solution to the epoxy reagent is 1:(0.05 - 0.8), the reaction temperature is 15 - 45 °C, and the reaction time is 0.5 - 4 h;

[0026] In step P2 - 2, the concentration of the immunoglobulin - binding protein solution is 5 - 20 mol / L; the solid salt is one or a mixture of several of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, and MnSO4.

[0027] As a further improvement of the present invention, the cleaning treatment in step P2-3 is to alternately clean with acid and alkali buffers for multiple times; the acid buffer is citric acid-sodium citrate buffer or glycine-hydrochloric acid buffer, and the alkali buffer is sodium carbonate-sodium bicarbonate buffer, Tris-hydrochloric acid buffer or phosphate buffer; the pH values of the acid buffer and the alkali buffer are 2-4 and 7-9 respectively; the number of alternate cleanings is 2-12 times;

[0028] The single cleaning dosage of the acid buffer is: 0.8-2 times the volume of the coupled product microspheres; the single cleaning dosage of the acid buffer is: 0.8-2 times the volume of the coupled product microspheres.

[0029] The cleaning treatment in step P2-3 is to clean with a citric acid-ethanol-ether mixed system. In the citric acid-ethanol-ether mixed system, the mass-volume ratio of citric acid, ethanol, and ether is: (5-10) g: (20-30) mL: (5-10) mL.

[0030] Alternatively, clean with a citric acid-ethanol-urea mixed system. In the citric acid-ethanol-urea mixed system, the mass-volume ratio of citric acid, ethanol, and urea is: (5-10) g: (20-30) mL: (5-10) g.

[0031] As a further improvement of the present invention, the mixed system may further include an aqueous solvent.

[0032] To achieve the above-mentioned invention purpose, the present invention also provides the application of the above-mentioned high-performance and low-risk protein A immunosorbent, and its application in the technical field of medical device blood purification.

[0033] The beneficial effects of the present invention are:

[0034] 1. The high-performance and low-risk protein A immunosorbent provided by the present invention simplifies the production process and reduces the production cost through the optimization of the preparation process and the independent research and development and production of recombinant proteins; it has high adsorption performance, and the treatment effect can be achieved with a single use, without the need for multiple elutions and recycling, shortening the treatment time, reducing the treatment risk, and alleviating the pain of patients and the burden on medical staff; there is no need to make the volume of the adsorption column very large, reducing the cost; at present, the adsorption capacity of commercial protein A immunosorbent columns in China for immunoglobulin (IgG) is about 50 mg / g, while the adsorption capacity of the protein A immunosorbent prepared by the present invention for IgG is 120 mg / g, with an adsorption performance more than twice that of existing products, which is easy to promote in actual production and has excellent clinical application value, and has a huge market prospect.

[0035] 2. The high-performance and low-risk protein A immunosorbent provided by the present invention, after the epoxy modification reaction is completed, adopts an alternating cleaning process with acidic and alkaline buffers for multiple times, and then uses a certain volume of physiological saline to wash away the acid and alkali. Only extremely trace amounts of protein residues (below 0.1 μg / L) are detected in the washing liquid. This process can almost completely elute the immunoglobulin-binding proteins non-specifically bound on the agarose gel microspheres, avoiding the protein from falling off into the plasma during subsequent treatment and causing severe allergic reactions; it overcomes the technical defect of the existing cleaning process that the unbound immunoglobulin-binding proteins cannot be effectively and fully eluted and removed after the coupling reaction, resulting in the safety hazard that the protein falls off into the plasma during subsequent treatment and causes severe allergic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the plasma separation and adsorption flow chart provided by the present invention ( Figure 1 a) and the schematic diagram of the protein A immunosorbent treatment process ( Figure 1 b).

[0037] Figure 2 is the schematic structural diagram of the vibrating screen device adopted by the present invention.

[0038] Figure 3 is the data comparison chart of the protein shedding amounts of Examples 2 - 3 and Comparative Example 1 of the present invention.

[0039] Figure 4 is the particle size distribution diagram of the agarose gel microspheres provided by Example 6 of the present invention (average particle size 74 μm).

[0040] Figure 5 is the characterization chart of the adsorption agent hemolysis experiment process and hemolysis rate results provided by the test example of the present invention.

[0041] Figure 6 is the physical diagram of the process of using the adsorption agent provided by the test example of the present invention for cell culture and cytotoxicity experiment.

[0042] Figure 7 is the cytotoxicity of the adsorption agent provided by the test example of the present invention at different concentrations.

[0043] Figure 8 is the on-site diagram of the whole blood perfusion experiment of the adsorption agent animal experiment provided by the test example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0044] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Here, it should also be noted that in order to avoid obscuring the present invention due to unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, while other details less related to the present invention are omitted.

[0046] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0047] The present invention provides a high-performance and low-risk protein A immunosorbent, which comprises an agarose gel porous microsphere carrier and an immunoglobulin-binding protein coupled to the agarose gel porous microsphere carrier; the average particle size of the agarose gel porous microsphere carrier is 30 - 200 μm, and the average pore size is 40 - 100 nm.

[0048] Preferably, the immunoglobulin-binding protein is one or a mixture of several of recombinant protein A, recombinant protein G, and recombinant protein L by genetic engineering.

[0049] Preferably, the agarose gel porous microsphere carrier is modified by the epoxy method to couple the immunoglobulin-binding protein;

[0050] The epoxy reagent used in the epoxy modification is one or a mixture of epibromohydrin, epichlorohydrin, and diepoxy reagents.

[0051] Preferably, the preparation method of the agarose gel porous microsphere carrier comprises the following steps:

[0052] P1, preparing and screening an agarose gel porous microsphere carrier with an average particle size of 45 - 165 μm;

[0053] P2, modifying the agarose gel porous microsphere carrier by the epoxy method to obtain a modified carrier;

[0054] Mixing the modified carrier with an immunoglobulin-binding protein solution to couple the protein to the agarose gel, and performing post-treatment to obtain a high-performance and low-risk immunosorbent.

[0055] Preferably, the specific process of the epoxy method in step P2 is as follows:

[0056] P2-1, mixing the agarose gel porous microsphere carrier, the epoxy reagent and the NaOH solution to modify the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain an activated carrier;

[0057] P2-2. Mix the activated carrier with the immunoglobulin-binding protein solution, adjust the pH value of the mixed solution to 6 - 11, and add solid salt to conjugate the protein onto the carrier to obtain a conjugate product.

[0058] P2-3. Wash the conjugate product multiple times alternately with acidic buffer and alkaline buffer, then wash with physiological saline, and after draining, obtain a high-performance and low-risk immunosorbent prepared by the epoxy method, and store it with a storage solution.

[0059] Preferably, in step P2-1, the concentration of the NaOH solution is 0.1 - 1.5 mol / L, and the epoxy reagent is one or a mixture of epibromohydrin, epichlorohydrin, and diepoxy reagent; the ratio of the NaOH solution to the epoxy reagent is 1:(0.05 - 0.8), the reaction temperature is 15 - 45 °C, and the reaction time is 0.5 - 4 h.

[0060] In step P2-2, the concentration of the immunoglobulin-binding protein solution is 5 - 20 mol / L; the solid salt is one or a mixture of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, and MnSO4.

[0061] In step P2-3, the acidic buffer is citric acid-sodium citrate buffer or glycine-hydrochloric acid buffer, and the alkaline solution is sodium carbonate-sodium bicarbonate buffer, Tris-hydrochloric acid buffer, or phosphate buffer; the pH values of the acid buffer and the base buffer are 2 - 4 and 7 - 9 respectively; the number of alternate washings is 2 - 12 times.

[0062] The single-use amount of the acid buffer for washing is 0.8 - 2 times the volume of the conjugate product microspheres; the single-use amount of the acid buffer for washing is 0.8 - 2 times the volume of the conjugate product microspheres.

[0063] The preferred carrier material of the present invention is spherical agarose gel porous microspheres. Due to its good biocompatibility and containing a large number of reactive sites. In addition, the particle size of the agarose gel porous microspheres is easy to screen. The particle size of the microspheres is closely related to the adsorption performance. If the particle size is too large, the specific surface area of the microspheres is too small, which will directly affect the adsorption efficiency; if the particle size is too small, they will be closely packed in the adsorption column, which will hinder the flow rate of plasma and there will also be a serious risk of leakage. Generally speaking, there are filters at both ends of the adsorption column, and the pore size of the filter is smaller than the particle size of the adsorbent microspheres, so as to prevent the adsorbent microspheres from entering the human body without affecting the normal flow of blood. However, there are often small-sized microspheres in the microsphere raw materials, and the small-sized microspheres will pass through the adsorption column filter and enter the human body, which may block capillaries or cause allergic reactions in the human body. Therefore, it is necessary to strictly screen the particle size of the agarose gel porous microspheres.

[0064] When screening agarose gels in the present invention, a self-made microsphere screening device is used for repeated screening to strictly control the particle size of agarose microspheres and avoid the existence of too small particles. Generally, it is considered that 30 - 200 μm is more appropriate, and 45 - 165 μm is a better choice.

[0065] Please refer to Figure 2 As shown, the microsphere screening device uses a vibrating screen with a conventional predetermined screening particle size setting, which is composed of multi-stage sieves, vibrating motors, heavy hammers, feeding and discharging ports, etc.; a vertical vibrating motor is used as the exciter, and eccentric heavy hammers are installed at the upper and lower ends of the vibrating motor to convert the rotational motion of the vibrating motor into three-dimensional motions in the horizontal, vertical, and inclined directions, and then transmit this motion to the sieve surface; by adjusting the phase angles at the upper and lower ends, the movement trajectory of the material on the sieve surface can be changed. Through the cooperation of vibration and multi-stage sieves, the screening of microsphere particle sizes is achieved. Based on this vibrating screen device, the specific screening process is as follows:

[0066] First, open the feeding port and inject the mixed liquid of microspheres and water (the mixing ratio of microspheres to water is 1:(10 - 20)) into the vibrating screen. Then, start the vibrating motor and the flushing water simultaneously (there are annularly arranged flushing water pipes on the dust cover, and several annular spray nozzles facing the inner cavity of the vibrating screen are arranged on the flushing water pipes, and the flushing water is injected into the mixed liquid in the inner cavity from the spray nozzles, and the volume of the flushing water is set to 15 - 50 times the volume of the microspheres). Under the action of the flushing water flow, the agarose gel microspheres are subjected to multi-stage screening, and different particle sizes are discharged and collected through different discharging ports, thereby achieving precise screening of the particle size of the gel microspheres, and the screening effect is 98% and above.

[0067] Those skilled in the art know that based on the above vibrating screen device, when screening is carried out by using the current existing conventional method (process parameter setting: the mixing ratio of microspheres to water is 1:(1 - 5). The volume of the flushing water used is: 5 - 10 times the volume of the microspheres), the actual screening effect can only reach about 80 - 87%.

[0068] In one embodiment, when preparing the mixed liquid of microspheres and water, a surfactant (concentration is 0.1% - 0.5%) is added.

[0069] Further, the surfactant is a quaternary ammonium salt cationic monomer, preferably (3-acrylamidopropyl) trimethyl ammonium chloride.

[0070] Further, before the mixed liquid of microspheres and water added with the surfactant enters the vibrating screen, ultrasonic pretreatment is carried out.

[0071] In another embodiment, before screening, a pretreatment of spraying a superhydrophilic self-cleaning coating on the multi-stage sieves is carried out. While preventing the microspheres from adhering and blocking the sieve holes, based on the superhydrophilic property of the sieve surface, a water film can also be formed to capture the gel microspheres into the sieve holes, improving the screening effect.

[0072] The superhydrophilic self-cleaning coating is an amphoteric ion polyacrylamide coating with superhydrophilicity (water contact angle ≤ 10°).

[0073] Compared with the screening process using a conventional sieve in this application (screening effect of 98% or more), when using a superhydrophilic self-cleaning coating sieve and / or adding a surfactant, the interception and screening effect of small-sized microspheres after screening reaches 99.5% or more.

[0074] Example 1

[0075] In Example 1 of the present invention, a method for preparing an agarose gel porous microsphere carrier includes the following steps:

[0076] P1. Using a microsphere sieve to strictly screen the particle size of agarose gel porous microspheres to obtain an agarose gel porous microsphere carrier with a particle size of 45 - 165 μm;

[0077] P2. Decorating the agarose gel porous microsphere carrier by the epoxy method to obtain a modified carrier; (in some embodiments, this step can be directly combined with step S4 for processing)

[0078] P2-1. Mixing the agarose gel porous microsphere carrier, the epoxy reagent epibromohydrin, and a 1.0 mol / L NaOH solution to modify the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain an activated carrier; the ratio of the NaOH solution to the epoxy reagent is 1:0.5, the reaction temperature is 35°C, and the reaction time is 1 h;

[0079] P2-2. Mixing the activated carrier with a 14 mol / L immunoglobulin-binding protein solution, adjusting the pH value of the mixed solution to 8, and adding the solid salt (NH4)2SO4 to couple the protein to the carrier to obtain a coupling product; the immunoglobulin-binding protein is a genetically engineered recombinant protein A;

[0080] P2-3. Cleaning the coupling product with a citric acid-sodium citrate buffer solution (pH = 2.5) with a volume 1 time that of the microspheres, then cleaning with a phosphate buffer solution (pH = 8.0) with a volume 1 time that of the microspheres, repeating 6 times, and finally cleaning with 3 volumes of physiological saline, and drying by suction to obtain a high-performance and low-risk immunosorbent prepared by the epoxy method, which is stored with a storage solution.

[0081] Refer to Figure 1 As shown, before the high-performance and low-risk protein A immunosorbent prepared by the present invention is used for treatment, the adsorbent is filled in a specific container to form an adsorption column with a volume of about 230 mL, and immunosorption treatment is carried out through the process shown in the figure. It can meet the clinical requirements with one use, without multiple elutions and recycling, reducing the treatment time and cumbersome operations.

[0082] Example 2 (Optimization of the number of cycle washes, Q1 - Q13)

[0083] The difference from Example 1 is that: the number of cycles in step P2 - 3 is changed to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times. The physiological saline from the last wash is collected, and its protein elution content is measured by ELISA. The results are as follows Figure 3 shown. Here, 0 times means not washing with the acid - base buffer solution, but directly washing with physiological saline at 3 times the volume of the microspheres.

[0084] Comparative Example 1

[0085] To verify that alternating washing with acid and base buffer solutions can effectively remove non - specifically bound immunoglobulin - binding proteins, Comparative Example 1 is added. The difference from Example 1 is that: instead of using the acid - base buffer solutions in the original step P2 - 3, it is directly changed to washing with physiological saline at multiple volumes. The physiological saline from the last wash is collected, and its protein elution content is measured by ELISA. The results are as follows Figure 3 shown.

[0086] From this Figure 3 it can be seen that the process of alternating washing with acid and base buffer solutions in the present invention can effectively remove non - specifically bound immunoglobulin - binding proteins. When the number of washing cycles is 3 times, the unbound proteins are basically removed. When the number of washing cycles is 6 times, the protein elution amount has been reduced to 0.12 - 0.14 μg / L.

[0087] However, when only rinsing with physiological saline in Comparative Example 1, the proteins cannot be effectively removed. When the total volume of physiological saline is 20 times, the protein elution amount is still about 12 μg / L. The above results show that the washing process in the method provided by the present invention can effectively remove the unbound proteins, thereby avoiding serious adverse events caused by their elution into the plasma during the treatment process, significantly improving the safety and reliability of the adsorbent, and reducing the treatment risk.

[0088] Example 3

[0089] The difference from Example 1 is that: the washing treatment in step P2 - 3 is as follows: a citric acid - ethanol - ether mixed system is used for washing. In the citric acid - ethanol - ether mixed system, the mass - to - volume ratio of citric acid, ethanol, and ether is: 5 g: 25 mL: 5 mL. Subsequently, washing is carried out multiple times with ethanol at 15 times the volume to remove the residue of the mixed solution, and finally washed with physiological saline at 15 - 20 times the volume to remove the ethanol. The physiological saline from the last wash is collected, and its protein elution content is measured by ELISA. The results are as follows Figure 3 shown.

[0090] Through this Figure 3 It can be seen that when the cleaning volume of the mixed solution is 4 times, the protein shedding amount is reduced to an extremely small amount of 0.083 μg / L.

[0091] Example 4 (Optimization of epoxy reagent ratio, test examples H1-H9)

[0092] The difference from Example 1 is that the ratio of the NaOH solution to the epoxy reagent in step P2-1 is changed to 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8 respectively. Others are the same as Example 1 and will not be elaborated here. The prepared adsorbents are numbered as H-0.05, H-0.1, H-0.2, H-0.3, H-0.4, H-0.5, H-0.6, H-0.7, H-0.8 in sequence.

[0093] Example 5 (Optimization of protein solution concentration, test examples C1-C8)

[0094] The difference from Example 1 is that the concentration of the immunoglobulin-binding protein solution in step P2-2 is changed to 6 mol / L, 8 mol / L, 10 mol / L, 12 mol / L, 14 mol / L, 16 mol / L, 18 mol / L, 20 mol / L respectively. Others are the same as Example 1 and will not be elaborated here. The prepared adsorbents are numbered as C-6, C-8, C-10, C-12, C-14, C-16, C-18, C-20 in sequence.

[0095] Example 6 (Optimization of coupling pH, test examples B1-B8)

[0096] The difference from Example 1 is that the pH of the mixed solution in step P2-2 is changed to 6, 7, 8, 9, 10, 11, 12 respectively. Others are the same as Example 1 and will not be elaborated here. The prepared adsorbents are numbered as B-6, B-7, B-8, B-9, B-10, B-11, B-12 in sequence.

[0097] Example 7 (Optimization of salt type, experimental examples Y1-Y6)

[0098] The difference from Example 1 is that the types of solid salts added in step P2-2 are changed to NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, MnSO4 respectively. Others are the same as Example 1 and will not be elaborated here. The prepared adsorbents are numbered as Y-NaCl, Y-KCl, Y-Na2SO4, Y-K2SO4, Y-(NH4)2SO4, Y-MnSO4 in sequence.

[0099] Example 8 (Optimization of average particle size, Test Examples A1 - A7)

[0100] It is different from Example 1 in that: in step P1, agarose gel microspheres with particle sizes of 0 - 45μm, 45 - 165μm, 100 - 200μm, 200 - 300μm, 300 - 400μm, 400 - 500μm, and 500 - 600μm are respectively prepared by using a microsphere sieve. Others are the same as in Example 1 and will not be elaborated here. The average particle sizes of the agarose microsphere gels obtained in Test Examples A1 - A7 are 31μm, 74μm, 152μm, 254μm, 367μm, 438μm, and 552μm, and the finally prepared adsorbents are sequentially numbered as A - 31, A - 74, A - 152, A - 254, A - 367, A - 438, and A - 552.

[0101] Example 9 (Comparison of self - made protein types, Test Examples G1 - G3, L1 - L2)

[0102] It is different from Example 1 in that: in step P2 - 2, the immunoglobulin - binding proteins are genetically engineered recombinant protein G1, genetically engineered recombinant protein G2, genetically engineered recombinant protein G3, genetically engineered recombinant protein L1, and genetically engineered recombinant protein L2. Others are the same as in Example 1 and will not be elaborated here. The adsorbents prepared in Test Examples G1 - G3, L1 - L2 are sequentially named as G1 - 74, G2 - 74, G3 - 74, L1 - 74, and L2 - 74.

[0103] Comparative Example 2

[0104] Comparative Example 2 uses a commercially available protein A immunoadsorption column in China at present.

[0105] Adsorption performance test:

[0106] 2g of the adsorbents in Comparative Example 2, Example 4 (Test Examples H1 - H9), Example 5 (Test Examples C1 - C8), Example 6 (Test Examples B1 - B8), Example 7 (Test Examples Y1 - Y6), Example 8 (Test Examples A1 - A7), and Example 9 (Test Examples G1 - G3, L1 - L2) are respectively loaded into a chromatography column with an inner diameter of 10mm and a length of 20cm. 30mL of healthy adult plasma is passed through the chromatography column at a flow rate of 1.7mL / min by a peristaltic pump for 1h. The IgG adsorption of various adsorbents is respectively detected to investigate the adsorption levels of different adsorbents. The results are shown in Table 1.

[0107] Blank blood is the blank control example, and the commercial adsorption column is Comparative Example 2.

[0108] Table 1 shows the comparison of the IgG adsorption performance of various adsorbents

[0109]

[0110]

[0111] It can be seen from Table 1 that:

[0112] ① It can be seen from Test Examples H1 - H9 that the adsorption performance of the adsorbent first increases and then decreases with the increase of the proportion of the epoxy reagent, and the optimal proportion is 1:0.5.

[0113] ② It can be seen from Test Examples C1 - C8 that the adsorption performance of the adsorbent first increases and then remains unchanged with the increase of the concentration of the immunoglobulin - binding protein, and the most cost - effective concentration is 14 mol / L.

[0114] ③ It can be seen from Test Examples B1 - B8 that the adsorption performance of the adsorbent first increases and then decreases with the increase of the pH during coupling, and the optimal pH during coupling is 8.

[0115] ④ It can be seen from Test Examples Y1 - Y6 that when the salt added is (NH4)2SO4, the adsorption performance of the adsorbent is optimal.

[0116] ⑤ It can be seen from Test Examples G1 - G3 and L1 - L2 that the self - made recombinant protein A / G / L of the present invention has excellent adsorption performance.

[0117] ⑥ It can be seen from Test Examples A1 - A7 that as the particle size of the agarose gel microspheres increases, the specific surface area of the microspheres gradually decreases, and the corresponding adsorption capacity gradually decreases. When the average particle size is greater than 367 μm, the adsorption performance significantly decreases and it is not very suitable for clinical application. When the average particle size is 152 μm, the mass of IgG adsorbed per 1 g of the adsorbent still remains above 100 mg, which is about 2 times that of the commercial adsorption column in Comparative Example 2, indicating that the adsorption performance of the adsorbent of the present invention is very high. When the average particle size is 31 μm, the particle size of the microspheres is too small, which is not conducive to plasma perfusion, the pressure in the adsorption column is high, and there is a serious risk of leakage. Therefore, agarose gel microspheres with a particle size range of 45 - 165 μm (average particle size 74 μm) are selected as the carrier material for the high - performance and low - risk protein A immunosorbent, and its particle size distribution is as Figure 4 shown.

[0118] Blood compatibility performance test:

[0119] Take the A - 74 adsorbent prepared in Example 8 above for hemolysis experiment to investigate its blood compatibility. The specific operation is as follows:

[0120] Take 5 mL of fresh anticoagulated rabbit blood, centrifuge at 1000 rpm for 10 min to remove plasma. Add approximately 10 times the volume of physiological saline to the precipitated red blood cells, gently shake well, and then centrifuge at 1000 rpm for 10 min to discard the supernatant. Repeat this process 2 - 4 times until the supernatant is no longer red. Prepare the obtained red blood cell precipitate into a 2% suspension with physiological saline (if the precipitate is 2 mL, then add 98 mL of physiological saline) for standby. Add physiological saline to the dried adsorbent A - 74 to prepare a 2 mg / mL mixture. Take 0.9 mL of the mixture into a 2 mL centrifuge tube, add 0.1 mL of the rabbit blood suspension to each tube, gently shake well, incubate in a 37°C water bath for 2 h, and observe the hemolysis phenomenon after centrifuging at 1000 rpm for 10 min. After observation, take the supernatant and place it in a 96 - well plate, and measure the absorbance at 545 nm with an enzyme - linked immunosorbent assay (ELISA) reader, and compare it with physiological saline (negative control) and 1% Triton - X - 100 (positive control). The results of the hemolysis experiment are as Figure 4 shown.

[0121] It can be seen that Figure 5 after the rabbit blood suspension is incubated with the adsorbent and centrifuged, all the blood cells and the adsorbent precipitate, and the supernatant is clear, transparent and almost colorless, indicating that hemolysis has not occurred, which shows that the blood compatibility of the adsorbent is very good. The data from the ELISA reader shows that the hemolysis rate of the A - 74 adsorbent is less than 5%, indicating that this material meets the requirements of medical biomaterials.

[0122] Biocompatibility performance test:

[0123] Take the A - 74 adsorbent prepared in Example 8 above to conduct a cytotoxicity experiment to investigate its biocompatibility. The specific operation is as follows:

[0124] ① The culture of hCMEC / D3 (immortalized human cerebral microvascular endothelial cells) is as follows: Inoculate hCMEC / D3 cells into a cell - specific culture flask, add an appropriate amount of hCMEC / D3 cell basal medium, and add 5% fetal bovine serum (FBS) and 1% penicillin - streptomycin (PS). Place it in a special incubator with sufficient oxygen, a constant temperature of 37°C, constant humidity, and a CO2 content of 5% for culture. When the cell density reaches 75 - 85%, cell passage or experiments can be carried out, as Figure 6 shown.

[0125] ② When the hCMEC / D3 cells grow to about 80% in the culture flask, remove the medium, wash twice with PBS, digest the cells with 0.25% EDTA trypsin for 4 min, add medium to terminate digestion and wash the cells off the bottom of the flask. Transfer the cell suspension to a 15 mL cell - specific centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, add an appropriate amount of medium, gently disperse the cell precipitate until it is uniform, and after volume - fixing, take 100 μL of the suspension (the number of cells is about 104 Add them into a 96-well plate one by one. Add 100 μL of PBS to each well in the outermost circle of the 96-well plate. After covering the plate lid, place it in a dedicated incubator and culture until the density is moderate. Take out the 96-well plate, carefully aspirate the cell culture medium with a pipette, and add 100 μL of adsorbents with different concentrations (prepared with culture medium) to each well according to the grouping requirements, and continue to culture for 24 h. Finally, take out the culture medium in each well, wash the cells three times with PBS, add 90 μL of culture medium and 10 μL of CCK-8 solution to each well, place it in the incubator and culture for 4 h, and measure the absorbance at 450 nm of each well with an enzyme-linked immunosorbent assay (ELISA) reader. The calculation formula for cell viability V(%) is: V(%) = (A s - A0) / (A c - A0)*100. Where A s is the absorbance of the experimental group, A c is the absorbance of cells incubated only with CCK-8, and A0 is the absorbance of the culture medium containing 10% CCK-8. The results are as Figure 7 shown

[0126] As Figure 7 shown, when the adsorbent A-74 was incubated with hCMEC / D3 cells, no obvious apoptosis occurred in the cells. Even when the adsorbent concentration was as high as 600 μg / mL, the cell survival rate remained above 80%, indicating that the A-74 adsorbent has good biocompatibility.

[0127] In vivo (animal experiment) test:

[0128] Put the adsorbent A-74 prepared in Example 8 above into a special container to make an adsorption column, and conduct an in vivo blood purification experiment on 18-month-old experimental pigs. In the in vivo experiment, all operations were carried out in a dedicated venue under the guidance of professional veterinarians in accordance with the "Guide for the Care and Use of Laboratory Animals", without any harm, abuse, intimidation, disability, etc. to the experimental pigs, and fully followed the animal ethics principles.

[0129] Conduct an immunoadsorption experiment on the experimental pigs, and detect the changes in their blood routine, blood biochemistry, coagulation indexes, and immunoglobulin content before and after the experiment. The experimental process is as Figure 8 shown, and the results are shown in the following table.

[0130] Table 2 shows the changes in blood routine indexes before and after animal immunoadsorption therapy

[0131]

[0132]

[0133] Table 3 shows the changes in blood biochemical indexes before and after animal immunoadsorption therapy

[0134]

[0135]

[0136] Table 4 shows the changes in coagulation indexes before and after animal immunoadsorption therapy

[0137]

[0138] Table 5 shows the changes in immunoglobulins before and after animal immunoadsorption therapy

[0139] Project Name Detection Method Unit Before Treatment After Treatment Quantitative Determination of Immunoglobulin A (IgA) Immunoturbidimetry g / L 0.14 <0.1 Quantitative Determination of Immunoglobulin G (IgG) Immunoturbidimetry g / L 6.29 1.55 Quantitative Determination of Immunoglobulin M (IgM) Immunoturbidimetry g / L 0.83 0.71

[0140] From the data in Tables 2 - 5, it can be seen that after the immunoadsorption test of adsorbent A - 74 on experimental pigs, there were no obvious abnormalities in its blood routine indexes. The white blood cell content increased slightly, with a slight inflammatory reaction, which was within a reasonable range; there were no obvious abnormalities in the blood biochemical indexes; the coagulation indexes changed greatly because anticoagulants needed to be continuously added when the blood was drawn out of the body to establish a circulation during treatment, resulting in an increase in coagulation time, which returned to normal after 24 h; the content of immunoglobulin G decreased significantly, and the IgG clearance rate reached 75.3% after one treatment.

[0141] Those skilled in the art know that in other embodiments of the present invention, according to the actual application needs, the adsorbents used in the experiments can also be G1 - 74, G2 - 74, G3 - 74, L1 - 74, L2 - 74, all of which can achieve excellent immunotherapy functions.

[0142] In summary, the present invention provides a protein A immunoadsorbent with high performance and low risk and its application. The immunoadsorbent includes an agarose gel porous microsphere carrier and an immunoglobulin - binding protein coupled to the agarose gel porous microsphere carrier; the particle size of the agarose gel porous microsphere carrier is 45 - 165 μm. The immunoadsorbent of the present invention can greatly improve the adsorption effect and shorten the treatment time, has good blood compatibility and biocompatibility, the preparation method of the adsorbent is simple, the cost is low, and it has great market prospects and economic value.

[0143] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A high-performance and low-risk protein A immunosorbent, characterized in that: The high-performance and low-risk immunosorbent includes an agarose gel porous microsphere carrier and an immunoglobulin-binding protein conjugated to the agarose gel porous microsphere carrier; the particle size of the agarose gel porous microsphere carrier is 30-200 μm, and the average pore size is 40-100 nm.

2. The high-performance and low-risk Protein A immunosorbent according to claim 1, wherein: The immunoglobulin-binding protein is one or a mixture of several of recombinant protein A, recombinant protein G, and recombinant protein L by genetic engineering.

3. The high-performance and low-risk protein A immunosorbent according to claim 1, wherein: The agarose gel porous microsphere carrier is modified by the epoxy method to conjugate the immunoglobulin-binding protein; The epoxy reagent used in the epoxy modification is one or a mixture of several of epibromohydrin, epichlorohydrin, and diepoxy reagent.

4. The high-performance and low-risk protein A immunosorbent according to claim 3, wherein: The preparation method of modifying the agarose gel porous microsphere carrier by the epoxy method includes the following steps: P1. Prepare and screen an agarose gel porous microsphere carrier with a particle size range of 45-165 μm; P2. Modify the agarose gel porous microsphere carrier by the epoxy method to obtain a modified carrier; mix the modified carrier with an immunoglobulin-binding protein solution to conjugate the protein to the agarose gel, and perform post-treatment to obtain a high-performance and low-risk immunosorbent.

5. The high-performance and low-risk protein A immunosorbent according to claim 4, wherein: The method for screening the agarose gel porous microsphere carrier with a particle size range of 45-165 μm in step P1 is: Screening is carried out by the rotary vibrating screen process, and the specific process settings are: the mixing ratio of microspheres and water is 1:(10-20); the volume of the rinsing water is set to 15-50 times the volume of the microspheres.

6. The high-performance and low-risk protein A immunosorbent according to claim 4, wherein: The specific process of the epoxy method in step P2 is: P2-1. Mix the agarose gel porous microsphere carrier, the epoxy reagent, and the NaOH solution to modify the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain an activated carrier; P2-2. Mix the activated carrier with an immunoglobulin-binding protein solution, adjust the pH value of the mixed solution to 6-11, and add solid salt to conjugate the protein to the carrier to obtain a conjugate product; P2-3. Wash the conjugate product, then wash it with physiological saline to remove acid and alkali, and dry it by suction to obtain a high-performance and low-risk immunosorbent prepared by the epoxy method, and store it with a storage solution.

7. The high-performance and low-risk protein A immunosorbent according to claim 6, wherein: The washing treatment in step P2-3 is: wash it alternately with acid and alkali buffer solutions for multiple times; the acid buffer solution is citric acid-sodium citrate buffer solution or glycine-hydrochloric acid buffer solution, and the alkali buffer solution is sodium carbonate-sodium bicarbonate buffer solution, Tris-hydrochloric acid buffer solution, or phosphate buffer solution; The pH values of the acid buffer solution and the alkali buffer solution are 2-4 and 7-9 respectively; the number of alternate washings is 2-12 times; The single-use amount of the acid buffer solution for washing is: 0.8-2 times the volume of the conjugate product microspheres; the single-use amount of the acid buffer solution for washing is: 0.8-2 times the volume of the conjugate product microspheres.

8. The high-performance and low-risk protein A immunosorbent according to claim 6, wherein: In step P2-1, the concentration of the NaOH solution is 0.1-1.5 mol / L, and the epoxy reagent is one or a mixture of several of epibromohydrin, epichlorohydrin, and diepoxy reagent; the ratio of the NaOH solution to the epoxy reagent is 1:(0.05-0.8), the reaction temperature is 15-45 °C, and the reaction time is 0.5-4 h; In step P2-2, the concentration of the immunoglobulin-binding protein solution is 5 to 20 mol / L; the solid salt is one or a mixture of several of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, and MnSO4; The average particle size of the agarose gel porous microsphere carrier is 74 μm.

9. The high-performance and low-risk protein A immunosorbent according to claim 6, wherein: The cleaning treatment in step P2-3 is: cleaning with a citric acid-ethanol-ether mixed system, in which the mass-volume ratio of citric acid, ethanol, and ether in the citric acid-ethanol-ether mixed system is (5 to 10) g : (20 to 30) mL : (5 to 10) mL.

10. Use of the high-performance and low-risk protein A immunosorbent according to any one of claims 1 to 9, characterized in that: The application of the high-performance and low-risk protein A immunosorbent in the field of medical device blood purification technology.

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