Protein A immunoadsorbent capable of being used for whole blood perfusion as well as preparation method and application of protein A immunoadsorbent

Abstract

The invention provides a protein A immunoadsorbent for whole blood perfusion as well as a preparation method and application of the protein A immunoadsorbent. The immunoadsorbent comprises an agarose gel porous microsphere carrier and an immunoglobulin binding protein coupled with the agarose gel porous microsphere carrier, the average particle size of the agarose gel porous microsphere carrier is 100-400 [mu] m, and the average pore size of the agarose gel porous microsphere carrier is 40-100 nm. The immunoadsorbent provided by the invention can greatly improve the adsorption effect and shorten the treatment time, realizes the first case of immunoadsorbent for whole blood perfusion in the world, and overcomes the defects that the adsorption performance is poor due to overlarge particle size of microspheres, the microspheres are easy to leak and enter human blood due to oversmall particle size of the microspheres, blood cells cannot pass through easily, and the treatment effect is poor in the prior art. And meanwhile, a plasma separator is not needed, so that the use cost is reduced. The preparation method disclosed by the invention is simple and low in cost, and has huge market prospect and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of medical device blood purification, and particularly relates to a protein A immunosorbent for whole blood perfusion, a preparation method thereof, and an application thereof. Background Art

[0002] Immunosorption therapy is a blood purification technology that began to emerge in the early 21st century. It uses highly specific antibodies, antigens, or ligands with specific physicochemical affinity properties to combine with a carrier (adsorbent material) to form an adsorbent, specifically removing pathogenic substances in the blood, thereby treating some diseases that cannot be cured by traditional therapies. 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 for patients. Immunosorption therapy can be divided into two modes according to the way of treating blood: plasma separation adsorption and whole blood adsorption.

[0003] Plasma separation adsorption means that the patient's blood is drawn out of the body, an extracorporeal circulation is established through a specific device and an anticoagulant, then the blood is separated into plasma and blood cells by a plasma separator, and then the plasma is introduced into an immunosorption device to specifically adsorb pathogenic factors in the plasma. Finally, the purified plasma is mixed with the blood cells and transfused back into the human body to achieve the treatment purpose. Due to the large plasma volume to be processed, the amount of blood outside the body is too much, and the patient's tolerance is very poor, and even severe hypotension occurs, which is very difficult to handle clinically. In addition, plasma separation adsorption must have two blood pump devices, one is a blood circulation pump, and the other is a plasma separation pump, and the pipelines used are also very many (as shown in the attachment Figure 1 ), the process is complex, and the equipment cost is high.

[0004] Whole blood adsorption, on the other hand, does not require plasma separation. The whole blood is directly introduced into an immunosorption column for adsorption and then transfused back into the human body. Whole blood adsorption only requires one extracorporeal circulation device, and the operation is simple; the treatment time is short, the amount of blood drawn out is small, and the patient's tolerance is good; the treatment cost and expenses are relatively low. Since the adsorbent used is usually a broad-spectrum adsorbent, the same adsorption column can be used for the treatment of various diseases. Two conditions must be met for whole blood adsorption. One is that the adsorbent must have a certain porosity to allow the blood to pass through smoothly without damaging blood cells; the other is that the outside of the adsorbent needs to be coated with a polymer material with good biocompatibility to improve the biocompatibility of the adsorbent.

[0005] Currently, the commercially available plasma adsorption immunoadsorption column in the German market is priced at 10,000 euros, which is extremely expensive. There is only one company in China that produces a commercially available protein A immunoadsorption column. The cost for patients to purchase it from the hospital is between 40,000 and 50,000 yuan, and an additional treatment fee of 3,000 yuan is required for each treatment. And so far, there has been no commercially available whole blood perfusion immunoadsorbent globally. Therefore, there is an urgent need to develop a safe, efficient, low-cost, and easy-to-operate immunoadsorbent (column) for whole blood perfusion to reduce the overall treatment cost and significantly reduce the treatment time.

[0006] The whole blood adsorption technology uses a whole blood adsorption column to draw blood out of the body and flow it through the adsorption column. Adsorbent microspheres are set inside the adsorption column, and the adsorbent microspheres bind to the target harmful substances in the blood, thereby achieving the purpose of blood purification. 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 tightly accumulate inside the adsorption column, which will hinder the blood perfusion speed, is not conducive to the passage of blood cells, and there is also a serious risk of leakage. Generally speaking, there are filter meshes at both ends of the adsorption column, and the aperture of the filter mesh is smaller than the particle size of the adsorbent microspheres, so as to be able to block the adsorbent microspheres from entering the human body without affecting the normal flow of blood. However, in the existing commercial microsphere raw materials, there are often inevitably small-sized microspheres that do not meet the particle size range. The small-sized microspheres will pass through the filter mesh of the adsorption column 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 gel microspheres. The existing screening method is to place the gel microspheres on a multi-layer stacked screening device with the required particle size and continuously rinse with water for screening. However, due to the elastic characteristics of the gel microspheres and the phenomenon that the microspheres are prone to adhesion and aggregation, the existing technical processes (for example: a microsphere screening device disclosed in CN217888274U) have the technical defect that the small-sized gel microspheres that adhere together during screening cannot be screened out and will also block the sieve holes. And for the existing screening process, the interception and screening effect of the small-sized gel microspheres can only reach 80% - 90% at most, and there are great potential safety hazards in actual application. Summary of the Invention

[0007] The purpose of the present invention is to provide a protein A immunoadsorbent for whole blood perfusion, its preparation method and application.

[0008] To achieve the above-mentioned invention purpose, the present invention provides a protein A immunoadsorbent for whole blood perfusion, which comprises an agarose gel porous microsphere carrier and an immunoglobulin-binding protein coupled to the agarose gel porous microsphere carrier;

[0009] The average particle size of the agarose gel porous microsphere carrier is 100 - 400 μm, and the average pore size is 40 - 100 nm.

[0010] As a further improvement of the present invention, the agarose gel porous microsphere carrier is sieved by a rotary vibrating screen device, and the screening process is set as follows: the mixing ratio of microspheres to water is 1:(10-20); the volume of flushing water is set to 15-50 times the volume of the microspheres.

[0011] As a further improvement of the present invention, when preparing the mixed solution of microspheres and water, a surfactant (concentration 0.1%-0.5%) is added.

[0012] As a further improvement of the present invention, the surfactant is a quaternary ammonium salt cationic monomer, preferably (3-acrylamidopropyl) trimethyl ammonium chloride.

[0013] As a further improvement of the present invention, the mixed solution of microspheres and water added with the surfactant is pretreated by ultrasonic waves before entering the rotary vibrating screen.

[0014] As a further improvement of the present invention, before screening, the multi-stage sieve mesh is pretreated by spraying a super-hydrophilic self-cleaning coating.

[0015] As a further improvement of the present invention, the immunoglobulin-binding protein is one or a mixture of several of genetically engineered recombinant protein A, genetically engineered recombinant protein G, and genetically engineered recombinant protein L.

[0016] As a further improvement of the present invention, the agarose gel porous microsphere carrier is modified by the periodate oxidation method or the epoxy method to couple the immunoglobulin-binding protein;

[0017] The periodate used in the periodate oxidation method modification is one or a mixture of potassium periodate and sodium periodate;

[0018] The epoxy reagent used in the epoxy method modification is one or a mixture of epibromohydrin, epichlorohydrin, and bis-epoxy reagents.

[0019] To achieve the above invention object, the present invention also provides a preparation method of the above protein A immunoadsorbent for whole blood perfusion, including the following steps:

[0020] P1, preparing an agarose gel porous microsphere carrier with an average particle size of 200-300 μm and an average pore size of 40-100 nm;

[0021] P2, modifying the agarose gel porous microsphere carrier by the periodate oxidation method or the epoxy method to obtain a modified carrier;

[0022] Mixing the modified carrier with the immunoglobulin-binding protein solution to couple the protein to the agarose gel, and performing post-treatment to obtain the immunoadsorbent for whole blood perfusion.

[0023] As a further improvement of the present invention, the specific process of the periodate oxidation method in step P2 is as follows:

[0024] P21, oxidize the hydroxyl groups of the agarose gel porous microsphere carrier with a periodate solution having a concentration of 0.1 to 2.5 mol / L to aldehyde groups to obtain an activated carrier;

[0025] P22, mix the activated carrier with an immunoglobulin-binding protein solution to couple the protein to the carrier;

[0026] P23, adjust the pH value of the mixed solution obtained in step P22, and then add a reducing agent for reduction to obtain a coupling product;

[0027] P24, wash the coupling product repeatedly with an acidic buffer solution and an alkaline buffer solution in alternation, and then wash with physiological saline. After draining, an immunosorbent for whole blood perfusion prepared by the periodate oxidation method is obtained and stored with a storage solution.

[0028] As a further improvement of the present invention, the reaction temperature in step P21 is 25 to 40 °C, and the reaction time is 2 to 6 h;

[0029] In step P22, the concentration of the immunoglobulin-binding protein solution is 7 to 11 mol / L, the volume ratio of the activated carrier to the volume of the immunoglobulin-binding protein solution is 1:(0.2 to 4), the reaction temperature is 25 to 40 °C, and the reaction time is 6 to 24 h;

[0030] The reducing agent in step P23 is sodium cyanoborohydride, sodium borohydride, or a mixture of the two in any ratio;

[0031] The acidic buffer solution in step P24 is a citric acid-sodium citrate buffer solution or a glycine-hydrochloric acid buffer solution, and the alkaline solution is a sodium carbonate-sodium bicarbonate buffer solution, a Tris-hydrochloric acid buffer solution, or a phosphate buffer solution.

[0032] As a further improvement of the present invention, the specific process of the epoxy method in step P2 is as follows:

[0033] P2-1, mix the agarose gel porous microsphere carrier, an epoxy reagent, and an NaOH solution to modify the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain an activated carrier;

[0034] P2-2, mix the activated carrier with an immunoglobulin-binding protein solution, adjust the pH value of the mixed solution to 6 to 11, and add a solid salt to couple the protein to the carrier to obtain a coupling product;

[0035] P2-3. The conjugate product was washed alternately with acidic buffer and alkaline buffer for multiple times, then washed with physiological saline, and dried to obtain the immunosorbent for whole blood perfusion prepared by the epoxy method, which was stored with the storage solution.

[0036] 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 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.

[0037] In step P2-2, the concentration of the immunoglobulin-binding protein solution is 7 - 11 mol / L; the solid salt is one or a mixture of several of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, and MnSO4.

[0038] 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.

[0039] To achieve the above-mentioned invention purpose, the present invention also provides the application of the above-mentioned protein A immunosorbent for whole blood perfusion, and its application in the technical field of medical device blood purification.

[0040] The beneficial effects of the present invention are as follows:

[0041] 1. The protein A immunosorbent for whole blood perfusion provided by the present invention simplifies the production process, reduces the production cost, is easy to promote in actual production, and has excellent clinical application value through the optimization of the preparation process and the independent research and development and production of recombinant proteins. This immunosorbent greatly shortens the treatment time, and realizes the world's first immunosorbent for whole blood perfusion, with huge market prospects and economic value. It has excellent adsorption performance, so there is no need to make the volume of the adsorption column very large, reducing the production cost; and the whole blood adsorption process does not require a plasma separation pump and additional special pipelines, reducing the clinical use cost; the whole blood adsorption treatment time is short, with high safety, and there is no need to pay excessive nursing fees, so it can reduce the overall treatment cost and significantly reduce the treatment time. At present, the only commercial protein A immunosorbent column in China has an adsorption capacity of immunoglobulin (IgG) of 50 - 55 mg / g, while the protein A immunosorbent prepared by the present invention has an adsorption capacity of IgG of 80 - 90 mg / g, with an adsorption performance 1.5 times higher than that of existing products.

[0042] 2. The protein A immunosorbent provided by the present invention for whole blood perfusion has a relatively high adsorption rate during whole blood adsorption. The adsorbent is nearly saturated after 2 hours of perfusion, and the clearance rate is significantly reduced, so the treatment can be stopped. The treatment can be carried out again after 8 - 10 hours or the next day. Compared with plasma adsorption in the prior art, the treatment time is significantly shortened, the treatment risk is reduced, and the burden on medical staff is also reduced.

[0043] 3. For the protein A immunosorbent provided by the present invention for whole blood perfusion, the particle size of the agarose microspheres is precisely controlled through a screening process. The interception and screening effect of small-sized microspheres reaches 98% or above, far higher than the controllable particle size standard of commercial agarose gel microspheres. It overcomes the technical defect that the interception and screening effect of small-sized microspheres can only reach about 80% - 90% by using the conventional screening process in the prior art, resulting in potential safety hazards in whole blood adsorption and blockage of sieve pores. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the plasma separation adsorption flow chart and whole blood adsorption flow chart provided by the present invention.

[0045] Figure 2 It is the vibrating screen device adopted by the present invention ( Figure 2 in which a is the structural schematic diagram; Figure 2 in which b is the sectional schematic diagram; Figure 2 in which c is the particle size distribution characterization of the conventional screening process; Figure 2 in which d is the particle size distribution characterization of the screening in Example 1 of the present invention).

[0046] Figure 3 It is the device diagram for testing the performance of the adsorbent (small sample) in extracorporeal blood circulation provided by Test Examples A1 - A7 and Test Examples B1 - B7 of the present invention.

[0047] Figure 4 It is the particle size distribution diagram of the agarose gel microspheres (average particle size 254μm) provided by Test Examples A1 - A7 and Test Examples B1 - B7 of the present invention.

[0048] Figure 5 It is the characterization diagram of the process and hemolysis rate result of the hemolysis experiment of the adsorbent provided by the test examples of the present invention.

[0049] Figure 6 It is the physical diagram of the process of using the adsorbent provided by the test examples of the present invention for cell culture and cytotoxicity experiment.

[0050] Figure 7 It is the cytotoxicity of the adsorbent provided by the test examples of the present invention at different concentrations.

[0051] Figure 8 It is the on-site diagram of the whole blood perfusion experiment of the adsorbent in the animal experiment provided by the test examples of the present invention. Detailed implementation manners

[0052] 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.

[0053] 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.

[0054] In addition, it should also 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 further includes elements inherent to such process, method, article or device.

[0055] The present invention provides a protein A immunosorbent that can be used for whole blood perfusion, which includes an agarose gel porous microsphere carrier and an immunoglobulin-binding protein coupled to the agarose gel porous microsphere carrier;

[0056] The average particle size of the agarose gel porous microsphere carrier is 100 - 400 μm, and the average pore size is 40 - 100 nm.

[0057] 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.

[0058] Preferably, the agarose gel porous microsphere carrier is modified by the periodate oxidation method or the epoxy method to couple the immunoglobulin-binding protein;

[0059] The periodate used in the periodate oxidation method modification is one or a mixture of potassium periodate and sodium periodate;

[0060] The epoxy reagent used in the epoxy method modification is one or a mixture of epibromohydrin, epichlorohydrin, and diepoxy reagent.

[0061] The present invention also provides a preparation method of the above-mentioned protein A immunosorbent that can be used for whole blood perfusion, including the following steps:

[0062] P1, preparing an agarose gel porous microsphere carrier with an average particle size of 200 - 300 μm and an average pore size of 40 - 100 nm;

[0063] P2, modifying the agarose gel porous microsphere carrier by the periodate oxidation method or the epoxy method to obtain a modified carrier;

[0064] Mix the modified carrier with the immunoglobulin-binding protein solution to conjugate the protein to the agarose gel, and then perform post-treatment to obtain an immunosorbent for whole blood perfusion.

[0065] Preferably, the specific process of the periodate oxidation method in step P2 is as follows:

[0066] P21, Oxidize the hydroxyl groups of the agarose gel porous microsphere carrier to aldehyde groups with a periodate solution having a concentration of 0.1 - 2.5 mol / L to obtain an activated carrier;

[0067] P22, Mix the activated carrier with the immunoglobulin-binding protein solution to conjugate the protein to the carrier;

[0068] P23, Adjust the pH value of the mixed solution obtained in step P22, and then add a reducing agent for reduction to obtain a conjugate product;

[0069] P24, Wash the conjugate product multiple times alternately with an acidic buffer solution and a basic buffer solution, then wash with physiological saline, drain, and obtain an immunosorbent for whole blood perfusion prepared by the periodate oxidation method, and store it with a storage solution.

[0070] Preferably, the reaction temperature in step P21 is 25 - 40 °C, and the reaction time is 2 - 6 h;

[0071] In step P22, the concentration of the immunoglobulin-binding protein solution is 7 - 11 mol / L, the volume ratio of the activated carrier to the volume of the immunoglobulin-binding protein solution is 1:(0.2 - 4), the reaction temperature is 25 - 40 °C, and the reaction time is 6 - 24 h;

[0072] The reducing agent in step P23 is sodium cyanoborohydride, sodium borohydride, or a mixture of the two in any ratio;

[0073] The acidic buffer solution in step P24 is citric acid - sodium citrate buffer solution or glycine - hydrochloric acid buffer solution, and the basic solution is sodium carbonate - sodium bicarbonate buffer solution, Tris - hydrochloric acid buffer solution, or phosphate buffer solution.

[0074] Preferably, the specific process of the epoxy method in step P2 is as follows:

[0075] 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;

[0076] 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 a solid salt to conjugate the protein to the carrier to obtain a conjugate product;

[0077] P2-3. The coupled product was washed repeatedly with acidic buffer and alkaline buffer alternately, and then washed with physiological saline. After being drained, the immunosorbent for whole blood perfusion prepared by the epoxy method was obtained and stored with the storage solution.

[0078] 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 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.

[0079] In step P2-2, the concentration of the immunoglobulin-binding protein solution is 7-11 mol / L; the solid salt is one or a mixture of several of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, and MnSO4.

[0080] 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.

[0081] The preferred carrier material of the present invention is spherical agarose gel porous microspheres. Due to its good biocompatibility, it contains a large number of reactive sites. When screening the agarose gel microspheres in the present invention, a microsphere screening device is used for repeated screening to strictly control the particle size of the agarose gel microspheres and avoid the existence of microspheres with too small sizes to meet the standards for whole blood perfusion. Generally, it is considered that 100-600 μm is more suitable, 100-400 μm is a better choice; 200-300 μm is the optimal choice.

[0082] Please refer to Figure 2 As shown, the microsphere screening device uses a vibrating screen, which is composed of multi-stage screens, vibrating motors, heavy hammers, inlet and outlet 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 transfer this motion to the screen surface; by adjusting the phase angles at the upper and lower ends, the motion trajectory of the material on the screen surface can be changed. Through the cooperation of vibration and multi-stage screens, the screening of the microsphere particle size is achieved. Based on this vibrating screen device, the specific screening process is as follows:

[0083] First, open the feed inlet and inject the mixed solution 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. The flushing water is injected into the mixed solution in the inner cavity from the spray nozzles, and the volume of the flushing water is set to be 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 discharge ports, thereby achieving precise screening of the particle size of the gel microspheres.

[0084] In one embodiment, when preparing the mixed solution of microspheres and water, a surfactant (concentration: 0.1% - 0.5%) is added.

[0085] Furthermore, the surfactant is a quaternary ammonium salt cationic monomer, preferably (3 - acrylamidopropyl) trimethyl ammonium chloride.

[0086] Furthermore, before the mixed solution of microspheres and water with added surfactant enters the vibrating screen, it is pretreated by ultrasound.

[0087] In another embodiment, before screening, a pretreatment of spraying a superhydrophilic self - cleaning coating on the multi - stage sieve mesh is carried out. While preventing the microspheres from sticking and blocking the sieve holes, based on the superhydrophilic property of the coating on the sieve mesh surface, a water film can also be formed to capture the gel microspheres into the sieve holes, improving the screening effect. The superhydrophilic self - cleaning coating is an amphoteric ion - type polyacrylamide coating with superhydrophilicity (water contact angle ≤ 10°).

[0088] Compared with the screening process using a conventional sieve mesh in this application (screening effect 98% and above), when using a superhydrophilic self - cleaning coating sieve mesh and / or adding a surfactant, the interception screening effect of small - sized microspheres after screening reaches 99.5% and above.

[0089] Example 1

[0090] Example 1 of the present invention provides a preparation method of a protein A immunosorbent for whole - blood perfusion, including the following steps:

[0091] P1. Use a microsphere screening device to strictly screen the particle size of agarose gel porous microspheres, and prepare an agarose gel porous microsphere carrier with an average particle size of 200 - 300 μm and an average pore size of 40 - 100 nm;

[0092] As Figure 2 shown, based on the vibrating screen device, the specific screening process is as follows:

[0093] First, open the feed inlet and inject the mixture of microspheres and water (the mixing ratio of microspheres to water is set to 1:20) into the rotary vibrating screen. Then, start the vibrating motor and the flushing water simultaneously (the volume of the flushing water is set to 40 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 through different discharge ports, thereby achieving precise screening of the particle size.

[0094] After screening, the interception screening effect of small-sized microspheres (<200 μm) reaches 98% and above, effectively avoiding the technical defect that small-sized microspheres are difficult to screen due to adhesion and aggregation in the prior art, resulting in poor screening effect.

[0095] P2, Modify the agarose gel porous microsphere carrier by the periodate oxidation method to obtain the modified carrier;

[0096] P21, Oxidize the hydroxyl groups of the agarose gel porous microsphere carrier to aldehyde groups with a 1.5 mol / L periodate solution, the reaction temperature is 25 - 40 °C, and the reaction time is 2 - 6 h to obtain the activated carrier;

[0097] P22, Mix the activated carrier with the immunoglobulin-binding protein solution to couple the protein to the carrier; the concentration of the immunoglobulin-binding protein solution is 8 mol / L, the volume ratio of the activated carrier to the immunoglobulin-binding protein solution is 1:1, the reaction temperature is 25 - 40 °C, and the reaction time is 6 - 24 h; the immunoglobulin-binding protein is genetically engineered recombinant protein A;

[0098] P23, Adjust the pH value of the mixed solution obtained in step P22, and then add the reducing agent sodium cyanoborohydride for reduction to obtain the coupling product;

[0099] P24, Wash the coupling product multiple times alternately with citrate-sodium citrate buffer and sodium carbonate-sodium bicarbonate buffer, then wash with physiological saline, and after drying by suction, obtain the immunoadsorbent prepared by the periodate oxidation method for whole blood perfusion, and store it with the storage solution.

[0100] Example 2

[0101] The second embodiment of the present invention provides a preparation method of a protein A immunoadsorbent that can be used for whole blood perfusion, including the following steps:

[0102] P1, Use a microsphere screening device to strictly screen the particle size of the agarose gel porous microspheres to prepare an agarose gel porous microsphere carrier with an average particle size of 200 - 300 μm and an average pore size of 40 - 100 nm;

[0103] Among them, the mixing ratio of microspheres and water is set to 1:20; the volume of the rinsing water is set to 50 times the volume of the microspheres; after screening, the interception screening effect of the small-sized microspheres reaches 98% or more.

[0104] P2, using the epoxy method to decorate the agarose gel porous microsphere carrier to obtain the modified carrier.

[0105] P2-1, mixing the agarose gel porous microsphere carrier, the epoxy reagent epibromohydrin and 1.0 mol / L NaOH solution to modify the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain the 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.

[0106] P2-2, mixing the activated carrier with 8 mol / L immunoglobulin-binding protein solution, adjusting the pH value of the mixed solution to 7, and adding solid salt NaCl to couple the protein to the carrier to obtain the coupling product; the immunoglobulin-binding protein is a genetically engineered recombinant protein A.

[0107] P2-3, washing the coupling product alternately with acidic buffer and basic buffer for multiple times, then washing with physiological saline, and drying by suction to obtain the immunoadsorbent prepared by the epoxy method for whole blood perfusion, which is stored with the storage solution.

[0108] Among them, the acidic buffer is citric acid-sodium citrate buffer or glycine-hydrochloric acid buffer, and the basic solution is sodium carbonate-sodium bicarbonate buffer, Tris-hydrochloric acid buffer or phosphate buffer.

[0109] When the whole blood perfusion protein A immunoadsorbent prepared by the present invention is used for whole blood adsorption, if the whole blood flow rate is too fast, the adsorption rate is low; if the flow rate is too slow, it is easy to coagulate, which will increase the usage amount of anticoagulants. Therefore, it is generally controlled at 100-150 mL / min. Since the adsorption rate of most adsorption columns is relatively high, the adsorbent is almost saturated after 2 hours of perfusion, and the clearance rate is significantly reduced, so the treatment can be stopped, and the treatment can be carried out again after 8-10 hours or the next day. Compared with plasma adsorption, the treatment time is significantly shortened, and the burden on medical staff is also reduced.

[0110] Blank control group

[0111] Based on the rotary vibrating screen device, the agarose gel microspheres are screened by the existing conventional method. The difference is that the existing conventional screening process is as follows:

[0112] The mixing ratio of microspheres and water is 1:(1-5). The volume of the rinsing water used is 5-10 times the volume of the microspheres. After screening, the interception screening effect of the small-sized microspheres can only reach 80%-90%.

[0113] Those skilled in the art are aware that the models of agarose gel microspheres widely used in the market include 4FF / 6FF (45 - 165μm), 4BB / 6BB (100 - 300μm), etc. However, the control of their particle size range is not strict enough, and the proportion of small particle microspheres still does not meet the requirements of whole blood perfusion adsorption. Specifically, in the blank control group, conventional screening methods in the market were used (the screening particle size was set at 100 - 300μm). From Figure 2 As can be seen from c in

[0114] , there are 13% of small-sized gel microspheres below 100μm and 8.5% of gel microspheres above 300μm. That is, its interception effect on small-sized gel microspheres is less than 90%. Figure 2 As can be seen from d in

[0115] , there are 1.76% of gel microspheres below 200μm and 0.51% of gel microspheres above 300μm. That is, the screening process of Example 1 has an interception effect on small-sized gel microspheres reaching 98.24%, which is far higher than the screening interception effect in the blank control group.

[0116] Control Example 1

[0117] Control Example 1 uses a currently commercial protein A immunosorbent column.

[0118] Test Examples A1 - A7

[0119] The differences from Example 1 are as follows: In step P1, agarose gel microspheres with average particle sizes of 31μm, 74μm, 152μm, 254μm, 367μm, 438μm, and 552μm were prepared respectively using a microsphere sieve (with different particle size settings for the screening filter mesh), and the rest were the same as Example 1, which will not be elaborated here. The adsorbents prepared in Test Examples A1 - A7 are numbered A - 31, A - 74, A - 152, A - 254, A - 367, A - 438, and A - 552.

[0120] Test Examples B1 - B7

[0121] The difference from Example 2 is as follows: In step P1, agarose gel microspheres with average particle sizes of 31 μm, 74 μm, 152 μm, 254 μm, 367 μm, 438 μm, and 552 μm were respectively prepared using a microsphere sieve (with different particle size settings for the screening filter mesh), and the rest was the same as in Example 2, which will not be elaborated here. The adsorbents prepared in Test Examples B1 - B7 were numbered B-31, B-74, B-152, B-254, B-367, B-438, and B-552.

[0122] Adsorption performance test:

[0123] Take 2 g of the adsorbents in Comparative Example 1, Test Examples A1 - A7, and Test Examples B1 - B7 respectively and load them into a chromatography column with an inner diameter of 10 mm and a length of 20 cm. Use a peristaltic pump to pass 30 mL of healthy adult whole blood through the chromatography column at a flow rate of 3.4 mL / min for 2 h. Detect the IgG adsorption and blood routine changes of various adsorbents respectively to investigate the adsorption levels and blood cell permeability of different adsorbents. The experimental device is as Figure 3 shown, and the results are shown in Table 1 and Table 2.

[0124] The blank blood was the blank control example.

[0125] Table 1 shows the comparison of the IgG adsorption performance of various adsorbents

[0126]

[0127]

[0128] It can be seen from Table 1 that all the adsorbents prepared in Test Examples A1 - A7 and Test Examples B1 - B7 have no obvious effect on albumin in the blood and only specifically adsorb IgG. 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. Moreover, the adsorbents obtained by the two preparation methods provided in the present invention have almost no difference in performance. When the average particle size is 254 μm, the mass of IgG adsorbed by each 1 g of the adsorbent still remains above 80 mg, which is more than 1.5 times that of Comparative Example 1.

[0129] Table 2 shows the experimental results of the blood cell permeability of various adsorbents

[0130]

[0131]

[0132] As can be seen from Table 2, the smaller the average particle size of the adsorbent, the worse the permeability to blood cells. When the average particle size is less than 152 μm, obvious loss of blood cells begins to occur; when the average particle size is less than 74 μm, it can be considered that blood cells cannot pass through normally.

[0133] Taking into comprehensive consideration the content of Table 1 and Table 2, a smaller average particle size is beneficial to improving the adsorption performance, while a larger average particle size is beneficial to the blood cell passing rate. Therefore, agarose gel microspheres with a particle size range of 200 - 300 μm (average particle size 254 μm) are selected as the carrier material of the protein A immunosorbent for whole blood perfusion, and its particle size distribution is as Figure 4 shown.

[0134] Blood compatibility performance test:

[0135] Take the two adsorbents A - 254 and B - 254 prepared in the above test examples for hemolysis experiments to investigate their blood compatibility. The specific operation is as follows:

[0136] Take 5 mL of fresh anticoagulated rabbit blood, centrifuge at 1000 rpm for 10 min to remove plasma, add about 10 times the volume of normal saline to the precipitated red blood cells, shake gently, and then centrifuge at 1000 rpm for 10 min to remove the supernatant. Repeat this 2 - 4 times until the supernatant is no longer red. Prepare the obtained red blood cell precipitate into a 2% suspension (if the precipitate is 2 mL, then add 98 mL of normal saline) for standby. Add normal saline to the dried adsorbents A - 254 and B - 254 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, shake gently, incubate in a water bath at 37 °C 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 normal saline (negative control) and 1% Triton - X - 100 (positive control). The results of the hemolysis experiment are as Figure 5 shown.

[0137] Through Figure 5 it can be seen that 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 no hemolysis phenomenon has occurred, showing that the blood compatibility of the adsorbent is very good. The data from the ELISA reader shows that the hemolysis rate of both adsorbents is less than 5%, indicating that this material meets the requirements of medical biomaterials.

[0138] Biocompatibility performance test:

[0139] Take the two adsorbents A - 254 and B - 254 prepared in the above test examples for cytotoxicity experiments to investigate their biocompatibility. The specific operation is as follows:

[0140] 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). Incubate in a dedicated incubator with sufficient oxygen, a constant temperature of 37 °C, constant humidity, and a CO2 content of 5%. When the cell density reaches 75 - 85%, cell passage or experiments can be carried out, as Figure 6 shown.

[0141] 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, remove the supernatant, add an appropriate amount of medium, gently disperse the cell pellet until uniform, and after volume fixation, take 100 μL of the suspension (the number of cells is about 10 4 cells) and add them to a 96-well plate one by one. Add 100 μL of PBS to each well in the outermost circle of the 96-well plate. Cover the plate and incubate in a dedicated incubator until the density is appropriate. Take out the 96-well plate, carefully aspirate the cell medium with a pipette, and add 100 μL of different concentrations of adsorbent (prepared with medium) to each well according to the grouping requirements and continue to incubate for 24 h. Finally, take out the medium in each well, wash the cells three times with PBS, add 90 μL of medium and 10 μL of CCK-8 solution to each well, incubate in the incubator for 4 h, and measure the absorbance of each well at 450 nm 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 medium containing 10% CCK-8. The results are as Figure 7 shown

[0142] As Figure 7 shown, when adsorbents A-254 and B-254 are incubated with hCMEC / D3 cells, no obvious apoptosis phenomenon occurs in the cells. Even when the adsorbent concentration is as high as 600 μg / mL, the cell survival rate still remains above 80%, indicating that the adsorbents prepared by the two preparation methods in the present invention have good biocompatibility.

[0143] In vivo (animal experiment) test:

[0144] The adsorbent B-254 prepared in the above test example was loaded into a special container to make an adsorption column, and a live blood purification experiment was carried out on 18-month-old experimental pigs. In the live experiment, all operations were carried out in a special 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 and other behaviors to the experimental pigs, and fully followed the principles of animal ethics.

[0145] A whole blood perfusion experiment was carried out on the experimental pigs, and the changes in their blood routine, blood biochemistry, coagulation indexes, and immunoglobulin content were detected before and after the experiment. The experimental process is as Figure 8 shown, and the results are shown in Table 3 below.

[0146] Table 3 shows the changes in blood routine indexes before and after animal whole blood perfusion treatment

[0147]

[0148]

[0149] Table 4 shows the changes in blood biochemical indexes before and after animal whole blood perfusion treatment

[0150]

[0151]

[0152] Table 5 shows the changes in coagulation indexes before and after animal whole blood perfusion treatment

[0153]

[0154] Table 6 shows the changes in immunoglobulins before and after animal whole blood perfusion treatment

[0155] 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 2.31 Quantitative Determination of Immunoglobulin M (IgM) Immunoturbidimetry g / L 0.76 0.51

[0156] It can be seen from the data in Tables 3-6 that after the whole blood perfusion test of the experimental pigs with the adsorbent B-254 in the test example, there were no obvious abnormalities in its blood routine indexes, the white blood cell content increased slightly, and there was 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 establishing a circulation by drawing blood out of the body during whole blood perfusion, resulting in an increase in coagulation time, which would return to normal after 24 hours; the content of immunoglobulin G decreased significantly, and the IgG clearance rate reached 63.3% after one treatment.

[0157] Those skilled in the art know that in other embodiments of the present invention, according to the actual application needs, the immunoglobulin-binding protein can also be one or a mixture of several of recombinant protein G and recombinant protein L by genetic engineering, and all can achieve excellent whole blood adsorption function.

[0158] In summary, the present invention provides a protein A immunosorbent that can be used for whole blood perfusion, its preparation method and application. The immunosorbent includes 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 100 - 400 μm, and the average pore size is 40 - 100 nm. The immunosorbent of the present invention can greatly improve the adsorption effect and shorten the treatment time, achieving the world's first immunosorbent that can be used for whole blood perfusion, overcoming the technical defects in the prior art that the microspheres with too large particle size have poor adsorption performance, and the microspheres with too small particle size are prone to leakage and enter the human blood, which is not conducive to the passage of blood cells and even causes damage to blood cells and platelets. The preparation method of the present invention is simple and low in cost, and has great market prospects and economic value.

[0159] The above embodiments are only used to illustrate the technical solutions of the present invention and not 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 protein A immunosorbent that can be used for whole blood perfusion, characterized in that: The immunosorbent for whole blood perfusion 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 100-400 μm, and the average pore size is 40-100 nm.

2. The protein A immunosorbent for whole blood perfusion according to claim 1, characterized in that: The agarose gel porous microsphere carrier is screened by a rotary vibrating sieve, and the screening process is set as follows: the mixing ratio of microspheres and water is 1: (10-20); the volume of flushing water is set to 15-50 times the volume of the microspheres.

3. The protein A immunosorbent usable for whole blood perfusion according to claim 1, wherein: The immunoglobulin binding protein is one or a mixture of genetically engineered recombinant protein A, genetically engineered recombinant protein G, and genetically engineered recombinant protein L.

4. The protein A immunosorbent for whole blood perfusion according to claim 1, characterized in that: Sepharose porous microsphere carriers are modified by periodate oxidation or epoxidation to couple immunoglobulin binding proteins; The periodate used in the periodate oxidation modification method is potassium periodate, sodium periodate, or a mixture of the two; The epoxy reagent used in the epoxy modification is one or a mixture of epibromohydrin, epichlorohydrin and diepoxy reagents.

5. The preparation method of the protein A immunosorbent usable for whole blood perfusion according to any one of claims 1 to 4, characterized in that: The steps include: P1, preparing agarose gel porous microsphere carriers with an average particle size of 200-300 μm and an average pore size of 40-100 nm; P2, using periodate oxidation or epoxy method to modify the agarose gel porous microsphere carrier to obtain a modified carrier; the modified carrier is mixed with an immunoglobulin binding protein solution to couple the protein to the agarose gel, and post-processed to obtain an immunosorbent for whole blood perfusion.

6. The method for preparing a protein A immunosorbent for whole blood perfusion according to claim 5, characterized in that: The specific process of the periodate oxidation method in step P2 is: P21, using a periodate solution with a concentration of 0.1 to 2.5 mol / L to oxidize the hydroxyl groups of the agarose gel porous microsphere carrier to aldehyde groups to obtain an activated carrier; P22, mixing the activated carrier with the immunoglobulin binding protein solution to couple the protein to the carrier; P23, adjusting the pH value of the mixed solution obtained in step P22, and then adding a reducing agent for reduction to obtain a coupling product; P24, the coupling product is washed and dried to obtain an immunosorbent for whole blood perfusion prepared by the periodate oxidation method, which is stored in a storage solution.

7. The preparation method of the protein A immunosorbent usable for whole blood perfusion according to claim 6, wherein: The reaction temperature in step P21 is 25-40°C, and the reaction time is 2-6 hours; The concentration of the immunoglobulin binding protein solution in step P22 is 7 to 11 mol / L, the volume ratio of the activated carrier to the immunoglobulin binding protein solution is 1:(0.2 to 4), the reaction temperature is 25 to 40° C., and the reaction time is 6 to 24 hours; The reducing agent in step P23 is sodium cyanoborohydride, sodium borohydride, or a mixture of the two in any ratio.

8. The method for preparing a protein A immunosorbent for whole blood perfusion according to claim 5, characterized in that: The specific process of the epoxy method described in step P2 is: P2-1, mixing agarose gel porous microsphere carrier, epoxy reagent and NaOH solution, modifying the surface of the agarose gel porous microsphere carrier with epoxy groups to obtain an activated carrier; P2-2, mixing the activated carrier with the immunoglobulin binding protein solution, adjusting the pH of the mixed solution to 6-11, and adding a solid salt to couple the protein to the carrier to obtain a coupling product; P2-3, the coupling product is washed and dried to obtain an immunosorbent for whole blood perfusion prepared by the epoxy method, which is stored in a storage solution.

9. The preparation method of the protein A immunosorbent usable for whole blood perfusion according to claim 8, characterized in that: In step P2-1, the concentration of the NaOH solution is 0.1 to 1.5 mol / L, 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 to 0.8), the reaction temperature is 15 to 45° C., and the reaction time is 0.5 to 4 h; The concentration of the immunoglobulin binding protein solution in step P2-2 is 7-11 mol / L; the solid salt is one or a mixture of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4, and MnSO4.

10. Use of the protein A immunoadsorbent for whole blood perfusion according to any one of claims 1 to 4, or the protein A immunoadsorbent for whole blood perfusion prepared by the method for preparing the protein A immunoadsorbent for whole blood perfusion according to any one of claims 5 to 9, characterized in that: Application of the protein A immunosorbent that can be used for whole blood perfusion in the technical field of medical device blood purification.

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