Adsorbing material as well as preparation method and application thereof

Abstract

The invention provides an adsorption material and a preparation method and application thereof, the adsorption material comprises a carrier and an endotoxin affinity binding ligand, the endotoxin affinity binding ligand comprises a recombinant C factor protein, and the carrier and the recombinant C factor protein are connected through a spacer arm; wherein the carrier comprises at least one of resin, chitosan, cellulose, agarose and glucan; the spacer arm comprises at least one of modified polyethylene glycol, polyvinyl pyrrolidone, polymethylacrylic acid-2-hydroxyethyl ester, polyvinyl alcohol, polyacrylic acid, hexamethylenediamine, divinyl sulfone and glutaraldehyde, and at least one end of the modified polyethylene glycol is provided with carboxyl, aldehyde or amino. The adsorption material provided by the invention can efficiently adsorb endotoxin, has the characteristics of low toxicity risk and excellent blood compatibility, and can improve the safety of blood purification.

Description

Technical Field

[0001] This invention relates to the field of blood purification technology, and more specifically, to an adsorbent material, its preparation method, and its application. Background Technology

[0002] Endotoxins are lipopolysaccharides (LPS) found in the cell walls of Gram-negative bacteria, released when bacteria die or lyse. Endotoxins bind to Toll-like receptor 4 (TLR4), activating the NF-κB pathway, leading to overactivation of the immune system and the release of pro-inflammatory factors such as TNF-α and IL-1β, triggering a "cytokine storm." Simultaneously, the complement and coagulation systems are activated, resulting in microthrombus formation (DIC) and bleeding tendencies. Timely removal of endotoxins from a patient's body can prevent a vicious cycle of inflammation, prevent further disease progression, and reduce the occurrence of multiple organ failure or death. This not only helps alleviate patient suffering but also reduces the consumption of medical resources.

[0003] Currently, blood purification methods are being increasingly used to adsorb endotoxins from the blood in order to reduce or eliminate them. However, endotoxin adsorption products in related technologies (such as Toray's PMX fiber columns or Baxter's Oxiris membranes) use electrostatic adsorption, which suffers from low adsorption capacity, slow adsorption rate, and unstable adsorption effect. Furthermore, polymyxin B attached to the PMX fiber columns is highly toxic to the human kidneys and nervous system, while the ligands attached to the Oxiris membrane through multiple coatings have a significant risk of detachment. Moreover, because these endotoxin adsorption products adsorb endotoxins through electrostatic adsorption, their surfaces are usually positively charged, easily triggering adverse reactions such as platelet activation, cell adhesion, and blood protein adsorption, resulting in poor compatibility between these endotoxin adsorption products and blood. Summary of the Invention

[0004] The present invention aims to provide an adsorbent material, its preparation method and application. The adsorbent material can efficiently adsorb endotoxins and has the characteristics of low toxicity risk and excellent blood compatibility, which can improve the safety of blood purification.

[0005] To address the aforementioned problems, a first aspect of the present invention provides an adsorbent material comprising a carrier and an endotoxin affinity-binding ligand, wherein the endotoxin affinity-binding ligand comprises a recombinant factor C protein, and the carrier and the recombinant factor C protein are connected by a spacer arm.

[0006] The carrier includes at least one of resin, chitosan, cellulose, agarose, and dextran;

[0007] The spacer arm comprises at least one of modified polyethylene glycol, polyvinylpyrrolidone, poly(2-hydroxyethyl methacrylate), polyvinyl alcohol, polyacrylic acid, hexamethylenediamine, divinyl sulfone, and glutaraldehyde, wherein at least one end of the modified polyethylene glycol has a carboxyl group, an aldehyde group, or an amino group.

[0008] A second aspect of the present invention provides a method for preparing an adsorbent material as described in the first aspect, comprising the following steps:

[0009] Preparation of recombinant factor C protein;

[0010] The carrier is activated to obtain a carrier containing functionalized groups;

[0011] The carrier containing the functionalized group and the spacer arm are connected by a covalent coupling reaction to obtain a carrier with grafted spacer arm.

[0012] The carrier grafted with the spacer arm and the recombinant C factor protein are covalently linked to obtain the adsorbent material.

[0013] The third aspect of the present invention provides the application of an adsorbent material as described in the first aspect, or an adsorbent material prepared by the preparation method described in the second aspect, in blood purification materials or devices.

[0014] The adsorbent material of this invention, by immobilizing recombinant C protein on a carrier, allows the recombinant C protein to specifically recognize lipid A in endotoxins through its active structure. This enables the adsorbent material to specifically and stably adsorb endotoxins from the blood. Furthermore, the recombinant C factor protein has extremely low toxicity risks from contact with blood or detachment, exhibiting good blood compatibility. Adsorbing endotoxins from the blood using the recombinant C factor protein not only achieves highly efficient adsorption of endotoxins but also improves the safety of the adsorption process. The recombinant C factor protein and the carrier are connected by spacer arms. This creates a stable "carrier-spacer arm-recombinant C factor protein" connection structure, increasing the tightness of the connection between the recombinant C factor protein and the carrier, reducing the risk of detachment, and improving the safety of the adsorbent material. Additionally, the spacer arms have a degree of flexibility, reducing steric hindrance and non-specific adsorption of blood proteins, thus enhancing the adsorption capacity of the adsorbent material for endotoxins. Furthermore, the spacer arm selected in this invention does not affect the activity of the recombinant factor C protein or has a minimal impact on its activity when connecting it, which is beneficial for improving the recombinant factor C protein's adsorption capacity for endotoxins. The adsorption material provided in this invention overcomes the shortcomings of endotoxin adsorption products in related technologies. It can not only specifically adsorb endotoxins in the blood, but also the chemically bonded adsorption ligands are not easily detached, the adsorption ligands have low toxicity to the human body, and the adsorption of non-specific proteins is reduced, making it suitable for adsorbing endotoxins in the blood during whole blood perfusion.

[0015] The method for preparing the adsorbent material described in this invention, by functionalizing the outer layer of the carrier, allows modification of only the outer layer of the carrier. By grafting spacer arms and recombinant factor C protein onto the outer layer of the carrier, the hydrophobicity and pore structure stability of the carrier are preserved. This facilitates the movement of endotoxins containing hydrophobic lipid A in the blood toward the carrier, enabling the recombinant factor C protein immobilized on the carrier to specifically adsorb endotoxins in the blood, thereby improving the adsorption capacity for endotoxins and enhancing the adsorption efficiency. In this invention, a carrier containing functionalized groups and a spacer arm are covalently coupled to obtain a spacer arm-grafted carrier. The spacer arm-grafted carrier and recombinant factor C protein are then covalently coupled, with both ends of the spacer arm connected to the carrier and the recombinant factor C protein via chemical bonds. This enables the adsorbent material to form a stable "carrier-spacer arm-recombinant factor C protein" connection structure, improving the tightness of the connection between the recombinant factor C protein and the carrier, reducing the risk of recombinant factor C protein detachment, and enhancing the safety of the adsorbent material. Furthermore, the spacer arm's flexibility, connecting the recombinant factor C protein to the carrier via the spacer arm, reduces steric hindrance, facilitates the exposure of the recombinant factor C protein's active sites, reduces non-specific adsorption of blood proteins, and improves the adsorption capacity of the adsorbent material for endotoxins. In addition, the preparation method of the adsorbent material provided by this invention has a simple process flow, mild reaction conditions, and high safety during the reaction process, making it suitable for industrial production. Moreover, the preparation process has minimal impact on the activity of the recombinant factor C protein, ensuring that the recombinant factor C protein on the adsorbent material retains high activity, thus enabling the recombinant factor C protein to have a good adsorption capacity for endotoxins.

[0016] The adsorbent material described in this invention can be applied to blood purification materials or equipment (such as blood purifiers or hemoperfusion devices). Using the adsorbent material provided by this invention in blood purification materials or equipment allows for whole blood perfusion in patients with endotoxin infections. This adsorbent material can exert therapeutic and regulatory effects on endotoxins and cytokines in the patient's body, providing a more comprehensive and effective treatment method. Attached Figure Description

[0017] Figure 1 This is a process flow diagram for preparing adsorbent materials provided in an embodiment of the present invention;

[0018] Figure 2 This is a gel electrophoresis image of the rFC protein prepared according to Example 1 of the present invention. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0021] Furthermore, the terms "comprising," "including," "containing," and "having" are non-restrictive and can refer to the addition of other steps and components that do not affect the results. Unless otherwise specified, all materials, equipment, and reagents are commercially available.

[0022] Furthermore, although the present invention describes each step in the preparation process in the form of S110, S120, S130 and S140, this description is only for ease of understanding. The forms such as S110, S120, S130 and S140 do not indicate a limitation on the order of the steps.

[0023] A first aspect of this application provides an adsorbent material comprising a carrier and an endotoxin affinity-binding ligand, wherein the endotoxin affinity-binding ligand comprises a recombinant factor C protein, and the carrier and the recombinant factor C protein are connected by a spacer arm, thereby immobilizing the recombinant factor C protein on the carrier.

[0024] The carrier includes at least one of resin, chitosan, cellulose, agarose, and dextran;

[0025] The spacer arm comprises at least one of modified polyethylene glycol, polyvinylpyrrolidone (PVP), poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, polyacrylic acid, hexamethylenediamine, divinyl sulfone, and glutaraldehyde, wherein at least one end of the modified polyethylene glycol has a carboxyl group, an aldehyde group, or an amino group.

[0026] The adsorbent material provided in this embodiment, by immobilizing recombinant C protein on a carrier, allows the recombinant C protein to specifically recognize lipid A in endotoxins through its active structure. This enables the adsorbent material to specifically and stably adsorb endotoxins from the blood. Moreover, the recombinant C factor protein has extremely low toxicity risk from contact with blood or detachment, and its blood compatibility is good. Adsorbing endotoxins from the blood through recombinant C factor protein not only achieves efficient adsorption of endotoxins but also improves the safety of adsorption. The recombinant C factor protein and the carrier are connected by spacer arms. On the one hand, this allows the adsorbent material to form a stable "carrier-spacer arm-recombinant C factor protein" connection structure, improving the tightness of the connection between the recombinant C factor protein and the carrier, reducing the risk of detachment of the recombinant C factor protein, and improving the safety of the adsorbent material. On the other hand, the spacer arms have a certain degree of flexibility. The recombinant C factor protein is connected to the carrier through the spacer arms, which can reduce the influence of steric hindrance and reduce the non-specific adsorption of blood proteins, which is beneficial to improving the adsorption capacity of the adsorbent material for endotoxins. Furthermore, the spacer arm selected in this embodiment does not affect the activity of the recombinant factor C protein or has a minimal impact on its activity when connecting it, which is beneficial for improving the recombinant factor C protein's adsorption capacity for endotoxins. The adsorption material provided in this embodiment improves upon the deficiencies of endotoxin adsorption products in related technologies. It can not only specifically adsorb endotoxins in the blood, but also the adsorption ligands connected by chemical bonds are not easily detached, the adsorption ligands have low toxicity to the human body, and the adsorption of non-specific proteins is reduced, making it suitable for adsorbing endotoxins in the blood during whole blood perfusion.

[0027] In this embodiment, the endotoxin affinity ligand can be recombinant factor C protein, or other molecules or proteins that bind to endotoxins. The recombinant factor C protein in this embodiment is recombinant factor C (rFC) from horseshoe crabs. The rFC in this embodiment is obtained through molecular biology techniques, using eukaryotic or prokaryotic cell expression systems for expression and purification. As a preferred embodiment, the rFC in this embodiment is obtained through eukaryotic cell expression and purification, using pINFUSE-hIgG1-FC2 as the expression gene vector. Therefore, rFC has a bioaffinity for lipid A in the endotoxin structure, and can specifically recognize lipid A to bind to endotoxins in the blood, achieving efficient adsorption of endotoxins from the blood. In this embodiment, humanized rFC protein groups can be designed and synthesized using molecular biology techniques to avoid rejection reactions in the human body and prevent toxicity risks caused by rFC protein contact with blood or detachment, thus further improving the safety of the adsorbent material.

[0028] In this embodiment, the loading of recombinant factor C protein on each 1g of adsorbent material is between 0.4mg and 2.0mg. Therefore, by setting the loading of recombinant factor C protein on the adsorbent material within the above range, the influence of steric hindrance can be further reduced, which is beneficial to further improving the adsorption efficiency of the adsorbent material for endotoxins.

[0029] In this embodiment, at least one of resin, chitosan, cellulose, agarose, and dextran is used as the carrier, which possesses good chemical stability and biocompatibility. Based on the above embodiments, as a preferred embodiment, the carrier is a polystyrene resin, and the polystyrene resin has a particle size of 0.2 mm to 1.8 mm, an average pore size of 5 nm to 100 nm, and a pore volume of 0.3 cm³. 3 / g to 2.5cm 3 / g, with a specific surface area ranging from 200m² 2 / g to 1200m 2 / g. Preferably, the polystyrene resin has a particle size of 0.5 mm to 1.0 mm, an average pore size of 5 nm to 30 nm, and a pore volume of 0.8 cm³. 3 / g to 1.2cm 3 / g, with a specific surface area ranging from 300m² 2 / g to 800m 2 / g. Therefore, the above-mentioned polystyrene resin was selected as the carrier. Polystyrene resin has a rich pore structure and is a hydrophobic framework. The hydrophobicity and rich pore structure of polystyrene resin are conducive to the movement of endotoxins containing hydrophobic lipid A toward the carrier, which is conducive to the specific adsorption of endotoxins by recombinant factor C protein on the adsorption material, thereby improving the adsorption capacity of endotoxins and enhancing the adsorption efficiency.

[0030] In this embodiment, the carrier polystyrene resin can be polystyrene-divinylbenzene resin chlorine beads.

[0031] The polystyrene-divinylbenzene resin chlorospheres are prepared using the following steps: Suspension polymerization step: First, an aqueous phase and an oil phase are prepared separately at room temperature, with a volume ratio of oil phase to aqueous phase of 1:1.5 to 1:3. The oil phase comprises an organic mixture of styrene monomers, divinylbenzene monomers, an initiator, and a porogen, with a mass ratio of styrene monomers to divinylbenzene monomers of 5:1 to 12:1. The aqueous phase comprises an aqueous solution of a dispersant and water, with the dispersant used at 0.8% to 2% of the weight of the aqueous phase, the initiator used at 0.5% to 2% of the total monomer mass fraction, and the porogen used at 60% to 200% of the total monomer mass fraction. Then, the oil phase is poured into the aqueous phase and stirred to disperse. Next, the temperature is raised to 50°C. The reaction is carried out at 90°C for 6 to 20 hours. After the reaction system solidifies, the reaction is stopped to obtain polystyrene-based resin white spheres. The chloromethylation step involves using chloromethyl ether as the reaction solvent and zinc chloride or ferric chloride as the catalyst. The reaction system ratio is: for every 1g of the polystyrene-based resin white spheres added, 2ml to 7mL of chloromethyl ether and 0.2g to 0.5g of the catalyst are added. The dried polystyrene-based resin white spheres are added to the reaction vessel. Then, chloromethyl ether is added to swell the spheres for 1 to 5 hours. Next, the catalyst is added, and the temperature is raised to 45°C to 50°C, followed by a reaction for 20 to 24 hours. The mixture is then cooled to room temperature, the mother liquor is filtered out, and the mixture is extracted with methanol for 12 hours. The methanol is washed away with water, and the mixture is filtered, sieved, and the polystyrene-based resin chloro spheres within the desired particle size range are obtained. It is understood that those skilled in the art can adjust the reaction conditions according to actual conditions to obtain polystyrene-divinylbenzene resin chloro spheres within the above parameter range.

[0032] In addition, the carrier polystyrene resin in this embodiment can also be a secondary crosslinked resin prepared by a post-crosslinking reaction of polystyrene-divinylbenzene resin chlorospheres. The post-crosslinking reaction is as follows: take polystyrene-divinylbenzene resin chlorospheres, add 1,2-dichloroethane equivalent to 5 to 7 times the mass of the chlorospheres, let it stand at 35-45°C for 4 to 5 hours to swell, add anhydrous ferric chloride equivalent to 0.1 to 0.5 times the mass of the chlorospheres under mechanical stirring, and heat at 78-83°C for 10 to 16 hours to obtain the secondary crosslinked resin.

[0033] In this embodiment, both ends of the spacer arm have reactive groups. One end of the spacer arm reacts with the carrier, and the other end reacts with the recombinant C factor protein, so that one end of the spacer arm is connected to the carrier by a chemical bond, and the other end of the spacer arm is connected to the recombinant C factor protein by a chemical bond. The recombinant C reactive protein is immobilized on the carrier through the spacer arm, thereby forming a stable connection structure of "carrier-spacer arm-recombinant C factor protein" in the adsorbent material. Moreover, in this embodiment, the reaction conditions when the spacer arm reacts with the recombinant C factor protein are mild, which does not affect the activity of the recombinant C factor protein or has little impact on the activity of the recombinant C factor protein, so that the recombinant C factor protein has a good adsorption efficiency for endotoxin.

[0034] Based on the above embodiments, as a preferred embodiment, the spacer arm is modified polyethylene glycol (PEG). One end of the modified PEG is hydroxyl or amino, and the other end is carboxyl, aldehyde, or amino, and its main chain is composed of repeated ethylene oxide units. In this embodiment, modified PEG is used as the spacer arm. Modified PEG is a PEG derivative, which retains the good biocompatibility and flexibility of PEG. After the recombinant factor C protein is attached to the spacer arm, the recombinant factor C protein can move flexibly, making it less susceptible to steric hindrance, effectively reducing non-specific adsorption, and avoiding recognition by immune cells such as macrophages. Introducing carboxyl, aldehyde, or amino groups at the end of the PEG enables the grafting of recombinant factor C protein under mild conditions without affecting the adsorption efficiency of the recombinant factor C protein for endotoxins. Furthermore, modifying the end of the PEG can reduce rejection reactions with the organism, lower the risk of inflammatory reactions, and enhance the anticoagulant and antiprotein adsorption capacity of the adsorbent material, reducing immune responses triggered by foreign substances in the body.

[0035] Based on the above embodiments, as a preferred embodiment, the spacer arm is modified polyethylene glycol, with one end of the modified polyethylene glycol having a hydroxyl group and the other end having a carboxyl group, and its main chain being composed of repeating ethylene oxide units. Therefore, modifying only one end of the modified polyethylene glycol and introducing a carboxyl group is beneficial for improving the compatibility of the adsorbent material with blood, and the reaction conditions between the carboxyl group and the recombinant factor C protein are milder, having almost no impact on the activity of the recombinant factor C protein.

[0036] Based on the above embodiments, as an optional implementation, the molecular weight (number average molecular weight, the same below) of the modified polyethylene glycol is 800 Da to 10000 Da. Since the molecular weight of recombinant factor C protein is relatively large (approximately 42,000 Da), if the molecular weight of the modified polyethylene glycol is too small, the shielding effect against non-specific adsorption will be poor. When the recombinant factor C protein is grafted onto a nearby modified polyethylene glycol, a certain steric hindrance will occur, affecting the recombinant factor C protein's adsorption capacity for endotoxins. In other words, a small molecular weight of modified polyethylene glycol has a significant impact on the steric hindrance of the recombinant factor C protein, hindering the exposure of its active sites and affecting its adsorption capacity for endotoxins. Conversely, a large molecular weight of modified polyethylene glycol affects its grafting efficiency onto the carrier, resulting in a lower grafting efficiency of the recombinant factor C protein on the carrier. Therefore, by selecting modified polyethylene glycol within the above molecular weight range in this embodiment, on the one hand, steric hindrance can be reduced, and the active sites of recombinant factor C protein can be better exposed to improve the adsorption capacity for endotoxins. On the other hand, it can avoid affecting the grafting spacer arms on the carrier surface and avoid affecting the grafting efficiency of recombinant factor C protein on the carrier.

[0037] Based on the above embodiments, as a preferred embodiment, the molecular weight of the modified polyethylene glycol is between 2500 Da and 4000 Da. Therefore, with the molecular weight of the modified polyethylene glycol within this range, the influence of steric hindrance on the recombinant factor C protein can be further reduced, the wobble of the recombinant factor C protein can be improved, the active sites of the recombinant factor C protein can be fully exposed, and the range of endotoxin capture by the recombinant factor C protein can be expanded.

[0038] Figure 1 This is a process flow diagram for preparing the adsorbent material provided in the embodiments of this application. (In conjunction with...) Figure 1 As shown, a second aspect of this application provides a method for preparing an adsorbent material, used to prepare the adsorbent material described in the first aspect. The method for preparing the adsorbent material includes:

[0039] Step S110: Prepare recombinant factor C protein.

[0040] The recombinant C factor protein in this embodiment is a recombinant horseshoe crab C factor protein. In this embodiment, the recombinant horseshoe crab C factor protein with endotoxin binding ability is constructed by humanizing the gene sequence of the horseshoe crab C factor protein and using molecular biology techniques.

[0041] Specifically, the Limulus Amebocyte Lysate (LAL) C factor protein gene sequence was obtained, and the LAL C factor protein gene sequence was humanized to obtain a synthesized gene. The synthesized gene was amplified using polymerase chain reaction (PCR) to obtain the amplified gene. The amplified gene was then cleaved using restriction endonucleases. The digested gene fragment was mixed with an expression gene vector digested with the same restriction enzyme, and the gene fragment and expression gene vector were ligated using DNA ligase. Then, plate screening was performed. After positive screening and sequencing, the vector plasmid correctly embedded with the rFC gene was taken and introduced into cells (CHO, HEK293F, or S2, etc.). The cells with the introduced gene were cultured in shake flasks. After culture, the supernatant of the culture medium was collected, enriched using an affinity chromatography column, and the rFC protein was obtained after elution. Therefore, in this embodiment, a humanized rFC protein was designed and synthesized using molecular biology techniques to avoid rejection reactions in the human body and prevent toxicity risks caused by contact with blood or detachment of the rFC protein, thus further improving the safety of the adsorbent material.

[0042] The Limulus Amebocyte Lysate (LAL) C factor protein gene sequence in this embodiment can be obtained from professional websites (such as PDB, Uniprot, NCBI, etc.). The LAL C factor protein gene sequence is then humanized, and the synthesized gene can be sent to commercial companies (such as Nanjing Genscript, Shanghai Sangon Biotech, Shenzhen BGI Genomics, etc.) for synthesis.

[0043] As an optional implementation, in this embodiment, the Limulus amebocyte lysate (LAL) C factor protein gene sequence is humanized, and the synthesized gene sequence is as follows:

[0044] AGCGGTGAATGCCAGTGCAAAAACGGTGGCATCTGCGATCAACGTACCGGTGCTTGCGCTTGCCGTGACCGTTACGAGGGTGTTCACTGCGAAATCCTGAAAGGTTGCCCGCTGCTGCCGAGCGACAGCCAGGTGCAGGAAGTTCGTAACCCGCCGGATAACCCGCAGACCATCGACTACAGCTGTAGCCCGGGTT TCAAGCTGAAAGGCATGGCGCGTATCAGCTGCCTGCCGAACGGCCAGTGGAGCAACTTCCCGCCGAAGTGCATCCGTGAGTGCGCTATGGTGAGCAGCCCGGAACACGGCAAAGTTAACGCGCTGAGCGGTGATATGATCGAGGGCGCTACCCTGCGTTTCAGCTGCGACAGCCCGTACTACCTGATCGGTCAGGAA

[0045] The sequence of the rFC protein obtained in this embodiment is as follows:

[0046] SGECQCKNGGICDQRTGACACRDRYEGVHCEILKGCPLLPSDSQVQE

[0047] VRNPPDNPQTIDYSCSPGFKLKGMARISCLPNGQWSNFPPKCIRECAMVSS

[0048] PEHGKVNALSGDMIEGATLRFSCDSPYYLIGQE

[0049] Based on the above embodiments, as an optional implementation, the expression gene vector is a linear plasmid, which can be at least one of pINFUSE-hIgG-FC2, PTT5, pAc-V5, or pCDNA. Preferably, the expression gene vector is pINFUSE-hIgG-FC2, and the recombinant protein is expressed using CHO cells. Thus, the rFC protein expressed by the vector pINFUSE-hIgG-FC2 carries the FC2 fusion fragment of human IgG protein, composed of CH2 and CH3 structures on the IgG protein heavy chain, with a molecular weight of approximately 27 kDa.

[0050] Step S120: Activate the support to obtain a support containing functional groups.

[0051] Specifically, under the action of a catalyst, the support is functionalized by a modifying agent containing functionalized groups to obtain a support containing functionalized groups; wherein the functionalized groups include at least one selected from chloromethyl, hydroxy, amino, carboxyl, aldehyde, and amide groups. More specifically, the modifying agent containing functionalized groups and the support are mixed and reacted at 40°C to 80°C under the action of a catalyst to obtain a support containing functionalized groups. In this embodiment, the support is modified by functionalized groups, enabling the support to be tightly connected to the spacer arms through chemical bonds, thereby improving the bonding strength between the spacer arms and the support.

[0052] It is understood that the functionalized groups in this embodiment are selected according to the specific type of the spacer arm to ensure that the functionalized modified support can react with one end of the spacer arm, so that one end of the spacer arm is connected to the support through a chemical bond. This embodiment does not further limit the specific reaction temperature and reaction time; those skilled in the art can adjust them according to the selected functionalized groups.

[0053] Based on the above embodiments, as an optional implementation, the functionalized group is chloromethyl, and the modifying agent containing the functionalized group is chloromethyl ether. The following method can be used to prepare the chloromethyl-containing support: Under the action of a catalyst, the support is mixed with chloromethyl ether, and the reaction is continuously stirred at a reaction temperature of 50°C to 80°C. After the reaction is complete, the mixture is first extracted with methanol, then washed with water until no methanol remains, filtered, and dried to obtain the chloromethyl-containing support.

[0054] In this embodiment, the chlorine content in the carrier containing functionalized groups is 5% w / w to 25% w / w, and after the carrier containing functionalized groups is connected to the spacer arm, the chlorine content in the carrier does not exceed 3% w / w. Therefore, by adjusting the chlorine content in the carrier within the above range, the content of the grafted spacer arm on the carrier can be kept within a suitable range, thereby limiting the immobilization content of recombinant factor C protein. This helps to further reduce the influence of steric hindrance, thereby improving the adsorption efficiency of the adsorbent material for endotoxins. The chlorine content in the carrier not exceeding 3% w / w avoids adverse reactions caused by excessive chlorine content.

[0055] Based on the above embodiments, as an optional implementation method, the catalyst is at least one of zinc chloride, ferric chloride, manganese chloride and magnesium chloride.

[0056] Step S130: The support containing the functionalized group and the spacer arm are connected by a covalent coupling reaction to obtain the support with the grafted spacer arm.

[0057] Specifically, a support containing functionalized groups and a spacer arm are covalently coupled under the action of a first activator, so that the spacer arm and the functionalized groups on the support are connected by covalent bonds to obtain a support grafted with a spacer arm; wherein the temperature of the covalent coupling reaction is 60°C to 80°C. More specifically, after swelling the support containing functionalized groups and dissolving the spacer arm, the functionalized groups and the spacer arm are mixed evenly to obtain a first mixed solution; the first activator is added to the first mixed solution to cause the support containing functionalized groups and the spacer arm to undergo a covalent coupling reaction, and the reaction is continuously stirred until the reaction is completed to obtain a support grafted with a spacer arm. In this embodiment, the support containing functionalized groups and the spacer arm are connected by a covalent coupling reaction, which improves the connection stability between the spacer arm and the support, making the spacer arm and the support firmly connected, and the reaction conditions of the covalent coupling reaction are mild and do not affect the performance of the support or the spacer arm.

[0058] The covalent coupling reaction in this embodiment includes nucleophilic substitution, peracetic acid method, or ethylene oxide method. Preferably, the nucleophilic substitution method is used in this embodiment.

[0059] Based on the above embodiments, as an optional implementation, the temperature and time of the covalent coupling reaction are positively correlated with the molecular weight of the spacer arm; that is, the larger the molecular weight of the spacer arm, the higher the temperature of the covalent coupling reaction and the longer the reaction time. This ensures that the spacer arm and the support containing the functionalized groups react fully. In this embodiment, the temperature of the covalent coupling reaction is 60°C to 80°C, and the reaction time is 6 hours to 36 hours.

[0060] Based on the above embodiments, the molar ratio of the spacer arms to the functionalized groups on the support is 1 to 10:1. The amount of spacer arms used is higher than that of the functionalized groups on the support, ensuring sufficient reaction between the spacer arms and the functionalized groups on the support, which is beneficial for the functionalized groups on the support to connect to more spacer arms.

[0061] Based on the above embodiments, as an optional implementation, the first activator is sodium hydroxide, and the amount of sodium hydroxide used is 1 to 3 times the molar amount of the group at one end of the spacer arm. The group at one end of the spacer arm refers to the group at the end connected to the functionalized group on the support. For example, when the spacer arm is modified polyethylene glycol, and one end of the modified polyethylene glycol is hydroxyl or amino, and the other end is carboxyl, aldehyde, or amino, sodium hydroxide is added at 1 to 3 times the molar amount of the hydroxyl or amino group of the modified polyethylene glycol. Preferably, sodium hydroxide is added at 1.5 times the molar amount of the hydroxyl or amino group of the modified polyethylene glycol. Thus, when the amount of the first activator is within the above range, it can enhance nucleophilicity, optimize product selectivity, promote the activation of functional groups, and lower the reaction energy barrier.

[0062] Step S140: The carrier grafted with the spacer arm and the recombinant C factor protein are covalently linked to obtain the adsorbent material.

[0063] Specifically, under weakly acidic conditions, the carrier grafted with the spacer arm is activated with a second activator, thereby activating the free ends of the spacer arm. Under neutral or weakly alkaline conditions, the carrier with the activated free ends of the spacer arm undergoes a covalent reaction with recombinant factor C protein, allowing the recombinant factor C protein to connect to the carrier through the spacer arm, thus obtaining the adsorbent material. More specifically, the carrier grafted with the spacer arm is added to a weakly acidic first buffer solution to swell, thereby opening the pore structure of the carrier and promoting subsequent reactions. Then, the carrier grafted with the spacer arm is added to activate the free ends of the spacer arm, and then the carrier grafted with the spacer arm is washed to neutrality. In a neutral or weakly alkaline second buffer solution at room temperature, the carrier with the activated free ends of the spacer arm undergoes a covalent reaction with recombinant factor C protein, allowing the recombinant factor C protein to connect to the carrier through the spacer arm. After the reaction is complete, the unreacted residual groups are blocked with a capping agent, and then the mixture is washed with a neutral solution to obtain the adsorbent material. In this embodiment, the free end of the spacer arm is first activated under weakly acidic conditions, which is beneficial to improving the binding activity between the free end of the spacer arm and the recombinant factor C protein. Then, the carrier grafted with the spacer arm undergoes a covalent reaction with the recombinant factor C protein. Through the covalent reaction, the spacer arm and the recombinant factor C protein are linked by covalent bonds, which improves the connection stability between the spacer arm and the recombinant factor C protein and makes the spacer arm and the recombinant factor C protein firmly connected. The covalent reaction has excellent selectivity and can achieve precise connection between the spacer arm and the recombinant factor C protein. Moreover, the covalent reaction conditions are mild and have minimal impact on the activity of the recombinant factor C protein. In addition, in this embodiment, the recombinant factor C protein can maintain its activity under neutral or weakly alkaline conditions without affecting the recombinant factor C protein's adsorption capacity for endotoxins.

[0064] Based on the above embodiments, as a preferred embodiment, the spacer arm is modified polyethylene glycol (PEG). One end of the modified PEG is hydroxyl or amino, and the other end is carboxyl, aldehyde, or amino. The functionalized groups on the support are covalently coupled to the hydroxyl or amino group at one end of the modified PEG, thus immobilizing the spacer arm on the support. The carboxyl, aldehyde, or amino group at the other end of the modified PEG serves as the free end of the spacer arm. After activation, it undergoes a covalent reaction with the amino group on the recombinant factor C protein, thus immobilizing the recombinant factor C protein on the spacer arm, forming a stable "carrier-spacer arm-recombinant factor C protein" connection structure. Preferably, one end of the modified PEG is hydroxyl, and the other end is carboxyl. The functionalized groups on the support are covalently coupled to the hydroxyl group at one end of the modified PEG, and the carboxyl group at the other end of the modified PEG serves as the free end of the spacer arm. After activation, it undergoes a covalent reaction with the amino group on the recombinant factor C protein.

[0065] Based on the above embodiments, as an optional implementation, the pH of the first buffer solution is 4 to 6, and the first buffer solution is at least one selected from borate buffer (BB), phosphate buffer (PBS), 2-(N-morpholine)ethanesulfonic acid (MES), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), preferably 2-(N-morpholine)ethanesulfonic acid (MES). Thus, a first buffer solution with a pH of 4 to 6 can improve the activity of the free end of the spacer arm.

[0066] Based on the above embodiments, as an optional implementation, the second activator is at least one selected from carbodiimide, hydroxysuccinimide ester, glutaraldehyde, diethylene sulfone, and maleimide. The carbodiimide includes, but is not limited to, at least one selected from N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride (EDC); the hydroxysuccinimide ester includes, but is not limited to, at least one selected from N-hydroxysuccinimide (NHS), 1-pyrenebutyric acid-N-hydroxysuccinimide ester, sulfonic acid-N-hydroxysuccinimide ester, lysine-N-hydroxysuccinimide ester, and palmitic acid-N-hydroxysuccinimide ester.

[0067] Based on the above embodiments, as a preferred embodiment, the second activator is carbodiimide and hydroxysuccinimide ester, wherein the final concentration of carbodiimide is 2 mmol / L to 100 mmol / L, and the final concentration of hydroxysuccinimide ester is 5 mmol / L to 250 mmol / L. Preferably, the second activator is EDC and NHS, and the molar ratio of EDC to NHS is 1:2.5. Thus, by using carbodiimide and hydroxysuccinimide ester in combination, the activity of the terminal groups of the spacer arm can be activated, especially the activity of the carboxyl groups on the spacer arm, and the terminal groups of the spacer arm can be kept stable for a longer period of time, which is beneficial to improving the grafting efficiency of recombinant factor C protein onto the spacer arm.

[0068] Based on the above embodiments, as an optional implementation, the pH of the second buffer is 7 to 8, and the second buffer can be PBS buffer. Therefore, a pH of 7 to 8 for the second buffer ensures the activity of the recombinant factor C protein.

[0069] Based on the above embodiments, as an optional implementation, when the carrier grafted with the spacer arm and the recombinant C factor protein undergo a covalent reaction, the reaction is carried out with a molar ratio of the free end of the spacer arm to the recombinant C factor protein of 1:1 to 1:4. Preferably, the reaction is carried out with a molar ratio of the free end of the spacer arm to the recombinant C factor protein of 1:2. Thus, the amount of recombinant C factor protein is excessive compared to the amount of the free end of the spacer arm, which is beneficial for grafting the recombinant C factor protein onto the spacer arm of the carrier, thereby improving the grafting efficiency of the recombinant C factor protein in the adsorbent material. Simultaneously, it reduces the influence of steric hindrance during endotoxin adsorption, which is beneficial for improving the efficiency of endotoxin adsorption.

[0070] Based on the above embodiments, as an optional implementation, the capping agent is a reagent containing a primary amino group. This reagent can be a glycine buffer solution with a final concentration of 50 mmol / L to 2000 mmol / L, a tris(hydroxymethyl)aminomethane (Tris) buffer solution with a final concentration of 50 mmol / L to 1000 mmol / L, or an ethanolamine solution with a final concentration of 20 mmol / L to 200 mmol / L. Preferably, the capping agent is a glycine buffer solution with a final concentration of 500 mmol / L. Therefore, using a reagent containing a primary amino group as the capping agent can effectively block residual carboxyl groups and create steric hindrance, preventing non-specific adsorption of substances in the blood. Furthermore, the reaction conditions between the reagent containing the primary amino group and the carboxyl group are mild, requiring no complex protecting groups to assist the reaction. This not only makes the operation convenient but also better maintains the activity of the recombinant factor C protein, with minimal impact on its activity.

[0071] The method for preparing the adsorbent material provided in this embodiment can achieve modification of only the outer layer of the carrier by functionalizing the outer layer of the carrier. By grafting spacer arms and recombinant factor C protein onto the outer layer of the carrier, the hydrophobicity and pore structure stability of the carrier are preserved. This is conducive to the movement of endotoxins containing hydrophobic lipid A in the blood toward the carrier, and the recombinant factor C protein immobilized on the carrier specifically adsorbs endotoxins in the blood, thereby improving the adsorption capacity for endotoxins and enhancing the adsorption efficiency. In this embodiment, a carrier containing functionalized groups and a spacer arm are covalently coupled to obtain a spacer arm-grafted carrier. The spacer arm-grafted carrier and recombinant factor C protein are then covalently coupled, with both ends of the spacer arm connected to the carrier and the recombinant factor C protein via chemical bonds. This allows the adsorbent material to form a stable "carrier-spacer arm-recombinant factor C protein" connection structure, improving the tightness of the connection between the recombinant factor C protein and the carrier, reducing the risk of recombinant factor C protein detachment, and enhancing the safety of the adsorbent material. Furthermore, the spacer arm's flexibility reduces steric hindrance, allowing the recombinant factor C protein to be connected to the carrier, thus facilitating the exposure of its active sites and reducing non-specific adsorption of blood proteins, thereby improving the adsorbent material's adsorption capacity for endotoxins. In addition, the preparation method of the adsorbent material provided in this embodiment has a simple process flow, mild reaction conditions, and high safety during the reaction process, making it suitable for industrial production. Moreover, the preparation process has minimal impact on the activity of the recombinant factor C protein, ensuring that the recombinant factor C protein on the adsorbent material retains high activity, thereby enabling the recombinant factor C protein to have a good adsorption capacity for endotoxins.

[0072] A third aspect of this application provides an application of an adsorbent material that can be used in blood purification materials or devices (such as blood purifiers or hemoperfusion devices). Using the adsorbent material provided in this application in blood purification materials or devices allows for whole blood perfusion in patients with endotoxin infection. This adsorbent material can exert therapeutic and regulatory effects on endotoxins and cytokines in the patient's body, providing a more comprehensive and effective treatment method.

[0073] To provide a more detailed description of the present invention, specific embodiments will be used to further illustrate the invention. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods; the materials and reagents used in the embodiments of the present invention are commercially available unless otherwise specified. To avoid the influence of various impurities and metal ions in the water, the water used in the preparation of the adsorbent material must be purified water or water for injection, and the water must meet the water quality requirements for purified water or water for injection in the Pharmacopoeia of the People's Republic of China to ensure safe use for medical device disinfection.

[0074] Example 1

[0075] This embodiment provides an adsorbent material, which is prepared by the following method:

[0076] (1) Preparation of recombinant factor C protein: Gene sequence 1 and gene sequence 2 were expressed using the CHO eukaryotic cell expression system to obtain recombinant factor C protein. Specifically: the Limulus amebocyte lysate (LAL) factor C protein gene sequence was obtained, and the LAL factor C protein gene sequence was humanized to obtain the synthesized gene (as shown in the synthesized gene sequence above). The amplified synthesized gene was cleaved with restriction endonuclease and mixed with the linear plasmid pINFUSE-hIgG1-FC2 digested with the same enzyme. The mixture was ligated using T4 DNA ligase at 16–22℃ for 1–3 h, and then plated for screening. After positive screening test and measurement... Following this, the plasmid correctly embedded with the rFC gene (pINFUSE-hIgG1-FC2-rFC) was introduced into CHO cells and cultured in shake flasks at 37°C and 100 rpm for 10 days. After the culture was completed, the supernatant of the culture medium was collected, and affinity chromatography was performed using a Protein A / G column to obtain rFC protein samples (as shown in the sequence of the rFC protein obtained above). The purity of the rFC protein samples was then confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). When the purity of the rFC protein samples was not less than 95%, the rFC protein was used as the target recombinant factor C protein.

[0077] (2) Functional modification of the carrier: 50g of polystyrene-divinylbenzene resin (polystyrene-divinylbenzene white spheres obtained by suspension polymerization) was weighed as the carrier. The polystyrene-divinylbenzene resin had a particle size of 0.5-1.0mm, an average pore size range of 8.79nm, and a pore volume of 1.1cm³. 3 / g, specific surface area 700m² 2 / g; Add 350mL of chloromethyl ether to polystyrene-divinylbenzene resin and let stand at room temperature for 1.5 hours; start stirring and maintain, add 50g of zinc chloride as a catalyst, heat to 50℃, and react for 24 hours; after the reaction is complete, filter off the mother liquor, extract with methanol for 12 hours, wash with ultrapure water until no methanol is present, and then filter and dry to obtain pale yellow chlorospheres, i.e., chloromethylated resin. The chlorine content in the chlorospheres was determined to be 10.24% by the Volhard method.

[0078] (3) Preparation of the graft spacer: The chloromethylated resin obtained in step (2) was soaked in N,N-dimethylformamide (DMF) and allowed to stand at room temperature for 2 hours to remove the solvent; modified polyethylene glycol 3350 (which contains a carboxyl group at one end and has the structure HO-PEG-COOH) was weighed and dissolved in DMF and stirred until completely dissolved. The swollen resin was added to the modified polyethylene glycol 3350 solution according to the molar ratio of modified polyethylene glycol 3350 to chloromethyl groups of 1.5:1. Add sodium hydroxide at 1.5 times the molar amount of hydroxyl groups of 3350 and stir for 10 min to activate the hydroxyl groups; heat to 70℃ and continue stirring for 18 h; cool the reaction solution to room temperature and add dilute hydrochloric acid to neutralize to pH 7; after separating the resin, wash 3-6 times with DMF, deionized water, ethanol, and acetone respectively, and filter until the filtrate is colorless and transparent. Place it in a vacuum drying oven and dry at 40-50℃ to constant weight to obtain the modified resin (i.e., the carrier for grafting spacer arms). The chlorine content of the modified resin is 2.56%, and the grafting rate is calculated to be 75%.

[0079] (4) Preparation of adsorbent material: Disperse 10g of the modified resin obtained in step (3) in a 25mmol / L MES buffer solution with a pH of 5.0, and allow it to swell at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. Wash the resin three times with the same buffer solution. Dissolve 10mmol / L of EDC and 20mmol / L of NHS in a 5.0 MES buffer solution (as above), mix them thoroughly, and use the mixture as an activator. Add the activator to the modified resin solution and allow it to react with shaking at room temperature for 15 minutes. After the reaction is complete, wash the resin with a PBS buffer solution with a pH of 7.4 to obtain the activated modified resin. The target rFC protein from step (1) was dissolved in PBS buffer at pH 7.4. The modified resin was then added to the activated resin at a molar ratio of 1:1 (carboxyl groups on the modified resin to rFC protein) to achieve a final rFC protein concentration of 1 mg / mL. The mixture was reacted at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, resulting in a final concentration of 500 mmol / L. The mixture was then reacted at room temperature with shaking for 1 hour to quench residual activating groups. The resin was then washed sequentially with a buffer containing Tween-20, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, thus obtaining the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 65.9%.

[0080] Example 2

[0081] This embodiment provides an adsorbent material, the preparation method of which is basically the same as that in Example 1, except that the adsorbent material is prepared according to the following steps:

[0082] The 10g modified resin obtained in step (3) of Example 1 was dispersed in a 25mmol / L MES buffer solution with a pH of 5.0 and swollen at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer solution. A final EDC concentration of 10mmol / L and a final NHS concentration of 20mmol / L were dissolved in a 5.0 MES buffer solution (as described above), mixed thoroughly, and used as an activator. This activator was added to the modified resin solution, and the mixture was shaken at room temperature for 15 minutes. After the reaction, the resin was washed clean with a PBS buffer solution with a pH of 7.4 to obtain the activated modified resin. The target rFC protein in step (1) was dissolved in PBS buffer at pH 7.4. The modified resin was added to the activated resin at a molar ratio of 1:2 (carboxyl groups on the modified resin to rFC protein) to achieve a final rFC protein concentration of 1 mg / mL. The mixture was reacted at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, resulting in a final concentration of 500 mmol / L. The mixture was then reacted at room temperature with shaking for 1 hour to quench any residual activating groups. The resin was then washed sequentially with a buffer containing Tween-20, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, thus obtaining the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 78.7%.

[0083] Example 3

[0084] This embodiment provides an adsorbent material, the preparation method of which is basically the same as that in Example 1, except that the adsorbent material is prepared according to the following steps:

[0085] The 10g modified resin obtained in step (3) of Example 1 was dispersed in a 25mmol / L MES buffer solution with a pH of 5.0 and swollen at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer solution. A final EDC concentration of 10mmol / L and a final NHS concentration of 20mmol / L were dissolved in a 5.0 MES buffer solution (as described above), mixed thoroughly, and used as an activator. This activator was added to the modified resin solution, and the mixture was shaken at room temperature for 15 minutes. After the reaction, the resin was washed clean with a PBS buffer solution with a pH of 7.4 to obtain the activated modified resin. In step (1), the target rFC protein was dissolved in PBS buffer at pH 7.4. The modified resin was then added to the activated resin at a molar ratio of 1:4 (carboxyl groups on the modified resin to rFC protein), resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was then shaken at room temperature for 1 hour to quench any residual activating groups. The resin was then washed sequentially with a Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, yielding the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 85.3%.

[0086] Example 4

[0087] This embodiment provides an adsorbent material, which is prepared by the following method:

[0088] (1) The preparation method of recombinant factor C protein is the same as that in Example 1.

[0089] (2) Functional modification of the carrier: 50g of polystyrene-divinylbenzene resin (polystyrene-divinylbenzene white spheres obtained by suspension polymerization) was weighed as the carrier. The polystyrene-divinylbenzene resin had a particle size of 0.5-1.0mm, an average pore size range of 26.54nm, and a pore volume of 0.9cm³. 3 / g, specific surface area 500m² 2 / g; Add 350mL of chloromethyl ether to polystyrene-divinylbenzene resin and let it stand at room temperature for 1.5 hours; start stirring and maintain, add 50g of zinc chloride as a catalyst, heat to 50℃, and react for 24 hours; after the reaction is complete, filter off the mother liquor, extract with methanol for 12 hours, wash with ultrapure water until no methanol is present, and then filter and dry to obtain pale yellow chlorospheres, i.e., chloromethylated resin. The chlorine content in the chlorospheres was determined to be 9.97% by the Volhard method.

[0090] (3) Preparation of the graft spacer: The chloromethylated resin obtained in step (2) was soaked in N,N-dimethylformamide (DMF) and allowed to stand at room temperature for 2 hours to remove the solvent; modified polyethylene glycol 3350 (which contains a carboxyl group at one end) was weighed and dissolved in DMF and stirred until completely dissolved. The swollen resin was added to the modified polyethylene glycol 3350 solution according to the molar ratio of modified polyethylene glycol 3350 to chloromethyl groups of 1.5:1. Add 1.5 times the amount of sodium hydroxide and stir for 10 min to activate the hydroxyl groups; heat to 70℃ and continue stirring for 18 h; cool the reaction solution to room temperature and add dilute hydrochloric acid to neutralize to pH 7; after separating the resin, wash 3-6 times with DMF, deionized water, ethanol, and acetone respectively, and filter until the filtrate is colorless and transparent. Place it in a vacuum drying oven and dry at 40-50℃ to constant weight to obtain the modified resin (i.e., the carrier for grafting spacers). The chlorine content of the modified resin is 2.99%, and the grafting rate is calculated to be 70%.

[0091] (4) Preparation of adsorbent material: Disperse 10g of the modified resin obtained in step (3) in a 25mmol / L MES buffer solution with a pH of 5.0, and allow it to swell at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. Wash the resin three times with the same buffer solution. Dissolve 10mmol / L of EDC and 20mmol / L of NHS in a 5.0 MES buffer solution (as above), mix them thoroughly, and use the mixture as an activator. Add the activator to the modified resin solution and allow it to react with shaking at room temperature for 15 minutes. After the reaction is complete, wash the resin with a PBS buffer solution with a pH of 7.4 to obtain the activated modified resin. The target rFC protein from step (1) was dissolved in PBS buffer at pH 7.4. The modified resin was then added to the activated resin at a molar ratio of 1:1 (carboxyl groups on the modified resin to rFC protein), resulting in a final rFC protein concentration of 1 mg / mL. The mixture was reacted at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, resulting in a final concentration of 500 mmol / L. The mixture was then reacted at room temperature with shaking for 1 hour to quench residual activating groups. The resin was then washed sequentially with a Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, thus obtaining the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 60.6%.

[0092] Example 5

[0093] This embodiment provides an adsorbent material, which is prepared using the same method as that in Example 4, except that the adsorbent material is prepared according to the following steps:

[0094] The 10g modified resin obtained in step (3) of Example 4 was dispersed in a 25mmol / L MES buffer solution at pH 5.0 and swollen at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer solution. A final EDC concentration of 10mmol / L and a final NHS concentration of 20mmol / L were dissolved in a 5.0 MES buffer solution (as described above), mixed thoroughly, and used as an activator. This activator was added to the modified resin solution, and the mixture was shaken at room temperature for 15 minutes. After the reaction, the resin was washed thoroughly with a PBS buffer solution at pH 7.4 to obtain the activated modified resin. In step (1), the target rFC protein was dissolved in PBS buffer at pH 7.4. The modified resin was then added to the activated resin at a molar ratio of 1:2 (carboxyl groups on the modified resin to rFC protein), resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was then shaken at room temperature for 1 hour to quench any residual activating groups. The resin was then washed sequentially with a Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, yielding the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 72.1%.

[0095] Example 6

[0096] This embodiment provides an adsorbent material, which is prepared by a method that is basically the same as that in Example 4, except that the adsorbent material is prepared according to the following steps:

[0097] The 10g modified resin obtained in step (3) of Example 4 was dispersed in a 25mmol / L MES buffer solution at pH 5.0 and swollen at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer solution. A final EDC concentration of 10mmol / L and a final NHS concentration of 20mmol / L were dissolved in a 5.0 MES buffer solution (as described above), mixed thoroughly, and used as an activator. This activator was added to the modified resin solution, and the mixture was shaken at room temperature for 15 minutes. After the reaction, the resin was washed thoroughly with a PBS buffer solution at pH 7.4 to obtain the activated modified resin. In step (1), the target rFC protein was dissolved in PBS buffer at pH 7.4. The modified resin was then added to the activated resin at a molar ratio of 1:4 (carboxyl groups on the modified resin to rFC protein), resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was then shaken at room temperature for 1 hour to quench any residual activating groups. The resin was then washed sequentially with Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, yielding the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 81.4%.

[0098] Comparative Example 1

[0099] This comparative example provides a method for preparing an adsorbent material. The preparation methods of steps (1) and (2) of this adsorbent material are basically the same as those in Example 1, except that steps (3) and (4) are prepared according to the following steps:

[0100] Preparation of the graft spacer arm carrier: Take 10g of the chloromethylated resin obtained in step (2) of Example 1, add 10mL of peracetic acid solution (30% w / v), place it in a 500mL Erlenmeyer flask, and shake at 25℃ (200rpm) for 6 hours. After the reaction, discard the supernatant, wash the resin 3 times with deionized water, and vacuum dry the resin (40℃, 2 hours) to obtain epoxidized polystyrene resin; add 200mL of borate buffer (pH 9.0), shake at room temperature (150rpm) for 30 minutes to fully open the epoxy groups; mix the activated resin with 200mL of modified polyethylene glycol 3350 (which contains a carboxyl group at one end) solution, place it in a flask, and shake slowly at 4℃ (100rpm) for 12 hours. Centrifuge (3000rpm, 5 minutes) and collect the supernatant; add 200mL of glycine solution (100mol / L, pH 9.0) to the resin. 8.0) End capping, shaking at room temperature (150 rpm) for 2 hours, centrifugation to discard the supernatant, washing the resin 3 times with PBS buffer to obtain the modified resin, which is the carrier for grafting spacer arms.

[0101] Preparation of adsorbent material: 10g of the modified resin obtained in the previous step was dispersed in a 25mmol / L MES buffer solution with a pH of 5.0 and allowed to swell at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer solution. A final EDC concentration of 10mmol / L and a final NHS concentration of 20mmol / L were dissolved in a MES buffer solution with a pH of 5.0 (as described above). After thorough mixing, the mixture was used as an activator. The activator was added to the modified resin solution and the mixture was shaken at room temperature for 15 minutes. After the reaction, the resin was washed with a PBS buffer solution with a pH of 7.4 to obtain the activated modified resin. The steps of Example 1 (1) were then... The target rFC protein was dissolved in PBS buffer at pH 7.4. The modified resin was added to the activated resin at a molar ratio of carboxyl groups on the modified resin to rFC protein of 1:2, resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4–12 hours (the reaction time can be extended to improve the grafting rate). Glycine buffer was added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was then shaken at room temperature for 1 hour to quench residual activating groups. The resin was then washed sequentially with Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, yielding the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 71%.

[0102] Comparative Example 2

[0103] This comparative example provides a method for preparing an adsorbent material. The preparation methods of steps (1) and (2) of this adsorbent material are basically the same as those in Example 4, except that steps (3) and (4) are prepared according to the following steps:

[0104] Preparation of the graft spacer arm carrier: Take 10 g of the chloromethylated resin obtained in step (2) of Example 4, add 10 mL of peracetic acid solution (30% w / v), place it in a 500 mL Erlenmeyer flask, and shake the reaction at 25 °C (200 rpm) for 6 hours. After the reaction is complete, discard the supernatant, wash the resin 3 times with deionized water, and vacuum dry the resin (40 °C, 2 hours) to obtain epoxidized polystyrene resin; add 200 mL of borate buffer (pH 10). 9.0), shake at room temperature (150 rpm) for 30 minutes to fully open the epoxy groups; mix the activated resin with 200 mL of modified polyethylene glycol 3350 (which contains a carboxyl group at one end) solution, place in a flask, and react slowly at 4°C (100 rpm) for 12 hours (avoid vigorous shaking to prevent protein denaturation), centrifuge (3000 rpm, 5 minutes), and collect the supernatant; add 200 mL of glycine solution (100 mol / L, pH 8.0) to the resin for end-capping, shake at room temperature (150 rpm) for 2 hours, centrifuge and discard the supernatant, wash the resin 3 times with PBS buffer to obtain the modified resin, which is the carrier for grafting spacer arms.

[0105] Preparation of adsorbent material: 10g of the modified resin obtained in the previous step was dispersed in PBS buffer (pH 6.0, 25 mmol / L) and swollen at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer. 10 mmol / L EDC and 20 mmol / L NHS were dissolved in PBS buffer (pH 6.0, as above) and mixed thoroughly. The mixture was then used as an activator. The activator was added to the modified resin solution and the mixture was shaken at room temperature for 15 minutes to obtain the activated modified resin. The target rFC protein from step (1) of Example 4 was dissolved in PBS buffer (pH 6.0). PBS buffer with H=7.4 was added to the activated modified resin at a molar ratio of carboxyl groups on the modified resin to rFC protein of 1:2, resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4–12 hours (the reaction time can be extended to improve the grafting rate). Glycine buffer was added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was then shaken at room temperature for 1 hour to quench any residual activating groups. The resin was then washed sequentially with Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, yielding the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 71%.

[0106] Comparative Example 3

[0107] This comparative example provides an adsorbent material prepared by the following method:

[0108] (1) The preparation method of recombinant factor C protein is the same as that in Example 1.

[0109] (2) Carboxylation modification of the carrier: 10g of polystyrene-divinylbenzene resin was weighed as the carrier. The polystyrene-divinylbenzene resin had a particle size of 0.5-1.0mm, an average pore size range of 26.54nm, and a pore volume of 0.9cm³. 3 / g, specific surface area 500m² 2 / g; Polystyrene-divinylbenzene resin was added to 50g of an acidic potassium permanganate solution (which can be sulfuric acid or potassium permanganate solution) with a mass concentration of 5% w / v and a pH of 2. The acidic potassium permanganate solution and polystyrene-divinylbenzene resin were subjected to an oxidation reaction at a reaction temperature of 25℃ for 0.5h. After the reaction was completed, the oxidation reaction product was obtained. The oxidation reaction product was washed with a mass concentration of 1wt% hydrochloric acid solution to remove the by-products generated by the oxidation reaction. Then, it was washed with water until neutral to obtain polystyrene-divinylbenzene resin containing carboxyl groups, which was used as the modified resin.

[0110] (3) Preparation of adsorbent material: The modified resin was dispersed in PBS buffer at pH 6 with a solvent of 25 mmol / L and swollen at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer. EDC (final concentration 10 mmol / L) and NHS (final concentration 20 mmol / L) were dissolved in PBS buffer at pH 6.0 (as above), mixed thoroughly, and used as an activator. The activator was added to the modified resin solution and reacted with shaking at room temperature for 15 minutes to obtain the activated modified resin. The target rFC protein was dissolved in PBS buffer at pH 6.0. PBS buffer at 7.4 (7.0–8.0) was added to the activated modified resin at a molar ratio of 1:2 (carboxyl groups on the resin to rFC protein), resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4 hours. Glycine buffer was then added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was shaken at room temperature for 1 hour to quench any residual activating groups. The resin was then washed sequentially with Tween-20 buffer, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts. The modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material, was obtained and stored at 4°C for later use. The concentration of unbound protein in the supernatant was measured, and the grafting rate was calculated to be 54%.

[0111] Comparative Example 4

[0112] This comparative example provides a method for preparing an adsorbent material. The preparation methods of steps (1) and (2) of this adsorbent material are basically the same as those in Example 4, except that steps (3) and (4) are prepared according to the following steps:

[0113] Preparation of the graft spacer carrier: The chloromethylated resin obtained in step (2) of Example 4 was soaked in N,N-dimethylformamide (DMF) and allowed to stand at room temperature for 2 hours to remove the solvent; modified polyethylene glycol 750 (which contains a carboxyl group at one end) was weighed and dissolved in DMF and stirred until completely dissolved. The swollen resin was added to the modified polyethylene glycol 750 solution according to the molar ratio of modified polyethylene glycol 750 to chloromethyl groups of 1.5:1. Add 1.5 times the amount of sodium hydroxide and stir for 10 min to activate the hydroxyl groups; heat to 70℃ and continue stirring for 18 h; cool the reaction solution to room temperature and add dilute hydrochloric acid to neutralize to pH 7; after separating the resin, wash 3 to 6 times with DMF, deionized water, ethanol, and acetone respectively, and filter until the filtrate is colorless and transparent. Place it in a vacuum drying oven and dry at 40 to 50℃ to constant weight to obtain the modified resin (i.e., the carrier for grafting spacers). The chlorine content of the modified resin is 2.84%, and the grafting rate is calculated to be 70%.

[0114] Preparation of adsorbent material: 10g of the modified resin obtained in the previous step was dispersed in a 25mmol / L MES buffer solution at pH 5.0 and allowed to swell at room temperature for 1-2 hours to open the pores of the modified resin and promote subsequent reactions. The resin was then washed three times with the same buffer solution. A final EDC concentration of 10mmol / L and a final NHS concentration of 20mmol / L were dissolved in a 5.0 MES buffer solution (as described above), and the mixture was thoroughly mixed. This mixture was then used as an activator. The activator was added to the modified resin solution, and the mixture was shaken at room temperature for 15 minutes. After the reaction, the resin was washed thoroughly with a PBS buffer solution at pH 7.4 to obtain the activated modified resin. The following steps were then performed. In step (1) of Example 4, the target rFC protein was dissolved in PBS buffer at pH 7.4. The modified resin was added to the activated resin at a molar ratio of 1:2 (carboxyl groups on the modified resin to rFC protein), resulting in a final rFC protein concentration of 1 mg / mL. The reaction was carried out at room temperature or 4°C with shaking for 4 hours. Glycine buffer was added for end-capping, bringing the final concentration to 500 mmol / L. The mixture was then shaken at room temperature for 1 hour to quench any residual activating groups. The resin was then washed sequentially with a buffer containing Tween-20, deionized water, and ethanol by centrifugation to remove unbound protein and byproducts, yielding the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. This adsorbent material was stored at 4°C for later use. The concentration of unbound rFC protein in the supernatant was measured, and the grafting rate was calculated to be 79%.

[0115] Comparative Example 5

[0116] This comparative example provides an adsorbent material prepared by the following method:

[0117] (1) The preparation method of recombinant factor C protein is the same as that in Example 1.

[0118] (2) Functional modification of the carrier: Measure 200 mL of sodium carbonate solution (300 mmol / L, pH 10.5-12.5), add 10 g of 4% w / v agarose microspheres 6B-CL, and finally add 7 mL of diethylene sulfone. Stir continuously for 12 h, filter under vacuum, and wash with ultrapure water to obtain modified agarose microspheres, which are carriers containing functional groups. Store at 4 °C.

[0119] (3) Preparation of the carrier for grafting spacer arms: Weigh 1g of the modified agarose microspheres obtained in step (2) and immerse them in 2mmol / L ethylamine solution (pH 10.05). React at room temperature for 12h, wash them with a large amount of ultrapure water, rinse them with acetone, and dry them at 50℃ for 24h to obtain modified agarose microspheres connected to ethylamine. Perform elemental analysis on the proportion of each element in CHON and find that the proportion of active sites is 58.6%.

[0120] (4) Preparation of adsorbent material: Prepare a 50 mmol / L sodium carbonate solution (pH 10.0) as a reaction buffer. For every 1 g of the modified agarose microspheres linked with ethylamine obtained in step (3), add 5 mL of the target rFC protein solution (containing 0.5% v / v Tween 20) from step (1) at a concentration of 1 mg / mL. React at room temperature for 8 h with gentle stirring. After the reaction, vacuum filter the reaction system, wash it with ultrapure water, and resuspend it in a 500 mmol / L glycine solution (1 mL glycine solution for every 1 g of immobilized microspheres) to block excess diethylene sulfone groups. After vacuum filtration again, wash it with ultrapure water to obtain the modified resin immobilized with recombinant factor C protein, i.e., the adsorbent material. Store the adsorbent material in a 4°C refrigerator for later use. Detect the concentration of unbound protein in the supernatant and calculate the grafting rate as 57%.

[0121] Comparative Example 6

[0122] This comparative example provides an adsorbent material prepared by the following method:

[0123] (1) The preparation method of recombinant factor C protein is the same as that in Example 1.

[0124] (2) Functional modification of the carrier: Weigh 40g of agarose microspheres 6B-CL (wet weight), wash three times with ultrapure water to remove impurities, and dehydrate sequentially with 30%→50%→70%→100% ethanol gradient, soaking for 30 minutes each time. Replace the ethanol with acetone, filter, and vacuum dry until the microspheres are in a loose state. Prepare an alkaline reaction solution with the following components: 0.9mol / L NaOH solution (200mL) + 40% (v / v) epichlorohydrin (80mL) + 0.5g / L NaBH4 (stabilizer to prevent epoxy group hydrolysis). Add the dried microspheres to the reaction solution and purge with nitrogen for 10 minutes to remove oxygen. Shake the reaction in a 40℃ water bath for 4 hours (150rpm), and maintain the pH at 12-13 using NaOH. Neutralize to pH using 0.1mol / L HCl. 7.0, terminate the reaction, wash successively with ultrapure water → 30% ethanol → 50% ethanol → 70% ethanol, 500 mL each time, filter, and store in 20% ethanol at 4°C to obtain epoxidized agarose microspheres, which are stored in 20% ethanol at 4°C (avoid freezing).

[0125] (3) Preparation of the graft spacer carrier: Take 10g of epoxidized agarose microspheres (wet weight), wash 3 times with 0.1mol / L PBS buffer (pH 9.0) (100mL each time, centrifuged at 3000rpm×5min) to remove the storage solvent; dissolve COOH-m polyethylene glycol 3350-NH2 (i.e., modified polyethylene glycol 3350 has a carboxyl group at one end and an amino group at the other end) in PBS buffer to prepare a 67mg / mL modified polyethylene glycol solution (excess), and purge with nitrogen for 10 minutes to remove oxygen (to prevent oxidation side reactions); add the pretreated microspheres to the modified polyethylene glycol solution, shake at 25℃ for 48 hours at 120rpm, maintain pH 9.0±0.2 (monitor every 6 hours and adjust with 0.1mol / L NaOH), replenish nitrogen every 12 hours, and operate in the dark during the reaction; after the reaction, add 1mol / L ethanolamine solution (final concentration 0.1mol / L, pH 9.0). 8.5), continue shaking at 25℃ for 6 hours to block unreacted epoxy groups; collect microspheres by filtration, and wash three times with 100mL of the following solutions: PBS buffer (pH 9.0), high-salt PBS (containing 1mol / L NaCl), ultrapure water, and 20% ethanol; prepare modified agarose, and store at 4℃ in 20% ethanol + 0.02% NaN3 (antibacterial), avoiding freezing.

[0126] (4) Preparation of adsorbent material: Modified agarose was dispersed in 25 mmol / L pH 7.6 PBS buffer and swollen at room temperature for 1-2 hours to open the channels of modified agarose and promote subsequent reactions. The mixture was then washed three times with the same buffer. Glutaraldehyde was added to a final concentration of 0.5% v / v, and the mixture was shaken at 4°C and 100 rpm for 2 hours. The mixture was then washed three times with PBS solution to remove unreacted glutaraldehyde. Protein rFC was dissolved in pH 7.6 PBS buffer. 7.6% PBS buffer was added to activated modified agarose at a molar ratio of 1:2 (carboxyl group on polyethylene glycol to rFC protein), resulting in a final protein concentration of 1 mg / mL. The reaction was carried out at 4°C and 50 rpm for 2 hours with shaking. Ethanolamine buffer was then added for end-capping, bringing the final concentration to 200 mmol / L. The mixture was then blocked at 4°C with shaking for 1 hour to quench residual activating groups. The agarose-polyethylene glycol-rFC product was obtained by centrifugation and washing sequentially with Tween-20 buffer, deionized water, and ethanol to remove unbound rFC protein and byproducts. The product was stored at 4°C for later use. The concentration of unbound protein in the supernatant was measured, and the grafting rate was calculated to be 77%.

[0127] Experimental Example 1

[0128] The adsorption performance of the adsorbent materials in each embodiment and comparative example for endotoxins in a human plasma environment was tested. The specific test methods are as follows:

[0129] A plasma solution with an initial endotoxin concentration of 10 EU / mL was obtained by external addition. Using the adsorbents from the above examples and comparative examples as adsorbents, 1 g of each adsorbent was placed in a 25 mL Erlenmeyer flask. The supernatant was removed with a dropper, and physiological saline was added to a depth of 5 mL for washing. The flask was then allowed to stand, and the washing was repeated 5 times. 20 mL of the prepared plasma solution was added, ensuring the adsorbent was completely submerged in the plasma and not adhering to the flask wall. The flask was then sealed tightly with sealing film and incubated in a constant-temperature shaker at 37°C at a rate of 60 r / min for 2 hours. The concentrations of endotoxin and interleukin-6 before and after adsorption were measured. Three to six parallel experiments were conducted for each example or comparative example, and the corresponding adsorption rate was calculated. The endotoxin concentration was detected using the Limulus Amebocyte Lysate (LAL) assay. All consumables and containers used in the experiment were pyrogen-free treated and the experiment was conducted in a clean environment. The results are shown in Table 1. The adsorption performance was calculated using the following formula:

[0130] Cr (%) = (C0 - C) t ) / C0×100%Cr----The rate of decrease in the concentration of the target substance;

[0131] C0----Detection concentration of the target substance before adsorption;

[0132] C t----The concentration of the target substance after 2 hours of adsorption.

[0133] Table 1

[0134] sample Endotoxin adsorption performance Example 1 79.5％ Example 2 86.8％ Example 3 78.3％ Example 4 83.8％ Example 5 96.2％ Example 6 81.7％ Comparative Example 1 68.8％ Comparative Example 2 71.4％ Comparative Example 3 59.3％ Comparative Example 4 65.4％ Comparative Example 5 53.7％ Comparative Example 6 72.6％

[0135] As shown in Table 1, in Examples 1 to 6, polystyrene-divinylbenzene resin was used as the carrier. The carrier was first modified with chloromethylation to obtain a resin containing chloromethyl groups. Then, modified polyethylene glycol with a carboxyl group at one end was grafted onto the chloromethyl-containing resin as a spacer arm. The hydroxyl group at one end of the modified polyethylene glycol was linked to the chloromethyl group via a nucleophilic substitution reaction, while the carboxyl group at the other end of the modified polyethylene glycol served as the free end of the spacer arm. After activation, it underwent a covalent reaction with the amino group on the rFC protein. Examples 1 to 3 and Examples 4 to 6 show that as the molar ratio of the carboxyl group at the modified polyethylene glycol end to the rFC protein increased, the grafting rate of the rFC protein increased. However, the adsorption capacity of the adsorbent material for endotoxins showed a trend of first increasing and then decreasing. This is because both the rFC protein and endotoxins have a certain volume. If too much rFC protein is grafted onto the adsorbent material, the steric hindrance during endotoxin adsorption becomes more pronounced, which is detrimental to the adsorption of endotoxins by the adsorbent material. When the molar ratio of the carboxyl groups at the modified polyethylene glycol end to rFC protein is 1:2, the grafting rate of rFC protein is suitable, and the adsorption capacity of the corresponding adsorbent material for endotoxins is optimal. This indicates that appropriately increasing the molar ratio of the carboxyl groups at the modified polyethylene glycol end to rFC protein is beneficial to improving the adsorption capacity of the adsorbent material for endotoxins. Furthermore, a comparison between Examples 1 to 3 and Examples 4 to 6 shows that the polystyrene-divinylbenzene resin has a smaller pore size and a larger specific surface area, which is beneficial for grafting modified polyethylene glycol and rFC protein onto the carrier, thereby improving the grafting rate of rFC protein. However, excessive rFC protein leads to a more significant steric hindrance effect during endotoxin adsorption, which is detrimental to the adsorption of endotoxins by the adsorbent material. This indicates that appropriately increasing the pore size of the polystyrene-divinylbenzene resin is beneficial to improving the adsorption capacity of the adsorbent material for endotoxins. As can be seen from the comparison between Example 2 and Comparative Example 1, and between Example 5 and Comparative Example 2, this invention first modifies the support by chloromethylation to obtain a resin containing chloromethyl groups, and then links the hydroxyl group at one end of the modified polyethylene glycol to the chloromethyl group via a nucleophilic substitution reaction to graft a spacer arm onto the resin. Compared with the method used in Comparative Examples 1 and 2, which uses epoxidation to graft modified polyethylene glycol as a spacer arm onto the support, Examples 1 to 6 can improve the grafting rate of rFC protein and significantly enhance the adsorption capacity of the corresponding adsorbent materials for endotoxins. This is because the epoxidation method is prone to side reactions, and the product is easily affected by the environment and undergoes ring-opening. In contrast, the covalent coupling reaction method such as nucleophilic substitution used in this embodiment can improve the selectivity of the reaction and accurately achieve the substitution reaction, thereby improving the adsorption capacity of the adsorbent material for endotoxins.As can be seen from Examples 1 to 6 compared to Comparative Example 3, compared to directly grafting rFC protein onto the support in Comparative Example 3, connecting the support and rFC protein through spacer arms in Examples 1 to 6 can significantly improve the grafting rate of rFC protein and enhance the connection stability between the support and rFC protein, which is beneficial for significantly improving the adsorption capacity of the adsorbent material for endotoxins. As can be seen from the comparison between Examples 1 to 6 and Comparative Example 4, compared to using low molecular weight modified polyethylene glycol as the spacer arm in Comparative Example 4, selecting a higher molecular weight modified polyethylene glycol as the spacer arm in Examples 1 to 6 can reduce the influence of steric hindrance on the rFC protein, improve the wobble of the rFC protein, and facilitate the full exposure of the active sites of the rFC protein, thereby significantly improving the adsorption capacity of the adsorbent material for endotoxins. Comparative Examples 5 and 6 both used agarose microspheres as a carrier. Comparing Examples 1 to 6 with Comparative Example 6, it can be seen that, compared with Comparative Example 6, which used agarose microspheres as a carrier and grafted modified polyethylene glycol onto the agarose microspheres as spacers using an epoxidation method, Examples 1 to 6 used polystyrene-divinylbenzene resin as a carrier. After chloromethylation modification, the resin was linked through a nucleophilic substitution reaction. In Examples 1 to 6, the hydrophobicity and abundant pore structure of polystyrene resin facilitated the movement of endotoxins containing hydrophobic lipid A toward the carrier, which was beneficial for the specific adsorption of endotoxins by the recombinant factor C protein on the adsorbent material, thus significantly improving the adsorption capacity of the adsorbent material for endotoxins. As can be seen from the comparison between Comparative Examples 5 and 6, compared with the direct grafting of rFC protein onto agarose microspheres in Comparative Example 5, the connection between agarose microspheres and rFC protein via spacer arms in Comparative Example 6 can significantly improve the grafting rate of rFC protein and enhance the connection stability between agarose microspheres and rFC protein, which is beneficial to significantly improving the adsorption capacity of the adsorbent material for endotoxins.

[0136] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. An adsorbent material, characterized in that, It includes a carrier and an endotoxin affinity-binding ligand, the endotoxin affinity-binding ligand comprising a recombinant factor C protein, the carrier and the recombinant factor C protein being connected by a spacer arm; The carrier includes at least one of resin, chitosan, cellulose, agarose, and dextran; The spacer arm comprises at least one of modified polyethylene glycol, polyvinylpyrrolidone, poly(2-hydroxyethyl methacrylate), polyvinyl alcohol, polyacrylic acid, hexamethylenediamine, divinyl sulfone, and glutaraldehyde, wherein at least one end of the modified polyethylene glycol has a carboxyl group, an aldehyde group, or an amino group.

2. The adsorbent material according to claim 1, characterized in that, The spacer arm is modified polyethylene glycol, one end of which is hydroxyl or amino, and the other end is carboxyl, aldehyde or amino, and the main chain is composed of repeated oxyethylene units; The modified polyethylene glycol has a molecular weight of 800 Da to 10000 Da.

3. The adsorbent material according to claim 1 or 2, characterized in that, The modified polyethylene glycol has a molecular weight of 2500 Da to 4000 Da.

4. The adsorbent material according to claim 1, characterized in that, The carrier is a polystyrene resin with a particle size of 0.2 mm to 1.8 mm, an average pore size of 5 nm to 100 nm, and a pore volume of 0.3 cm³. 3 / g to 2.5cm 3 / g, with a specific surface area ranging from 200m² 2 / g to 1200m 2 / g.

5. The adsorbent material according to claim 1, characterized in that, The recombinant C factor protein is recombinant horseshoe crab C factor protein, and the loading of the recombinant C factor protein on each 1g of the adsorbent material is 0.4mg to 2.0mg.

6. A method for preparing an adsorbent material as described in any one of claims 1 to 5, characterized in that, Includes the following steps: Provides recombinant factor C protein; The carrier is activated to obtain a carrier containing functionalized groups; The carrier containing the functionalized group and the spacer arm are connected by a covalent coupling reaction to obtain a carrier with grafted spacer arm. The carrier grafted with the spacer arm and the recombinant C factor protein are covalently linked to obtain the adsorbent material.

7. The method for preparing the adsorbent material according to claim 6, characterized in that, The activation of the carrier to obtain a carrier containing functionalized groups includes: Under the action of a catalyst, the support is functionalized by a modifying agent containing functionalized groups to obtain a support containing functionalized groups. The functionalized groups include at least one of chloromethyl, hydroxy, amino, carboxyl, aldehyde and amide groups.

8. The method for preparing the adsorbent material according to claim 6, characterized in that, The support containing functionalized groups and the spacer arm are connected by a covalent coupling reaction to obtain a support with grafted spacer arms, comprising: The carrier containing the functionalized group and the spacer arm are subjected to a covalent coupling reaction under the action of a first activator, so that the spacer arm is connected to the functionalized group on the carrier by a covalent bond, thereby obtaining a carrier grafted with a spacer arm. The temperature of the covalent coupling reaction is 60°C to 80°C, and the temperature of the covalent coupling reaction is positively correlated with the molecular weight of the spacer arm.

9. The method for preparing the adsorbent material according to claim 6, characterized in that, The carrier with the grafted spacer arm and the recombinant C factor protein are covalently linked to obtain the adsorbent material, comprising: Under weakly acidic conditions, the carrier of the grafted spacer arm is activated with a second activator to activate the free end of the spacer arm. Under neutral or weakly alkaline conditions, the carrier activated at the free end of the spacer arm is covalently reacted with the recombinant C factor protein, so that the recombinant C factor protein is linked to the carrier through the spacer arm, thereby obtaining the adsorbent material.

10. The adsorbent material according to any one of claims 1 to 5, or the adsorbent material prepared by the preparation method according to any one of claims 6 to 9, is used in blood purification materials or devices.

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