Biological component adsorption material

Abstract

The purpose of the present invention is to provide a biological component adsorption material that has high adsorption performance with respect to biological components, particularly with respect to cytokines, independently from the content and / or chemical structure of a ligand. The present invention provides a biological component adsorption material comprising water-insoluble material which comprises a base material that includes a scaffold polymer, a linker that is bound to the base material, and ligand that is bound to the linker, wherein the water-insoluble material exhibits a T2 relaxation time of retrained amorphous component of 0.0210-0.0270 ms, as seen in free induction decay (FID) observed with 1H-pulse NMR using a solid echo method.

Description

Biocomponent adsorption materials

[0001] This invention relates to a material for adsorbing biological components.

[0002] In recent years, the importance of separation and analysis techniques for biological components has been increasing in the fields of medicine and biotechnology. In particular, technologies are being developed to efficiently separate and adsorb specific proteins or cellular components, with the aim of treating inflammatory diseases, suppressing immunosuppression before and after transplantation, and suppressing side effects such as fever and infection caused by blood products. Among these, technologies related to blood purification therapy that adsorb and remove biological components such as cytokines and leukocytes are attracting attention, especially technologies that adsorb and remove interleukin-6 and / or interleukin-8, which are biomarkers for inflammatory diseases.

[0003] Water-insoluble materials containing substrates to which charged ligands are bound exhibit excellent interaction with living cells such as leukocytes or cytokines, and biocomponent adsorption materials using these materials have been developed.

[0004] Patent Document 1 discloses a bio-component adsorption material that is a water-insoluble material to which ligands having amide groups and amino groups are bound, and in which the blood purification performance is improved by increasing the content of amide groups and amino groups compared to conventional materials.

[0005] Patent Document 2 discloses a water-insoluble carrier to which a compound containing a charged functional group is bonded, and which improves the adsorption performance of activated leukocyte-activated platelet complexes by setting the surface development length ratio within a certain range, thereby providing a material for removing activated leukocyte-activated platelet complexes.

[0006] Patent No. 6589993 Patent No. 6699731

[0007] Charged ligands adsorb biological components through electrostatic interactions. However, in blood purification therapy, if ligands elute from the biological component adsorption material, there is a concern that the biological components adsorbed to the eluted ligands may cause side effects. Therefore, there is generally an upper limit to the ligand content in biological component adsorption materials, and restrictions on the chemical structure of ligands that can be immobilized on the substrate.

[0008] Therefore, the present invention aims to provide a biocomponent adsorption material that exhibits high adsorption performance for biological components, particularly cytokines, regardless of the ligand content and / or chemical structure.

[0009] The present inventors have diligently pursued studies to solve the above problems and have found a water-insoluble material comprising a substrate containing a scaffold polymer, a linker bonded to the substrate, and a ligand bonded to the linker, wherein the water-insoluble material is obtained by the solid echo method １ T of the constrained amorphous component in FID observed by H-pulse NMR ２ We discovered that bio-component adsorption materials with a relaxation time within a certain range exhibit high bio-component adsorption performance, which led to the completion of this invention.

[0010] Although the detailed mechanism for the high biocomponent adsorption performance is unknown, it is presumed that if the scaffold polymer is somewhat soft, when biocomponents collide with the water-insoluble material, minute changes in the shape of the substrate become possible. As a result, the contact area between the ligand and the biocomponents increases, making them less likely to detach.

[0011] In other words, the present invention encompasses the following (1) to (7): (1) A water-insoluble material comprising a substrate containing a scaffold polymer, a linker bonded to the substrate, and a ligand bonded to the linker, wherein the water-insoluble material is obtained by solid echo method １ In free induction decay (FID) observed by H-pulse NMR, the constrained amorphous component of T ２ A biocomponent adsorption material having a relaxation time of 0.0210 to 0.0270 ms. (2) The biocomponent adsorption material according to (1), wherein the linker contains a carbonyl bond or an ether bond. (3) The biocomponent adsorption material according to (1), wherein the linker contains an amide bond. (4) The biocomponent adsorption material according to (3), wherein the linker has a chemical structure represented by the following general formula (A). (In the formula, the dashed line indicates the binding position with the substrate, and X represents the ligand.) (5) The scaffold polymer is a biocomponent adsorption material according to any one of (1) to (4), wherein the scaffold polymer includes a crosslinked structure. (6) The biocomponent adsorption material according to any one of (1) to (5), wherein the content of charged functional groups contained in the ligand is 0.2 to 4.0 mmol per 1 g of dry weight of the water-insoluble material. (7) A biocomponent adsorption column comprising the biocomponent adsorption material according to any one of (1) to (6).

[0012] The biocomponent adsorption material of the present invention has high biocomponent adsorption performance, and therefore can adsorb biocomponents with high efficiency.

[0013] The biocomponent adsorption material of the present invention comprises a water-insoluble material consisting of a substrate containing a scaffold polymer, a linker bound to the substrate, and a ligand bound to the linker, wherein the water-insoluble material is obtained by the solid echo method １ T of the constrained amorphous component in FID observed by H-pulse NMR ２ It is characterized by a relaxation time of 0.0210 to 0.0270 ms.

[0014] A "biometabolite adsorbent" refers to a material used to adsorb biological components. While there are no particular limitations on the composition of a biometabolite adsorbent other than the inclusion of a water-insoluble material, it can consist solely of a water-insoluble material or a mixture of a water-insoluble material and a reinforcing material. From the viewpoint of high functionality, a biometabolite adsorbent consisting solely of a water-insoluble material is preferable.

[0015] A "water-insoluble material" refers to a material that consists of a substrate, a linker, and a ligand, and is water-insoluble. From the viewpoint of exhibiting water insolubility and controlling the mobility of the constrained amorphous components, it is preferable that the scaffold polymer contained in the substrate includes a cross-linked structure.

[0016] By "water-insoluble" is meant that the amount of a material dissolved in water is 1% by mass or less. Immerse 1 g of the dried material in 10 mL of pure water at 37°C. After 24 hours, lift only the material with tweezers, dry the remaining water in a vacuum dryer at 50°C, and if the mass of the remaining solid content is 10 mg or less, the dissolution amount is 1% by mass or less, and it can be said that the material is water-insoluble. If the material is not insoluble, there is a risk of an increase in eluate when actually used, which is not preferable in terms of safety.

[0017] "Substrate" means a polymer material to which a ligand can be immobilized by chemical modification. The substrate includes a scaffold polymer and may further include a support polymer for maintaining the strength of the material, but it is preferable that at least half of the surface of the substrate is a scaffold polymer.

[0018] "Scaffold polymer" means a polymer having a functional group (hereinafter referred to as a scaffold functional group) to which a linker can be bonded as a chemical structure of a repeating unit. The scaffold polymer is not particularly limited except that it has a scaffold functional group, and may be a homopolymer composed of repeating units of a single chemical structure or a copolymer composed of repeating units of a plurality of chemical structures.

[0019] The scaffold functional group is preferably a neutral functional group from the viewpoint of suppressing interference with the ligand. For example, an aromatic functional group (aryl group or arylene group), a hydroxy group, a vinyl group, an acetylene group, an aldehyde group, a ketone group, a thiol group, a cyano group, or a halogenated alkyl group is preferable, and an aromatic functional group, a halogenated alkyl group, or a hydroxy group is more preferable because of high decomposition resistance.

[0020] The aryl group, which is an aromatic functional group as a scaffold functional group, means a phenyl group, a naphthyl group, or an anthracenyl group, and the arylene group means a phenylene group, a naphthylene group, or an anthracenylene group. As the aromatic functional group as a scaffold functional group, a phenyl group or a phenylene group is preferable because of the large number of reaction points.

[0021] Examples of the scaffold polymer having an aromatic functional group as a scaffold functional group include, for example, from the viewpoints of ease of handling and availability, as a scaffold polymer having a phenyl group as a scaffold functional group, polystyrene and its derivatives [for example, poly(p-chloromethylstyrene), poly(α-methylstyrene), poly(β-methylstyrene), poly(p-tert-butoxystyrene), poly(p-acetoxystyrene) and poly(p-(1-ethoxy)styrene)], and as a scaffold polymer having a phenylene group as a scaffold functional group, polysulfone and its derivatives, polyether ketone, polyphenylene sulfide, polyethylene terephthalate, polyether ether ketone, etc. From the viewpoints of the application results to blood purification and biocompatibility, polystyrene or its derivatives, polysulfone or its derivatives, polyethylene terephthalate, polyether ketone or polyether ether ketone are preferable. From the viewpoint of less deterioration due to decomposition, polystyrene or its derivatives, polysulfone or its derivatives, polyether ketone or polyether ether ketone are more preferable. From the viewpoint of ease of reaction, polystyrene or its derivatives, or polysulfone or its derivatives are even more preferable. Among these, polystyrene or its derivatives having an aromatic ring in the side chain where the biological components can easily access are particularly preferable. The substitution position of the substituent for each aromatic ring is not limited.

[0022] Examples of the scaffold polymer having a halogenated alkyl group as a scaffold functional group include, for example, vinylidene chloride, its copolymers, etc.

[0023] Examples of the scaffold polymer having a hydroxy group as a scaffold functional group include, for example, saccharides (for example, cellulose, dextran, chitosan, etc.), polyvinyl alcohol, polyvinyl alcohol-ethylene copolymer, etc.

[0024] From the viewpoints of suppressing the generation of eluate and controlling the motility of the scaffold polymer, it is preferable that the scaffold polymer contains a crosslinked structure.

[0025] A "crosslinked structure" refers to a chemical bond formed between the scaffolding functional groups of a scaffolding polymer. Examples of crosslinked structures include chemical structures derived from divinyl compounds and chemical structures derived from aldehydes. From the viewpoint of being able to introduce the crosslinked structure after the polymer has been molded and ease of processing, a crosslinked structure derived from an aldehyde is preferred. As an aldehyde-derived crosslinked structure, a crosslinked structure derived from formaldehyde or a substituted benzaldehyde [for example, benzaldehyde, p-diethylaminobenzaldehyde (DEAP), p-dimethylaminobenzaldehyde (DMAP), or p-isopropylbenzaldehyde (IPP)] is preferred, and from the viewpoint of avoiding discoloration, formaldehyde is more preferred. When paraformaldehyde is used as a crosslinking agent, the introduced crosslinked structure is derived from its decomposition product, formaldehyde.

[0026] A "support polymer" refers to a polymer that does not contain scaffolding functional groups and is appropriately included in the substrate to maintain the strength of the material. While there are no particular limitations on the support polymer as long as it does not contain scaffolding functional groups in its repeating units, polyolefins (e.g., polyethylene, polypropylene, etc.) are suitable from the viewpoint of strength and chemical resistance.

[0027] "Linker" refers to the chemical structure between the scaffold polymer and the ligand. The linker originates from a reactive functional group. The chemical structure of the linker is not particularly limited, but from the viewpoint of increasing the mobility of the scaffold polymer, it is preferable to include heteroatoms (e.g., oxygen, nitrogen, and chlorine atoms) that disrupt the orientation derived from intermolecular interactions, and to include electrically neutral bonds, and may include multiple electrically neutral bonds. The linker preferably includes carbonyl bonds or ether bonds, which are known to disrupt the orientation derived from intermolecular interactions, more preferably includes urea bonds, amide bonds, ester bonds, urethane bonds, or ether bonds, and even more preferably includes amide bonds. The linker preferably further includes alkyl groups. The linker most preferably has a structure represented by the following general formula (A). The fact that carbonyl bonds (urea bonds, amide bonds, ester bonds, or urethane bonds) are chemical structures that disrupt orientation derived from intermolecular interactions has been shown in a paper reporting on the stacking changes of aromatic rings due to the introduction of carbonyl bonds (Do et al., J. Am. Chem. Soc., 2025, Vol. 147, pp. 32273-32286). The fact that ether bonds are chemical structures that disrupt orientation derived from intermolecular interactions has been shown in a paper reporting on the crystallization changes of aromatic polymers (Perez-Martin et al., Compos. B Eng., 2021, Vol. 223, 109127, Section 2.1). (In the formula, the wavy line indicates the binding site with the substrate, and X represents the ligand.)

[0028] Examples of reactive functional groups that give rise to the linker include active halogen groups such as haloalkyl groups (e.g., halomethyl and haloethyl groups), haloacyl groups (e.g., haloacetyl and halopropionyl groups), and haloacetamidoalkyl groups (e.g., haloacetamidomethyl and haloacetamidoethyl groups), as well as epoxy groups, carboxyl groups, isocyanate groups, thioisocyanate groups, and acid anhydride groups. From the viewpoint of having a moderate level of reactivity, active halogen groups are preferred as reactive functional groups, haloacetamidoalkyl groups are more preferred, and haloacetamidomethyl groups are even more preferred.

[0029] The linker content per gram of dry weight of a water-insoluble material can be measured by hydrolyzing the water-insoluble material with hydrochloric acid, desalting the resulting decomposed water-insoluble material with an aqueous sodium hydroxide solution, neutralizing it by contacting it with hydrochloric acid, and then quantifying the remaining hydrochloric acid concentration by acid-base titration.

[0030] The linker content per gram of dry weight of the water-insoluble material is not particularly limited, but if it is too low, it is difficult to disrupt the orientation of the scaffold polymer, and if it is too high, the volume of the scaffold polymer increases and the water-insoluble material deforms. Therefore, 0.2 to 5.0 mmol is preferred, and 2.0 to 4.0 mmol is more preferred.

[0031] "Ligand" refers to a chemical structure that contributes to the adsorption of biological components. The ligand of the present invention is bonded to a substrate via a linker and includes a chemical structure containing a charged functional group (hereinafter referred to as a charged chemical structure). The chemical structure of the ligand is not particularly limited as long as it includes a charged chemical structure, and from the viewpoint of controlling the performance of the biological component adsorption material and / or the selectivity of the adsorbed components by hydrophilicity, a hydrophobic functional group may be bonded to the charged chemical structure via a spacer.

[0032] A charged chemical structure is not particularly limited as long as it contains a charged functional group, but from the viewpoint of not inhibiting the biocomponent adsorption performance by the charged functional group, it is preferable that it is composed of a charged functional group and a hydrocarbon group.

[0033] A hydrocarbon group refers to a chemical structure composed of carbon atoms and hydrogen atoms. Examples include saturated alkyl groups, saturated alkylene groups, unsaturated alkyl groups, unsaturated alkylene groups, aryl groups, and arylene groups, with saturated alkyl groups or saturated alkylene groups being preferred.

[0034] The number of carbon atoms in the hydrocarbon group is not particularly limited, but if there are too few, the mobility as a ligand will be low, and if there are too many, steric hindrance will inhibit the adsorption performance of biological components. Therefore, it is preferable that there be 1 to 20 carbon atoms, and more preferably 1 to 5 carbon atoms, per charged functional group.

[0035] Examples of charged functional groups include sulfonic acid groups and carboxyl groups, which are negatively charged (also called acidic functional groups), and amino groups, which are positively charged (also called basic functional groups). From the viewpoint of biocompatibility, amino groups are preferred.

[0036] Compounds containing charged functional groups that give rise to charged chemical structures include, for example, compounds containing amino groups (hereinafter referred to as amine compounds), compounds containing sulfonic acid groups (hereinafter referred to as sulfonic acid compounds), and compounds containing carboxylic acid groups (hereinafter referred to as carboxylic acid compounds). The chemical structure of compounds containing charged functional groups is not particularly limited, but from the viewpoint of not inhibiting the adsorption performance of biological components by the charged functional group and from the viewpoint of bonding with linkers, it is preferable that amine compounds consist of an amino group and a hydrocarbon group, sulfonic acid compounds consist of a sulfonic acid group, a functional group that bonds with a reactive functional group, and a hydrocarbon group, and carboxylic acid compounds consist of a carboxylic acid group, a functional group that bonds with a reactive functional group, and a hydrocarbon group.

[0037] Amine compounds composed of an amino group and a hydrocarbon group include, for example, primary amine compounds such as ammonia, methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, and dodecylamine; secondary amine compounds such as methylhexylamine, diphenylmethylamine, and dimethylamine; amine compounds having unsaturated alkyl chains such as allylamine; tertiary amine compounds such as trimethylamine, triethylamine, dimethylethylamine, phenyldimethylamine, and dimethylhexylamine; amine compounds having aromatic rings such as 1-(3-aminopropyl)imidazole, pyridine-2-amine, and 3-sulfoaniline; and tris(2-aminoethyl)amine and ethylene. Examples of polyamines include compounds in which two or more amino groups are bonded to alkyl chains, aromatic compounds, heterocyclic compounds, monocyclic compounds, etc., such as diamines, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, dipropylenetriamine, polyethyleneimine, N-methyl-2,2'-diaminodiethylamine, N-acetylethylenediamine, and 1,2-bis(2-aminoethoxyethane). Propylamine or polyamine is preferred, propylamine, ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, or polyethyleneimine is more preferred, and propylamine, diethylenetriamine, tetraethylenepentamine, or polyethyleneimine is even more preferred.

[0038] Examples of sulfonic acid compounds composed of a sulfonic acid group, a functional group that bonds to a reactive functional group, and a hydrocarbon group include sulfonic acid compounds composed of a sulfonic acid group, a hydroxyl group, and a hydrocarbon group, such as hydroxymethanesulfonic acid, 2-hydroxyethanesulfonic acid, and p-hydroxybenzenesulfonic acid, and sulfonic acid compounds composed of a sulfonic acid group, an amino group, and a hydrocarbon group, such as 2-aminoethanesulfonic acid and p-aminobenzenesulfonic acid. From the viewpoint of not inhibiting charge, sulfonic acid compounds composed of a sulfonic acid group, a hydroxyl group, and a hydrocarbon group are preferred.

[0039] Examples of carboxylic acid compounds composed of a carboxylic acid group, a functional group bonded to a reactive functional group, and a hydrocarbon group include carboxylic acid compounds composed of a carboxylic acid group, a hydroxyl group, and a hydrocarbon group, such as hydroxyacetic acid, 2-hydroxybutyric acid, and 3-hydroxypropionic acid, and carboxylic acid compounds composed of a carboxylic acid group, an amino group, and a hydrocarbon group, such as glycine and p-aminobenzoic acid. From the viewpoint of not inhibiting charge, carboxylic acid compounds composed of a carboxylic acid group, a hydroxyl group, and a hydrocarbon group are preferred.

[0040] The content of charged functional groups per gram of dry weight of a water-insoluble material can be measured by acid-base titration using hydrochloric acid and sodium hydroxide aqueous solutions.

[0041] The content of charged functional groups per gram of dry weight of a water-insoluble material is preferably 0.2 to 4.0 mmol, more preferably 0.4 to 3.0 mmol, and even more preferably 0.5 to 2.5 mmol, because if it is too low, it cannot interact with biological components, and if it is too high, the amount of eluted material increases, making it more likely to become toxic and reducing its stability during storage.

[0042] A spacer is a neutral chemical bond that connects a charged chemical structure to a hydrophobic functional group. Examples of spacers include urea bonds, amide bonds, urethane bonds, ether bonds, and ester bonds. From the viewpoint of controlling the adsorption performance of biological components, urea bonds, amide bonds, urethane bonds, or ester bonds that have hydrogen bonds are preferred, and urea bonds, amide bonds, or urethane bonds are more preferred.

[0043] A hydrophobic functional group is a chemical structure consisting of a carbon atom, which may be substituted with a halogen atom, and a hydrogen atom. The number of carbon atoms in a hydrophobic functional group is preferably 3 to 15, and more preferably 3 to 10, because if it is too small, the hydrophobic adjustment function will not be expressed even when bonded, and if it is too large, the hydrophobicity will increase too much and the biocomponent adsorption function will not be expressed.

[0044] Examples of hydrophobic functional groups include alkyl groups (e.g., methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, etc.), phenyl groups, and alkyl-substituted phenyl groups (e.g., para(p)-methylphenyl group, meta(m)-methylphenyl group, ortho(o)-methylphenyl group, para(p)-ethylphenyl group, meta(m)-ethylphenyl group, ortho(o)-ethylphenyl group, etc.), and halogen-substituted phenyl groups (e.g., para(p)-fluorophenyl group, meta(m)-fluorophenyl group, ortho(o)-fluorophenyl group, para(p)-chlorophenyl group, meta(m)-chlorophenyl group, ortho(o)-chlorophenyl group, etc.).

[0045] The positions of the hydrophobic functional group and spacer within the ligand are not particularly limited, but from the viewpoint of controlling the adsorption performance of biological components, it is preferable that they be located on the surface side of the ligand. That is, it is preferable that the ligand from the scaffold polymer is composed in the order of "scaffold polymer - linker - charged chemical structure - spacer - hydrophobic functional group". Note that "-" indicates a chemical bond.

[0046] The content of hydrophobic functional groups in a ligand is determined by hydrolyzing 1 g of water-insoluble material by dry weight with a specified amount of hydrochloric acid, adding an internal standard of known concentration to the resulting eluate, and neutralizing it with sodium hydroxide. １ This can be calculated by performing 1H-NMR measurements and determining the ratio of the integral value of the peak derived from the chemical structure originating from the hydrophobic functional group to the integral value of the internal standard.

[0047] Since charged functional groups need to interact with the substances to be removed from the blood, they must be present on at least the side of the water-insoluble material's surface that comes into contact with the blood. If the water-insoluble material is a sea-island type composite fiber, charged functional groups must be present on at least the surface of the sea component that comes into contact with the blood. Here, if the surface of the water-insoluble material has an uneven shape, the outermost layer along that unevenness is also considered to be the surface of the water-insoluble material. Furthermore, if the water-insoluble material has through holes inside, the outer layer of the internal through holes is also considered to be the surface of the water-insoluble material.

[0048] Examples of water-insoluble material shapes include fiber shapes, flat membrane shapes, hollow fiber membrane shapes, and particle shapes. From the viewpoint of not creating stagnation areas when in contact with blood, fiber shapes or particle shapes are preferred for water-insoluble materials. Furthermore, among fiber shapes, processed fiber bundles, yarns, felts, nets, knitted fabrics, or woven fabrics are preferred, and from the viewpoint of large surface area and low flow resistance, yarn bundles, knitted fabrics, or woven fabrics are more preferred.

[0049] When the water-insoluble material is in the form of particles, the particle size may be of any size, but from the viewpoint of improving the contact area and suppressing clogging, 1 μm to 1 mm is preferred, and 20 μm to 500 μm is more preferred.

[0050] "Particle diameter" refers to the average value of measurements taken when 10 particles are randomly selected as a sample, a photograph of each sample is taken using a scanning electron microscope or similar device, and the diameter of the particles is measured at 10 locations in each photograph (a total of 100 locations for all samples).

[0051] When the water-insoluble material is in the form of fibers, the diameter of the single fibers may be any thickness, but from the viewpoint of improving the contact area and maintaining the strength of the material, it is preferably 3 to 200 μm, more preferably 5 to 50 μm, and even more preferably 10 to 40 μm.

[0052] "Single fiber diameter" refers to the average value of measurements taken when 10 small fiber samples are randomly selected, and a photograph of each sample is taken using a scanning electron microscope or similar device. The diameter of the fiber is measured at 10 locations in each photograph (a total of 100 locations for all samples).

[0053] When the water-insoluble material is in the form of fibers, the cross-sectional structure of the fibers can include, for example, single fibers made of one type of polymer, core-sheath type, sea-island type, or side-by-side type composite fibers. However, from the viewpoint of maintaining the strength of the material during blood purification, core-sheath type composite fibers or sea-island type composite fibers are preferred.

[0054] Water-insoluble materials with a knitted, felted or netted shape can be manufactured by known methods using fibrous base materials or water-insoluble materials as raw materials. Examples of felt manufacturing methods include, for example, the wet method, the carding method, the air-laying method, the spunbond method, and the meltblowing method. Also, examples of knitted and net manufacturing methods include, for example, the plain weaving method and the circular knitting method. In particular, from the perspective of a large filling mass per unit volume and filling in a blood purifier, a knitted fabric manufactured by the circular knitting method is preferred.

[0055] １ H-Pulse NMR is １ a method of measuring the H nucleus (hydrogen nucleus) and observing the free induction decay (FID) of the whole substance, also called TD-NMR (Time-Domain NMR, time-domain nuclear magnetic resonance method). It is a method specialized for measuring the relaxation time of the whole substance, and can measure the relaxation time with higher reproducibility and accuracy than using general high-resolution NMR. There are various pulse sequences for obtaining FID, but when evaluating the mobility of a solid such as a resin, the solid echo method, which does not generate the measurement-inaccessible time (also called Dead Time) of the initial spectrum, is preferred for rigid molecules.

[0056] １ Relaxation in H-Pulse NMR means the process by which the NMR signal (FID) emitted by the spin system excited by the pulse irradiation of radio waves decays. The relaxation time means the time until the FID decays from the initial value to a certain value. The relaxation time mainly includes the longitudinal relaxation time (T １ ) and the transverse relaxation time (T ２ ), each reflecting different physical processes.

[0057] T １ The relaxation time is also called the spin-lattice relaxation time and is the time until the spin system exchanges energy with the surrounding lattice (environment) and returns to the equilibrium state.

[0058] T ２Relaxation time, also known as spin-spin relaxation time, is defined as the time it takes for the FID emitted by radio wave pulse irradiation to decay to 36.8%. This represents the time it takes for the excited spin system to lose its transverse magnetization and return to its original state. Generally, the more mobile a molecule is, the longer it takes to lose its transverse magnetization. Therefore, T ２ Substances with longer relaxation times are generally considered to have higher molecular mobility and a softer structure. ２ Solids with relaxation times exceeding 0.1 milliseconds (ms) exhibit rubber-like properties, while solids with relaxation times shorter than 0.001 ms are treated as crystalline parts of a polymer. (Water-soluble component T) ２ The relaxation time exceeds 1 ms.

[0059] FID is observed as the sum of the FIDs of multiple components, depending on the physical structure of the object being measured. However, by performing component fitting using analysis software, it can be separated into its individual components. Component fitting is a method of separating the FID, which is observed as the sum of multiple components, into its individual components according to a model equation. For example, in the case of the FID of a crystalline polymer, it is generally known that two-component fitting can separate it into a crystalline component to which the crystalline structure belongs and an amorphous component to which the amorphous structure belongs. Furthermore, when measuring a crystalline polymer in a water-soaked state, three-component fitting including the water peak is performed, and it can be separated into a crystalline component to which the crystalline structure belongs, an amorphous component to which the amorphous structure of the crystalline polymer belongs (a constrained amorphous component), and an amorphous component to which water belongs (an unconstrained amorphous component).

[0060] In the water-insoluble material of the present invention １ H-pulse NMR must be evaluated in a wet state to prevent loss of ligand properties. Specifically, water-insoluble material stored in water should be removed from the water and immediately wiped dry three times using Kimwipes (manufactured by Nippon Paper Crecia Co., Ltd.). Then, the water-insoluble material should be filled to the upper limit of the inner tube of the sample tube used for NMR measurement, the sample tube should be sealed with silicone rubber, and the sample tube should be left standing in a 37°C probe for 10 minutes to stabilize the temperature before measurement using the solid echo method.

[0061] The water-insoluble material of the present invention contains a scaffold polymer and a ligand that is soluble in water, therefore, in a wet state １ When H-pulse NMR is performed, the FID is observed as the sum of a crystalline component [hereinafter, component (I)], a constrained amorphous component [hereinafter, component (II)], and an unconstrained amorphous component [hereinafter, component (III)]. Each component can be separated by software fitting the Weibull function as a model equation to the observed FID. ２ The relaxation time increases in this order.

[0062] FID analysis is, １ This can be performed using general analysis software that is typically included with H-pulse NMR measuring instruments. In the case of the water-insoluble material contained in the biocomponent adsorption material of the present invention, the observed FID can be separated by a three-component analysis (without offset components) using a nonlinear least squares method. Component (I) can be separated by fitting an exponential function with a Weibull coefficient of 1, i.e., representing a crystalline material, and components (II) and (III) can be separated by fitting a Gaussian function with a Weibull coefficient of 2, i.e., representing an amorphous material. Furthermore, if separation is not possible with the above fitting, i.e., if the analysis does not converge, components (II) and (III) can be separated by a two-component analysis (without offset components) using a Weibull coefficient of 2. If the analysis does not converge even with the above two-component analysis, it can be determined that the water-insoluble material has a different structure from the water-insoluble material of the present invention. Furthermore, if the analysis converges in both the three-component analysis and the two-component analysis described above, the analysis with the smaller sum of squared residuals (SSR) in each analysis can be considered more appropriate.

[0063] Component (I) of the water-insoluble material of the present invention is presumed to originate from the most strongly oriented structure with restricted mobility, specifically, a scaffold polymer or support polymer with unbound ligands. If the biocomponent adsorption material of the present invention does not contain a scaffold polymer or support polymer with unbound ligands, then component (I) is absent. １When performing a three-component analysis of FID observed by H-pulse NMR, if the analysis does not converge, or if the SSR in the three-component analysis is greater than the SSR in the two-component analysis, it can be determined that component (I) does not exist.

[0064] Component (II) of the water-insoluble material of the present invention is presumed to originate from an insoluble and amorphous structure, specifically from a scaffold polymer to which a ligand is bound. Furthermore, component (II) is also presumed to originate from linkers bound to the scaffold polymer and from the cross-linking structure contained in the scaffold polymer.

[0065] The component (III) of the water-insoluble material of the present invention is the most T ２ The long relaxation time indicates a dissolved structure, which is specifically presumed to originate from water adhering to the surface of the biocomponent adsorbent material (adherent water). Furthermore, component (III) is also presumed to originate from a ligand that is locally dissolved in the adherent water. If component (III) is not detected, it can be determined that the material is dry and no water is adhering to it, and therefore remeasurement is necessary.

[0066] In other words, the water-insoluble material of the present invention is "by the solid echo method" １ In free induction decay (FID) observed by H-pulse NMR, the constrained amorphous component of T ２ "Relaxation time" refers to the measurement of a water-insoluble material in a wet state using the solid echo method. １ H-pulse NMR is performed to observe the FID, and the observed FID is analyzed using a nonlinear least squares method for three-component analysis or two-component analysis with the Weibull function as the model equation. (i) Of the components in the three-component analysis, T ２ The component with the second shortest relaxation time is T ２ Relaxation time, or (ii) among the components in the two-component analysis, T ２ The component with the shorter relaxation time T ２ This is the relaxation time. Here, T in (i) above. ２ The relaxation time is used when the analysis converges using only the three-component analysis, or when the analysis converges using both the three-component and two-component analysis, and the sum of squared residuals for each component is smaller in the three-component analysis, as described in (ii) above. ２Relaxation time is used when the analysis converges using only two-component analysis, or when the analysis converges using both three-component and two-component analysis, and the sum of squared residuals for each component is smaller in the two-component analysis.

[0067] The T of component (I) of the water-insoluble material of the present invention ２ The relaxation time is not particularly limited, but if it is too long, the crystallinity will be too low and the material strength will not be maintained, which is undesirable. If it is too short, the adhesion with component (III) will decrease and peeling may occur, raising concerns that fine particles may dissolve. Therefore, 0.0050 to 0.0200 ms is preferred, and 0.0060 to 0.0150 ms is more preferred.

[0068] Component (II) of the water-insoluble material of the present invention is T ２ If the relaxation time is too long, the mobility is too high, the surface of the substrate deforms freely, the mobility of the water also increases, and the effect of the biocomponent adsorption on properly enclosing the water is lost, so the adsorption performance of the biocomponent does not manifest. If it is too short, the mobility of the substrate surface is lost, so the adsorption performance of the biocomponent does not manifest. Therefore, the relaxation time is 0.0210 to 0.0270 ms, preferably 0.0220 to 0.0260 ms, and more preferably 0.0221 to 0.0260 ms.

[0069] The relative abundance of component (I) and component (II) is not particularly limited. However, if the abundance of component (I) is too high, the layer of the scaffold polymer to which the ligand is bound, which is presumed to be the origin of component (II), becomes thin, and the performance cannot be fully expressed. If the abundance of component (I) is too low, the strength cannot be guaranteed, and there is a concern about the elution of fine particles. For this reason, the abundance of component (I) is preferably 1 to 90%, more preferably 30 to 80%, and even more preferably 55 to 75%.

[0070] The component (III) in the water-insoluble material of the present invention varies depending on the degree of water adhesion, therefore, T ２ The relaxation time and abundance ratio are not particularly limited. However, this does not apply to water-insoluble materials. １ In H-pulse NMR measurements, too much water adhesion causes noise in the measurement, while too little water adhesion causes the water-insoluble material to dry out, resulting in a loss of ligand properties.１ In H-pulse NMR measurements, when the sum of components (I) and (II) is taken as 100%, the abundance ratio of component (III) should be between 50% and 300%. １ The method for H-pulse NMR measurement needs to be adjusted.

[0071] The relative abundance of component (III) can be adjusted by the time spent wiping off the water from the water-insoluble material using Kimwipes before NMR measurement. Specifically, shortening the time increases the relative abundance of component (III), while lengthening the time decreases the relative abundance of component (III).

[0072] A "reinforcement material" refers to a material that does not possess the ability to adsorb biological components on its own. Reinforcement materials are mixed in to maintain the shape of water-insoluble materials and to control their contact state with biological components.

[0073] The material of the reinforcing material is not particularly limited, but from the viewpoint of maintaining strength, a highly crystalline polymer material is preferred. Examples of highly crystalline polymer materials include polyamide, polyacrylonitrile, polyethylene, polypropylene, nylon, polymethyl methacrylate, and polytetrafluoroethylene, as well as copolymers thereof and blends thereof. As a highly crystalline polymer material, polyethylene or polypropylene, which have repeating units consisting only of carbon atoms and hydrogen atoms and have low reactivity, are preferred.

[0074] If a biocomponent adsorbent contains a reinforcing material, the water-insoluble material can be extracted by adding the biocomponent adsorbent to a solvent in which only the reinforcing material dissolves, or by physically separating the reinforcing material from the biocomponent adsorbent.

[0075] "Biocomponents" refer to substances or cells present in living organisms, such as those in tissues, organs, and blood. Biocomponents in blood include blood components that naturally make up blood, biocomponents released from other bodily fluids, tissues, organs, etc., that enter the bloodstream, and foreign pathogens, pathogenic microorganisms, and their secretions that enter the bloodstream. Blood components are further classified into humoral components and cellular components of blood.

[0076] "Cellular components in the blood" refers to the floating cells contained in the blood, such as white blood cells, red blood cells, and platelets. White blood cells are further classified into granulocytes, monocytes, and lymphocytes. Granulocytes are classified into activated and inactivated granulocytes, and monocytes are classified into activated and inactivated monocytes. Platelets are classified into activated and inactivated platelets. In addition, there are activated granulocyte-activated platelet complexes and activated monocyte-activated platelet complexes, which are combinations of white blood cells and platelets.

[0077] "Activated granulocytes" and "activated monocytes" refer to granulocytes and monocytes, respectively, that have been activated by stimulation such as cytokines and lipopolisaccharides (LPS). Activated granulocytes and activated monocytes release cytokines, reactive oxygen species, etc. The activation of granulocytes or monocytes can be determined by measuring the amount of reactive oxygen species released by the activated granulocytes or monocytes, or by measuring the expression of surface antigens using flow cytometry or other methods.

[0078] "Activated platelets" refer to platelets that have been activated by stimuli such as cytokines and LPS. Activated platelets release cytokines, reactive oxygen species, etc. The activation of platelets can be determined by measuring the expression of surface antigens using flow cytometry or other methods.

[0079] "Activated granulocyte-activated platelet complexes" and "activated monocyte-activated platelet complexes" refer to complexes in which activated granulocytes or activated monocytes are bound to activated platelets, which then exhibit phagocytic activity towards the body's own tissues.

[0080] "Hymolytic components of blood" refer to organic substances dissolved in the blood. Specifically, these include low-molecular-weight organic compounds such as urea and amino acids, proteins such as β2-microglobulin, cytokines, IgE, and IgG, polysaccharides such as LPS, and lipids such as LDL cholesterol and HDL cholesterol.

[0081] "Cytokines" refer to a group of proteins produced by various cells, including immune cells, in response to stimuli such as infection or trauma, and released extracellularly to exert their effects. Cytokines are classified into several groups according to their function, and examples include interferons (interferon-α, interferon-β, and interferon-γ), interleukins (interleukin-1 to interleukin-15), chemokines (CXCL8, CCL2, CCL3, and CCL5), tumor necrosis factors (TNF-α and TNF-β), cell growth factors (TGF-β, etc.), high mobility group box-1, and colony-stimulating factors (erythropoietin and G-CSF).

[0082] Among cytokines, those that induce or promote inflammatory responses are called inflammatory cytokines. Examples of inflammatory cytokines include interleukin-1, interleukin-6, interleukin-10, high mobility group box-1, and TNF-α. On the other hand, cytokines that suppress inflammatory responses are called anti-inflammatory cytokines. Examples of anti-inflammatory cytokines include interleukin-10 and TGF-β. A cytokine storm is a condition in which inflammatory cytokines become excessive, mainly due to external factors (viruses, trauma, drug administration, etc.), and the balance between inflammatory and anti-inflammatory cytokines is disrupted. This is thought to be the starting point of inflammatory diseases.

[0083] "Inflammatory diseases" refer to all diseases in which an inflammatory response is triggered in the body. Examples of inflammatory diseases include systemic lupus erythematosus, malignant rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, drug-induced hepatitis, alcoholic hepatitis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, sepsis (e.g., sepsis caused by Gram-negative bacteria, sepsis caused by Gram-positive bacteria, culture-negative sepsis, and fungal sepsis), influenza, acute respiratory distress syndrome (ARDS; also called acute respiratory distress syndrome or acute respiratory acceleration syndrome), acute lung injury (ALI), pancreatitis, and idiopathic interstitial pneumonia. Examples include fibrosis (IPF), reperfusion injury after organ transplantation, cholecystitis, cholangitis, and neonatal blood group incompatibility.

[0084] The biocomponent adsorption material of the present invention is used for the purpose of adsorbing and separating unwanted biocomponents from a solution, and is preferably used in blood purification therapy to remove unwanted biocomponents from blood.

[0085] In blood purification therapy, blood components are preferred as the adsorption targets for biocomponent adsorption materials.

[0086] In blood purification therapy, the cellular components in the blood that are adsorbed by the biocomponent adsorption material are preferably leukocyte components, and among leukocyte components, activated granulocytes, activated monocytes, activated granulocyte-activated platelet complexes and / or activated monocyte-activated platelet complexes that release inflammatory cytokines are more preferably, and activated granulocytes and / or activated granulocyte-activated platelet complexes that are thought to be directly involved in the pathogenesis of inflammatory diseases are even more preferably.

[0087] In blood purification therapy, preferred adsorption targets for biocomponent adsorption materials include β2-microglobulin, cytokines, proteins such as IgE and IgG, polysaccharides such as LPS, LDL cholesterol, or HDL cholesterol. More preferably, cytokines are involved in signal transduction and whose overproduction directly causes disease, and even more preferably, interleukin-6 (hereinafter referred to as IL-6) or interleukin-8 (hereinafter referred to as IL-8), which are inflammatory cytokines.

[0088] Biocomponent adsorbents capable of adsorbing excessively produced inflammatory cytokines (especially IL-6 and IL-8) can be used in the treatment of inflammatory diseases as blood purification therapy. Preferred inflammatory diseases for treatment with biocomponent adsorbents include drug-induced hepatitis, alcoholic hepatitis, hepatitis A, B, C, D, E, sepsis (e.g., sepsis from Gram-negative bacteria, sepsis from Gram-positive bacteria, culture-negative sepsis, or fungal sepsis), influenza, acute respiratory distress syndrome, acute lung injury, pancreatitis, or idiopathic interstitial pneumonia, where causative substances are released into the bloodstream and therapeutic effects through blood purification are particularly expected. Furthermore, inflammatory diseases for treatment with biocomponent adsorbents that are difficult to treat with drugs alone and are thought to involve both cytokines and activated leukocytes-activated platelets are even more preferred, including sepsis, influenza, acute respiratory distress syndrome, acute lung injury, or idiopathic interstitial pneumonia.

[0089] "Adsorption" refers to a state in which a specific substance adheres to a material and does not easily detach. Principles of adsorption include, for example, ionic interactions such as electrostatic interactions, van der Waals forces such as hydrophobic interactions or hydrogen bonds, and biological adhesion such as cell adhesion and phagocytosis of white blood cells.

[0090] One method for evaluating the adsorption capacity of biocomponent adsorbents to liquid components in blood is to impregnate fetal bovine serum (FBS) containing dissolved cytokines with the biocomponent adsorbent, measure the decrease in cytokine concentration in the FBS before and after impregnation, and calculate the cytokine adsorption rate. Examples of cytokines to be evaluated include IL-6 and IL-8.

[0091] Since the adsorption of IL-6 or IL-8 onto biocomponent adsorption materials is considered to be a Langmuir-type equilibrium reaction that coats the surface with a single layer, it is thought that adsorption equilibrium can be reached by performing the adsorption treatment for about 4 hours, regardless of the concentration of IL-6 or IL-8.

[0092] For the reasons stated above, it is preferable that the adsorption rate of IL-6 or IL-8 in the biocomponent adsorption material be 100% after 4 hours of adsorption treatment. However, since it is time-dependent, if the adsorption rate is 50% or more after 2 hours of adsorption treatment, it can be considered to have sufficient adsorption capacity, and if it is 60% or more, it is considered that IL-6 or IL-8 produced during extracorporeal circulation of blood can be sufficiently removed.

[0093] A "biometabolism adsorption column" refers to a column equipped with a biometabolism adsorption material. The biometabolism adsorption material of the present invention is preferably used as a material to fill a container that forms the outer shape of the biometabolism adsorption column.

[0094] The shape of the container that forms the outer shape of the biocomponent adsorption column is not particularly limited as long as it can be filled with biocomponent adsorption material and has an inlet and outlet for a solution containing biocomponents. Examples of such container shapes include cylindrical containers and prismatic containers such as triangular, square, hexagonal, and octagonal prismatic containers.

[0095] Furthermore, while the applications of the biocomponent adsorption column are not particularly limited, applications for removing unwanted biocomponents from a solution are preferred, and applications for removing proteins (e.g., cytokines) from a solution are more preferred. Specifically, applications include blood purification therapy, which removes pathogenic substances from the blood (e.g., treatment by removing cytokines from the blood of patients with inflammatory diseases), and applications for removing unwanted biocomponents from solutions containing biocomponents (e.g., removal of cytokines from organ preservation solutions to maintain the preservation state of organs extracted for transplantation, removal of cytokines in the manufacturing process of cell products such as blood products, removal of biocomponents other than T cells when T cells are extracted in CAR-T therapy, removal of cytokines and cell membranes from iPS cell-derived platelets, regeneration of dialysate by treating unwanted proteins and urea in liquids such as dialysate, and removal of unwanted bacterial proteins in the manufacturing process of biopharmaceuticals). Among these, it can be suitably used in blood purification therapy, where the removal of inflammatory cytokines is effective. When using a biocomponent adsorption column in blood purification therapy, an extracorporeal circulation method is preferred, in which the biocomponent adsorption column and the patient are connected by a blood circuit, blood taken from the patient is passed through the biocomponent adsorption column, and then returned to the patient. The processing time for blood in blood purification therapy is preferably long, more preferably 4 hours or more, and even more preferably 24 hours or more, from the viewpoint of suppressing the induction of further inflammatory responses by biocomponents.

[0096] The biocomponent adsorption column may be used in combination with other body fluid treatment methods or medical devices. Examples of other body fluid treatment methods or medical devices include plasma exchange, peritoneal dialysis, plasma separators, hemofilters, cardiopulmonary bypass, and Extracorporeal Membrane Oxygenation (ECMO). Furthermore, the biocomponent adsorption material or biocomponent adsorption column may be incorporated as a component of other medical devices.

[0097] The bio-component adsorption material of the present invention can be manufactured, for example, by the following method, but is not limited thereto.

[0098] The base material can be a commercially available one. If you wish to process the base material into any shape, such as fiber, flat film, hollow fiber film, or particle, you can purchase a commercially available resin, heat and melt it and extrude it from a nozzle, or dissolve it in a good solvent and then extrude it from a nozzle into a poor solvent or the atmosphere to form the desired shape. Alternatively, particle-shaped base materials can be produced by dissolving the polymer material that will become the base material in a good solvent, dropping the solution of the polymer material into a poor solvent, and then desolvating it to aggregate the polymer, or by suspension polymerization, which causes polymerization and precipitation simultaneously. When mixing reinforcing materials into the base material, they can be mixed, for example, by mixing and molding during the processing of the base material, or by physically mixing them in by needle punching after processing.

[0099] A substrate in which a reactive functional group is bonded to a scaffold polymer can be produced by adding the substrate, or the substrate and reinforcing material, to a solution in which a linker compound, a chloroacetamide having a haloalkyl group and a hydroxyl group, and a catalyst are dissolved in an aprotic solvent, and then reacting the mixture.

[0100] For the reaction to add reactive functional groups to a scaffold polymer [a reaction to introduce a linker into the substrate (hereinafter referred to as the linker reaction)], for example, sulfuric acid, hydrochloric acid, nitric acid, aluminum(III) chloride, or iron(III) chloride can be used as a catalyst, with sulfuric acid or iron(III) chloride being preferred.

[0101] As a chloroacetamide having a haloalkyl group and a hydroxyl group, for example, N-hydroxymethyl-2-chloroacetamide (hereinafter referred to as NMCA) can be used.

[0102] Examples of aprotic solvents used in the linker reaction include nitrobenzene, nitropropane, chlorobenzene, toluene, and xylene, with nitrobenzene or nitropropane being preferred.

[0103] The concentration of the catalyst in the reaction solution during the linker reaction is preferably 5 to 80 wt%, and more preferably 30 to 70 wt%.

[0104] The reaction temperature for the linker reaction is preferably 5 to 25°C, and more preferably 7 to 18°C, because if it is too low the linker will not react, and if it is too high there is a concern about side reactions. The reaction time for the linker reaction is preferably 1 minute to 120 hours, more preferably 5 minutes to 24 hours, and even more preferably 30 minutes to 12 hours.

[0105] A substrate in which a cross-linked structure is bonded to a scaffolding polymer can be manufactured by adding the substrate, or the substrate and reinforcing material, to a solution obtained by dissolving a cross-linking agent and a catalyst in an aprotic solvent and allowing it to react.

[0106] For example, paraformaldehyde (hereinafter referred to as PFA), acetaldehyde, or benzaldehyde can be used as the crosslinking agent in the reaction that introduces a crosslinked structure into the substrate (hereinafter referred to as the crosslinking reaction). The timing of the crosslinking reaction is not particularly limited, but it is preferable to carry it out simultaneously with the linker reaction. Specifically, the linker reaction and the crosslinking reaction can be carried out simultaneously by adding the crosslinking agent to the linker compound solution.

[0107] Examples of catalysts used in the crosslinking reaction include sulfuric acid, hydrochloric acid, nitric acid, aluminum(III) chloride, and iron(III) chloride, with sulfuric acid or iron(III) chloride being preferred.

[0108] Examples of aprotic solvents used in the crosslinking reaction include nitrobenzene, nitropropane, chlorobenzene, toluene, and xylene, with nitrobenzene or nitropropane being preferred.

[0109] The concentration of the catalyst in the reaction solution during the crosslinking reaction is preferably 5 to 80 wt%, and more preferably 30 to 70 wt%.

[0110] When the linker reaction and crosslinking reaction are carried out simultaneously, the time from the addition of the crosslinking agent to the linker compound solution to the start of the linker reaction (addition of the base material) should be 1 to 30 minutes, and more preferably 1 to 5 minutes, because if it is too long, side reactions will occur and the crosslinking reaction will be hindered. Furthermore, from the viewpoint of reaction control, it is preferable to dissolve the crosslinking agent in advance before adding it to the linker compound solution.

[0111] If the dissolution temperature of the crosslinking agent is too low, there is a concern that the crosslinking reaction will not proceed properly due to insufficient dissolution, and if it is too high, there is a concern that the reaction will not proceed properly due to decomposition, oxidation, etc. of the crosslinking agent. Therefore, a dissolution temperature of 30 to 70°C is preferred, and 35 to 60°C is more preferred.

[0112] The dissolution time for the crosslinking agent is preferably 45 to 120 minutes. If it is too short, there is a concern that the crosslinking reaction will not proceed properly due to insufficient dissolution, and if it is too long, there is a concern that the reaction will not proceed properly due to decomposition, oxidation, etc. of the crosslinking agent.

[0113] The reaction temperature for the crosslinking reaction is preferably 5 to 25°C, and more preferably 7 to 18°C. If it is too low, the reaction proceeds slowly, the scaffold polymer with disrupted orientation elutes, and only the components with low mobility become water-insoluble materials. If it is too high, crosslinking becomes excessive, and the mobility of the substrate decreases. The reaction time for the crosslinking reaction is preferably 1 minute to 120 hours, more preferably 5 minutes to 24 hours, and even more preferably 30 minutes to 12 hours.

[0114] A substrate in which a ligand is bonded to a scaffold polymer via a linker can be produced by adding a substrate to which a reactive functional group is bonded to a polar solvent containing a charged compound and allowing it to react. Furthermore, by subsequently adding a polar solvent containing a compound from which a spacer and a hydrophobic functional group are derived to a substrate in which a charged chemical structure is bonded as a ligand to a scaffold polymer via a linker and allowing it to react, it is possible to further bond spacers and hydrophobic functional groups to the bonded ligand.

[0115] Examples of compounds containing charged functional groups used in the reaction to immobilize a ligand on a linker-bound substrate (hereinafter referred to as the ligand reaction) include amine compounds (e.g., polyethyleneimine, hexaethylenetetramine, tetraethylenepentamine, triethylenetetramine, diethylenetriamine, and ethylenediamine), sulfonic acid compounds (e.g., sodium 2-hydroxyethanesulfonate, 2-aminoethanesulfonic acid), and carboxylic acid compounds (e.g., glycine and sodium 3-hydroxypropionate).

[0116] Examples of polar solvents used in the ligand reaction include N,N-dimethylformamide, diethyl ether, dioxane, tetrahydrofuran, and dimethyl sulfoxide, with N,N-dimethylformamide or dimethyl sulfoxide being preferred.

[0117] The reaction temperature for the ligand reaction is preferably 10 to 90°C, and more preferably 30 to 60°C. The reaction time for the ligand reaction is preferably 1 minute to 120 hours, and more preferably 5 minutes to 24 hours.

[0118] Examples of compounds from which spacers and hydrophobic functional groups are derived, used in the reaction to further attach spacers and hydrophobic functional groups to a ligand (hereinafter referred to as the spacer reaction), include methyl isocyanate, ethyl isocyanate, propyl isocyanate, phenyl isocyanate, 4-fluorophenyl isocyanate, 4-chlorophenyl isocyanate, 4-bromophenyl isocyanate, 4-iodophenyl isocyanate, acetate chloride, butyrate chloride, propionic acid chloride, benzoate chloride, 4-fluorobenzoate chloride, 4-chlorobenzoate chloride, 4-bromobenzoate chloride, and 4-iodobenzoate chloride.

[0119] Examples of polar solvents used in the spacer reaction include N,N-dimethylformamide, diethyl ether, dioxane, tetrahydrofuran, and dimethyl sulfoxide, with N,N-dimethylformamide or dimethyl sulfoxide being preferred.

[0120] The reaction temperature for the spacer reaction is preferably 10 to 90°C, and more preferably 30 to 60°C. The reaction time for the spacer reaction is preferably 1 minute to 120 hours, and more preferably 5 minutes to 24 hours.

[0121] T of component (II) of water-insoluble material ２ The relaxation time can be extended, for example, by increasing the dissolution temperature of the crosslinking agent and the reaction temperature of the linker reaction.

[0122] The content of charged functional groups in water-insoluble materials can be increased in ligand reactions by methods such as increasing the concentration of the compound containing the charged functional group, increasing the reaction temperature, or increasing the reaction time.

[0123] Methods for producing biocomponent adsorption materials by mixing a reinforcing material with a water-insoluble material include, for example, needle punching the reinforcing material with the water-insoluble material, and finely cutting the water-insoluble material and mixing it with the reinforcing material.

[0124] The bio-component adsorption material of the present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the examples, wt% means weight%, M means mol / L, mM means millimol / L, s means seconds, and ms means milliseconds. Unless otherwise specified, the weight of the base material, water-insoluble material, or bio-component adsorption material is the dry weight. Fineness is the weight (grams) per 10,000 m of fiber and is denoted as dtex. Also, in the following examples, no reinforcing material was used, so the bio-component adsorption material is also a water-insoluble material.

[0125] (Reference Example 1) Preparation of base material A: Polystyrene as the sea component and polypropylene as the island component were melted and weighed separately. These were then fed into a spinning pack incorporating a sea-island composite die with 704 island component distribution holes per discharge hole, creating a sea-island composite flow, which was then melted and extruded. By controlling the island ratio to 50 wt% during melting and extrusion, a sea-island type solid composite fiber with a single fineness of 3.0 dtex (fiber diameter 20 μm) was produced. From this sea-island type solid composite fiber, the gauge adjustment scale was adjusted using a tubular knitting machine (model name: circular knitting machine MR-1, Maruzen Sangyo Co., Ltd.) to obtain a basis weight of 55 g / m². ２ A tubular knitted fabric (hereinafter referred to as base material A) was prepared.

[0126] (Comparative Example 1) Preparation of the biocomponent adsorption material of Comparative Example 1 Following the method for producing the blood purification material 3 in Patent Document 1 (Patent No. 6589993), the biocomponent adsorption material of Comparative Example 1 was prepared as follows.

[0127] The following linker and crosslinking reactions were carried out on substrate A. 23 g of NMCA was added to a mixed solution of 310 g of nitrobenzene and 310 g of 98 wt% sulfuric acid, and the mixture was stirred at 10°C until the NMCA dissolved to obtain an NMCA solution. Next, 2 g of PFA was added to a mixed solution of 20 g of nitrobenzene and 20 g of 98 wt% sulfuric acid, and the mixture was stirred at 20°C for 15 minutes until the PFA dissolved to obtain a PFA solution. 42 g of the PFA solution cooled to 5°C was mixed with 643 g of the NMCA solution, stirred for 5 minutes, and then 10 g of substrate A was added and impregnated at 5°C for 2 hours. After impregnation, substrate A was immersed in 2000 mL of nitrobenzene at 0°C to stop the reaction, and then the nitrobenzene adhering to substrate A was extracted and removed with methanol.

[0128] Next, the ligand reaction was carried out as follows: 1.5 g of tetraethylenepentamine (hereinafter referred to as TEPA) and 4 g of triethylamine were dissolved in 510 g of dimethyl sulfoxide (hereinafter referred to as DMSO), and base material A, which had undergone the linker reaction and crosslinking reaction, was added to this solution and impregnated at 40°C for 3 hours. After impregnation, base material A was filtered off onto a glass filter and washed with 5000 mL of DMSO.

[0129] 470 g of DMSO, which had been previously dehydrated and dried with activated molecular sieves 3A, was mixed with 0.75 g of p-chlorophenyl isocyanate under a nitrogen atmosphere and heated to 30°C. Substrate A, which had undergone ligand reaction, was then impregnated in this mixture for 1 hour. After impregnation, substrate A was filtered off onto a glass filter to obtain the biocomponent adsorption material of Comparative Example 1.

[0130] (Comparative Example 2) Preparation of the biocomponent adsorption material of Comparative Example 2 The biocomponent adsorption material of Comparative Example 2 was obtained in the same manner as in Comparative Example 1, except that the amount of PFA added to the linker reaction was changed from 2 g to 1 g.

[0131] (Example 1) The linker reaction and crosslinking reaction were carried out on substrate A of the biocomponent adsorption material of Example 1 as follows. 23 g of NMCA was added to a mixed solution of 310 g of nitrobenzene and 310 g of 98 wt% sulfuric acid, and stirred at 10°C until the NMCA dissolved to obtain an NMCA solution. Next, 2 g of PFA was added to a mixed solution of 20 g of nitrobenzene and 20 g of 98 wt% sulfuric acid, and stirred at 40°C for 45 minutes until the PFA dissolved to obtain a PFA solution. 42 g of the PFA solution cooled to 5°C was mixed with the total amount of NMCA solution prepared, stirred for 5 minutes, and then 10 g of substrate A was added and impregnated at 5°C for 2 hours. After impregnation, substrate A was immersed in 2000 mL of nitrobenzene at 5°C to stop the reaction, and then the nitrobenzene adhering to substrate A was extracted and removed with methanol.

[0132] Next, the ligand reaction was carried out as follows: 1.5 g of TEPA and 4 g of triethylamine were dissolved in 510 g of DMSO, and base material A, which had undergone the linker reaction and crosslinking reaction, was added to this solution and impregnated at 40°C for 3 hours. After impregnation, base material A was filtered off onto a glass filter and washed with 5000 mL of DMSO.

[0133] 470 g of DMSO, which had been previously dehydrated and dried with activated molecular sieves 3A, was mixed with 0.75 g of p-chlorophenyl isocyanate under a nitrogen atmosphere and heated to 30°C. Substrate A, which had undergone ligand reaction, was then impregnated in this mixture for 1 hour. After impregnation, substrate A was filtered off onto a glass filter to obtain the bio-component adsorption material of Example 1 (support polymer: polypropylene; scaffold polymer: polystyrene; linker: structure of general formula (A); ligand: chemical structure in which tetraethylenepentamine, as a charged compound, is bonded to a urea bond as a spacer and a 4-chlorophenyl group as a hydrophobic functional group).

[0134] (Example 2) Preparation of the biocomponent adsorption material of Example 2 The biocomponent adsorption material of Example 2 was obtained in the same manner as in Example 1, except that the amount of PFA added to the linker reaction was changed from 2 g to 3 g and the reaction temperature of the linker reaction was changed from 5°C to 10°C.

[0135] (Example 3) Preparation of the biocomponent adsorption material of Example 3 The biocomponent adsorption material of Example 3 was obtained in the same manner as in Example 1, except that the reaction temperature of the linker reaction was changed from 5°C to 10°C.

[0136] (Example 4) Preparation of the biocomponent adsorption material of Example 4 The biocomponent adsorption material of Example 4 was obtained in the same manner as in Example 1, except that the reaction temperature of the linker reaction was changed from 5°C to 15°C.

[0137] (Example 5) Preparation of the biocomponent adsorption material of Example 5 The biocomponent adsorption material of Example 5 was obtained in the same manner as in Example 1, except that the reaction temperature of the linker reaction was changed from 5°C to 25°C.

[0138] (Comparative Example 3) Preparation of the biocomponent adsorption material of Comparative Example 3 The biocomponent adsorption material of Comparative Example 3 was obtained in the same manner as in Example 1, except that the amount of PFA added to the linker reaction was changed from 2 g to 0.1 g and the reaction temperature of the linker reaction was changed from 5°C to 10°C.

[0139] (Example 6) Preparation of the biocomponent adsorbent material of Example 6 The biocomponent adsorbent material of Example 6 was obtained in the same manner as in Example 1, except that the reaction temperature of the linker reaction was changed from 5°C to 10°C and the amount of TEPA added to the ligand reaction was changed from 1.5 g to 7.5 g.

[0140] (Example 7) Preparation of the biocomponent adsorption material of Example 7 The biocomponent adsorption material of Example 7 was obtained by the same method as in Example 1, except that the reaction temperature of the linker reaction was changed from 5°C to 10°C and the amount of p-chlorophenyl isocyanate added was changed from 0.75 g to 0 g. (Support polymer: polypropylene; scaffold polymer: polystyrene; linker: structure of general formula (A); ligand: tetraethylenepentamine as a charged compound)

[0141] (Comparative Example 4) Preparation of the biocomponent adsorption material of Comparative Example 4 The biocomponent adsorption material of Comparative Example 4 was obtained in the same manner as in Example 1, except that the reaction temperature of the linker reaction was changed from 5°C to 10°C and the amount of TEPA added to the ligand reaction was changed from 1.5 g to 0 g.

[0142] (Comparative Example 5) The biocomponent adsorption material of Comparative Example 5 was prepared by washing the substrate A with water.

[0143] (Example 8) Preparation of the biocomponent adsorption material of Example 8 A linker reaction and a crosslinking reaction were carried out on substrate A in the same manner as in Example 1. Next, the ligand reaction was carried out as follows: 1.5 g of propylamine and 4 g of triethylamine were dissolved in 510 g of DMSO, and substrate A after the linker reaction and crosslinking reaction was added to this, and impregnated at 40°C for 3 hours. After impregnation, substrate A was filtered off onto a glass filter and washed with 5000 mL of DMSO. Substrate A was then filtered off onto a glass filter to obtain the biocomponent adsorption material of Example 8 (support polymer: polypropylene; scaffold polymer: polystyrene; linker: structure of general formula (A); ligand: propylamine as a charged compound).

[0144] (Example 9) Preparation of the biocomponent adsorbent material of Example 9 The biocomponent adsorbent material of Example 9 (support polymer: polypropylene; scaffold polymer: polystyrene; linker: structure of general formula (A); ligand: diethylenetriamine as a charged compound) was obtained by the same method as in Example 8, except that the propylamine used in the ligand reaction was changed to diethylenetriamine.

[0145] (Example 10) Preparation of the biocomponent adsorption material of Example 10 The biocomponent adsorption material of Example 10 was obtained by the same method as in Example 8, except that the propylamine used in the ligand reaction was changed to polyethyleneimine with a mass-average molecular weight of 1800. (Support polymer: polypropylene; scaffold polymer: polystyrene; linker: structure of general formula (A); ligand: polyethyleneimine as a charged compound)

[0146] (Example 11) Evaluation of the content of charged functional groups (amino groups) in biocomponent adsorbent material <Calibration of pH meter> A benchtop pH meter (F-74BW, with standard ToupH electrode 9615S-10D, manufactured by Horiba, Ltd.) was calibrated using a pH standard solution set [phthalate standard solution (pH 4.01, manufactured by Horiba, Ltd.), neutral phosphate standard solution (pH 6.86, manufactured by Horiba, Ltd.), borate standard solution (pH 9.18, manufactured by Horiba, Ltd.)]. <Measurement of amino group content> Approximately 2 g of biocomponent adsorbent material and 50 mL of 6 M sodium hydroxide aqueous solution were added to a polypropylene container and stirred for 30 minutes. After stirring, the biocomponent adsorbent material was filtered off using filter paper. Next, 50 mL of ion-exchanged water and the filtered biocomponent adsorbent material were added to the polypropylene container and stirred for 30 minutes, after which the biocomponent adsorbent material was filtered off using filter paper. Using a pH meter, the pH of ion-exchanged water to which the biocomponent adsorbent material had been added was measured. The addition and filtration of the biocomponent adsorbent material to ion-exchanged water was repeated until the pH was 6 or less, thereby obtaining the desalted biocomponent adsorbent material. After the desalted biocomponent adsorbent material was allowed to stand for 48 hours at 80°C and atmospheric pressure, 1.0 g of the biocomponent adsorbent material and 30 mL of 0.1 M hydrochloric acid were added to a polypropylene container and stirred for 10 minutes. After stirring, 5 mL of the solution was withdrawn and transferred to another polypropylene container. Next, 0.1 mL of 0.1 M sodium hydroxide aqueous solution was added dropwise to the obtained solution. After addition, the mixture was stirred for 10 seconds using a vortex mixer, and the pH of the solution was measured using a pH meter. The stirring and pH measurement after adding the sodium hydroxide aqueous solution were repeated, and the amount of sodium hydroxide aqueous solution added when the pH of the solution exceeded 8.5 was defined as the titration volume. The amino group content in the ligand per gram of dry weight of the biocomponent adsorbent material (water-insoluble material) was calculated by rounding the titration volume and the value obtained using the following formula 1 to two decimal places. The results are shown in Tables 1 and 2. Amino group content in ligand per gram of water-insoluble material (moles / g) = [{Volume of 0.1 M hydrochloric acid added (30 mL) / Volume of hydrochloric acid withdrawn (5 mL)} × {Volume of neutralization solution (5 mL) - Titration volume (mL)} × Concentration of sodium hydroxide aqueous solution (0.1 M)] / {Dry weight of added water-insoluble material (1 g) ... Formula 1

[0147] (Example 12) Evaluation of linker content in biocomponent adsorbent material <pH meter calibration> The pH meter was calibrated in the same manner as in Example 11. <Measuring linker content> 1.0 g of biocomponent adsorbent material and 100 mL of 6 M hydrochloric acid were added to a 200 mL round-bottom flask and refluxed at 120°C for 24 hours. After refluxing, the biocomponent adsorbent material was recovered by filtering with filter paper to obtain the decomposed biocomponent adsorbent material. Next, the decomposed biocomponent adsorbent material and 50 mL of 6 M sodium hydroxide aqueous solution were added to a polypropylene container and stirred for 30 minutes, after which the decomposed biocomponent adsorbent material was filtered using filter paper. Next, 50 mL of ion-exchanged water and the filtered biocomponent adsorbent material were added to a polypropylene container and stirred for 30 minutes, after which the biocomponent adsorbent material was filtered using filter paper. The addition of the biocomponent adsorbent material to the ion-exchanged water and filtering were repeated until the pH of the ion-exchanged water containing the biocomponent adsorbent material was 6 or less, to obtain the decomposed and desalted biocomponent adsorbent material. The biocomponent adsorbent material, after decomposition and desalting, was left to stand for 48 hours at 80°C and atmospheric pressure. Next, 60 mL of 0.1 M hydrochloric acid was added to the biocomponent adsorbent material in a polypropylene container and stirred for 10 minutes. After stirring, 5 mL of the solution was withdrawn and transferred to a polypropylene container. Next, 0.1 mL of 0.1 M sodium hydroxide aqueous solution was added dropwise to the obtained solution. After adding the solution, the mixture was stirred for 10 minutes and the pH of the solution was measured. The stirring for 10 minutes after adding the solution and the pH measurement were repeated 100 times in the same manner. The amount of sodium hydroxide aqueous solution added when the pH of the solution exceeded 8.5 was defined as the titration volume per gram. The linker content per gram of dry weight of the biocomponent adsorbent material (water-insoluble material) was calculated by rounding the titration volume per gram and the value obtained using the following formula 2 to two decimal places. The results are shown in Tables 1 and 2. Linker content per gram of dry weight of water-insoluble material (moles / g) = {Volume of 0.1 M hydrochloric acid added (60 mL) / Volume of hydrochloric acid withdrawn (5 mL)} × Titration volume per gram (mL) × Concentration of sodium hydroxide aqueous solution (0.1 M) ...Equation 2

[0148] (Example 13) Biocomponent Adsorption Material １Analysis using H-pulse NMR measurement: The biocomponent adsorbent material, which had been stored in water, was removed from the water. Immediately after removal, the water was wiped off three times by sandwiching it between Kimwipes (manufactured by Nippon Paper Crecia Co., Ltd.), then cut with scissors, and the biocomponent adsorbent material was filled to the upper limit of the inner tube of the sample tube used for NMR measurement, and the sample tube was sealed with a silicone rubber cap. Subsequently, under the following conditions, １ H-pulse NMR measurements were performed, and the FID was observed. Equipment: Minispec mq20 (Bruker Japan Co., Ltd.) Measurement temperature: 37°C Sample temperature stabilization time: 10 minutes after filling the sample tube with the biocomponent adsorption material Observation frequency: 19.95 MHz 90° pulse width: 2.24 μs Pulse mode: Solid echo method Scans: 64 Recycle Delay: 3 s Acquisition Scale: 0.4 ms

[0149] The observed FID, １ The analysis was performed using the analysis software included with the instrument used for H-pulse NMR measurement [TDNMR-A software (Version 8.1, Rev 0.2), manufactured by Bruker Japan Co., Ltd.]. Specifically, a "seek" was performed, followed by "optimize," and then fitting was performed with settings 1 and 2 below. The fitting that did not produce an "error" was adopted. If no "error" occurred in either setting, the fitting with the smaller SSR was adopted. If an "error" occurred in both fittings, it was determined that the configuration differed from the biocomponent adsorption material of the present invention. In this embodiment, setting 1 fitting was actually adopted for all biocomponent adsorption materials evaluated. Setting 1 Fitting Formula: 1 / w Number of fitting components: 3 Weibull factor of fitting component 1: 1 Weibull factor of fitting component 2: 2 Weibull factor of fitting component 3: 2 offset: none Measurement target: T ２Setting 2 Fitting Formula: 1 / w Number of fitting components: 2 Weibull factor of fitting component 1: 2 Weibull factor of fitting component 2: 2 offset: none Measurement target: T ２

[0150] Next, the T of each component ２ The relaxation time was analyzed using similar analysis software. Note that each component is T ２ The components are listed in order from shortest relaxation time to longest: Component (I), Component (II), and Component (III). The ratio of each component was calculated with the sum of Component (I) and Component (II) set to 100%. The results are shown in Tables 1 and 2.

[0151] (Example 14) Evaluation of IL-6 adsorption rate of biocomponent adsorbent material To confirm the IL-6 adsorption capacity of the biocomponent adsorbent material, the biocomponent adsorbent material was impregnated in a liquid containing IL-6 for a predetermined time, then removed, and the IL-6 adsorption rate was measured from the difference in the amount of IL-6 in the liquid before and after impregnation. The detailed measurement method is as follows.

[0152] Four 6 mm diameter discs were cut from the biocomponent adsorbent material, their thickness was measured with a micrometer, and the total volume of the four discs was calculated and placed in a polypropylene container. In this container, an FBS solution prepared to have an IL-6 concentration of 2000 pg / mL was added to the total volume of the biocomponent adsorbent material (1 cm³). ３It was added at a solid-liquid ratio of 30 mL. After inversion mixing in a 37°C incubator for 2 hours, the IL-6 concentration in the FBS solution was measured by ELISA. The IL-6 concentration was calculated from the absorbance of the FBS solution. For absorbance measurement, a microplate reader (Spectra Max M5, Molecular Devices Japan Co., Ltd.) was used, with a measurement wavelength of 450 nm and a reference wavelength of 570 nm, after performing a blank measurement beforehand. The IL-6 adsorption rate was calculated by rounding the value obtained from the IL-6 concentrations before and after inversion mixing to the first decimal place using the following formula 3. The results are shown in Tables 1 and 2. IL-6 adsorption rate (%) = 100 × {IL-6 concentration before inversion mixing (pg / mL) - IL-6 concentration after inversion mixing (pg / mL)} / IL-6 concentration before inversion mixing (pg / mL) ...Formula 3

[0153] (Example 15) Evaluation of IL-8 adsorption rate of biocomponent adsorbent material The IL-8 adsorption rate of Comparative Examples 1 and 3-5, and the biocomponent adsorbent materials of Examples 1-3 were measured using the same method as in Example 14, except that IL-6 was replaced with IL-8. The results are shown in Table 1.

[0154]

[0155]

[0156] As shown in Tables 1 and 2, the T of component (II) ２ It was found that biocomponent adsorbent materials with a relaxation time of 0.0210 to 0.0270 ms have a high adsorption rate of biocomponents (IL-6 and IL-8). Note that the biocomponent adsorbent material of Comparative Example 4 had a T of component (II). ２ Although the relaxation time was within the above range, it hardly adsorbed any biological components because it contained no ligands, i.e., charged functional groups. Furthermore, as is clear from the comparison between the biological component adsorption material of Example 6 and the biological component adsorption materials of the other examples, the biological component adsorption material of the present invention had a high adsorption rate of biological components, regardless of the content of charged functional groups (amino groups).

[0157] The biocomponent adsorption material of the present invention can be used as an adsorption material for biocomponent adsorption columns aimed at separating biocomponents, particularly for extracorporeal circulation columns in blood purification therapy.

Claims

1. The material comprises a water-insoluble material consisting of a substrate containing a scaffold polymer, a linker bonded to the substrate, and a ligand bonded to the linker, wherein the water-insoluble material is obtained by solid echo method １ In free induction decay (FID) observed by H-pulse NMR, the constrained amorphous component of T ２ A bio-component adsorption material having a relaxation time of 0.0210 to 0.0270 ms.

2. The biocomponent adsorption material according to claim 1, wherein the linker includes a carbonyl bond or an ether bond.

3. The bio-component adsorption material according to claim 1, wherein the linker includes an amide bond.

4. The biosorbent material according to claim 3, wherein the linker has a chemical structure represented by the following general formula (A). (In the formula, the wavy line represents the bonding position with the substrate, and X represents a ligand.) 5. The scaffold polymer includes a crosslinked structure, the bio-component adsorption material according to any one of claims 1 to 4.

6. The biocomponent adsorption material according to any one of claims 1 to 5, wherein the content of charged functional groups contained in the ligand is 0.2 to 4.0 mmol per gram of dry weight of the water-insoluble material.

7. A biocomponent adsorption column comprising a biocomponent adsorption material according to any one of claims 1 to 6.

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