Blood purification column

Abstract

To provide a blood purification column that enables suppression of lymphocyte adsorption while allowing adsorption of cytokines and neutrophils.SOLUTION: A blood purification column is provided in which an adsorbent containing a water-insoluble material is packed and the maximum length of an adsorbent layer composed of the adsorbent is 6.5 mm or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a column for blood purification. [Background technology]

[0002] Chimeric antigen receptor T-cell therapy (CAR-T cell therapy), which has recently attracted attention as a cancer treatment, has a problematic side effect of cytokine release syndrome (CRS) (Non-Patent Document 1). Treatments for CRS include the administration of anti-interleukin-6 receptor antibodies, steroids, and other pharmaceuticals. However, pharmaceutical treatments may not be effective enough due to factors such as attenuation of therapeutic efficacy with continuous administration and the existence of drug-resistant patients. Furthermore, depending on the type of pharmaceutical, there are concerns about side effects such as increased neurotoxicity and decreased immune function.

[0003] One way to solve the problems of CRS treatment with pharmaceuticals is to treat CRS using extracorporeal circulation therapy. Extracorporeal circulation therapy is used, for example, as a treatment method for inflammatory diseases, in which cytokines and neutrophils, a source of cytokines, are adsorbed and removed from the body. In the treatment of inflammatory diseases, it is desirable to adsorb and remove both cytokines and neutrophils, because the therapeutic effect of only cytokines or neutrophils alone is limited.

[0004] Furthermore, in extracorporeal circulation therapy, if lymphocytes are adsorbed and removed, the patient's immune function may be weakened, and they may develop infections during or after treatment. In particular, in CRS treatment, which occurs as a side effect of CAR-T cell therapy, if CAR-T cells, which are derived from T cells (a type of lymphocyte), are adsorbed and removed, the effectiveness of cancer treatment will be reduced.

[0005] Therefore, there is a need to develop a blood purification column for extracorporeal circulation to treat CRS and other side effects of CAR-T cell therapy that adsorbs cytokines and neutrophils while suppressing lymphocyte adsorption.

[0006] As a material that selectively removes neutrophils, for example, Patent Document 1 discloses a material for removing activated granulocyte-activated platelet complexes in which the average open area of ​​the stitches of the knitted fabric or the weave of the woven fabric is specified.

[0007] Furthermore, Patent Document 2 discloses an adsorption carrier for removing leukocytes and cytokines, which is characterized by having a two-layer structure of at least a net and a nonwoven fabric.

[0008] Furthermore, Patent Document 3 discloses a blood purification column having two packing chambers therein, each packed with adsorption particles of a different particle size. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2021-145918 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-305342 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-19685 [Non-patent literature]

[0010] [Non-Patent Document 1] Justin G Yoon et al., Am J Roentgenol., 2021, Vol. 217, 1461-1474 Summary of the Invention [Problem to be solved by the invention]

[0011] However, while the removal material described in Patent Document 1 is a material in which the average open pore area of ​​the knitted or woven fabric is specified, Patent Document 1 does not disclose the idea of ​​inhibiting lymphocyte adsorption by specifying the method of packing into a column, nor does it disclose anything about cytokine removal. Furthermore, since the open pore rate in the average open pore area is 30.2 to 35.0%, which means the density of the knitted or woven fabric is low, it is thought that this removal material would be difficult to pack into a column.

[0012] Patent Document 2 lists white blood cells (a broader concept than lymphocytes) as the target for adsorption by the adsorption carrier described in Patent Document 2, discloses a fiber diameter suitable for adsorbing lymphocytes, and further discloses in the examples that the adsorption carrier adsorbs lymphocytes.

[0013] Patent document 3 lists inflammatory cells such as white blood cells (a broader concept than lymphocytes) as targets for adsorption by the adsorption particles described in Patent document 3, and also discloses in the examples that a blood purification column filled with the adsorption particles adsorbs inflammatory cells.

[0014] Therefore, an object of the present invention is to provide a blood purification column that adsorbs cytokines and neutrophils while suppressing the adsorption of lymphocytes. [Means for solving the problem]

[0015] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that a blood purification column packed with an adsorbent containing a water-insoluble material and having an adsorbent layer composed of said adsorbent with a maximum length of 6.5 mm or less can adsorb cytokines and neutrophils, while inhibiting the adsorption of lymphocytes.

[0016] That is, the present invention provides the following (1) to (5). (1) A column for blood purification, packed with an adsorbent containing a water-insoluble material, the maximum length of the adsorbent layer made of said adsorbent being 6.5 mm or less. (2) The blood purification column according to (1), which is packed with spacers, and in which spacer layers composed of the spacers and the adsorbent layers are alternately laminated. (3) The blood purification column according to (1) or (2), wherein the water-insoluble material contains a ligand having an amide group and an amino group, the content of the amide group being 3.0 to 7.0 mmol per gram of dry weight of the water-insoluble material, and the content of the amino group being 1.0 to 7.0 mmol per gram of dry weight of the water-insoluble material. (4) The blood purification column according to any one of (1) to (3), wherein the adsorbent is a knitted or woven fabric. (5) The blood purification column according to any one of (1) to (3), which is used for treating cytokine release syndrome. [Effects of the Invention]

[0017] The blood purification column of the present invention adsorbs cytokines and neutrophils while inhibiting the adsorption of lymphocytes, and therefore can be used in the treatment of inflammatory diseases, particularly CRS. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 2 is a diagram showing the adsorbent layer of the adsorbent and spacer material packed in a column, and the lengths of the spacer material layer and the adsorbent layer. [Figure 2] FIG. 2 is a diagram showing the configuration of the adsorbent and spacer materials packed in columns A to G. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below.

[0020] The blood purification column of the present invention is packed with an adsorbent containing a water-insoluble material, and the maximum length of the adsorbent layer made of the adsorbent is 6.5 mm or less.

[0021] The term "adsorbent" refers to a material that can adsorb a substance to be adsorbed in a solution by contacting the solution, thereby reducing the concentration of the substance to be adsorbed in the solution.

[0022] The shape of the adsorbent is not particularly limited, but is preferably a film, particle, or fiber shape, and more preferably a fiber shape. Considering use in blood purification, the shape of the adsorbent is more preferably an islands-in-the-sea fiber shape, which has a large specific surface area, is flexibly deformable, and is easy to handle. Furthermore, as shapes obtained by processing the above-mentioned fiber shapes, fiber bundles, knitted fabrics, woven fabrics, and nonwoven fabrics are preferred, with knitted fabrics, woven fabrics, and nonwoven fabrics being more preferred, which have a large specific surface area and low flow path resistance, and knitted fabrics and woven fabrics being even more preferred from the viewpoints of ease of filling the adsorbent and uniformity of the flow paths.

[0023] "Water-insoluble material" refers to a material that is insoluble in water, including a substrate. Here, "water-insoluble" means that the change in dry weight of the water-insoluble material before and after immersion in water is 1% or less. This change in dry weight is determined by immersing the water-insoluble material in 37°C water in an amount 9 times its dry weight for 1 hour, then removing it with tweezers or the like, and vacuum-drying the remaining water at 50°C or less, and then measuring the dry weight of the remaining solids relative to the dry weight of the water-insoluble material before immersion. If the material is not insolubilized, there is a risk of excessive elution during actual use, which is undesirable from a safety standpoint.

[0024] The term "substrate" refers to a material to which a ligand can be immobilized by chemical modification and which is water-insoluble after immobilization. From the viewpoint of stability in blood, the substrate is preferably a polymeric compound. There are no particular restrictions on the polymeric compound used as the substrate, but since it is used for blood purification, a sterilization-resistant polymeric compound is preferred. Examples of sterilization-resistant polymeric compounds include polystyrene, polypropylene, polyvinyl chloride, polymethyl methacrylic acid, cellulose, polyarylate, and polyethylene terephthalate.

[0025] The term "adsorbent containing a water-insoluble material" refers to an adsorbent containing a water-insoluble material as at least a part of its material, and includes the water-insoluble material alone or immobilized on or mixed with a suitable reinforcing material.

[0026] The material of the reinforcing material is not particularly limited, but examples thereof include polymeric materials that do not contain aromatic rings or hydroxyl groups in the repeating structure, such as homopolymers or copolymers of polyamide, polyethylene, polypropylene, nylon, or polymethyl methacrylate, or materials obtained by physically blending the above homopolymers or copolymers. Among these, polyethylene or polypropylene is preferred.

[0027] The term "ligand" refers to a chemical structure contained in a water-insoluble material to exhibit adsorptive properties for cytokines and neutrophils.

[0028] The term "amide group" refers to an amide bond contained in the ligand. From the viewpoint of exhibiting neutrophil adsorption performance, it is preferable that there is little steric hindrance around the amide group. Therefore, it is preferable that the chemical structure adjacent to the amide group does not contain a cyclic structure, and is preferably, for example, an alkylene group such as a methylene group or ethylene.

[0029] The term "amino group" refers to a chemical structure contained in a ligand and containing one or more amines as partial structures. From the viewpoint of exhibiting cytokine adsorption performance, the amino group is preferably an amino group derived from a polyamine, and the polyamine is preferably an ethylenediamine polymer such as diethyltriamine, tetraethylenepentamine, or polyethyleneimine.

[0030] The term "ligand having an amide group and an amino group" refers to a chemical structure containing one or more amide groups and one or more amino groups. There are no particular limitations on the ligand having an amide group and an amino group, but it is preferred that the amino group is a polyamine and the chemical structure adjacent to the amide group is an alkylene group.

[0031] A preferred mode of binding a ligand having an amide group and an amino group to a substrate in a water-insoluble material includes, for example, a structure represented by the following general formula (I): [ka] [In the formula, X represents an amino group, and the wavy line represents the bonding position to the substrate.]

[0032] Furthermore, the ligand having an amide group and an amino group preferably has a phenyl group and is bound to the substrate in a structure represented by the following general formula (II), which can further inhibit platelet adhesion. [ka] [In the formula, X represents an amino group, A represents a linker, B represents a hydrogen atom or a halogen atom, and the wavy line represents the bonding position to the substrate.]

[0033] The term "phenyl group" refers to a phenyl group derived from unsubstituted benzene or a substituted benzene compound, such as benzene, fluorobenzene, chlorobenzene, bromobenzene, 1,2-difluorobenzene, 1,2-dichlorobenzene, 1,2-bromobenzene, 1,3-difluorobenzene, 1,3-dichlorobenzene, 1,3-dibromobenzene, 1,4-difluorobenzene, 1,4-dichlorobenzene, and 1,4-dibromobenzene. To control the charge of the amino group, a phenyl group derived from a halogenated benzene to which an electron-withdrawing group has been added is preferred, and a chlorophenyl group derived from chlorobenzene is particularly preferred. The electron-withdrawing group is preferably added at the para position in terms of resonance structure. In particular, in the case of a phenyl group derived from chlorobenzene, a parachlorophenyl group in which the linker and the chlorine atom are substituted at the para position is preferred.

[0034] The term "linker" refers to a chemical bond between the amino group and the phenyl group, and examples thereof include electrically neutral chemical bonds such as an amide bond, a urea bond, an ether bond, and an ester bond, with an amide bond or a urea bond being preferred.

[0035] "Dry weight" refers to the weight of a solid in a dry state. Here, a water-insoluble material in a dry state refers to a state in which the amount of liquid components contained in the water-insoluble material is 1% by weight or less.

[0036] From the viewpoint of exhibiting cytokine adsorption performance, the content of amino groups per gram of dry weight of the water-insoluble material is preferably 1.0 to 7.0 mmol.

[0037] From the viewpoint of exhibiting neutrophil adsorption performance, the content of amide groups per 1 g of dry weight of the water-insoluble material is preferably 3.0 to 7.0 mmol.

[0038] The amide group content of the water-insoluble material contained in the adsorbent can be determined, for example, by dissolving the reinforcing material contained in the blood purification material in a solvent, extracting only the water-insoluble material, measuring the dry weight, and then hydrolyzing the water-insoluble material by heating it in hydrochloric acid, ion-exchanging the generated amino groups with hydrochloric acid, and back-titrating with sodium hydroxide. In the case of an adsorbent that does not contain a reinforcing material, the step of dissolving the reinforcing material in a solvent is not necessary.

[0039] The amino group content of the water-insoluble material contained in the adsorbent can be determined, for example, by dissolving the reinforcing material contained in the blood purification material in a solvent, extracting only the water-insoluble material, measuring the dry weight, and then ion-exchanging the amino groups in the water-insoluble material with hydrochloric acid and back-titrating with sodium hydroxide. In the case of an adsorbent that does not contain a reinforcing material, the step of dissolving the reinforcing material in a solvent is not necessary.

[0040] An "adsorbent layer" refers to an adsorbent layer stacked in a continuous manner within a column. That is, an adsorbent layer is composed of adsorbents. An adsorbent layer may be composed of only one adsorbent.

[0041] The "length of the adsorbent layer" refers to the length of the adsorbent layer in the direction parallel to the direction of liquid flow in the column.

[0042] The "maximum length of the adsorbent layer" refers to the maximum length of one or more adsorbent layers present in a column.

[0043] It is thought that if the maximum length of the adsorbent layer is long, physical adsorption is more likely to occur due to the physical overlap of the adsorbents.From the perspective of suppressing lymphocyte adsorption, the maximum length of the adsorbent layer is 6.5 mm or less.

[0044] The adsorbent packing volume is the sum of the volumes of the adsorbents present in the column.

[0045] The volume of the adsorbent is calculated according to the shape of the adsorbent. For example, if the adsorbent is in the shape of a knitted fabric, the volume can be calculated by multiplying the area of ​​the knitted fabric, the thickness of the knitted fabric, and the number of layers of knitted fabric.

[0046] From the viewpoint of highly efficient adsorption of the target substance, it is preferable that the ratio of the adsorbent filling volume to the solution volume is large. When the solution is blood and the target substances are cytokines and neutrophils, the adsorbent filling volume should be 2.0 cm for 1 L of blood. 3 It is preferable that this is equal to or greater than this.

[0047] The blood purification column of the present invention is preferably packed with spacers in addition to the adsorbent, and spacer layers composed of the spacers and adsorbent layers are alternately laminated.

[0048] The "spacer" is a material packed into a column to prevent the adsorbents from overlapping with each other, and preferably has a higher porosity than the adsorbents.

[0049] There are no particular restrictions on the shape of the spacer, but from the viewpoint of achieving both high porosity and high mechanical strength, a ring shape or a mesh shape is preferred.

[0050] From the viewpoint of stability in blood, the spacer is preferably a polymer compound. There are no particular limitations on the polymer compound used as the spacer, but since it is used for blood purification, a sterilization-resistant polymer compound is preferred. Examples of sterilization-resistant polymer compounds include polystyrene, polypropylene, polyvinyl chloride, polymethyl methacrylic acid, cellulose, polyarylate, and polyethylene terephthalate.

[0051] The term "spacer layer" refers to spacers stacked continuously within a column. That is, the spacer layer is composed of spacers. The spacer layer may be composed of only one spacer.

[0052] The phrase "adsorbent layers and spacer layers are stacked alternately" refers to adsorbent layers and spacer layers being stacked alternately. When a column contains multiple adsorbent layers and / or spacer layers, multiple adsorbent layers and spacer layers may be stacked alternately.

[0053] A schematic diagram of the adsorbent layer, spacer layer, and length of the adsorbent layer is shown in Figure 1. The adsorbent layer 3 is composed of continuously stacked adsorbents 1, and the spacer layer 4 is composed of continuously stacked spacers 2. The length 5 of the adsorbent layer corresponds to the total thickness of the adsorbents 1 that make up the adsorbent layer 3.

[0054] "Cytokines" are a group of proteins produced by various cells, including immune cells, in response to stimuli such as infection or trauma, and released extracellularly to act. Examples of cytokines include interferon-α, interferon-β, interferon-γ, interleukin-1 to -15, tumor necrosis factor-α, tumor necrosis factor-β, high mobility group box-1, erythropoietin, and monocyte chemotactic factor. Among cytokines, interleukin-6 (IL-6) in particular is believed to be a causative agent of inflammatory diseases such as CRS.

[0055] A column used in extracorporeal circulation therapy for inflammatory diseases preferably has a higher lymphocyte adsorption rate than a neutrophil adsorption rate, and the adsorption rate ratio, obtained by dividing the neutrophil adsorption rate by the lymphocyte adsorption rate, can be used as an indicator of this. When the inflammatory disease is CRS, which occurs as a side effect of CAR-T cell therapy, the above adsorption rate ratio is preferably 4.0 or higher to prevent a decrease in the therapeutic effect due to lymphocyte adsorption removal.

[0056] A method for evaluating the adsorption performance of a blood purification column for neutrophils, lymphocytes, or cytokines can be, for example, by filling a container having an inlet and an outlet with an adsorbent and a spacer, passing a liquid containing neutrophils, lymphocytes, and / or cytokines through the container, and calculating the adsorption rates of the neutrophils, lymphocytes, and / or cytokines from the changes in their concentrations at the inlet and outlet.

[0057] The neutrophil concentration can be measured, for example, by reacting a neutrophil-containing liquid with a neutrophil detection reagent and measuring the fraction of blood cells bound to the reagent. This measurement can be performed using a flow cytometer or a hemocytometer.

[0058] The lymphocyte concentration can be measured, for example, by reacting a lymphocyte-containing liquid with a lymphocyte detection reagent and measuring the fraction of blood cells bound to the reagent. For this measurement, a flow cytometer or a hemocytometer can be used.

[0059] The concentration of a cytokine can be measured, for example, by reacting a cytokine-containing liquid with a cytokine detection reagent and measuring the amount of cytokine bound to the reagent, using an enzyme-linked immunosorbent assay (ELISA) or the like. [Example]

[0060] The blood purification column of the present invention will be specifically described below using examples, but the present invention is not limited to these examples. The methods for producing adsorbents A and B described below are in accordance with Japanese Patent No. 6,589,993.

[0061] (Production of Fiber A) Using a spinneret with discharge holes designed to form a fiber cross section with 256 island components in the sea component, polystyrene was used for the sea component and polypropylene for the island components, and melt spinning was performed so that the discharge ratio of the sea component to the island components was 50:50 by volume, to obtain Fiber A, which was a bundle of 36 sea-island composite fibers, each with a fineness of 3 dex (the total fineness of Fiber A was 108 dex).

[0062] (Production of knitted fabric A) Fiber A was knitted using a cylindrical knitting machine (boiler diameter: 7 inches, number of needles: 480, needle density: 8.6 / cm) with the stitch density adjustment scale set to 3.5, to obtain knitted fabric A.

[0063] (Production of knitted fabric B) Fiber A was knitted using a cylindrical knitting machine (boiler diameter: 7 inches, number of needles: 480, needle density: 8.6 / cm) with the stitch density adjustment scale set to 5.0, to obtain knitted fabric B.

[0064] (Preparation of adsorbent intermediate A) A reaction solution was prepared by mixing, stirring, and dissolving 46 wt% nitrobenzene, 46 wt% sulfuric acid, 1 wt% paraformaldehyde, and 7 wt% N-methylol-α-chloroacetamide at 10°C or below. 1.0 g of knitted fabric A was immersed in 40 mL of the reaction solution cooled to 5°C, and the reaction was carried out for 2 hours while maintaining the temperature of the reaction solution at 5°C. After that, knitted fabric A was removed from the reaction solution and immersed and washed in 40 mL of nitrobenzene. Next, knitted fabric A was removed and immersed and washed in methanol to obtain adsorbent intermediate A.

[0065] (Preparation of adsorbent A) 10 g of adsorbent intermediate A was immersed in a reaction solution prepared by dissolving tetraethylenepentamine and triethylamine in 500 mL of dimethyl sulfoxide to a concentration of 3 mM and 474 mM, respectively, and reacted at 40°C for 3 hours. The post-reaction adsorbent intermediate A was then removed from the reaction solution and immersion-washed in 500 mL of dimethyl sulfoxide, then in 500 mL of methanol, and further in 500 mL of water to obtain adsorbent A. The thickness of adsorbent A was 0.213 mm.

[0066] (Preparation of adsorbent intermediate B) Adsorbent intermediate B was obtained by carrying out the same procedure as in the preparation of adsorbent intermediate A, except that knitted fabric B was used instead of knitted fabric A.

[0067] (Preparation of adsorbent B) Adsorbent B was obtained by carrying out the same procedure as in the preparation of adsorbent A, except that adsorbent intermediate B was used instead of adsorbent intermediate A. Adsorbent B had a thickness of 0.265 mm.

[0068] (Preparation of Column A) Adsorbent A was cut into a 10 mm diameter disk, and spacers (polypropylene mesh, 200 μm opening, 0.56 mm thick) were cut into an 8 mm diameter disk. Hereinafter, the "disk-shaped adsorbent" and "disk-shaped spacer" will be simply referred to as "adsorbent" and "spacer," respectively. A cylindrical container (inner diameter 10 mm × height 12 mm, made of polycarbonate) with a liquid inlet at one end and a liquid outlet at the other end was packed with adsorbent layers consisting of 4 or 5 layers of adsorbent A and one spacer alternately stacked so that the total number of adsorbent A packed was 38 and the total number of spacers packed was 8, to obtain column A. The maximum length of the adsorbent layer in column A was 1.07 mm, and the packed volume was 6.36 mm. 3 It was.

[0069] (Preparation of Column B) Column B was obtained by the same procedure as in the preparation of Column A, except that the method of packing the adsorbent and spacers into the container was changed to stacking 7 or 8 layers of adsorbent A alternately with one spacer 5 times, followed by packing with 3 spacers. The maximum length of the adsorbent layer in Column B was 1.70 mm, and the packing volume was 6.36 mm. 3 It was.

[0070] (Preparation of Column C) Column C was obtained by the same procedure as in the preparation of Column A, except that the method of packing the adsorbent and spacers into the container was changed to packing 38 sheets of adsorbent A followed by 8 spacers. The maximum length of the adsorbent layer of Column C was 8.09 mm, and the packing volume was 6.36 mm. 3 It was.

[0071] (Preparation of Column D) Using the preparation method for column A as a reference, adsorbent B was used instead of adsorbent A, and two or three layers of adsorbent B were alternately stacked with one spacer so that the total number of adsorbent B sheets packed in the container was 23 and the total number of spacers was 10, to obtain column D. The maximum length of the adsorbent layer of column D was 0.80 mm, and the packed volume was 4.79 mm. 3 It was.

[0072] (Preparation of Column E) Column E was obtained by the same procedure as in the preparation of Column D, except that the method of packing the adsorbent and spacers into the container was to pack 23 sheets of adsorbent B followed by 10 spacers. The maximum length of the adsorbent layer of Column E was 6.10 mm, and the packing volume was 4.79 mm. 3 It was.

[0073] (Preparation of Column F) Column F was obtained by carrying out the same procedure as in Column D, except that 35 adsorbent sheets and 5 spacers were packed into the column after 35 adsorbent sheets had been packed, followed by 5 spacers. The maximum length of the adsorbent layer in Column F was 9.28 mm, and the packed volume was 7.28 mm. 3 It was.

[0074] (Preparation of Column G) Column G was obtained by the same procedure as in Column D, except that 46 adsorbent sheets were packed and no spacers were packed. The maximum length of the adsorbent layer in Column G was 12.19 mm, and the packed volume was 9.57 mm. 3 It was.

[0075] The configurations of columns A to G are shown in FIG.

[0076] [Table 1]

[0077] (Measurement of the amino group content of water-insoluble materials contained in adsorbents) The content of amino groups in the water-insoluble material contained in the adsorbent was determined by measuring the amount of amino groups in the water-insoluble material by acid-base back titration, as described in detail below.

[0078] 5.0 g of the adsorbent was placed in a 200 mL recovery flask, 100 mL of toluene was added, and the mixture was refluxed at 150 °C for 24 hours to remove the polypropylene reinforcing material. The refluxed solution was quickly added to 2 L of toluene heated to 100 °C and washed. The insoluble components were filtered through filter paper, washed with methanol, and then placed in a dryer at 60 °C for 4 hours to obtain a water-insoluble material. Next, 1.0 g of the water-insoluble material and 50 mL of 6 M sodium hydroxide solution were added to a polypropylene container and stirred for 30 minutes. The water-insoluble material was then filtered through filter paper. The filtered water-insoluble material was then added to 50 mL of ion-exchanged water, stirred for 30 minutes, and filtered through filter paper. This process of adding the water-insoluble material to ion-exchanged water and filtering was repeated until the pH of the ion-exchanged water reached 7, yielding a desalted water-insoluble material. The desalted water-insoluble material was allowed to stand at 80°C under normal pressure for 48 hours, after which 1.0 g of the water-insoluble 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 removed and transferred to a polypropylene container. 0.1 mL of 0.1 M aqueous sodium hydroxide solution was added dropwise to the solution. After addition, the solution was stirred for 10 minutes and the pH of the solution was measured. The dropwise addition and subsequent stirring were repeated, and the amount of aqueous sodium hydroxide solution added when the solution pH exceeded 8.5 was taken as the titer per gram. The amino group content per gram of dry weight of the water-insoluble material was calculated using the appropriate amount per gram and the following formula 1.

[0079] Amino group content per 1 g of dry weight of water-insoluble material (mmol / g) = {volume of 0.1 M hydrochloric acid added (30 mL) / volume of hydrochloric acid removed (5 mL)} × titration amount per 1 g (mL) × concentration of sodium hydroxide solution (0.1 M) Equation 1

[0080] Measurements of adsorbent A and B showed that the amino group content per 1 g of dry weight of the water-insoluble material contained in adsorbent A was 1.6 mmol, and the amino group content per 1.0 g of dry weight of the water-insoluble material contained in adsorbent B was 1.6 mmol.

[0081] (Measurement of the amide group content of water-insoluble materials contained in adsorbents) The amount of amide groups contained in the water-insoluble carrier A contained in the adsorbent A was determined by measuring the amount of amino groups generated by hydrolysis of the amide groups in the water-insoluble material A by acid-base back titration. The detailed method is described below.

[0082] 5.0 g of the adsorbent was placed in a 200 mL recovery flask, 100 mL of toluene was added, and the mixture was refluxed at 150 °C for 24 hours to remove the polypropylene reinforcing material. The refluxed solution was quickly added to 2 L of toluene heated to 100 °C and washed. The insoluble components were filtered off using filter paper, washed with methanol, and left to stand in a dryer at 60 °C for 4 hours to obtain a water-insoluble material. Next, 1.0 g of the water-insoluble material and 100 mL of 6 M hydrochloric acid were added to a 200 mL recovery flask and refluxed at 130 °C for 24 hours. After refluxing, the water-insoluble material was recovered by filtering using filter paper to obtain the water-insoluble material after acid hydrolysis. Next, the entire amount of the obtained water-insoluble material after acid hydrolysis was added to a polypropylene container, 50 mL of 6 M sodium hydroxide aqueous solution was added, stirred for 30 minutes, and then filtered using filter paper. The filtered water-insoluble material after acid hydrolysis was then added to 50 mL of ion-exchanged water, stirred for 30 minutes, and filtered using filter paper. Addition to ion-exchanged water and filtration were repeated until the pH of the ion-exchanged water containing the water-insoluble material after acid hydrolysis reached 7. After the pH of the ion-exchanged water containing the water-insoluble material after acid hydrolysis reached 7, the water-insoluble material after acid hydrolysis was allowed to stand at 80°C under normal pressure for 48 hours and dried. Next, the entire amount of the water-insoluble material and 60 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 removed and transferred to a polypropylene container. 0.1 mL of 0.1 M aqueous sodium hydroxide solution was added dropwise to the solution. After addition, the solution was stirred for 10 minutes, and the pH of the solution was measured. The dropwise addition and subsequent stirring were repeated, and the amount of sodium hydroxide solution added when the solution pH exceeded 8.5 was defined as the titer per 1.0 g. The content of amide groups per 1.0 g of dry weight of the water-insoluble material was calculated using the titer per 1.0 g and the following formula 2:

[0083] Amide group content per 1 g of dry weight of water-insoluble material (mmol / g) = {volume of 0.1 M hydrochloric acid added (60 mL) / volume of hydrochloric acid removed (5 mL)} × titration amount per 1 g (mL) × concentration of sodium hydroxide solution (0.1 M) Equation 2

[0084] Measurements of adsorbent A and B showed that the amide group content per gram of dry weight of the water-insoluble material contained in adsorbent A was 6.2 mmol, and the amide group content per gram of dry weight of the water-insoluble material contained in adsorbent B was 6.2 mmol.

[0085] (Measurement of neutrophil and lymphocyte adsorption rates) Healthy human blood, to which lipopolysaccharide had been added at 70 EU / mL, was shaken at 65 rpm at 37°C for 30 minutes to activate blood cells. The blood was then passed through the column at a flow rate of 0.63 mL / min, and blood samples were collected at the inlet and outlet of the column. The time point at which blood entered the column was defined as 0 minutes, and blood samples were collected between 3.5 and 6.5 minutes after the time point. The collected blood was measured using a hemocytometer (XN-1000V, Sysmex Corporation), and the concentrations of neutrophils and lymphocytes were calculated. The neutrophil adsorption rate and lymphocyte adsorption rate during passage through the column were calculated using the following equations 3 and 4, respectively. Neutrophil adsorption rate (%) = {(neutrophil concentration at the column inlet) - (neutrophil concentration at the column outlet)} / (neutrophil concentration at the column inlet) × 100 Equation 3 Lymphocyte adsorption rate (%) = {(lymphocyte concentration at the column inlet) - (lymphocyte concentration at the column outlet)} / (lymphocyte concentration at the column inlet) × 100 Equation 4

[0086] In addition, the ratio of the adsorption rates of neutrophils and lymphocytes was calculated using the following formula 5. Adsorption rate ratio (neutrophils / lymphocytes) = (neutrophil adsorption rate) / (lymphocyte adsorption rate) × 100 Equation 5

[0087] (Measurement of IL-6 adsorption rate) Five mL of fetal bovine serum (FBS) adjusted to an IL-6 concentration of 2000 pg / mL was passed through the column at a flow rate of 0.63 mL / min. Samples were taken before and after passing the column, and the IL-6 concentration in the sampled FBS solution was measured. Measurements were performed using a Human IL-6 Quantikine ELISA Kit (R&D Systems). The IL-6 adsorption rate was calculated from the IL-6 concentrations before and after passing the column using the following formula (6), and the value was rounded to one decimal place. IL-6 adsorption rate (%) = {IL-6 concentration before flow (pg / mL) - IL-6 concentration after flow (pg / mL)} / IL-6 concentration before flow (pg / mL) × 100 Formula 6

[0088] (Examples 1 to 4 and Comparative Examples 1 to 3) The neutrophil adsorption rate, lymphocyte adsorption rate, and IL-6 adsorption rate were measured for columns A to G. The results are shown in Table 2.

[0089] [Table 2]

[0090] The results of Examples 1 to 4 and Comparative Examples 1 to 3 in Table 2 reveal that columns with a maximum adsorbent layer length of 6.5 mm or less adsorb neutrophils and IL-6, but have a low adsorption rate for lymphocytes.

[0091] In particular, the results of Examples 1 and 2 and Comparative Example 1 revealed that even when the adsorbent packing volume was the same, only columns with a maximum adsorbent layer length of 6.5 mm or less had a low lymphocyte adsorption rate. [Industrial Applicability]

[0092] The blood purification column of the present invention adsorbs cytokines and neutrophils while suppressing adsorption of lymphocytes, and therefore can be used to treat inflammatory diseases, and is particularly suitable for use in treating cytokine release syndrome, a side effect of CAR-T cell therapy. [Explanation of symbols]

[0093] 1. Adsorbent 2 spacers 3. Adsorbent layer 4 Spacer Layer 5. Length of the adsorbent layer

Claims

1. A blood purification column packed with an adsorbent containing a water-insoluble material, wherein the maximum length of an adsorbent layer made of said adsorbent is 6.5 mm or less.

2. 2. The blood purification column according to claim 1, wherein spacers are packed in the column, and spacer layers composed of the spacers and the adsorbent layers are alternately stacked.

3. the water-insoluble material includes a ligand having an amide group and an amino group; the content of the amide group is 3.0 to 7.0 mmol per 1 g of the dry weight of the water-insoluble material; 3. The blood purification column according to claim 1, wherein the content of the amino group is 1.0 to 7.0 mmol per 1 g of the dry weight of the water-insoluble material.

4. 4. The blood purification column according to claim 1, wherein the adsorbent is a knitted or woven fabric.

5. The blood purification column according to any one of claims 1 to 3, which is used for treating cytokine release syndrome.

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