Filter and method for manufacturing same

Abstract

The present disclosure provides a means for separating specific cells from among a plurality of types of cells having sizes close to those of the specific cells. Disclosed is a filter through which a liquid containing a cell or protein that is an object to be separated is allowed to pass to capture the object. The filter comprises: a substrate comprising a porous polyurethane in which a plurality of micropores are formed; and an active group fixed to the surface of the substrate and capable of specifically binding to the object. The active group contains: a constituent unit A bound to the substrate and derived from a compound A having an epoxy group and an ethylenically unsaturated group; and a constituent unit B bound to the constituent unit A and derived from first biotin.

Description

Filter and manufacturing method thereof

[0001] The present invention relates to a filter for capturing cells or proteins contained in a liquid and a method for producing the same.

[0002] Methods for separating cells from a cell suspension include a method using a filter, a centrifugation method, etc. For example, Japanese Patent Application Laid-Open No. 2011-24575 discloses a centrifugation method.

[0003] Some cell suspensions (cell solutions) contain multiple types of cells or proteins that are similar in size, and it is difficult to separate a specific cell or protein from multiple types of cells or proteins that are similar in size using methods that use fine-pore filters or centrifugation.

[0004] The present invention aims to solve the above-mentioned problems.

[0005] The filter of the present invention is (1) a filter that captures cells or proteins as separation targets by passing a liquid containing the cells or proteins as separation targets, and includes a substrate made of porous polyurethane having a plurality of micropores formed therein, and active groups that are fixed to the surface of the substrate and can specifically bind to the separation targets, and the active groups include a structural unit A derived from a compound A having an epoxy group and an ethylenically unsaturated group, which is bound to the substrate, and a first structural unit B derived from biotin, which is bound to the structural unit A.

[0006] According to the above configuration, it is possible to separate specific cells from multiple types of cells of similar size contained in the cell fluid, and similarly, it is possible to separate specific proteins from multiple types of proteins of similar size contained in the cell fluid.

[0007] (2) In the filter of (1) above, the active group preferably includes an antibody or an aptamer capable of specifically binding to an antigen contained in the object to be separated.

[0008] (3) In the filter of (2) above, the active group preferably includes a conjugate in which the antibody or the aptamer is bound to a second biotin, and a biotin-binding protein capable of binding to the second biotin.

[0009] (4) In the filter described in (2) or (3) above, the active group preferably includes a conjugate in which the antibody or the aptamer is bound to a second biotin; a constituent unit B derived from the first biotin; and a biotin-binding protein capable of binding to each of the constituent unit B and the second biotin-derived constituent unit C in the conjugate.

[0010] (5) In the filter according to any one of (1) to (4) above, it is preferable that the compound A is at least one selected from the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexylmethyl acrylate, 3,4-epoxycyclohexylmethyl methacrylate, β-methylglycidyl acrylate, and β-methylglycidyl methacrylate.

[0011] (6) The filter according to any one of (1) to (5) above preferably includes a container that contains a plurality of the substrates having the active groups fixed thereto, and the plurality of substrates are preferably stacked in the container from upstream to downstream of the flow of the liquid.

[0012] According to the above configuration, the cell liquid can be brought into contact with each substrate.

[0013] (7) The filter of (6) above preferably includes a plurality of the containers arranged in a line from upstream to downstream of the flow of the liquid, and the type of the active group fixed to the substrate differs for each of the containers.

[0014] According to the above configuration, multiple types of separation objects can be separated by passing the cell liquid through the filter once.

[0015] (8) In the filter according to any one of (1) to (7) above, it is preferable that the plurality of micropores formed in the substrate are larger than the object to be separated.

[0016] According to the above configuration, clogging of the filter can be prevented, and the recovery rate of the target cells can be improved.

[0017] (9) It is preferable that the filter described in any one of (1) to (8) above is made of a porous material and has a separate substrate other than the substrate, and that the multiple micropores formed in the separate substrate are larger than the object to be separated and smaller than the object to be separated other than the object to be separated.

[0018] According to the above configuration, it is possible to separate large-sized substances contained in the cellular fluid.

[0019] The method for producing a filter of the present invention includes: (i) irradiating with plasma at least a portion of the surface of a substrate made of porous polyurethane having a plurality of micropores formed therein; (ii) contacting the substrate after the plasma irradiation with a compound A having an epoxy group and an ethylenically unsaturated group, thereby binding the compound A to the substrate; and (iii) contacting with a first biotin with the substrate after the contact with the compound A, thereby binding the first biotin to the surface of the substrate via the structural unit A derived from the compound A.

[0020] FIG. 1 is a diagram showing the configuration of a filter. FIG. 2 is a partially enlarged view of a filter unit. FIG. 3 is a diagram showing the configuration of a separation apparatus. FIG. 4 is a flowchart of a filter preparation process and a separation process performed using the separation apparatus. FIG. 5 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in steps S1, S2, and S5 of FIG. 4. FIG. 6 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in step S1 of FIG. 4. FIG. 7 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in step S2 of FIG. 4. FIG. 8 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in step S3 of FIG. 4. FIG. 9 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in step S3 of FIG. 4. FIG. 10 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in step S4 of FIG. 4. FIG. 11 is a diagram showing the opening and closing of valves of the separation apparatus and the flow of liquid in the separation apparatus in step S4 of FIG. 4. Fig. 12 is a diagram showing the opening and closing of the valves of the separation device and the flow of liquid in the separation device in step S5 of Fig. 4. Fig. 13 is a diagram showing the opening and closing of the valves of the separation device and the flow of liquid in the separation device in the cell recovery process. Fig. 14 is a diagram for explaining the fixation of active groups capable of specifically binding to the object to be separated according to the present invention. Fig. 15 is a fluorescent photograph showing the presence of biotin in sample 1 (before sterilization) in Example 1. Fig. 16 is a fluorescent photograph showing the presence of biotin in sample 2 (after sterilization) in Example 2. Fig. 17 is a schematic diagram showing a test device for evaluating cell capture performance.

[0021] In one aspect of the present invention, a filter is provided that captures cells or proteins as separation targets by passing a liquid containing the cells or proteins through the filter. The filter comprises a substrate made of porous polyurethane having a plurality of micropores formed therein, and active groups immobilized on the surface of the substrate and capable of specifically binding to the cells or proteins, the active groups including a structural unit A derived from compound A having an epoxy group and an ethylenically unsaturated group and bound to the substrate, and a structural unit B derived from a first biotin and bound to the structural unit A. According to the present invention, a specific cell can be separated from multiple types of cells having similar sizes, and similarly, a specific protein can be separated from multiple types of proteins having similar sizes. Furthermore, according to the present invention, the structural unit B derived from first biotin is firmly immobilized on the surface of the substrate by a chemical bond via the structural unit A derived from compound A having an epoxy group and an ethylenically unsaturated group. As a result, according to the present invention, the dissociation of the active groups in the filter can be suppressed.

[0022] Hereinafter, embodiments for carrying out the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the claims. Furthermore, the embodiments described in this specification can be arbitrarily combined to form other embodiments.

[0023] Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to be used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will prevail.

[0024] In this specification, the range "X to Y" includes X and Y and means "X or more and Y or less." Unless otherwise specified, the operations and measurements of physical properties are performed under the conditions of room temperature (20 to 25°C) and a relative humidity of 40 to 60% RH.

[0025] As used herein, the term "(meth)acrylic" encompasses both acrylic and methacrylic. Thus, for example, the term "(meth)acrylic acid" encompasses both acrylic acid and methacrylic acid. Similarly, the term "(meth)acryloyl" encompasses both acryloyl and methacryloyl. Thus, for example, the term "(meth)acryloyl group" encompasses both acryloyl and methacryloyl groups.

[0026] In this specification, "A and / or B" means both A and B, or either A or B.

[0027] Furthermore, in this specification, when a certain structural unit is defined as being "derived from" a certain monomer, it means that the structural unit is a structural unit that is generated by cleavage of one bond of an ethylenically unsaturated group (polymerizable unsaturated double bond) and / or is generated by a condensation reaction of a reactive group possessed by the corresponding monomer.

[0028] [1. Structure of Filter 10] Fig. 1 is a diagram showing the structure of filter 10. Filter 10 introduces a liquid from an inlet, captures one or more types of separation target substances contained in the liquid, and discharges the liquid from an outlet. The liquid is, for example, a cell suspension (cell fluid). The separation target substances are cells or proteins. Filter 10 includes one or more filter units 12. Filter 10 shown in Fig. 1 includes two filter units 12 (a first filter unit 12a and a second filter unit 12b).

[0029] The two filter units 12 are arranged in a line from upstream to downstream in the direction of liquid flow so that liquid introduced into the filter 10 passes through each filter unit 12 in turn. In Fig. 1, the liquid flows from bottom to top. The separation target captured by the first filter unit 12a is different from the separation target captured by the second filter unit 12b. Each filter unit 12 includes a container 18 and a plurality of substrates 20.

[0030] The container 18 contains a plurality of substrates 20. The container 18 has an inlet port 22 and an outlet port 24. The inlet port 22 allows liquid to be introduced from the outside of the container 18 to the inside. The inlet port 22 of the first filter unit 12a corresponds to the inlet of the filter 10. The outlet port 24 allows liquid to be discharged from the inside of the container 18 to the outside. The outlet port 24 of the second filter unit 12b corresponds to the outlet of the filter 10. The outlet port 24 of the first filter unit 12a and the inlet port 22 of the second filter unit 12b are connected to each other by a connecting flow path 14.

[0031] 3, when another flow path (first bypass flow path 88) joins the connecting flow path 14, the inlet port 22 of the second filter unit 12b can become the inlet of the filter 10. Also, when another flow path (second bypass flow path 90) branches off from the connecting flow path 14, the outlet port 24 of the first filter unit 12a can become the outlet of the filter 10.

[0032] The plurality of substrates 20 are filled inside the container 18. The plurality of substrates 20 are stacked in the thickness direction of the substrates 20. Furthermore, the stacking direction of the plurality of substrates 20 (thickness direction of the substrates 20) is the same as the direction from the inlet port 22 toward the outlet port 24. In other words, the plurality of substrates 20 are arranged in a line from upstream to downstream in the flow of the liquid.

[0033] Figure 2 is a partially enlarged view of the filter unit 12. Figure 2 schematically shows the surface 26 and active groups 28 of the substrate 20. The surface 26 of the substrate 20 refers to the portion of the substrate 20 that can come into contact with the liquid passing through the filter unit 12. The substrate 20 is made of porous polyurethane having a plurality of micropores formed therein. The porous polyurethane allows the surface area of ​​the substrate 20 to be increased.

[0034] Active groups 28 capable of specifically binding to the substance to be separated are fixed to the surface 26 of the substrate 20. The active groups 28 include antibodies 30 capable of specifically binding to antigens possessed by the substance to be separated. The active groups 28 may include aptamers instead of the antibodies 30.

[0035] The filter unit 12 shown in Fig. 2 has a biotinylated antibody 36 bound to an antibody 30 and a second biotin 34b, avidin 32 (a biotin-binding protein), and a first biotin 34a. The biotinylated antibody 36 is immobilized on the surface 26 of the substrate 20 via the avidin 32 and the first biotin 34a. In the filter unit 12 shown in Fig. 2, the first biotin 34a is immobilized on the surface 26 of the substrate 20 by chemical bonding. Furthermore, the first biotin 34a and the avidin 32 are bound to each other. Furthermore, the avidin 32 and the biotinylated antibody 36 are bound to each other.

[0036] 2, when the cellular fluid flows from the inlet port 22 to the outlet port 24 of the filter unit 12, the antibodies 30 of the active groups 28 bind to antigens contained in the substances to be separated in the cellular fluid. The type of antibody 30 is selected in advance depending on the type of substance to be separated. As a result, the substances to be separated in the cellular fluid are captured by the substrate 20, while the cellular fluid containing substances other than the substances to be separated is discharged from the filter unit 12 through the outlet port 24.

[0037] [2. Method of Manufacturing Filter Unit 12] As described above, porous polyurethane is used as the substrate 20 of the filter 10. Generally, porous polyurethane is used as a leukocyte removal filter. The micropores of the porous polyurethane used as the substrate 20 are larger than the micropores of a leukocyte removal filter. The size of the micropores of the porous polyurethane is adjusted to be larger than the substances to be separated contained in the cell fluid. The porous polyurethane is cut to a size that can be contained in the container 18 to form the substrate 20. After cutting, active groups 28 are fixed to the surface 26 of the substrate 20 by the following manufacturing method.

[0038] The filter unit 12 shown in Fig. 2 can be produced, for example, as follows: In the filter unit 12 shown in Fig. 2, the active group includes a conjugate in which the antibody or the aptamer is bound to a second biotin, a first biotin immobilized on a substrate, and a biotin-binding protein capable of binding to each of the first biotin and the second biotin.

[0039] Here, the first biotin 34a in FIG. 2 is immobilized on the substrate via the structural unit A derived from the compound A having an epoxy group and an ethylenically unsaturated group, which is bound to the substrate.

[0040] The filter unit 12 having such a structure is preferably produced by the following steps: (i) irradiating at least a portion of the surface of a substrate with plasma (irradiation step); (ii) contacting the substrate after the plasma irradiation with a compound A having an epoxy group and an ethylenically unsaturated group, thereby binding (fixing) the compound A to the substrate (compound A immobilization step); and (iii) contacting the substrate after the contact with the compound A with a first biotin, thereby binding (fixing) the first biotin to the surface of the substrate via the structural unit A derived from the compound A (biotin immobilization step).

[0041] Therefore, the present invention also provides a method for producing a filter, comprising: (i) irradiating with plasma at least a portion of the surface of a substrate made of porous polyurethane having a plurality of micropores formed therein; (ii) contacting the substrate after the plasma irradiation with a compound A having an epoxy group and an ethylenically unsaturated group, thereby binding (fixing) the compound A to the substrate; and (iii) contacting a first biotin with the substrate after the contact with the compound A, thereby binding (fixing) the first biotin to the surface of the substrate via the structural unit A derived from the compound A.

[0042] The above manufacturing method will be described in detail below with reference to FIG.

[0043] (i) Irradiation Step In this step, plasma is irradiated onto at least a portion of the surface of a substrate (substrate 20) made of porous polyurethane with a plurality of micropores. The phrase "irradiating at least a portion of the substrate surface with plasma" refers to the fact that, depending on the application, it is not necessary to immobilize biotin (and therefore antibodies or aptamers) on the entire substrate surface (the entire surface); rather, biotin (and therefore antibodies or aptamers) need only be immobilized on the surface portion (which may be a portion or the entire surface) where desired cells are to be captured. Therefore, the plasma-irradiated area preferably includes at least the surface portion where desired cells are to be captured, and more preferably substantially includes the surface portion where desired cells are to be captured. Note that the phrase "substantially includes the surface portion where desired cells are to be captured" means that, in order to more reliably capture the desired cells, the area is preferably slightly larger than the surface where desired cells are to be captured, and more preferably 0.5 mm or more, and more preferably 10 to 20 mm larger than the edge (outermost periphery) of the surface.

[0044] The substrate 20 is cut to a predetermined size (substrate A-1). Here, the substrate is made of porous polyurethane with a plurality of micropores formed therein. The size of the substrate is not particularly limited and can be appropriately selected depending on the application.

[0045] Next, the cut substrate (substrate A-1) is subjected to plasma treatment (substrate A-2). This activates the surface of substrate A-1. Therefore, when the substrate A-1 is brought into contact with a compound A having an ethylenically unsaturated group in the next step, the compound A undergoes graft polymerization to the substrate A-1. This allows compound A to be firmly bonded (fixed) to the substrate 20. Note that the plasma irradiation step is preferably performed by ionized gas plasma treatment, since this allows the desired plasma treatment to be performed even on a small, narrow inner surface of a small diameter opening.

[0046] Before the surface of the substrate A-1 is irradiated with plasma, the surface of the substrate may be washed by an appropriate method. The method of the washing treatment is not particularly limited, and the surface may be washed with an appropriate solvent (for example, ultrasonic washing, a method of immersing in a washing solvent, a method of pouring a washing solvent, or the like).

[0047] The pressure conditions for the plasma treatment are not particularly limited, and either reduced pressure or atmospheric pressure is possible. In addition, when the substrate is tubular, the plasma gas may be irradiated while the plasma irradiation nozzle is rotated around the substrate (substrate to be treated) at the center, thereby uniformly and without unevenness around the entire circumference of the substrate (substrate to be treated).

[0048] The ionized gas that can be used in the plasma treatment is one or more gases selected from the group consisting of helium, neon, argon, krypton, air, oxygen, carbon dioxide, carbon monoxide, water vapor, nitrogen, and hydrogen. The plasma treatment is preferably carried out in an atmosphere that is substantially free of oxygen, and more preferably in an atmosphere of at least one inert gas selected from the group consisting of helium, argon, and nitrogen. This allows compound A to be more efficiently bonded (graft polymerized) to the substrate in the next step. Here, the "substantially oxygen-free atmosphere" means that the amount of oxygen in the atmosphere is less than 3% by volume, preferably 0.1% by volume or less. The above can be adjusted by introducing a sufficient amount of a predetermined gas into the plasma irradiation device.

[0049] The plasma treatment conditions (irradiation time, LF output, applied current, gas flow rate, inter-electrode gap, plasma irradiation distance) are not particularly limited and can be appropriately selected depending on the binding strength between compound A and the substrate (ease of immobilization), the type and area of ​​the substrate used, etc.

[0050] For example, the irradiation time in the plasma treatment is preferably 5 to 60 seconds or less, and particularly preferably 10 to 40 seconds, under such conditions, a sufficient amount of compound A can be bound (immobilized) to the surface of the substrate.

[0051] As an example, the LF output in the plasma treatment is preferably 100 to 500 W.

[0052] As an example, the gas pressure in the plasma treatment is preferably 20 to 60 Pa.

[0053] The temperature of the object to be treated (porous polyurethane substrate) in the plasma treatment is not particularly limited, and may be room temperature or may be heated or cooled. From an economical viewpoint, it is preferable to perform the treatment at a temperature (e.g., 5 to 35°C) at which a heating or cooling device is not required.

[0054] Plasma irradiation devices (systems) that can be used for plasma treatment are not particularly limited, and examples include plasma irradiation devices (systems) that have a plasma generating tube that introduces gas molecules and excites them to generate plasma, and an electrode that excites the gas molecules in the plasma generating tube, and that emit plasma from one end of the plasma generating tube. However, the present invention is not limited to such configurations (systems). Examples include devices that employ a high-frequency induction system, a capacitively coupled electrode system, a corona discharge electrode-plasma jet system, a parallel plate system, a remote plasma system, an atmospheric pressure plasma system, a low-pressure plasma system, an ICP-type high-density plasma system, and the like. Additionally, commercially available ionized gas plasma irradiation devices (systems), particularly atmospheric pressure plasma irradiation devices (systems), can be used. Specifically, a plasma irradiation device manufactured by TRI-STAR TECHNOLOGIES: DURADYNE (product name or trademark name), a plasma irradiation device manufactured by DIENER ELECTRONIC: PLASMABEAM, a plasma irradiation device manufactured by DIENER ELECTRONIC: Pico Full PC, etc. can be used, but the present invention is not limited to these.

[0055] (ii) Compound A Immobilization Step In this step, compound A having an epoxy group and an ethylenically unsaturated group is brought into contact with the substrate (substrate A-2) that has been plasma-irradiated in the irradiation step. As a result, compound A is graft-polymerized onto the activated substrate, and a substrate (substrate A-3) in which compound A is bound (immobilized) to substrate A-2 is obtained. In this specification, compound A having an epoxy group and an ethylenically unsaturated group is also simply referred to as "compound A."

[0056] Compound A may be any compound as long as it has an epoxy group and an ethylenically unsaturated group, and preferably has a glycidyl group and an ethylenically unsaturated group. Examples of such compound A include, but are not limited to, (meth)acrylic acid esters having a glycidyl group (epoxy group), such as glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexylmethyl acrylate, 3,4-epoxycyclohexylmethyl methacrylate, β-methylglycidyl acrylate, and β-methylglycidyl methacrylate; and vinyl ethers having a glycidyl group (epoxy group), such as allyl glycidyl ether. Among these, from the viewpoint of further improving the bonding property to the porous polyurethane substrate (graft polymerization property with the substrate), it is preferable that the monomer A is at least one selected from the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexylmethyl acrylate, 3,4-epoxycyclohexylmethyl methacrylate, β-methylglycidyl acrylate, and β-methylglycidyl methacrylate. It is more preferable that the monomer A contains at least one selected from the group consisting of glycidyl acrylate and glycidyl methacrylate, it is even more preferable that the monomer A contains glycidyl acrylate or glycidyl methacrylate, it is particularly preferable that the monomer A contains glycidyl methacrylate, and it is most preferable that the monomer A is glycidyl methacrylate. The above-mentioned compound A may be used alone or in combination of two or more types. Furthermore, when two or more types of compound A are used, the structural units A may be arranged in a block or random configuration.

[0057] In this step, a specific method for contacting the compound A with the plasma-irradiated substrate (substrate A-2) may be either a method in which the compound A is directly contacted with the substrate A-2, or a method in which a compound A solution is prepared by dissolving the compound A in a solvent and then the compound A solution is contacted with the substrate A-2. From the viewpoint of further improving the contact efficiency (graft polymerization efficiency) between the substrate A-2 and the compound A, it is preferable to directly contact the compound A with the substrate A-2, and it is more preferable to contact the gaseous compound A with the substrate A-2. In this case, the contact between the compound A and the substrate A-2 can be carried out, for example, by introducing the compound A, which has been separately kept under reduced pressure, into the plasma irradiation device while the plasma irradiation device is kept under reduced pressure.

[0058] Furthermore, in the method of contacting the compound A solution with the substrate A-2, the solvent used to dissolve the compound A is not particularly limited and is appropriately selected depending on the type of compound A. From the viewpoint of high solubility, water (reverse osmosis water (RO water), pure water, ion-exchanged water, distilled water, etc.); alcoholic solvents such as methanol, ethanol, isopropyl alcohol, and butanol; and organic solvents such as dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran (THF), dimethyl sulfoxide, N,N-dimethylformamide (DMF), acetone, dioxane, and benzene are preferably used. These solvents may be used alone or in combination of two or more (in the form of a mixed solvent). The concentration of the compound A in the compound A solution is not particularly limited. For example, the concentration of the compound A in the compound A solution is preferably 1 to 10% by mass, and more preferably 2 to 5% by mass. When the concentration of the compound A is within the above range, a sufficient amount of the compound A can be bound (immobilized) to the surface of the substrate.

[0059] Next, the compound A solution prepared as described above is brought into contact with the plasma-irradiated substrate A-2. Here, as a specific method for "contacting", a conventionally known method such as a method of applying the compound A solution to the surface of the substrate or a method of immersing the substrate in the compound A solution can be appropriately adopted.

[0060] The contact conditions between compound A or compound A solution and substrate A-2 may be any conditions that allow compound A to be sufficiently graft-polymerized onto substrate A-2. For example, the contact temperature between compound A or compound A solution and substrate A-2 is preferably 5 to 50°C, more preferably 20 to 40°C. The contact time between compound A or compound A solution and substrate A-2 is preferably 30 to 90 minutes, more preferably 45 to 70 minutes, and particularly preferably 50 to 60 minutes. This results in a substrate (substrate A-3) on which compound A has been graft-polymerized.

[0061] In this step, if necessary, the substrate (substrate A-3) after contact with compound A may be washed. The method of washing is not particularly limited, and can be performed by washing with an appropriate solvent (e.g., water such as ultrafiltered water) (e.g., ultrasonic washing, immersion in a washing solvent, roller press washing, pouring the washing solvent, etc.). The washing time is not particularly limited, but is preferably 5 to 15 hours. If necessary, the washing treatment may be repeated multiple times (e.g., 2 to 5 times). When the washing treatment is repeated multiple times, each washing treatment may be the same or different.

[0062] (iii) Biotin Immobilization Step In this step, biotin is brought into contact with the surface of the substrate (substrate A-3) that has been contacted with compound A obtained in the compound A immobilization step. This causes a reaction between the carboxyl group (—COOH) of biotin and the epoxy group of compound A, resulting in a substrate (substrate A-4) with biotin bound (immobilized) to the surface via the structural unit A derived from compound A.

[0063] In this step, a specific method for contacting the biotin with the substrate (substrate A-3) after contacting with the compound A may be either a method of contacting biotin directly with substrate A-3 or a method of preparing a biotin solution by dissolving biotin in a solvent and then contacting the biotin solution with substrate A-3. From the viewpoint of further improving the contact efficiency between substrate A-3 and biotin (reactivity between epoxy groups and carboxyl groups), it is preferable to employ a method of contacting a biotin solution with substrate A-3.

[0064] The solvent used to prepare the biotin solution is not particularly limited as long as it can dissolve biotin. Examples include, but are not limited to, one or more solvents selected from water (e.g., reverse osmosis water (RO water), ultrafiltered water, pure water, ion-exchanged water, distilled water, etc.); alcohols such as ethanol, isopropanol, n-propanol, n-butanol, isobutanol, sec-butanol, t-butanol, ethylene glycol, diethylene glycol, propylene glycol, and dipropylene glycol; and organic solvents such as chloroform, tetrahydrofuran, acetone, dioxane, pyridine, and benzene. A mixed solvent of water and an alcohol, or a mixed solvent of water, an alcohol, and an organic solvent may also be used. The concentration of biotin contained in the biotin solution is not particularly limited. For example, the concentration of biotin in the biotin solution is preferably 0.01 to 5 g / 100 mL of solvent, and more preferably 0.05 to 0.15 g / 100 mL of solvent. At such a concentration, the epoxy group of the structural unit A and the carboxyl group of biotin are in sufficient contact with each other, and therefore the epoxy group of the structural unit A and the carboxyl group of biotin can react more efficiently.

[0065] The biotin solution preferably further contains a reaction accelerator and an epoxy group ring-opening accelerator for the purpose of improving the reaction with the epoxy group. When a reaction accelerator is used, the reaction accelerator is not particularly limited, and examples thereof include carbodiimides such as 1-ethyl-(3-dimethylaminopropyl)-carbodiimide.HCl (EDC), dicyclohexylcarbodiimide (DDC), N,N'-diisopropylcarbodiimide (DIC), N,N'-di-tert.butylcarbodiimide, N,N'-dicyclohexylcarbodiimide (DCC), and N-tert.butyl-N-ethylcarbodiimide. When a reaction accelerator is used, the amount of the reaction accelerator added may be any amount that can accelerate the reaction between the epoxy group of the structural unit A and the carboxyl group of biotin, and can be appropriately selected depending on the structure of the structural unit A, the type of reaction accelerator, and the like. For example, the biotin solution preferably contains 100 to 250 parts by mass, more preferably 150 to 200 parts by mass, of the reaction accelerator relative to 100 parts by mass of biotin.

[0066] When an epoxy group ring-opening accelerator is used, the ring-opening accelerator is not particularly limited, and examples include pyridine, pyrimidine, quinoline, and tetrahydroxyquinoline. When a ring-opening accelerator is used, the amount of ring-opening accelerator added may be any amount that can accelerate the ring-opening reaction of the epoxy group in the structural unit A, and can be appropriately selected depending on the structure of the structural unit A, the type of ring-opening accelerator, and the like. For example, the biotin solution preferably contains 50 to 300 parts by mass, and more preferably 100 to 200 parts by mass, of the ring-opening accelerator per 100 parts by mass of biotin.

[0067] Next, the biotin solution prepared as described above is brought into contact with the substrate A-3. Here, as a specific method for "contacting", a conventionally known method such as a method of applying the biotin solution to the surface of the substrate or a method of immersing the substrate in the biotin solution can be appropriately adopted.

[0068] When contacting by the immersion method, the biotin solution may be stirred. This can further promote contact between the biotin solution and the substrate A-3. The stirring can be performed using a known device such as a stirrer, an orbital shaker, a rotary shaker, or a paint shaker. The stirring conditions are not particularly limited. From the viewpoint of more efficient contact with the workpiece, the stirring speed is, for example, 200 to 600 rpm, preferably 300 to 400 rpm. From the same viewpoint, the stirring time is, for example, 0.5 to 24 hours, preferably 1 to 12 hours.

[0069] Furthermore, when biotin is bound to only a portion of the substrate, biotin can be bound to a desired surface portion of the substrate by immersing only a portion of the substrate in a biotin solution.

[0070] When it is difficult to immerse only a portion of the substrate in the biotin solution, the surface portion of the substrate that does not need to be bound with biotin can be protected (coated, etc.) with a suitable detachable (removable) member or material in advance, and then the substrate is immersed in the biotin solution, and the protective member (material) can be removed, thereby binding biotin to the desired surface portion of the substrate. However, the present invention is not limited to these formation methods, and biotin can be bound using any conventionally known method as appropriate. For example, when it is difficult to immerse only a portion of the substrate in the biotin solution, other coating methods (e.g., a method of applying the biotin solution to a predetermined surface portion of the substrate using an application device such as a sprayer, bar coater, die coater, reverse coater, comma coater, gravure coater, spray coater, or doctor knife) may be used instead of the immersion method.

[0071] The contact conditions for biotin or a biotin solution with substrate A-3 may be any conditions that allow biotin to sufficiently react with the structural unit A. For example, the contact temperature for biotin or a biotin solution with substrate A-3 is preferably 20 to 70°C, more preferably 30 to 65°C. The contact time for biotin or a biotin solution with substrate A-3 is preferably 3 to 20 hours, more preferably 5 to 15 hours. This provides a substrate (substrate A-4) to which biotin is bound (immobilized) via structural unit A derived from compound A.

[0072] In this step, if necessary, the substrate (substrate A-4) after contact with biotin may be washed. The washing method is not particularly limited, and can be performed by washing with an appropriate solvent (e.g., water such as ultrafiltered water, aqueous hydrochloric acid) (e.g., ultrasonic washing, immersion in a washing solvent, roller press washing, pouring the washing solvent, etc.). The washing time is not particularly limited, but is preferably 5 to 15 hours. If necessary, the washing treatment may be repeated multiple times (e.g., 2 to 5 times). When the washing treatment is repeated multiple times, each washing treatment may be the same or different.

[0073] A filter is obtained by the above steps. The filter thus obtained can be sterilized by a conventionally known method such as high-pressure steam sterilization, radiation sterilization, ethylene oxide sterilization, or nitrogen dioxide sterilization. According to the present invention, as described above, the first biotin-derived structural unit B is firmly fixed to the surface of the substrate by chemical bonding via the structural unit A derived from the compound A having an epoxy group and an ethylenically unsaturated group, thereby preventing dissociation of the active group due to sterilization.

[0074] [3. Configuration of Separation Apparatus 40] By passing the cellular fluid through the filter 10 shown in Fig. 1, it is possible to separate the desired separation target from the cellular fluid. The apparatus that performs such a process is called a separation apparatus 40.

[0075] Fig. 3 is a diagram showing the configuration of a separation device 40. The separation device 40 can separate a desired separation target from a cell solution, and further, the separation device 40 can perform a part of the process of the method for producing the filter unit 12 shown in Fig. 2. The separation device 40 includes a separation circuit 42 and a controller 44.

[0076] The separation circuit 42 includes a supply unit 46, a separation unit 48, and a discharge unit 50. The supply unit 46 includes a cell fluid container 52, an avidin fluid container 54, a first antibody fluid container 56, a second antibody fluid container 58, a detachment fluid container 60, a washing fluid container 62, first supply flow paths 64 to sixth supply flow paths 74, valves 76a to 76f, and a pump 78. Each of the cell fluid container 52 to washing fluid container 62 is, for example, a medical bag formed into a bag shape from a soft resin material. Each of the cell fluid container 52 to washing fluid container 62 may also be a tank or the like made of a hard material. Each of the cell fluid container 52 to washing fluid container 62 is detachable from the separation device 40.

[0077] The cell fluid container 52 is filled with cell fluid. The cell fluid is a solution containing two types of separation targets. In this embodiment, the two types of separation targets are first cells and second cells. The avidin fluid container 54 is filled with avidin fluid. The avidin fluid is a solution containing avidin 32. The first antibody fluid container 56 is filled with a first antibody fluid. The first antibody fluid is a solution containing a first biotinylated antibody 36a (FIG. 2) in which a first antibody 30a and a second biotin 34b are bound. The first antibody 30a can specifically bind to an antigen possessed by the first cell. The second antibody fluid container 58 is filled with a second antibody fluid. The second antibody fluid is a solution containing a second biotinylated antibody 36b (FIG. 2) in which a second antibody 30b different from the first antibody 30a and a second biotin 34b are bound. The second antibody 30b can specifically bind to an antigen possessed by the second cell. The detachment liquid container 60 is filled with a detachment liquid. The detachment liquid is, for example, a deoxyribonuclease solution. The washing liquid container 62 is filled with a washing liquid. The washing liquid is, for example, water, phosphate buffered saline (PBS), etc.

[0078] The cell solution container 52 is connected to the suction port 80 of the pump 78 via a first supply flow path 64. A valve 76a is provided in the first supply flow path 64. The avidin solution container 54 is connected to the suction port 80 of the pump 78 via a second supply flow path 66. A valve 76b is provided in the second supply flow path 66. The first antibody solution container 56 is connected to the suction port 80 of the pump 78 via a third supply flow path 68. A valve 76c is provided in the third supply flow path 68. The second antibody solution container 58 is connected to the suction port 80 of the pump 78 via a fourth supply flow path 70. A valve 76d is provided in the fourth supply flow path 70. The stripping solution container 60 is connected to the suction port 80 of the pump 78 via a fifth supply flow path 72. A valve 76e is provided in the fifth supply flow path 72. The washing solution container 62 is connected to the suction port 80 of the pump 78 via a sixth supply flow path 74. The sixth supply flow path 74 is provided with a valve 76f.

[0079] The separation section 48 includes a filter 10, an upstream flow path 84, a downstream flow path 86, a first bypass flow path 88, a second bypass flow path 90, and valves 92a to 92d. The filter 10 shown in FIG. 3 has two filter units 12. Each filter unit 12 is detachable from the separation device 40. That is, the filter units 12 are replaceable depending on the type of material to be separated. The separation section 48 may be detachable from the separation device 40. Alternatively, the filter 10 may be detachable from the separation device 40.

[0080] As described above, the filter 10 includes a first filter unit 12a, a second filter unit 12b, and a connecting flow path 14. The inlet port 22 of the first filter unit 12a is connected to the outlet port 82 of the pump 78 via an upstream flow path 84. A valve 92a is provided in the upstream flow path 84. The upstream flow path 84, located upstream of the valve 92a, communicates with the connecting flow path 14 via a first bypass flow path 88. A valve 92b is provided in the first bypass flow path 88. The outlet port 24 of the second filter unit 12b is connected to the downstream flow path 86. A valve 92c is provided in the downstream flow path 86. The downstream flow path 86, located downstream of the valve 92c, communicates with the connecting flow path 14 via a second bypass flow path 90. A valve 92d is provided in the second bypass flow path 90.

[0081] The discharge unit 50 includes a collection container 94, a waste liquid container 96, a collection flow path 98, a waste liquid flow path 100, and valves 102a and 102b. The collection flow path 98 and the waste liquid flow path 100 are each connected to the downstream flow path 86 of the separation unit 48. The collection flow path 98 is provided with a valve 102a. The waste liquid flow path 100 is provided with a valve 102b.

[0082] The collection container 94 is connected to the outlet port 24 of the second filter unit 12b via the collection flow path 98 and the downstream flow path 86. The collection container 94 is also connected to the outlet port 24 of the first filter unit 12a via the collection flow path 98, the downstream flow path 86, and the second bypass flow path 90.

[0083] The waste liquid container 96 is connected to the outlet port 24 of the second filter unit 12b via a waste liquid flow path 100 and the downstream flow path 86. The waste liquid container 96 is also connected to the outlet port 24 of the first filter unit 12a via the waste liquid flow path 100, the downstream flow path 86, and the second bypass flow path 90.

[0084] The controller 44 has a processing circuit and various memories (volatile memory, non-volatile memory, etc.). The processing circuit may be a processor such as a CPU. The processing circuit may be an integrated circuit such as an ASIC or FPGA. The processor can perform various processes by executing programs stored in the non-volatile memory. Examples of volatile memory include RAM. The volatile memory is used as the working memory of the processor. The volatile memory temporarily stores data required for processing or calculation. Examples of non-volatile memory include ROM and flash memory. The non-volatile memory is used as storage memory. The non-volatile memory stores programs, tables, maps, etc.

[0085] The controller 44 controls the opening and closing of each valve in the separation circuit 42. The controller 44 also controls the operation of the pump 78. The controller 44 is capable of automatically performing the series of processes shown in Fig. 4 using a program.

[0086] [4 Operation of Separation Apparatus 40] Fig. 4 is a flowchart of the filter production process and separation process performed using the separation apparatus 40. Figs. 5 to 13 are diagrams showing the opening and closing of valves in the separation apparatus 40 and the flow of liquid in the separation apparatus 40 in each step. In Figs. 5 to 13, valves painted black indicate a closed state, and valves painted white indicate an open state. Each step shown in Fig. 4 is performed by the controller 44 controlling each valve and pump 78.

[0087] Before the series of processes shown in FIG. 4 , the user performs the following preparatory steps. The user fills each filter unit 12 with a substrate 20 to which a first biotin 34a is immobilized. The user prepares a first antibody solution containing a first biotinylated antibody 36a and fills the first antibody solution container 56. The user prepares a second antibody solution containing a second biotinylated antibody 36b and fills the second antibody solution container 58. In the process described with reference to FIG. 4 , the first biotinylated antibody 36a and the second biotinylated antibody 36b are substances capable of binding to unwanted separation targets (first cells, second cells) in the cell solution. The user attaches the cell solution container 52 to the washing solution container 62 to the separation device 40. When the user operates the controller 44, the series of processes shown in FIG. 4 begin.

[0088] In step S1, the controller 44 performs priming. The controller 44 places the separation circuit 42 in the state shown in FIG. 5. That is, the controller 44 opens valves 76f, 92a, 92c, and 102b and closes the other valves. Furthermore, the controller 44 operates the pump 78. As a result, the cleaning liquid in the cleaning liquid container 62 flows along the path indicated by the arrows in FIG. 5 and is collected in the waste liquid container 96.

[0089] Next, the controller 44 sets the separation circuit 42 to the state shown in Figure 6. That is, the controller 44 opens valves 76f, 92b, 92d, and 102b, and closes the other valves. Furthermore, the controller 44 operates the pump 78. As a result, the cleaning liquid in the cleaning liquid container 62 flows through the path indicated by the arrows in Figure 6 and is collected in the waste liquid container 96. The separation unit 48 is cleaned by the processing of step S1.

[0090] In step S2, the controller 44 performs a precoat (first precoat) with avidin 32. The controller 44 places the separation circuit 42 in the state shown in FIG. 7 . That is, the controller 44 opens valves 76b, 92a, 92c, and 102b and closes the other valves. Furthermore, the controller 44 operates the pump 78. The avidin solution in the avidin solution container 54 then flows through the path indicated by the arrows in FIG. 7 and is collected in the waste solution container 96. The controller 44 operates the pump 78 for a predetermined time or longer and then stops the pump 78 for a predetermined time or longer. By the process of step S2, the avidin solution is supplied to each filter unit 12, and the substrate 20 in the filter unit 12 is immersed in the avidin solution. The first biotin 34a immobilized on the substrate 20 binds to the avidin 32 in the avidin solution. This immobilizes the avidin 32 on the substrate 20.

[0091] Next, the controller 44 sets the separation circuit 42 to the state shown in Fig. 5. This causes the area through which the avidin solution has flowed to be washed.

[0092] In step S3, the controller 44 performs precoating with the first biotinylated antibody 36a (second precoating). The controller 44 places the separation circuit 42 in the state shown in FIG. 8 . That is, the controller 44 opens valves 76c, 92a, 92d, and 102b and closes the other valves. Furthermore, the controller 44 operates the pump 78. The first antibody liquid in the first antibody liquid container 56 then flows through the path indicated by the arrows in FIG. 8 and is collected in the waste liquid container 96. The controller 44 operates the pump 78 for a predetermined time or longer and then stops the pump 78 for a predetermined time or longer. By the process of step S3, the first antibody liquid is supplied to the first filter unit 12a, and the substrate 20 in the first filter unit 12a is immersed in the first antibody liquid. The avidin 32 immobilized on the substrate 20 binds to the first biotinylated antibody 36a in the first antibody liquid. As a result, in the first filter unit 12a, as shown in FIG. 2, active groups 28 (first biotin 34a, avidin 32, first biotinylated antibody 36a) are immobilized on the surface 26 of the substrate 20.

[0093] Next, the controller 44 sets the separation circuit 42 to the state shown in Fig. 9. That is, the controller 44 opens valves 76f, 92a, 92d, and 102b, and closes the other valves. Furthermore, the controller 44 operates the pump 78. Then, the cleaning solution in the cleaning solution container 62 flows through the path indicated by the arrows in Fig. 9 and is collected in the waste solution container 96. This process cleans the area through which the first antibody solution has flowed.

[0094] In step S4, the controller 44 performs precoating with the second biotinylated antibody 36b (third precoat). The controller 44 places the separation circuit 42 in the state shown in FIG. 10 . That is, the controller 44 opens valves 76d, 92b, 92c, and 102b and closes the other valves. Furthermore, the controller 44 operates the pump 78. The second antibody liquid in the second antibody liquid container 58 then flows through the path indicated by the arrows in FIG. 10 and is collected in the waste liquid container 96. The controller 44 operates the pump 78 for a predetermined time or longer and then stops the pump 78 for a predetermined time or longer. By the processing of step S4, the second antibody liquid is supplied to the second filter unit 12b, and the substrate 20 in the second filter unit 12b is immersed in the second antibody liquid. The avidin 32 immobilized on the substrate 20 binds to the second biotinylated antibody 36b in the second antibody liquid. As a result, in the second filter unit 12b, as shown in FIG. 2, active groups 28 (first biotin 34a, avidin 32, second biotinylated antibody 36b) are immobilized on the surface 26 of the substrate 20.

[0095] Next, the controller 44 sets the separation circuit 42 to the state shown in Figure 11. That is, the controller 44 opens valves 76f, 92b, 92c, and 102b, and closes the other valves. Furthermore, the controller 44 operates the pump 78. Then, the cleaning solution in the cleaning solution container 62 flows through the path indicated by the arrows in Figure 11 and is collected in the waste solution container 96. This process cleans the area through which the second antibody solution has flowed.

[0096] In step S5, the controller 44 separates the desired cells from the cell solution. The controller 44 sets the separation circuit 42 to the state shown in FIG. 12 . That is, the controller 44 opens valves 76a, 92a, 92c, and 102a, and closes the other valves. Furthermore, the controller 44 operates the pump 78. The cell solution in the cell solution container 52 then flows along the path indicated by the arrows in FIG. 12 and is collected in the collection container 94. The controller 44 operates the pump 78 until the cell solution in the cell solution container 52 is depleted, and then stops the pump 78. Through the processing of step S5, the cell solution is supplied to each filter unit 12, and the substrate 20 in each filter unit 12 is immersed in the cell solution. The first biotinylated antibody 36a immobilized on the substrate 20 of the first filter unit 12a binds to the antigen of the first cell in the cell solution. The second biotinylated antibody 36b immobilized on the substrate 20 of the second filter unit 12b binds to the antigen of the second cell in the cell solution, and as a result, the cell solution from which the first cell and the second cell have been removed is collected in the collection container 94.

[0097] Next, the controller 44 sets the separation circuit 42 to the state shown in Fig. 13. This allows the cells remaining in the separation circuit 42 to be collected without waste. This completes the series of processes.

[0098] In the process described with reference to FIG. 4 , unnecessary separation subjects (first cells, second cells) are removed from the cell fluid, and the cell fluid containing the desired cells is recovered. That is, the separation device 40 performs so-called negative selection. Alternatively, it is also possible to extract the necessary separation subjects from the cell fluid. That is, the separation device 40 can also perform so-called positive selection. In this case, after the necessary separation subjects are captured by the filter 10, the stripping solution in the stripping solution container 60 is poured through the filter 10, and the stripping solution and the separation subjects are recovered in the recovery container 94.

[0099] [5. Other] The filter 10 may include a separate unit that physically separates the objects to be separated. The separate unit is made of a porous material and includes a separate substrate separate from the filter unit 12. The multiple micropores formed in the separate substrate are larger than the objects to be captured by the filter unit 12, but smaller than the objects to be captured by the separate unit.

[0100] The present invention will be described in detail below using examples, but the present invention is not limited to these examples. In each example, parts and percentages are all based on mass. Unless otherwise specified, the room temperature storage conditions below are all 23°C / 55% RH.

[0101] [Example 1] 1. Graft polymerization of GMA onto the surface of a polyurethane filter medium (PU membrane) 1) Preparation of substrate (substrate A-1) A polyurethane filter medium (manufactured by Terumo Corporation, product name: Imguard, thickness: 2 mm) was cut into a size of 15 cm x 5 cm to prepare a PU membrane (substrate A-1).

[0102] 2) Plasma Treatment (Substrate A-2) and GMA Immobilization (Substrate A-3) The PU film (Substrate A-1) obtained in 1) above was placed in a graft polymerization reaction chamber (plasma irradiation device, Pico Full PC, manufactured by DIENER ELECTRONIC, low-pressure type), the chamber was evacuated, and Ar gas (100%) was introduced into the chamber at 0.2 torr. Plasma irradiation (argon ionized gas plasma irradiation) was carried out for 40 seconds under conditions of LF output: 400 W, gas pressure: 45 Pa, to obtain a plasma-treated PU film (Substrate A-2).

[0103] Next, the valve between the chamber and the vacuum pump was closed, and the reservoir cock for glycidyl methacrylate (GMA), which had been previously evacuated, was opened to introduce GMA into the chamber. After leaving it in this state for 1 hour, the pressure inside the chamber was returned to normal, and the PU film was removed from the chamber. This yielded a PU film (GMA-PU film) (substrate A-3) with GMA graft-polymerized onto its surface.

[0104] 2. Immobilization of biotin on GMA-PU film The GMA-PU film (substrate A-3) obtained in 1 above was immersed in a biotin solution of the following composition and incubated at 60°C for 6 hours to react the epoxy group of GMA with the carboxyl group of biotin (biotin-GMA-PU film) (substrate A-4).

[0105] (Biotin solution composition) Ultrafiltered water: 180 mL Ethanol: 20 mL Biotin: 0.16 g 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide.HCl (EDC): 0.26 g Pyridine: 0.32 mL

[0106] After the incubation was completed, the biotin-GMA-PU membrane was immersed in 2 L of ultrafiltered water and washed by pressing with a roller.

[0107] After washing, the biotin-GMA-PU membrane was left overnight at room temperature (25°C) and thoroughly dried, thereby obtaining a polyurethane filter medium (Sample 1) to which biotin was bound via GMA.

[0108] 3. Fluorescence Observation Sample 1 obtained in 2 above was punched out to a diameter of 1 mm (φ1 mm).

[0109] Separately, 0.05 (w / v)% Tween-20 was added to 10 mL of PBS to prepare a Tween-20 / PBS solution.

[0110] Streptavidin-FITC (StA-F; manufactured by Funakoshi Co., Ltd., trade name: Streptavidin Conjugates) was added to PBS to a concentration of 5 μg / mL to prepare a StA-F / PBS solution.

[0111] The above-mentioned sample 1 having a diameter of 1 mm was set in a commercially available filter housing (manufactured by EMD Millipore Corp., trade name: SWINNEX), which was then thoroughly primed with a Tween-20 / PBS solution to remove air bubbles from within the housing.

[0112] Thereafter, 2 mL of StA-F / PBS solution was injected into the housing after the priming, and the housing was incubated at 37° C. for 30 minutes.

[0113] After StA-F that had not been immobilized on Sample 1 was washed away with 30 mL of Tween-20 / PBS solution, Sample 1 was removed from the housing (measurement sample 1).

[0114] Using a confocal laser scanning microscope (manufactured by Olympus Corporation, product name: FV3000), fluorescence observation of the measurement sample 1 obtained above was carried out under the following conditions.

[0115] (Observation conditions) Magnification: 1.25x Excitation wavelength: 488 nm Voltage: 700V

[0116] The results are shown in Figure 15. As shown in Figure 15, Sample 1 (before sterilization) emitted fluorescence uniformly. This result indicates that biotin was uniformly immobilized on the polyurethane filter medium in Sample 1.

[0117] Example 2 Example 2 was carried out in the same manner as in steps 1 to 3 of Example 1, except for the following changes. In step 2 above, Sample 1 was sealed in a sterilization bag and subjected to high-pressure steam sterilization at 121°C for 20 minutes. This resulted in Sample 2 after sterilization. Furthermore, in step 3 above, Sample 2 was used instead of Sample 1 to prepare Measurement Sample 2, and fluorescence observation was performed.

[0118] The results are shown in Figure 16. As shown in Figure 16, Sample 2 (after sterilization) emitted fluorescence uniformly, just like Sample 1 (before sterilization). This result demonstrates that biotin immobilized on the polyurethane filter media via GMA is maintained (hard to dissociate) even after sterilization.

[0119] [Example 3] 1. Assembling the filter 200 In step 1 of Example 2, five biotin-GMA-PU membranes (sample 3) were prepared after sterilization in the same manner as in Example 2, except that a polyurethane filter medium (manufactured by Terumo Corporation, product name: Imguard, thickness: 2 mm) was cut into a size with a diameter (Φ) of 25 mm.

[0120] For Sample 3, the cell trapping performance was evaluated using the filter 200 shown in FIG. 17 according to the following method.

[0121] Fig. 17 is a schematic diagram showing a test device for evaluating cell capture performance. In Fig. 17, each sample 3 220 was stacked via a silicone spacer 231 (donut-shaped, inner diameter: 24 mm, outer diameter: 29 mm, thickness: 1.0 mm), and then a polypropylene (PP) mesh (diameter (Φ): 21 mm, thickness: 0.5 mm, mesh size: 1.7 mm × 1.7 mm) 230 was placed in the center of the top sample 3 220a and the bottom sample 3 220b to produce a filter unit 212. This filter unit 212 was placed in a container 218, which had tubes 223 and 225 connected to the bottom and top via tube connectors 222 and 224, respectively, to assemble the filter 200.

[0122] 2. Preparation of Cell Capture Filter PBS buffer (phosphate buffer solution, composition: sodium chloride 136.89 mM, potassium chloride 2.68 mM, potassium dihydrogen phosphate 0.147 mM, disodium hydrogen phosphate (anhydrous) 9.58 mM) was injected into the lower tube 223 of the filter 200 using a syringe pump at a flow rate of 1 mL / min, thereby priming the inside of the filter 200 with approximately 10 mL of PBS buffer.

[0123] A 0.05% (w / v) streptavidin (hereinafter also referred to as "StA") / PBS solution (StA / PBS solution) was prepared. Five mL of this StA / PBS solution was injected into filter 200 through the lower tube 223 of filter 200 using a syringe pump at a flow rate of 1 mL / min. After the injection of the StA / PBS solution, filter 200's upper tube 225 and lower tube 223 were clamped. In this state, the filter was incubated at 37°C for 15 minutes.

[0124] A biotin-conjugated porcine CD8 antibody (Thermo Fisher Scientific, product name: Invitrogen, CD8 alpha Monoclonal Antibody (76-2-11), Biotin) was added to PBS to a concentration of 0.005% (w / v) to prepare a porcine CD8 antibody / PBS solution. Five mL of this porcine CD8 antibody / PBS solution was injected into the filter 200 through the lower tube 223 of the filter 200 using a syringe pump at a flow rate of 1 mL / min. After injection of the porcine CD8 antibody / PBS solution, the upper tube 225 and the lower tube 223 of the filter 200 were clamped. The filter was incubated in this state at 37°C for 15 minutes.

[0125] 3. Evaluation of Cell Capture Performance 1,000 mL of porcine blood was collected into 140 mL of CPD solution (composition: sodium citrate hydrate 26.30 g, citric acid hydrate 3.27 g, glucose 23.20 g, sodium dihydrogen phosphate 2.51 g / 1,000 mL, water for injection) to prepare porcine whole blood (CPD blood collection).

[0126] 2. After incubation for a predetermined time at 2°C, 5 mL of the above porcine whole blood (collected from CPD) was injected into filter 200 through lower tube 223 of filter 200 at a flow rate of 1 mL / min using a syringe pump. After the injection of porcine whole blood (collected from CPD), upper tube 225 and lower tube 223 of filter 200 were clamped. In this state, the filter was incubated at 37°C for 15 minutes.

[0127] After the incubation, PBS buffer was injected into the filter 200 from the lower tube 223 of the filter 200 using a syringe pump at a flow rate of 1 mL / min, and 5 mL of the porcine whole blood in the filter 200 was collected.

[0128] Before injecting the filter and for the porcine whole blood collected above, hematology and flow cytometry were performed to calculate the CD8-positive rate among total leukocytes in the porcine whole blood before and after injecting the filter. Based on these values, the CD8-positive cell capture rate by the filter was calculated according to the following formula.

[0129]

[0130] Comparative Example 1 A polyurethane filter medium (manufactured by Terumo Corporation, product name: Imguard, thickness: 2 mm) was cut into a size of 25 mm in diameter (Φ) to prepare five comparative PU membranes (Comparative Example 1).

[0131] The CD8-positive cell capture rate was calculated in the same manner as in Example 3, except that the comparative PU membrane was used instead of Sample 3.

[0132] The results are shown in Table 1 below.

[0133]

[0134] As shown in Table 1, it can be seen that Sample 3 (Example 3) has a remarkable cell trapping performance compared to the comparative PU membrane (Comparative Example 1).

[0135] The present invention is not limited to the above disclosure, and various configurations can be adopted without departing from the gist of the present invention.

[0136] This application is based on Japanese Patent Application No. 2023-126173, filed on August 2, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0137] REFERENCE SIGNS LIST 10 filter 18 container 20 substrate 26 surface 28 active group 30 antibody 30a first antibody 30b second antibody 32 avidin (biotin-binding protein) 34 biotin 34a first biotin 34b second biotin 36 biotinylated antibody 36a first biotinylated antibody 36b second biotinylated antibody

Claims

1. A filter for capturing an object to be separated by passing a liquid containing cells or proteins as the object to be separated through the filter, the filter comprising: a substrate made of porous polyurethane having a plurality of micropores formed therein; and an active group fixed to the surface of the substrate and capable of specifically binding to the object to be separated, the active group including: a structural unit A derived from a compound A having an epoxy group and an ethylenically unsaturated group, bound to the substrate; and a first structural unit B derived from biotin, bound to the structural unit A.

2. A filter according to claim 1, wherein the active group comprises an antibody or an aptamer capable of specifically binding to an antigen possessed by the object to be separated.

3. A filter according to claim 2, wherein the active groups include a conjugate of the antibody or the aptamer bound to a second biotin, and a biotin-binding protein capable of binding to the second biotin.

4. A filter as described in claim 2, wherein the active groups include a conjugate in which the antibody or aptamer is bound to a second biotin; a building block B derived from the first biotin; and a biotin-binding protein capable of binding to each of the building block B and the building block C derived from the second biotin in the conjugate.

5. A filter according to claim 1, wherein said compound A is at least one selected from the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexylmethyl acrylate, 3,4-epoxycyclohexylmethyl methacrylate, β-methylglycidyl acrylate, and β-methylglycidyl methacrylate.

6. A filter according to claim 1, comprising a container for accommodating a plurality of said substrates having said active groups fixed thereto, said plurality of substrates being stacked in said container from upstream to downstream in the flow of said liquid.

7. A filter according to claim 6, comprising a plurality of the containers arranged in a line from upstream to downstream in the flow of the liquid, and the type of the active group fixed to the substrate differs for each of the containers.

8. A filter according to claim 1, wherein the plurality of micropores formed in the substrate are larger than the object to be separated.

9. A filter as claimed in claim 1, comprising a porous member and a separate substrate separate from the substrate, wherein a plurality of micropores formed in the separate substrate are larger than the object to be separated and smaller than the separate object to be separated.

10. A method for producing a filter, comprising: (i) irradiating with plasma at least a portion of a surface of a substrate made of porous polyurethane having a plurality of micropores formed therein; (ii) contacting the substrate after the plasma irradiation with a compound A having an epoxy group and an ethylenically unsaturated group, thereby binding the compound A to the substrate; and (iii) contacting a first biotin with the substrate after the contact with the compound A, thereby binding the first biotin to the surface of the substrate via a structural unit A derived from the compound A.

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