WO2013047527A1 - Substrat pour l'immobilisation d'un ligand et procédé de fabrication de celui-ci, agent de séparation de cellule spécifique et unité de traitement du sang - Google Patents

Substrat pour l'immobilisation d'un ligand et procédé de fabrication de celui-ci, agent de séparation de cellule spécifique et unité de traitement du sang Download PDF

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Publication number
WO2013047527A1
WO2013047527A1 PCT/JP2012/074574 JP2012074574W WO2013047527A1 WO 2013047527 A1 WO2013047527 A1 WO 2013047527A1 JP 2012074574 W JP2012074574 W JP 2012074574W WO 2013047527 A1 WO2013047527 A1 WO 2013047527A1
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general formula
ligand
substrate
water
insoluble carrier
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PCT/JP2012/074574
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English (en)
Japanese (ja)
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伸一郎 長澤
正和 福島
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旭化成株式会社
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Priority to JP2013536303A priority Critical patent/JP5784132B2/ja
Publication of WO2013047527A1 publication Critical patent/WO2013047527A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3276—Copolymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3278—Polymers being grafted on the carrier

Definitions

  • the present invention relates to a ligand immobilization substrate and a method for producing the same, a specific cell separation material on which an affinity ligand is immobilized, and a blood treatment device.
  • a substance having affinity for the specific component that is, a separation material in which a ligand is covalently fixed to a water-insoluble carrier is widely used clinically.
  • separation materials are used in such a manner that patient blood is once taken out of the body, blood itself or plasma separated by a plasma separator is processed by flowing through the separating material, and then returned to the patient.
  • non-specific adsorption with components other than the separation target due to contact with the solution containing the separation target leads to a decrease in specific separation performance.
  • the selective separation material in order to selectively separate a specific component, it is desired that the non-specific adsorption of components other than the separation target hardly occur on the base material itself.
  • a material having a low-fouling and low-adsorbing zwitterionic group (betaine) has been proposed as a material for forming a substrate in which nonspecific adsorption is suppressed, and is resistant to protein adhesion.
  • a light-transmitting polymer material a polymer using a polymerizable monomer having a zwitterionic group is disclosed (Patent Document 1).
  • the base material which has the low fouling surface containing the monomolecular layer of sulfobetaine and carboxybetaine is disclosed (patent document 2).
  • Patent Document 3 A copolymer with a vinyl polymerizable monomer is disclosed (Patent Document 3).
  • cationic functional groups for example, primary amino group, secondary amino group, tertiary amino group, and Conventionally known is a technique for selectively adsorbing cells by immobilizing a quaternary ammonium group
  • anionic functional group for example, sulfate ester group, sulfonic acid group, carboxyl group, and phosphate ester group
  • an affinity ligand having a specific affinity for a protein or sugar chain present on the cell surface of the target cell is effective in order to obtain high cell selective separation.
  • an antibody having an amino acid sequence having a strong affinity for a cell surface protein or sugar chain, an antibody fragment (F (ab) ′, Fab, Fab ′, etc.) containing an antigen binding site of the antibody, and such an amino acid sequence Synthetic peptides having these or modified peptides thereof are effective, and a method of covalently immobilizing them on a substrate is considered effective.
  • the present invention relates to a substrate for immobilizing a ligand, which has excellent performance that there are few non-specific interactions with proteins, cells, etc., and can bind a ligand firmly by a covalent bond, and a method for producing the same.
  • the purpose is to provide.
  • the present invention also has excellent performance that there are few non-specific interactions with proteins and cells, etc., and the ligand is firmly bound by a covalent bond, and the specific interaction with the immobilized ligand is achieved.
  • An object of the present invention is to provide a specific cell separation material that can exhibit high adsorption performance.
  • the present inventors have excellent non-specific adsorption suppression ability, can be firmly bound to the ligand by covalent bond, have a low risk of elution of the ligand, and fully exert the performance of the immobilized ligand.
  • a base material for immobilizing a ligand formed by binding a copolymer of a polymerizable sulfobetaine monomer and a polymerizable monomer having an electrophilic functional group to the surface of a water-insoluble carrier is described above.
  • the present inventors have found that the above problems can be solved and have completed the present invention.
  • a substrate for immobilizing a ligand comprising: a water-insoluble carrier; and a copolymer represented by the following general formula (1) bonded to at least the surface of the water-insoluble carrier.
  • n and m show a positive integer, and the value of m / (n + m) is 0.05 or more and 0.50 or less.
  • R 1 is H or CH 3
  • R 2 is an organic group having an electrophilic functional group
  • R 3 is a group represented by the following general formula (2).
  • Y represents O or NH
  • p represents 1 or 2
  • q represents 2 or 3.
  • n and m show a positive integer, and the value of m / (n + m) is 0.05 or more and 0.50 or less.
  • R 1 is H or CH 3
  • R 2 is an organic group having an electrophilic functional group
  • R 3 is a group represented by the following general formula (2).
  • Y represents O or NH
  • p represents 1 or 2
  • q represents 2 or 3.
  • [8] The use according to [7], wherein the copolymer contains an epoxy group.
  • a substrate for immobilizing a ligand which has excellent performance with less non-specific interaction with proteins and cells, etc., and can bind a ligand firmly by covalent bond, and its A manufacturing method is provided.
  • it has excellent performance that there are few non-specific interactions with proteins, cells, etc., and the ligand is firmly bound by a covalent bond and specific with the immobilized ligand.
  • a specific cell separation material capable of exhibiting high adsorption performance based on interaction is provided.
  • the substrate for immobilizing a ligand includes a water-insoluble carrier and a copolymer represented by the following general formula (1) bonded to at least the surface of the water-insoluble carrier.
  • n and m show a positive integer, and the value of m / (n + m) is 0.05 or more and 0.50 or less.
  • R 1 is H or CH 3
  • R 2 is an organic group having an electrophilic functional group
  • R 3 is a group represented by the following general formula (2).
  • Y represents O or NH
  • p represents 1 or 2
  • q represents 2 or 3.
  • the copolymer represented by the general formula (1) is a random copolymer, an alternating copolymer, a periodic copolymer and a block copolymer of a repeating unit having a repeating number of m and a repeating unit having a repeating number of n. Any of polymers may be used.
  • the copolymer represented by the general formula (1) is, for example, polymerizable having a polymerizable sulfobetaine monomer represented by the general formula (3) and an electrophilic functional group represented by the general formula (4). It can be obtained by copolymerizing with a monomer.
  • Y represents O or NH
  • p represents 1 or 2
  • q represents 2 or 3.
  • R 1 represents H or CH 3
  • R 2 represents an organic group having an electrophilic functional group.
  • the repeating unit having the repeating number m is a structural unit derived from the monomer represented by the general formula (3)
  • the repeating unit having the repeating number n is the general formula (4). It is a structural unit derived from the monomer represented.
  • the polymerizable sulfobetaine monomer represented by the general formula (3) is a monomer (monomer) having a sulfonic acid group as an anionic group and a quaternary ammonium group as a cationic group in the same monomer unit.
  • polymerizable sulfobetaine monomer represented by the general formula (3) include, for example, N, N-dimethyl-N- (3-acrylamidopropyl) -N- (3-sulfopropyl) ammonium inner salt, N , N-dimethyl-N- (3-acrylamidopropyl) -N- (2-sulfoethyl) ammonium inner salt, N, N-dimethyl-N- (3-methacrylamidopropyl) -N- (3-sulfopropyl) ammonium Internal salt, N, N-dimethyl-N- (3-methacrylamidopropyl) -N- (2-sulfoethyl) ammonium internal salt, N, N-dimethyl-N- (2-acryloyloxyethyl) -N- (3 -Sulfopropyl) ammonium inner salt, N, N-dimethyl-N- (2-acryloyloxyethy
  • the “electrophilic functional group” means a functional group that reacts electrophilically
  • specific examples of the polymerizable monomer having an electrophilic functional group represented by the general formula (4) include: And monomers having an electrophilic functional group such as an epoxy group, an isocyanate group or an aldehyde group in the molecule (R 2 in the general formula (1) and R 5 in the general formula (4)).
  • the polymerizable monomer having an epoxy group in the molecule is not particularly limited, but glycidyl acrylate, glycidyl methacrylate, glycidyl acrylamide, allyl glycidyl ether, methacryl glycidyl ether, glycidyl sorbate, glycidyl metaitaconate, ethyl glycidyl maleate
  • Examples of the polymerizable monomer having an isocyanate group in the molecule include acryloyloxyethyl isocyanate, acryloyloxymethyl isocyanate, acryloyl isocyanate, methacryloyl isocyanate, and methacryloyl ethyl isocyanate.
  • polymerizable monomers having a group in the molecule examples include cinnamaldehyde and crotonaldehyde.
  • Dehydrogenase can be exemplified acrolein and methacrolein, easy availability among this, cost, ease of handling, the polymerizable monomer having an epoxy group in the molecule are preferred, particularly glycidyl methacrylate are preferable among them.
  • the manner in which the copolymer is bonded to at least the surface of the water-insoluble carrier is not particularly limited, and any bonding mode such as covalent bond, ionic bond, and physical adsorption may be used.
  • any known method such as a grafting method and an insolubilizing precipitation method can be used. Therefore, a method of modifying the surface by a known method such as graft polymerization or covalent bonding of at least one of the polymer compound or its monomer using radiation or plasma (Japanese Patent Laid-Open Nos. 1-249063 and 3). No. -502094) can be preferably used.
  • ionizing radiation for introducing an active site on a water-insoluble carrier includes ⁇ rays, ⁇ rays, ⁇ rays, accelerated electron rays, X rays and ultraviolet rays, and is accelerated from the viewpoint of being practical. Electron beams or ⁇ rays are preferred.
  • the irradiation amount of the accelerated electron beam or ⁇ -ray can be arbitrarily changed depending on the nature of the water-insoluble carrier, the nature of the monomer used, the amount of the copolymer immobilized, etc., but is preferably 10 kGy to 200 kGy.
  • a simultaneous irradiation method of irradiating ionizing radiation in the presence of a water-insoluble carrier and a monomer, or after irradiating only a water-insoluble carrier with ionizing radiation in advance Any of the pre-irradiation methods in which an insoluble carrier is brought into contact can be used, but the pre-irradiation method is preferable because it hardly generates a side reaction other than graft polymerization.
  • the contact between the water-insoluble carrier and the monomer can be performed, for example, by bringing a water-insoluble carrier and a solution containing the monomer into contact with each other.
  • the monomer concentration in the solution containing the monomer can usually be 1% by mass or more and 20% by mass or less, but is not limited to this range and may be set as appropriate.
  • the reaction between the monomer and the water-insoluble carrier can be carried out at a reaction temperature in the range of 5 ° C. or higher and 40 ° C. or lower, but is not limited to this range and may be set as appropriate.
  • the solvent used in the reaction may be the same as the solvent of the solution containing the monomer.
  • the monomer is dissolved or dissolved in no solvent, a solvent such as water, methanol, ethanol, other alcohols, and acetone, or a mixed solvent thereof. Any material that can be dispersed can be used.
  • the graft ratio (G) to the carrier is preferably 5% or more and 300% or less, more preferably 20% or more and 200% or less, and further preferably 50% or more and 150% or less. If the graft ratio is less than 5%, the support surface is likely to be insufficiently coated with graft chains, the surface may be insufficiently modified, and the amount of ligand immobilization may be insufficient. On the other hand, if the graft ratio exceeds 300%, the physical properties of the water-insoluble carrier itself may be lost, which is not preferable in designing the base material for immobilizing a ligand.
  • the graft ratio (G) referred to here is a value represented by the following formula (4).
  • G (%) [(weight of carrier after grafting ⁇ weight of carrier before grafting) / (weight of carrier before grafting)] ⁇ 100 (4)
  • the excess monomer and the ungrafted polymer produced by the chain transfer reaction may be sufficiently washed away with a suitable solvent.
  • the molar composition ratio of the polymerizable sulfobetaine monomer in the copolymer according to this embodiment is preferably 0.05 or more and 0.50 or less with respect to all monomers.
  • the molar composition ratio of the polymerizable sulfobetaine monomer is less than 0.05, the biocompatible property is not exhibited.
  • the molar composition ratio of the polymerizable sulfobetaine monomer exceeds 0.50, the biocompatibility property is sufficiently exhibited, but the content of the electrophilic functional group is inevitably low, and the ligand is not immobilized. It is not preferable because it is not performed sufficiently.
  • the amount of each monomer to be mixed during the reaction for forming the copolymer depends on the solvent to be selected and may be appropriately selected.
  • the coverage of the water-insoluble carrier with the copolymer is preferably 25% or more. If the abundance ratio on the surface is less than 25%, the surface properties of the carrier remain, nonspecific adsorption increases, and the ligand is not sufficiently immobilized. There is no restriction
  • Examples of the material forming the water-insoluble carrier according to the present embodiment include organic polymer compounds such as natural polymers, synthetic polymers, and regenerated polymers, inorganic compounds typified by glass and metal, and organic / inorganic hybrid compounds. There is no particular limitation.
  • organic polymer materials are particularly preferred.
  • water-insoluble carrier all known carriers for medical adsorbents (or separation materials) can be used, but porous membranes or particles are preferred, and porous particles, non-woven fabrics or hollow fiber membranes are used for body fluids during extracorporeal circulation. Most preferred from the viewpoint of flowability, and non-woven fabric or particles are preferred for the purpose of cell adsorption.
  • porous membrane examples include non-woven fabrics, woven fabrics, knitted fabrics, and meshes manufactured using fibrous materials (solid fibers and hollow fibers) obtained from these materials.
  • a foaming method from the state in which an organic polymer material or an inorganic polymer material is thermally melted, in a solution state dissolved with a solvent, or in a state of plasticization using a plasticizer, a foaming method, a phase separation method (thermally induced phase separation method and A sheet-like membrane (flat membrane) and an empty fiber membrane that can be obtained by a wet phase separation method), a stretching method, a sintering method, or the like can also be used.
  • the porous membrane may have any structure as long as it has communication holes and the target component is adsorbed by contact with an affinity ligand present at least on the surface of the communication holes.
  • the communication hole is a hole communicating from one membrane surface of the support porous membrane to the opposite membrane surface, and if the liquid can pass through the communication hole, the pore surface of the membrane.
  • non-woven fabrics, woven fabrics, knitted fabrics, meshes, and the like manufactured from various fibrous materials are preferable from the viewpoint of the permeability of the cell suspension and the ability to capture cells.
  • a nonwoven fabric is a porous film that is preferably used.
  • the average fiber diameter can be, for example, 0.1 ⁇ m to 50 ⁇ m.
  • the thickness is preferably 2 ⁇ m to 20 ⁇ m.
  • the average fiber diameter is more preferably 3 ⁇ m to 12 ⁇ m, still more preferably 4 ⁇ m to 7 ⁇ m.
  • the average fiber diameter is less than 0.1 ⁇ m, the mechanical strength tends to decrease. Further, as the fiber diameter increases, the surface area per unit volume decreases, and a large separation material volume is required, so 50 ⁇ m or less is appropriate.
  • the particles according to the present embodiment may be selected to be nonporous or porous depending on the component to be separated, and in the case of porous, the pore diameter may be selected depending on the component to be separated.
  • An average particle size of 25 ⁇ m or more and 2500 ⁇ m or less can be used, but in view of its specific surface area (adsorption ability as an adsorbent) and fluidity of body fluids, a particle size of 50 ⁇ m or more and 1500 ⁇ m or less is preferable.
  • FIG. 1 is a schematic cross-sectional view of a ligand-immobilizing base material according to an embodiment.
  • the substrate 100 for immobilizing a ligand includes a water-insoluble carrier 1 and a copolymer 2 represented by the general formula (1) bonded to at least the surface of the water-insoluble carrier 1.
  • the copolymer 2 is bonded to the water-insoluble carrier 1 at the binding portion 10, and has R 3 groups 20 and R 2 groups 30 on the surface of the ligand immobilizing substrate 100.
  • the R 3 group 20 and the R 2 group 30 have the same meaning as the R 3 group and the R 2 group in the general formula (1).
  • the ligand immobilization substrate can be produced, for example, by a production method having the following steps.
  • (I) A step of irradiating the water-insoluble carrier with ionizing radiation.
  • (Ii) A step of graft polymerization by immersing the water-insoluble carrier obtained in step (i) in a solution containing the monomer represented by the general formula (3) and the monomer represented by the general formula (4). .
  • the water-insoluble carrier, ionizing radiation, solution, graft polymerization and the like are as described above.
  • the specific cell separation material according to the present embodiment is obtained by binding a ligand to the ligand immobilization substrate.
  • the ligand immobilization substrate and the ligand chemically bond, such as a covalent bond, a coordinate bond, an ionic bond, and a hydrogen bond, between the electrophilic functional group in the ligand immobilization substrate and the functional group of the ligand.
  • bond it is preferable to couple
  • the ligand is preferably an affinity ligand, and can be selected, for example, from the group consisting of antibodies, proteins, peptides, and low molecular compounds.
  • the affinity ligand immobilized on the specific cell separation material is not particularly limited as long as it has a selective affinity for specific cells and chemically binds to an electrophilic functional group.
  • the molecular weight is 500 Da or less. If low molecular weight compounds, proteins, and excellent adsorption ability are expressed, antibodies and chimeric antibodies having extremely high affinity for the target component, complementarity determining regions of variable regions of heavy chains or light chains of antibodies
  • ligands of F (ab ′) 2 , Fab, Fab ′ having other amino acid sequences that can be formed, and other peptides or modified peptide types thereof is preferred.
  • FIG. 2 is a schematic cross-sectional view of a specific cell separation material according to an embodiment.
  • the specific cell separation material 110 is formed by chemically bonding the R 2 group 30 of the ligand immobilization substrate 100 and the ligand 40.
  • the ligand 40 those described above can be used.
  • the blood processing apparatus has the above-mentioned unique inside the container having an inlet (inlet port) for introducing blood into the inside and an outlet (outlet port) for discharging the internal blood to the outside.
  • the target cell separation material is filled. Since the specific cell separation material can selectively adsorb the component to be separated, it can be preferably used for the purpose of removing a specific component from blood, for example. Specific examples include a blood component adsorber for direct blood perfusion and a specific cell adsorber.
  • FIG. 3 is a schematic cross-sectional view of a blood processing apparatus according to an embodiment.
  • the blood treatment device 200 includes a container 50 and a plurality of specific cell separation materials 110 filled in the container 50. Header caps 60 a and 60 b are provided at both ends of the container 50.
  • the header cap 60a serves as an inlet (inlet) for introducing blood into the inside, and the header cap 60b serves as an outlet (outlet) for discharging blood to the outside.
  • the blood flowing into the blood treatment device 200 from the direction of the arrow F from the inlet of the header cap 60a comes into contact with the specific cell separation material 110, so that the components in the blood that interact with the ligand 40 are adsorbed. Thereby, when it flows out from the outlet of the header cap 60b, the above components are removed from the blood.
  • Example 1 The (A) The method (1) non-woven fabric made of polypropylene as ⁇ -irradiation carrier (average fiber diameter 3.8 ⁇ m basis weight 80 g / m 2) and 0.108M 2 encapsulated in low oxygen permeability bag together with a deoxygenating agent sufficiently oxygen After removal, 25 kGy of ⁇ -rays were irradiated at ⁇ 78 ° C.
  • the circular nonwoven fabric substrate on which the monoclonal antibody was immobilized (hereinafter referred to as “antibody-immobilized circular nonwoven fabric”) was washed with 2 ml of PBS ( ⁇ ).
  • the antibody-immobilized circular nonwoven fabric was immersed in a 0.2% polyoxyethylene sorbitan monolaurate / PBS ( ⁇ ) solution (hereinafter referred to as “Tween 20 solution”) at room temperature for 2.5 hours to perform blocking. .
  • the antibody-immobilized circular nonwoven fabric was washed with 2 ml of PBS ( ⁇ ) to prepare a cell adsorption filter.
  • a cell adsorber was prepared by filling a cell with a capacity of 1 ml having an inlet and an outlet with four cell adsorption filters and a PBS ( ⁇ ) solution as a filling liquid.
  • X x / (x + y + z) (5)
  • Y y / (x + y + z) (6)
  • Z z / (x + y + z) (7)
  • x C 3
  • y (C 2 -4x) / 3
  • z ⁇ C 1- (10x + 7y) ⁇ / A
  • the molar composition ratio R of SPB in the copolymer is represented by the following formula (8).
  • the immobilization amount of the anti-human CD4 monoclonal antibody in the cell adsorption filter obtained above was determined using a BCA protein quantification reagent (Micro BCA (registered trademark) Protein Assay Reagent Kit 23235 manufactured by PIERCE). The absorbance was measured with a microplate spectrophotometer (SPECTRA MAX340PC, analysis software SOFT max PRO, manufactured by Molecular Devices) and quantified. As a result, 10.3 ⁇ g of antibody was immobilized on the cell adsorption filter (4 sheets) before washing, and 9.8 ⁇ g of antibody was immobilized on the cell adsorption filter after SDS washing.
  • BCA protein quantification reagent Micro BCA (registered trademark) Protein Assay Reagent Kit 23235 manufactured by PIERCE.
  • the absorbance was measured with a microplate spectrophotometer (SPECTRA MAX340PC, analysis software SOFT max PRO, manufactured by Molecular Devices) and quantified.
  • SPECTRA MAX340PC microplate spectrop
  • Example 2 A substrate for immobilizing a ligand was prepared in the same manner as in Example 1 except that the reaction solution used for the graft reaction was replaced with a reaction solution obtained by dissolving 14.0 g of SPB and 26.3 mL of GMA in 500 mL of methanol. Produced.
  • the molar composition ratio R of SPB was 0.12, and the coverage S was 87%.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • 10.8 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 8.9 ⁇ g of antibody was immobilized on the cell adsorption filter after SDS washing.
  • a cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1 and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 94.1% (F1) and 65.3%. (F2).
  • the adsorption rate of platelets was 34.0% (8 mL average).
  • Example 3 The base material for ligand immobilization was prepared in the same manner as in Example 1 except that the reaction solution used for the grafting reaction was replaced with a reaction solution obtained by dissolving 27.9 g of SPB and 26.3 mL of GMA in 500 mL of methanol. Produced.
  • the molar composition ratio R of SPB was 0.24 and the coverage S was 83%. Moreover, it was 0.51 m ⁇ 2 > / g when the specific surface area was measured.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • 11.2 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 10.3 ⁇ g of antibody was immobilized on the cell adsorption filter after washing with SDS.
  • a cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1, and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 98.2% (F1) and 87.2%. (F2).
  • the adsorption rate of platelets was 23.1% (8 mL average).
  • Example 4 The substrate for immobilizing the ligand was prepared in the same manner as in Example 1 except that the reaction solution used for the graft reaction was replaced with a reaction solution obtained by dissolving 55.9 g of SPB and 26.3 mL of GMA in 500 mL of methanol. Produced.
  • the molar composition ratio R of SPB was 0.38, and the coverage S was 84%.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • 9.8 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 8.9 ⁇ g of antibody was immobilized on the cell adsorption filter after washing with SDS.
  • a cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1, and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 97.1% (F1) and 87.3%. (F2).
  • the adsorption rate of platelets was 17.5% (8 mL average).
  • Example 5 The substrate for immobilizing the ligand was prepared in the same manner as in Example 1 except that the reaction solution used for the graft reaction was replaced with a reaction solution obtained by dissolving 89.4 g of SPB and 21.1 mL of GMA in 400 mL of methanol. Produced.
  • the molar composition ratio R of SPB was 0.50, and the coverage S was 79%. Moreover, it was 0.48 m ⁇ 2 > / g when the specific surface area was measured.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • 9.4 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 8.0 ⁇ g of antibody was immobilized on the cell adsorption filter after SDS washing.
  • a cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1, and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 92.7% (F1) and 52.6%. (F2).
  • the adsorption rate of platelets was 5.8% (8 mL average).
  • Example 6 The base material for ligand immobilization was prepared in the same manner as in Example 1 except that the reaction solution used for the graft reaction was replaced with a reaction solution obtained by dissolving 14.0 g of SPB and 13.2 mL of GMA in 500 mL of methanol. Produced.
  • the molar composition ratio R of SPB was 0.25, and the coverage S was 25%. Moreover, it was 0.46 m ⁇ 2 > / g when the specific surface area was measured.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • 10.9 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 9.6 ⁇ g of antibody was immobilized on the cell adsorption filter after washing with SDS.
  • a cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1, and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 97.5% (F1) and 81.3%. (F2).
  • the adsorption rate of platelets was 25.2% (8 mL average).
  • Example 1 A substrate for immobilizing a ligand was prepared in the same manner as in Example 1 except that the reaction solution used for the graft reaction was changed to a reaction solution obtained by dissolving 26.3 mL of GMA in 500 mL of methanol.
  • the molar composition ratio R of SPB was 0 and the coverage S was 75%.
  • it was 0.51 m ⁇ 2 > / g when the specific surface area was measured.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • the base material for ligand immobilization was prepared in the same manner as in Example 1 except that the reaction solution used for the graft reaction was replaced with a reaction solution obtained by dissolving 134.1 g of SPB and 15.8 mL of GMA in 350 mL of methanol. Produced.
  • the molar composition ratio R of SPB was 0.59, and the coverage S was 80%.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • As a result of measuring the antibody immobilization amount 0.7 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 0.2 ⁇ g of antibody was immobilized on the cell adsorption filter after SDS washing.
  • a cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1, and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 21.7% (F1) and 5.2%. (F2).
  • the adsorption rate of platelets was 5.3% (8 mL average).
  • Example 7 A substrate for immobilizing a ligand was prepared in the same manner as in Example 3 except that the carrier was replaced with a nonwoven fabric made of polypropylene (average fiber diameter: 2.9 ⁇ m, basis weight: 80 g / m 2 ).
  • the molar composition ratio R of SPB was 0.23 and the coverage S was 87%.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • Example 8 A substrate for immobilizing a ligand was prepared in the same manner as in Example 3 except that the carrier was replaced with a nonwoven fabric made of polypropylene (average fiber diameter 20.9 ⁇ m, basis weight 80 g / m 2 ).
  • the molar composition ratio R of SPB was 0.25, and the coverage S was 84%.
  • a cell adsorption filter was produced from the ligand immobilization substrate in the same manner as in Example 1, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1.
  • Example 9 Other than replacing the carrier with particles having an average particle size of 320 ⁇ m made of polyethylene, and replacing the reaction solution used for the graft reaction with a reaction solution obtained by dissolving 27.9 g of SPB and 26.3 mL of GMA in 500 mL of methanol.
  • a ligand-immobilizing substrate ligand-immobilizing particles
  • the molar composition ratio R of SPB was 0.28, and the coverage S was 67%. Moreover, it was 0.03 m ⁇ 2 > / g when the specific surface area was measured.
  • Cell adsorption particles were produced from 360 ⁇ L of the ligand immobilization particles in the same manner as in Example 1, and the particle amount was adjusted so that the surface area was the same as in Example 1.
  • 10.8 ⁇ g of antibody was immobilized on the cell adsorbed particles before washing, and 10.2 ⁇ g of antibody was immobilized on the cell adsorbed particles after SDS washing.
  • a cell adsorber was prepared from the cell adsorbed particles in the same manner as in Example 1, and the cell adsorption rate was measured.
  • the adsorption rates of CD4 positive cells were 83.7% (F1) and 74.1%. (F2).
  • the adsorption rate of platelets was 4.8% (8 mL average).
  • Example 10 A cell adsorption filter was prepared in the same manner as in Example 1 except that the antibody was replaced with an anti-human KLRG1 antibody, and the amount of nonwoven fabric was adjusted so that the surface area was the same as in Example 1. As a result of measuring the amount of immobilized antibody, 11.0 ⁇ g of antibody was immobilized on the cell adsorption filter before washing, and 9.1 ⁇ g of antibody was immobilized on the cell adsorption filter after washing with SDS. A cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1, and the cell adsorption rate was measured. As a result, the adsorption rates of KLRG1-positive cells were 90.1% (F1) and 77.6%. (F2). Moreover, the adsorption rate of platelets was 26.1% (8 mL average).
  • Example 11 A cell adsorption filter was prepared in the same manner as in Example 5 except that the antibody was replaced with an anti-human KLRG1 antibody, and the amount of non-woven fabric was adjusted so that the surface area was the same as in Example 1. As a result of measuring the amount of the antibody immobilized, 10.1 ⁇ g of the antibody was immobilized on the cell adsorption filter before washing, and 8.2 ⁇ g of the antibody was immobilized on the cell adsorption filter after SDS washing. A cell adsorber was prepared from the cell adsorption filter in the same manner as in Example 1 and the cell adsorption rate was measured. As a result, the adsorption rate of KLRG1-positive cells was 94.6% (F1) and 84.4%. (F2). Further, the adsorption rate of platelets was 12.5% (8 mL average).
  • Example 12 A ligand fixing substrate was prepared and evaluated in the same manner as in Example 1 except that the graft reaction was performed by the following method and the surface analysis by XPS was performed by the following method.
  • (Graft reaction) 27.9 g of SPB and 44.3 mL of 2-[(3,5-dimethylpyrazolyl) carboxyamino] ethyl methacrylate (Karenz MOI-BP, Showa Denko) were dissolved in 500 mL of methanol to obtain a reaction solution. Nitrogen was bubbled through the reaction solution at 40 ° C. for 60 minutes.
  • the carrier (nonwoven fabric described in Example 1) was quickly put into a pressure-resistant glass container, and after reducing the pressure, the reaction solution was drawn and reacted at 40 ° C. for 1 hour. After the reaction, the taken-out non-woven fabric was washed with dimethylformamide and methanol and vacuum-dried at 120 ° C. to deprotect the isocyanate group, thereby obtaining a ligand-immobilizing base material.
  • surface analysis by XPS In order to determine the molar composition ratio and coverage of the polymerized SPB and methacryloyloxyethyl isocyanate, the isocyanate group was labeled with sulfur.
  • 1 water-insoluble carrier, 2 ... copolymer, 10 ... coupling portion, 20 ... R 3 groups, 30 ... R 2 group (electrophilic functional group), 40 ... ligand, 50 ... container, 60a, 60b ... header caps, F ... direction of blood flow, 100 ... base material for immobilizing ligand, 110 ... specific cell separation material, 200 ... blood treatment device.

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Abstract

La présente invention concerne un substrat pour une immobilisation de ligand, qui comprend un support insoluble dans l'eau et un copolymère représenté par la formule générale (1) lié à au moins la surface du support insoluble dans l'eau. [Dans la formule générale (1), n et m représentent des entiers positifs et la valeur de m/(n+m) est 0,05 à 0,50. De plus, dans la formule générale (1), R1 représente H ou CH3, R2 représente un groupe organique ayant des groupes fonctionnels électrophiles et R3 est un groupe représenté par la formule générale (2). Dans la formule générale (2), Y représente O ou NH, p est 1 ou 2 et q est 2 ou 3.]
PCT/JP2012/074574 2011-09-28 2012-09-25 Substrat pour l'immobilisation d'un ligand et procédé de fabrication de celui-ci, agent de séparation de cellule spécifique et unité de traitement du sang WO2013047527A1 (fr)

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WO2016039293A1 (fr) * 2014-09-08 2016-03-17 Jsr株式会社 Support en phase solide, support en phase solide lié à un ligand, procédé de détection ou de séparation d'une substance cible, et procédé de production d'un support en phase solide
JP2016057105A (ja) * 2014-09-08 2016-04-21 Jsr株式会社 固相担体、リガンド結合固相担体、標的物質の検出又は分離方法、及び前記固相担体の製造方法
CN106103515A (zh) * 2014-03-05 2016-11-09 Jsr株式会社 固相载体、配体键合固相载体、靶物质的检测或分离方法、固相载体的制造方法以及配体键合固相载体的制造方法

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JP2000226550A (ja) * 1991-07-05 2000-08-15 Biocompatibles Ltd ポリマーの表面コーティング
JP2004154613A (ja) * 2002-11-01 2004-06-03 Asahi Kasei Corp 機能性多孔膜の製造方法
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CN106103515A (zh) * 2014-03-05 2016-11-09 Jsr株式会社 固相载体、配体键合固相载体、靶物质的检测或分离方法、固相载体的制造方法以及配体键合固相载体的制造方法
EP3115384A4 (fr) * 2014-03-05 2017-12-06 JSR Corporation Support solide, support solide lié à un ligand, procédé de détection ou de séparation de substance cible, procédé de production du support solide et procédé de production du support solide lié à un ligand
US10466235B2 (en) 2014-03-05 2019-11-05 Jsr Corporation Solid support, ligand-bound solid support, detection or separation method for target substance, solid support production method, and ligand-bound solid support production method
WO2016039293A1 (fr) * 2014-09-08 2016-03-17 Jsr株式会社 Support en phase solide, support en phase solide lié à un ligand, procédé de détection ou de séparation d'une substance cible, et procédé de production d'un support en phase solide
JP2016057105A (ja) * 2014-09-08 2016-04-21 Jsr株式会社 固相担体、リガンド結合固相担体、標的物質の検出又は分離方法、及び前記固相担体の製造方法
CN106796225A (zh) * 2014-09-08 2017-05-31 Jsr株式会社 固相载体、配体键合固相载体、靶物质的检测或分离方法以及上述固相载体的制造方法
JPWO2016039293A1 (ja) * 2014-09-08 2017-06-15 Jsr株式会社 固相担体、リガンド結合固相担体、標的物質の検出又は分離方法、及び前記固相担体の製造方法
US11125747B2 (en) 2014-09-08 2021-09-21 Jsr Corporation Solid phase carrier, ligand-bound solid phase carrier, method for detecting or separating target substance, and method for producing solid phase carrier

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