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  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
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  • 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
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
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  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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  • 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
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3206—Organic carriers, supports or substrates
  • B01J20/3208—Polymeric carriers, supports or substrates
  • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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  • 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
  • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
  • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/02—Separating microorganisms from their culture media
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
  • C12N5/0075—General culture methods using substrates using microcarriers
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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  • C12N2531/00—Microcarriers

Definitions

• Patent Document 1 describes a method for obtaining extracellular membrane vesicles or viruses in a sample, which comprises a carrier (Tim carrier) to which a protein (Tim protein) selected from T cell immunoglobulin mucin domain-containing molecule 4 (Tim4) protein, T cell immunoglobulin mucin domain-containing molecule 3 (Tim3) protein, and T cell immunoglobulin mucin domain-containing molecule 1 (Tim1) protein is bound, and the method includes the following steps: (1) a step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (complex formation step), (2) a step of separating the complex from the sample (complex separation step), and (3) a step of separating the extracellular membrane vesicles or viruses from the complex to obtain the extracellular membrane vesicles or viruses (obtaining step).
• a carrier to which a protein (Tim protein) selected from T cell immunoglobulin mucin domain-containing
• Patent Document 2 describes a cancer diagnostic device comprising an immobilization carrier having one or more lectins immobilized thereon that can specifically bind to surface glycans possessed by extracellular vesicles derived from cancer cells, each of which corresponds to one or more of the surface glycans, an extracellular vesicle capture unit that captures the extracellular vesicles through specific binding between the surface glycans and the lectins, and a detection unit that detects microRNA contained in the extracellular vesicles.
• methods for separating and purifying extracellular vesicles include a combination of two or more of TFF (tangential flow filtration), SEC (size exclusion chromatography), ion exchange, etc.
• TFF tangential flow filtration
• SEC size exclusion chromatography
• ion exchange ion exchange
• ⁇ 11> A method for obtaining extracellular vesicles in a sample, using the porous carrier according to any one of ⁇ 1> to ⁇ 10>.
• ⁇ 12> A sample containing extracellular vesicles is contacted with the porous carrier according to any one of ⁇ 1> to ⁇ 10>, and a complex is formed between a substance having affinity for the extracellular vesicles bound to the porous carrier and the extracellular vesicles in the sample; Separating the complex and the sample; and A method for producing extracellular vesicles, comprising separating the extracellular vesicles from the complex and obtaining the extracellular vesicles.
• a sample containing extracellular vesicles is contacted with the porous carrier according to any one of ⁇ 1> to ⁇ 10>, and a complex is formed between a substance having affinity for the extracellular vesicles bound to the porous carrier and the extracellular vesicles in the sample; and A method for producing extracellular vesicles, comprising removing the above complex.
• a kit for obtaining extracellular vesicles comprising a porous carrier having an exclusion limit molecular weight of 800 to 60,000 kDa, and a substance having affinity for extracellular vesicles.
• the present invention provides a porous carrier that can separate and obtain extracellular vesicles with high purity according to size and affinity, a method for obtaining extracellular vesicles using the porous carrier, a method for producing extracellular vesicles using the porous carrier, and a kit for obtaining extracellular vesicles.
• the porous carrier of the present invention is a porous carrier having an exclusion limit molecular weight of 800 to 60,000 kDa to which a substance having affinity for extracellular vesicles is bound, and is a porous carrier for obtaining extracellular vesicles.
• the porous carrier of the present invention extracellular vesicles can be separated and obtained with high purity according to their size.
• the porous carrier of the present invention has an exclusion limit molecular weight of 800 to 60,000 kDa.
• This porous carrier is considered to have a large number of pores in order to achieve the above exclusion limit molecular weight.
• EVs extracellular vesicles
• the extracellular vesicles are believed to penetrate into the pores.
• substances having affinity for the EVs are more likely to be bound to the inside of the pores, which have a large surface area, and the EVs are believed to bind to the substances in the pores and tend to remain in the pores.
• EVs with small particle diameters are more likely to penetrate into pores than EVs with large particle diameters, and are more likely to bind to substances that have affinity for the EVs. Therefore, when the porous carrier of the present invention is contacted with a sample containing EVs to form a complex between the porous carrier and EVs, and then the complex is separated from the sample, it is considered that the sample side contains EVs with large particle diameters, and the complex side contains EVs with small particle diameters. Then, by separating the EVs from the complex, it is possible to separate and obtain EVs with small particle diameters from a sample containing EVs with large particle diameters.
• EVs can be collected by using a carrier to which Tim protein is bound.
• a method such as SEC.
• SEC size fractionation
• the porous particles of the present invention are used, the recovery of EVs and separation according to size can be carried out as a single process. Therefore, compared to a method in which EVs are recovered and then further separated using SEC, EV loss is reduced and separation and purification can be performed with excellent purification efficiency.
• the excellent purification efficiency as described above is preferable because it not only reduces time and costs, but also minimizes damage to EVs.
• mol/l is also written as M.
• mmol/l and ⁇ mol/l are also written as “mM” and “ ⁇ M”.
• room temperature is 23°C. In the present invention, combinations of preferred embodiments are more preferred embodiments.
• the exclusion limit molecular weight of the porous carrier is preferably 1,000 kDa or more, more preferably 1,500 kDa or more, even more preferably 10,000 kDa or more, and particularly preferably 29,000 kDa or more.
• the exclusion limit molecular weight is preferably 40,000 kDa or less, more preferably 35,000 kDa or less, even more preferably 31,000 kDa or less, and particularly preferably 30,000 kDa or less.
• the exclusion limit molecular weight is not limited to the above preferred range, and may be appropriately set depending on the particle size of the extracellular vesicles to be obtained.
• the exclusion limit molecular weight can be the catalog value described for the carrier before binding to a substance having affinity for extracellular vesicles (hereinafter also referred to as "unbound carrier"). If there is no catalog value, it can be obtained, for example, by the following method.
• retention rate “retained fraction” / "input”
• the method for binding a substance having affinity for unbound carriers and extracellular vesicles of the present invention to the unbound carrier of the present invention may be any method known per se for binding a protein to a carrier, and examples of such methods include a method of binding by affinity binding; a method of binding by chemical binding (e.g., the methods described in Japanese Patent No. 3269554, WO 2012/039395, and WO 2016/088689); a method of binding by physical adsorption (e.g., the method described in JP-B-5-41946), and the like, with a method of binding by chemical binding being preferred.
• the solution containing the substance having affinity for the extracellular vesicles according to the present invention may be any solution that can dissolve the substance having affinity for the extracellular vesicles according to the present invention in a stable state, and examples of such solutions include purified water and buffer solutions having a buffering effect at pH 6.0 to 9.5, preferably 7.0 to 8.0 (e.g., Good's buffer solution such as MOPS, phosphate buffer solution, carbonate buffer solution, PBS, TBS, HBS, etc.).
• the buffer concentration in these buffer solutions is preferably 5 to 100 mM, more preferably 10 to 50 mM. When NaCl is added, the concentration is usually preferably 100 to 200 mM, more preferably 140 to 160 mM.
• a blocking treatment that is usually performed in this field may be carried out.
• purification treatment that is usually performed in this field may be performed.
• the purification treatment may be performed by removing impurities attached to the surface of the porous carrier or the substance having affinity for extracellular vesicles weakly bound to the carrier, and may, for example, be performed by washing the porous carrier of the present invention with a known washing solution such as guanidine hydrochloride.
• the porous carrier can be used in various modes, such as in the form of particles (beads) of the porous carrier, in the form of the porous carrier packed in a column, or in the form of a membrane made of the porous carrier.
• the porous carrier of the present invention is a porous carrier for obtaining extracellular vesicles.
• Extracellular vesicles are small membrane vesicles derived from cells and composed of a lipid bilayer. Extracellular vesicles include, for example, those classified in various ways according to their origin and the size of small membrane vesicles, as described in Nature Reviews Immunology 9, 581-593 (March 2009) and "Obesity Research" Vol. 13 No. 2 2007 Topics by Naoto Aoki et al. Specific examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, adiposomes, etc.
• Ectosomes are small membrane vesicles derived from the plasma membrane, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface.
• the diameter of ectosomes is preferably 50 nm to 200 nm, more preferably 50 nm to 150 nm, and even more preferably 50 nm to 100 nm.
• Ectosomes are known to contain CR1 and proteolytic enzymes, but not CD63.
• Membrane particles are small membrane vesicles derived from the plasma membrane, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface.
• the diameter of the membrane particle is preferably 50 nm to 80 nm. It is known that membrane particles contain CD133 but not CD63.
• Exosome-like vesicles are small membrane vesicles derived from early endosomes, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface.
• the diameter of exosome-like vesicles is usually preferably 20 nm to 50 nm.
• Exosome-like vesicles are known to contain TNF-RI.
• Apoptotic vesicles are small membrane vesicles derived from apoptotic cells, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface.
• the diameter of apoptotic vesicles is preferably 50 nm to 500 nm, more preferably 50 nm to 300 nm, and even more preferably 50 nm to 200 nm.
• Apoptotic vesicles are known to contain histones.
• Adiposomes are small membrane vesicles derived from fat cells, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface.
• the diameter of adiposomes is preferably 100 nm to 1000 nm, more preferably 100 nm to 800 nm, and even more preferably 100 nm to 500 nm.
• Adiposomes are known to contain MFG-E8 (milk fat globule-EGF factor 8).
• the porous carrier of the present invention makes it possible to separate and obtain extracellular vesicles with high purity according to their size and affinity.
• the exclusion limit molecular weight of the porous carrier of the present invention so that extracellular vesicles with a particle size of 200 nm or less can be retained within the pores, it becomes possible to selectively obtain extracellular vesicles with a particle size of 200 nm or less and capable of binding to an affinity substance from a sample containing extracellular vesicles, or to selectively remove extracellular vesicles with a particle size of 200 nm or less and capable of binding to an affinity substance from a sample containing extracellular vesicles.
• the porous carrier of the present invention extracellular vesicles can be easily separated and obtained with high purity according to size and affinity even from a large amount of sample.
• the method for obtaining extracellular vesicles of the present invention is a method for obtaining extracellular vesicles in a sample using the porous carrier of the present invention.
• the method for obtaining extracellular vesicles of the present invention preferably includes contacting a sample with the porous carrier of the present invention, forming a complex between a substance having affinity for the extracellular vesicles bound to the porous carrier and the extracellular vesicles in the sample (complex formation step), separating the complex from the sample (complex separation step), and separating the extracellular vesicles from the complex to obtain the extracellular vesicles (obtaining step).
• the liquid may be any liquid that stably suspends the extracellular vesicles according to the present invention and does not prevent the formation of a complex with the extracellular vesicles in the sample by binding to the porous carrier, etc.
• examples thereof include water and buffer solutions (e.g., TBS, HBS, etc.) that have a buffering effect at pH 7.0 to 8.0, preferably 7.2 to 7.6.
• As the buffer solution a buffer solution that is unlikely to bind with calcium and cause precipitation is preferable.
• the buffer concentration in these buffer solutions is preferably 5 to 50 mM, more preferably 10 to 30 mM.
• the NaCl concentration is preferably 100 to 200 mM, more preferably 140 to 160 mM.
• This solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc.
• An example of the surfactant is Tween 20, and the concentration of the surfactant is preferably 0.00001 to 0.2%, more preferably 0.0005 to 0.1%.
• the complex is preferably formed in the presence of calcium ions.
• the calcium ion concentration when the sample according to the present invention is brought into contact with the porous carrier of the present invention is preferably 0.5 to 100 mM, more preferably 1.0 to 10 mM, and even more preferably 2.0 to 5.0 mM. It is preferable that calcium ions at the above-mentioned concentration are present in a solution containing a complex formed between the porous carrier of the present invention and extracellular vesicles in a sample of the present invention until the complex is subjected to the complex separation process.
• the source of the calcium ion is not particularly limited, and examples include calcium chloride, calcium hydroxide, calcium bicarbonate, calcium iodide, calcium bromide, calcium acetate, etc., with calcium chloride, calcium bicarbonate, or calcium iodide being preferred, and calcium chloride or calcium bicarbonate being more preferred.
• the amount of the sample to be contacted with 1 mg of the porous particles of the present invention is preferably 0.01 to 10 ml, more preferably 0.01 to 1 ml, and even more preferably 0.01 to 0.5 ml.
• the temperature at which the sample is brought into contact with the porous carrier of the present invention is preferably 4 to 37°C, more preferably 4 to 30°C, and even more preferably 15 to 30°C.
• the contact time between the sample and the porous carrier of the present invention is preferably 0.5 to 24 hours, more preferably 0.5 to 8 hours, and even more preferably 0.5 to 4 hours in the batch method, and is preferably 1 to 5 minutes, more preferably 1 to 3 minutes, and even more preferably 1 to 2 minutes in the column method.
• the solution containing the complex can be centrifuged to separate the complex from the sample other than the complex.
• the complex may be loaded onto a filter and centrifuged to separate the sample other than the complex as a residual liquid.
• the centrifugation conditions may be determined taking into consideration the particle size, density, etc. of the porous particles, and may be, for example, 1 to 1000 ⁇ g for 30 seconds to 30 minutes.
• a solution containing the complex may be subjected to a column treatment to separate the complex from a sample other than the complex.
• Examples of the obtaining step include a method using a protein denaturant and a method of reducing calcium ion concentration.
• Examples of methods using a protein denaturant include the methods described in paragraphs 0162 to 0169 of WO 2016/088689.
• the method for reducing the calcium ion concentration is to set the calcium ion concentration in a solution containing the complex of the present invention to less than 0.5 mM, preferably less than 0.4 mM, and more preferably less than 0.2 mM.
• Methods for lowering the calcium ion concentration include the use of a calcium ion chelating agent and the use of a solution that does not contain calcium ions.
• the method described in paragraphs 0174 to 0191 of WO 2016/088689 can be referred to.
• the calcium ion chelating agent may be any compound capable of chelating calcium ions, and examples thereof include EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), GLDA (L-glutamic acid diacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), GEDTA (ethylene glycol bis( ⁇ -aminoethyl ether)-N,N,N,N-tetraacetic acid), TTHA (triethylenetetramine-N,N,N',N'',N'',N''',N'''-hexaacetic acid), HIDA (2-hydroxyethyliminodi(acetic acid)), DHEG (N,N-bis(2-hydroxyethyl)glycine), CyDTA (trans-1,2-Diaminocyclohexane-N
• this is generally done by contacting a solution containing the calcium ion chelating agent (hereinafter sometimes abbreviated as "calcium ion chelating agent-containing solution") with the complex of the present invention, and causing a reaction between the calcium ions bound to the complex of the present invention and the calcium ion chelating agent in the calcium ion chelating agent-containing solution.
• a solution containing a calcium ion chelating agent can be carried out, for example, by suspending the complex in the solution.
• the solution containing the calcium ion chelating agent may be any solution capable of dissolving the calcium ion chelating agent, and examples thereof include purified water and buffer solutions.
• the buffer solution include buffer solutions having a buffering effect at a pH of 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., PBS, TBS, HBS, etc.).
• the buffer concentration in these buffer solutions is appropriately selected from the range of usually 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is appropriately selected from the range of usually 100 to 200 mM, preferably 140 to 160 mM.
• the calcium ion chelating agent-containing solution may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.
• the concentration of the calcium ion chelating agent in the calcium ion chelating agent-containing solution is, for example, preferably 0.5 to 500 mM, more preferably 0.5 to 100 mM, and even more preferably 0.5 to 50 mM.
• the temperature and time for allowing the calcium ion chelating agent to act (contact) on the complex are, for example, 4.0 to 37°C, preferably 10 to 30°C, and more preferably 20 to 30°C, for example, 5 to 60 minutes, preferably 10 to 30 minutes, and more preferably 15 to 25 minutes.
• ⁇ Removal process> The details of the removal step are the same as those of the above-mentioned complex separation step, except that what is recovered is either a complex or a sample other than the complex.
• a method in which the complex is loaded onto a filter and centrifuged to separate samples other than the complex as a residual liquid, and the residual liquid is collected or a method in which a solution containing the complex is loaded onto a column and samples other than the complex are separated as a residual liquid by gravity or pressure from a peristaltic pump.
• kits of the present invention may also include instructions for the acquisition method of the present invention, instructions for the production method of the present invention, etc.
• Instructions refers to an instruction manual, package insert, pamphlet (leaflet), etc. for the kit in which the features, principles, operating procedures, determination procedures, etc. of the above-mentioned method are substantially described in text or diagrams, etc.
• Tim4-immobilized beads were obtained by the following procedure. WorkBeads 40/100 ACT, 40/1000 ACT, 40/10000ACT, and 40/30000 ACT (all manufactured by Bio-Works, all beads with non-through holes) were prepared, and 100 ⁇ L of each was suspended in 1 mL of phosphate buffer (50 mM, pH 7.4) to obtain a suspension. 16 ⁇ g of Tim4-Fc protein (FUJIFILM Wako Pure Chemical #137-18511) was added to each of the above suspensions, and the suspensions were shaken (1400 rpm) at 4 ° C. for 24 hours.
• phosphate buffer 50 mM, pH 7.4
• the particle size and number of EV particles were measured by the following procedure. Among the above-mentioned recovered fractions, the "recovered fraction" obtained from the "300 ⁇ g treated supernatant sample” was measured using a NANOSIGHT NS300 (Malvern Panalytical) (Camera Level: 16 Detection Threshold: 8).
• an "unrecovered fraction” and a “recovered fraction” were recovered using the "0.22 ⁇ m filter-treated supernatant sample” by a method similar to that described above in "EV purification from MSC supernatant using MagCapture Exosome Isolation Kit PS.”
• a PS Capture Exosome ELISA kit Streptavidin HRP (Fujifilm Wako Pure Chemical Industries #298-80601) was used and operated according to the protocol. The absorbance was measured using a plate reader Spark (Tecan). The ratio (FT signal) of each "unrecovered fraction” to the "0.22 ⁇ m filter-treated sample” was calculated, and the capture rate was calculated by (1-FT signal) x 100, as shown in Table 6.

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