WO2023199678A1 - 分離対象物質の純度が高められた精製液を製造する方法及び分離対象物質を精製するための精製キット - Google Patents

分離対象物質の純度が高められた精製液を製造する方法及び分離対象物質を精製するための精製キット Download PDF

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WO2023199678A1
WO2023199678A1 PCT/JP2023/009693 JP2023009693W WO2023199678A1 WO 2023199678 A1 WO2023199678 A1 WO 2023199678A1 JP 2023009693 W JP2023009693 W JP 2023009693W WO 2023199678 A1 WO2023199678 A1 WO 2023199678A1
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substance
carrier
liquid
container
chamber
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English (en)
French (fr)
Japanese (ja)
Inventor
敦 久野
隆 佐藤
直樹 渡▲邊▼
善宏 瀬戸口
裕孝 武藤
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National Institute of Advanced Industrial Science and Technology AIST
Valqua Ltd
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National Institute of Advanced Industrial Science and Technology AIST
Valqua Ltd
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Priority to JP2024514858A priority Critical patent/JPWO2023199678A1/ja
Priority to EP23788100.8A priority patent/EP4509602A4/en
Publication of WO2023199678A1 publication Critical patent/WO2023199678A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • 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
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/12—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the preparation of the feed
    • B01D15/122—Solid phase extraction
    • 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
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • 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
    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • Chromatography which separates and purifies a target substance from a sample solution containing multiple substances, can be classified into two types: continuous chromatography using a packed column (hereinafter referred to as column chromatography) and batch chromatography. Can be done.
  • Column chromatography is a method in which a sample solution is brought into contact with a carrier packed in a column online, a target substance is captured on the carrier, and then released.
  • An example of using column chromatography to simultaneously separate and purify a target substance from a large number of sample solutions including proteins, nucleic acids, lipids, sugars, viruses, exosomes, etc. Examples include spin columns that use carriers with similar characteristics.
  • Batch chromatography is a method in which a carrier and a sample liquid are mixed in a container, and then the carrier that has captured the target substance and the sample liquid are separated.
  • the carrier can be separated from the sample solution by centrifuging a mixture of the sample solution and the carrier and separating the carrier as a precipitate, or by placing the mixture of the sample solution and carrier on a filter cartridge, centrifuging it, and filtering it.
  • nucleic acid amplification techniques such as the polymerase chain reaction method (PCR method) have made it possible to detect encapsulated nucleic acids even from sample solutions containing minute amounts of viruses and exosomes.
  • PCR method polymerase chain reaction method
  • the accuracy of detecting the nucleic acid by PCR method is not high, and false positives or false negatives are likely to occur. Therefore, in order to solve these problems, a method of increasing the purity of the target substance in the sample liquid is required.
  • spin columns are useful in that they can quickly purify the target substance from a small amount of sample solution and generate less infectious waste, but the process of using it centrifugation is essential. Centrifugation requires an expensive centrifuge, and there is also the risk that sample liquid may leak or scatter due to tube breakage or loosening of the lid during centrifugation, spreading contamination.
  • the sample solution is added to the top of the spin column, and the sample solution passes through the carrier and moves to the bottom of the container by centrifugation, but the target substance in the sample solution comes into contact with the carrier only once.
  • the carrier cannot sufficiently capture the target substance, and as a result, the purified target substance cannot be fully captured. In some cases, the amount and purity were not sufficient.
  • Patent Document 1 The apparatus and method described in Patent Document 1 are useful in that they can extract nucleic acids even from viscous sample liquids, but it is essential to pressurize and depressurize the inside of the container. Therefore, the risk of pathogenic substances leaking out due to pressurization increases.
  • the device not only needs to be equipped with pressure control means, but also the equipment used must be able to withstand pressure control, and the opening must be covered during use.
  • the device and method described in Patent Document 1 are not sufficiently easy to operate, and cannot be used with equipment that does not have a pressurization/depressurization means, so they are not sufficient in terms of compatibility with automated devices.
  • One aspect of the present invention provides a method and a purification kit for producing a purified liquid with increased purity of a substance to be separated from a sample liquid containing a substance to be separated or a substance containing the substance to be separated by a simple operation.
  • a method for producing a purified liquid with increased purity of substance ⁇ from a sample liquid containing a substance to be separated (substance ⁇ ) or a substance containing substance ⁇ (substance ⁇ ), comprising: Using a container (i) having a first chamber having an opening at the top, and a second chamber partitioned from the first chamber by a filter and having an opening at the top, (1) includes the following step 1 and step 2-1, or (2) includes the following step 1 and step 2-2 (provided that the substance ⁇ in the substance ⁇ is a protein, a nucleic acid, a lipid, or a saccharide), Method for producing purified liquid.
  • Step 1 Step of obtaining a container (ii) having a carrier in the first chamber to which the substance ⁇ or substance ⁇ in the sample liquid is adsorbed.
  • Step 2-1 In the container (ii), the substance ⁇ or the substance ⁇ is adsorbed.
  • Step 2-2 The substance ⁇ can be released from the carrier.
  • step 2-1 or step 2-2 is performed by pipetting.
  • step 3 The method for producing a purified liquid according to [1] or [2], wherein the container (ii) is obtained in any of the following steps a to d.
  • Step a After placing a carrier capable of adsorbing substance ⁇ or substance ⁇ into the first chamber, A step of adsorbing the substance ⁇ or the substance ⁇ onto the carrier by introducing the sample liquid into the second chamber and diffusing the substance ⁇ and substance ⁇ in the sample liquid (however, the filter does not absorb the substance ⁇ and substance ⁇ can pass through)
  • Step b After putting a carrier capable of adsorbing substance ⁇ or substance ⁇ into the first chamber, A step of adsorbing the substance ⁇ or the substance ⁇ onto the carrier by introducing the sample liquid into the first chamber and diffusing the substance ⁇ and substance ⁇ in the sample liquid.
  • Step c Putting the sample liquid into the upper part.
  • Step d By mixing the sample liquid and a carrier capable of adsorbing substance ⁇ or substance ⁇ and diffusing substance ⁇ and substance ⁇ in the sample liquid, the substance containing substance ⁇ or substance ⁇ is adsorbed.
  • Step [4] After obtaining the carrier with the substance ⁇ or the substance ⁇ adsorbed into the first chamber, the method of diffusing the substance ⁇ and the substance ⁇ in the sample liquid is to The method for producing a purified liquid according to [3], which is a method of raising and lowering the surface. [5] The method for producing a purified liquid according to [3] or [4], wherein the method of diffusing the substance ⁇ and substance ⁇ in the sample liquid is performed by pipetting operation.
  • [8] A method of diffusing the substance ⁇ and substance ⁇ in the sample liquid, removing the liquid present in the container (ii) after any of the steps a to d, and The method for producing a purified liquid according to [6] or [7], wherein either the step 2-1 or the step 2-2 is performed by a pipetting operation.
  • a method and a purification kit for producing a purified liquid with increased purity of a substance to be separated from a sample liquid containing a substance to be separated or a substance containing the substance to be separated by a simple operation. can do.
  • FIG. 1 shows an amplification curve in quantitative PCR targeting the M gene of influenza A virus in Example 2.
  • FIG. 2 shows an amplification curve in quantitative PCR targeting miR181a in Example 3.
  • FIG. 3 shows an amplification curve in quantitative PCR targeting the M gene of influenza A virus in Example 4.
  • FIG. 4 shows an amplification curve in quantitative PCR targeting the M gene of influenza A virus in Example 5.
  • FIG. 5 shows an amplification curve in quantitative PCR targeting miR181a in Example 6.
  • FIG. 6 shows an amplification curve in quantitative PCR targeting miR181a in Example 7.
  • FIG. 7 shows an amplification curve in quantitative PCR targeting miR181a in Example 8.
  • FIG. 8 relates to Example 9.
  • FIG. 8(a) is a diagram showing an outline of the experiment of Example 9.
  • FIG. 8(b) is a diagram showing the results of detecting CD9 by Western blotting in Example 9.
  • a to B regarding a numerical range means a range of A to B, unless otherwise specified.
  • % means mass %.
  • One aspect of the present invention is to determine the purity of substance ⁇ from a sample solution containing a substance to be separated (hereinafter also referred to as “substance ⁇ ”) or a substance containing the substance to be separated (hereinafter also referred to as “substance ⁇ ”).
  • a method for producing an enriched purified liquid using a container (i) having a first chamber having an opening at the top, and a second chamber partitioned from the first chamber by a filter and having an opening at the top. , a method for producing a purified liquid, including the following steps 1 and 2-1.
  • Step 1 Step of obtaining a container (ii) having a carrier on which substance ⁇ or substance ⁇ in the sample liquid is adsorbed in the first chamber
  • Step 2-2 A liquid capable of releasing substance ⁇ from the carrier
  • This manufacturing method is also referred to as "manufacturing method (X)-2" hereinafter.
  • Manufacturing method (X)-1 and “manufacturing method (X)-2” are collectively referred to as “manufacturing method (X)" hereinafter.
  • the substance ⁇ is a substance produced by the production method (X) and whose purity is increased in the purified liquid.
  • the substance ⁇ and the substance ⁇ contained in the sample liquid may be one type or two or more types.
  • the number of substances ⁇ contained in the purified liquid may be one or two or more.
  • the substance ⁇ is not particularly limited, and includes, for example, proteins, nucleic acids, lipids, sugars, and extracellular substances such as viruses; exosomes, microvesicles, and apoptotic vesicles. Vesicles; examples include DDS (Drug Delivery System) preparations such as liposomes and lipid nano particles (LNPs), which may be labeled with fluorescent dyes, biotin, reporter enzymes, radioactive isotopes, and the like.
  • the substance ⁇ is preferably a nucleic acid, a protein, or an exosome.
  • the protein is not particularly limited, and includes, for example, simple proteins such as albumin, globulin, keratin, collagen, and fibroin; and complex proteins such as glycoprotein, phosphoprotein, chromoprotein, and nuclear protein. Among these, simple proteins are preferred.
  • the protein may be an antibody, a contractile protein, an enzyme, a hormonal protein, a structural protein, a storage protein, a transport protein, preferably an antibody, a transport protein.
  • the lipids are not particularly limited, and include, for example, simple lipids such as glycerides, sterol esters, waxes, and ceramides; complex lipids such as phospholipids and glycolipids; and derived lipids such as fatty acids, terpenoids, steroids, and carotenoids.
  • the sugars are not particularly limited, and include, for example, monosaccharides such as glucose and fructose; disaccharides such as maltose, sucrose, and lactose; polysaccharides such as starch, cellulose, and glycogen; Examples include sugar chains that exist as constituent components of the complex.
  • the virus has a nucleic acid housed inside an encapsulating substance (eg, capsid, envelope) composed of at least one selected from proteins, lipids, and sugars.
  • an encapsulating substance eg, capsid, envelope
  • the viruses include Flaviviridae, which includes dengue virus, hepatitis C virus, and Japanese encephalitis virus; Orthomyxoviridae, which includes influenza virus; and Coronaviridae, which includes SARS coronavirus, SARS coronavirus 2, and MERS coronavirus.
  • the extracellular vesicle contains at least one selected from nucleic acids, proteins, lipids, and sugars inside an encapsulating substance made from at least one selected from proteins, lipids, and sugars.
  • Examples of the extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles, and among these, exosomes are preferred.
  • the liposome contains at least one selected from nucleic acids, proteins, lipids, and sugars inside an encapsulating substance (for example, a lipid bilayer membrane) composed of lipids.
  • the LNP contains nucleic acids, proteins, lipids, and sugars inside an encapsulating substance composed of lipids (e.g., a lipid monolayer membrane or a lipid bilayer membrane composed of ionized lipids, helper lipids, PEG lipids, etc.). At least one selected item is stored.
  • sample liquid is not particularly limited as long as it is a liquid that may contain substance ⁇ or substance ⁇ .
  • sample liquid include biological samples, foods, environmental samples, microorganisms, or cell samples, and liquids obtained by diluting these samples with diluents.
  • the microorganism or cell sample may be, for example, a naturally-derived microorganism or cell, a transformant into which a recombinant vector has been introduced to express a target protein, or a hybridoma in which a plurality of cells are fused. good.
  • the microorganisms or cells also include disrupted liquids and culture supernatants. Examples of the microorganisms or cells include cultured cells such as E. coli, yeast, plant cells, insect cells, and animal cells, and preferably E. coli and animal cells.
  • the amount of the sample liquid used in the manufacturing method (X) is not particularly limited, and may be determined depending on the concentration of the substance ⁇ or substance ⁇ expected to be contained in the sample liquid, the capacity of the container, etc.
  • the amount of sample solution when using a container with a capacity of about 15 mL, is preferably 0.5 mL to 14 mL, more preferably 1 mL to 12 mL, and when using a container with a capacity of about 2 mL, the amount of sample solution is , preferably 100 ⁇ L to 1.5 mL, more preferably 500 ⁇ L to 1.2 mL.
  • the amount of the sample liquid used in "Production method (X)-2" is, for example, when using a container with a capacity of about 50 mL (e.g., a cell culture flask with a culture area of 25 cm 2 ), the amount of the sample liquid is:
  • the volume of the sample solution is preferably 10 mL to 50 mL, more preferably 20 mL to 40 mL, and when using a container with a capacity of about 150 mL (e.g., a cell culture flask with a culture area of 75 cm 2 ), the amount of the sample solution is preferably 30 mL to 150 mL, More preferably 60 mL to 120 mL.
  • the protein concentration of the sample liquid is preferably 0.1 to 10000 ⁇ g/mL, more preferably 1 to 1000 ⁇ g/mL.
  • the virus concentration (titer) of the sample solution is a concentration at which viral nucleic acid can be detected by PCR etc. after concentration, preferably 200 TCID 50 /mL or more, more preferably 2000 TCID 50 /mL. It is mL.
  • the sample solution may contain protein (eg, BSA), preferably 0.1 to 100,000 ⁇ g/mL, more preferably 1 to 10,000 ⁇ g/mL.
  • the exosome concentration of the sample liquid is preferably 0.1 to 10.0 ⁇ g/mL, more preferably 1.0 to 10.0 ⁇ g/mL.
  • the sample solution used in "Production method (X)-2" is a cell culture supernatant
  • the cell culture obtained after culturing cells for 12 hours to 7 days A supernatant is preferred, and a cell culture supernatant obtained after culturing for 24 hours to 5 days is more preferred.
  • the cell culture supernatant may be used after being diluted, for example, 2 to 5 times with the medium used for cell culture.
  • the purified liquid manufactured by the manufacturing method (X) has a reduced amount of impurities and an increased purity of the substance ⁇ compared to the sample liquid, and therefore can be used for various purposes. For example, if the purified liquid is used in a method for detecting trace substances such as PCR, it becomes easier to detect trace substances with higher accuracy.
  • the production method (X) is suitable for increasing the purity of an expensive substance ⁇ having activity such as an antibody
  • the purified liquid can be used in the production of biopharmaceuticals such as antibody drugs, vaccines, and protein preparations. Can be used.
  • the purified liquid produced by production method (X) is a purified liquid in which the purity of substance ⁇ is increased from a sample liquid containing substance ⁇ or substance ⁇ .
  • the increased purity of the substance ⁇ means that the ratio of the substance ⁇ to substances other than the substance ⁇ is increased.
  • a purified liquid can be obtained that has a purity of preferably 400% or more, more preferably 500% or more.
  • the container preferably has an opening at the top for introducing the sample solution or the carrier, and the opening at the top is preferably closable to prevent contamination.
  • the material constituting the container may be selected as appropriate depending on the type of sample liquid, and there are no particular restrictions as long as it has non-adsorption properties for substance ⁇ or substance ⁇ and does not have reactivity for sample liquid.
  • examples include synthetic resins such as polystyrene, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate, polyetherimide, polyimide, ABS resin, polyvinyl chloride, polyvinylidene chloride, fluorine resin; glass; metals such as stainless steel; . These may be used alone or in combination of two or more.
  • the container is preferably a transparent or translucent container. Furthermore, it is preferable that the filter be more transparent than the filter described below.
  • the container is transparent or semi-transparent, it is easy to visually check the position of the pipette tip, how the sample liquid is sucked and discharged, how the sample liquid and carrier are stirred, etc. from the outside of the container. Therefore, it becomes easier to handle the situation with automated equipment.
  • the container having a first chamber having an opening at the top and a second chamber partitioned from the first chamber by a filter and having an opening at the top is also referred to as a "container (i)."
  • a container having a carrier on which the substance ⁇ or ⁇ in the sample liquid is adsorbed in the first chamber is also referred to as “container (ii)”.
  • a container having an opening at the top is also referred to as a "container (iii).”
  • the container (i) has a carrier to which the substance ⁇ or ⁇ in the sample liquid is adsorbed in the first chamber, it becomes the container (ii).
  • the container (iii) has a carrier capable of adsorbing the substance ⁇ or the substance ⁇ therein, and has a first chamber having an opening at the top, and a first chamber partitioned from the first chamber by a filter and having an opening at the top.
  • a second chamber having a second chamber is provided, the container (ii) is obtained.
  • the first chamber is a space in the container (i) that has an opening at the top and has a carrier to which the substance ⁇ or ⁇ in the sample liquid is adsorbed.
  • the shape of the first chamber is not particularly limited as long as it has an opening at the top, but it is preferably a shape that allows the removal process and/or cleaning process described below to be performed.
  • the opening may be at least a part of the upper part of the first chamber, but preferably the entire upper part of the first chamber is open.
  • the shape and size of the opening are not particularly limited either, but it is preferable that the opening has a shape and size that allow insertion of a pipette tip. It is preferable that the opening is located above the maximum height of the liquid level of the sample liquid, and it is preferable that the carrier and the sample liquid do not leak out from such an opening.
  • the volume of the first chamber is not particularly limited as long as it can accommodate therein a carrier to which the substance ⁇ or substance ⁇ in the sample liquid is adsorbed, but it is a volume that allows the removal process and/or washing process described below to be performed. It is preferable that there be.
  • the storage capacity of the carrier is not particularly limited, the volume of the carrier relative to the volume of the first chamber is preferably 1/10 to 500, more preferably 1/50 to 400, and even more preferably 100 to 200. It is 1/1. If it is within the above range, a sufficient space in which the carrier can be dispersed is ensured in the first chamber, so that the substance ⁇ or ⁇ is likely to be adsorbed onto the carrier.
  • the method of storing the carrier on which the substance ⁇ or ⁇ in the sample liquid is adsorbed inside the first chamber is not particularly limited as long as the carrier is stored so as to be immersed in the sample liquid.
  • the second chamber is a space inside the container (i) that is partitioned from the first chamber by a filter that does not allow the carrier to pass through, and has an opening at the top.
  • the filter will be described later.
  • the shape and volume of the second chamber are not particularly limited as long as it has an opening at the top, but it is preferably a shape and volume that allows the removal process and/or cleaning process described below to be performed.
  • the opening may be at least a part of the upper part of the second chamber, but preferably the entire upper part of the second chamber is open.
  • the shape and size of the opening are not particularly limited either, but it is preferable that the opening has a shape and size that allow insertion of a pipette. It is preferable that the opening is located above the maximum height of the liquid level of the sample liquid, and it is preferable that the sample liquid does not leak out from such an opening.
  • the partition between the first chamber and the second chamber only needs to have at least a part of the region made of the filter, a part of the region made of the filter, and the remaining region made of a material other than the filter. However, it is preferable that all regions be composed of the filter.
  • the position of the area constituted by the filter is not particularly limited as long as it is immersed in the sample liquid, and is, for example, the side or lower part of the first chamber.
  • the material other than the filter is not particularly limited as long as it has non-adsorption properties for the substance ⁇ or substance ⁇ and does not have reactivity to the sample liquid, and examples thereof include polystyrene, polycarbonate, polyethylene, polypropylene, and polyethylene.
  • examples include synthetic resins such as terephthalate, polyetherimide, polyimide, ABS resin, polyvinyl chloride, polyvinylidene chloride, and fluorine resin; glass; metals such as stainless steel; These may be used alone or in combination of two or more.
  • the material constituting the spacer is not particularly limited as long as it has non-adsorptive properties for the substance ⁇ or substance ⁇ and does not have reactivity to the sample liquid, but examples thereof include polystyrene, polycarbonate, polyethylene, polypropylene, Examples include synthetic resins such as polyethylene terephthalate, polyetherimide, polyimide, ABS resin, polyvinyl chloride, polyvinylidene chloride, and fluorine resin; glass; and metals such as stainless steel. These may be used alone or in combination of two or more.
  • the manufacturing method (X) can also be applied to an automated device that is equipped with only a pipetting means for suctioning and discharging a solution and a means for moving the pipetting means up and down and back and forth.
  • the number of processes performed by humans is reduced and the number of processes performed by machines is increased, increasing the safety of work.
  • sufficient space can be secured at the pipette tip insertion position by inserting the spacer. , can be compatible with automated equipment.
  • the filter is a partition for separating the first chamber and the second chamber, and is a filter that does not allow the carrier to pass through. Specifically, it is a filter that has holes large enough to not allow the carrier to pass through.
  • the pore size of the filter is not particularly limited as long as it does not allow the carrier to pass through, that is, the pore diameter is smaller than the size of the carrier.
  • the pore diameter of the filter can be appropriately selected in the range of 100 nm or more and less than 2 mm depending on the size of the carrier used, but is preferably 100 nm to 20 ⁇ m.
  • the diameter of the virus is generally about 100 to 200 nm
  • the diameter of the exosome is about 20 to 200 nm
  • the average particle diameter of the carrier is preferably 0.5 ⁇ m to 2 mm. Therefore, the pore diameter of the filter A can be appropriately selected in the range of about 200 nm to 2 mm depending on these diameters, but is preferably 250 nm to 20 ⁇ m.
  • the diameter of the protein is generally about 1 to 100 nm
  • the average particle diameter of the carrier is preferably 0.5 ⁇ m to 2 mm.
  • the filter A further removes impurities in the sample liquid. It is preferable to use a filter that does not allow it to pass through.
  • impurities remain in the second chamber,
  • a removal step is performed to remove the liquid present in the container (ii) after step a or step c, especially in the second chamber, contaminants in the sample liquid are removed during this removal step. Since most of the substances can be removed and the impurities can be sufficiently separated from the carrier on which substance ⁇ or substance ⁇ is adsorbed, a purified liquid with higher purity of substance ⁇ can be easily produced. be able to.
  • steps b and d there are no particular limitations as long as a filter that does not allow the carrier to pass through is used, but the substance ⁇ (e.g., nucleic acid) is removed from the substance ⁇ (e.g., an encapsulating substance containing nucleic acid) adsorbed on the carrier.
  • a filter hereinafter also referred to as "filter B"
  • the filter B is not limited in its pore size, etc., as long as it does not allow the substance ⁇ to pass through and the carrier and the substance ⁇ to pass through.
  • the diameter of the virus is generally about 100 to 200 nm
  • the diameter of the exosome is about 20 to 200 nm
  • the average particle diameter of the carrier is preferably 0.5 ⁇ m to 2 mm. Therefore, the pore diameter of filter B can be appropriately selected in the range of about 1 nm to 2 mm depending on these diameters, but is preferably 1 nm to 200 nm.
  • the material constituting the filter is not particularly limited, but a material to which substance ⁇ and substance ⁇ are difficult to adsorb is preferable.
  • the filter is preferably a hydrophilic filter since the substance ⁇ and substance ⁇ are difficult to adsorb.
  • Examples of such a filter having hydrophilicity include a filter made of a material having hydrophilicity, and a filter made of a material that is not sufficiently hydrophilic and subjected to a hydrophilic treatment.
  • FKM FKM, FFKM
  • pulp, hemp natural materials such as cellulose, kenaf, chitin, chitosan, and cotton
  • inorganic materials such as glass, silica, and metals.
  • fluororesins and fluoroelastomers are more preferable as materials constituting the filter. Further preferred is a filter composed of FKM).
  • the fluororesin is not particularly limited and includes, for example, polytetrafluoroethylene (PTFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), and ethylene.
  • PTFE polytetrafluoroethylene
  • PFA tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer
  • FEP tetrafluoroethylene-hexafluoropropylene copolymer
  • ethylene ethylene
  • ETFE tetrafluoroethylene copolymer
  • EPE tetrafluoroethylene-hexafluoropropylene-perfluoroalkyl vinyl ether copolymer
  • FFEVE fluoroethylene-vinyl ether copolymer
  • PCTFE poly(chlorotrifluoroethylene)
  • ECTFE ethylene-chlorotrifluoroethylene-copolymer
  • PVDF polyvinylidene fluoride
  • PVDF polyvinyl fluoride
  • PVDF-HFP copolymer vinylidene fluoride-hexafluoropropylene copolymer
  • fluoride Examples include vinylidene chloride-hexafluoropropylene-tetrafluoroethylene copolymer (VDF-HFP-TFE copolymer).
  • PTFE and PVDF are preferable because they exhibit the effects of the present invention more effectively and have excellent chemical resistance.
  • the fluoroelastomer is not particularly limited and includes, for example, perfluoro(alkyl vinyl ether), perfluoro(alkoxyalkyl vinyl ether), vinylidene fluoride-hexafluoropropylene polymer, and vinylidene fluoride-hexafluoropropylene-tetrafluoroethylene polymer.
  • Polymer tetrafluoroethylene-propylene polymer, vinylidene fluoride-propylene-tetrafluoroethylene polymer, ethylene-tetrafluoroethylene-perfluoromethyl vinyl ether polymer, vinylidene fluoride-tetrafluoroethylene-perfluoromethyl
  • vinyl ether polymers and vinylidene fluoride-perfluoromethyl vinyl ether polymers.
  • ternary FKM is preferred, and vinylidene fluoride-hexafluoropropylene-tetrafluoroethylene polymer is more preferred, from the standpoint of excellent heat resistance, chemical resistance, etc.
  • the hydrophilic treatment preferably includes a step of coating the filter with a compound having a hydrophilic group (hereinafter also referred to as "hydrophilic compound"), and more preferably includes immersing the filter in a solution of the hydrophilic compound. , comprising coating the filter with a hydrophilic compound and then crosslinking the hydrophilic compound. Specific examples of such steps include steps described in International Publication No. 2014/021167 and Japanese Patent Publication No. 4-075051. In “manufacturing method (X)-2", the hydrophilic treatment may be repeated multiple times.
  • the filter is immersed in a solution of a hydrophilic compound, the filter is coated with a hydrophilic compound, the hydrophilic compound is then crosslinked, and then the filter is further immersed in a solution of a hydrophilic compound that is the same as or different from the hydrophilic compound already used.
  • the filter may be coated with the hydrophilic compound.
  • the hydroxyl group-containing compound is not particularly limited, but includes, for example, polyvinyl alcohol (PVA) and modified products thereof (e.g., ethylene oxide group-modified PVA, carboxyl group-modified PVA, sulfonic acid group-modified PVA, quaternary ammonium-modified PVA); agarose , dextran, chitosan, cellulose, heparin and other polysaccharides and their derivatives; collagen; gelatin; copolymers of vinyl alcohol and vinyl group-containing monomers (e.g.
  • PVA polyvinyl alcohol
  • modified products thereof e.g., ethylene oxide group-modified PVA, carboxyl group-modified PVA, sulfonic acid group-modified PVA, quaternary ammonium-modified PVA
  • agarose dextran, chitosan, cellulose, heparin and other polysaccharides and their derivatives
  • collagen gelatin
  • vinyl alcohol-vinyl acetate copolymers ethylene-vinyl alcohol copolymers
  • vinyl alcohol-polyvinylpyrrolidone copolymer vinyl alcohol-polyvinylpyrrolidone copolymer
  • (meth)acrylic polyol, fluorine-containing polyol, polyoxyalkylene e.g. polyethylene glycol, copolymer of polyethylene glycol and polypropylene glycol [e.g.
  • the carboxylic acid group-containing compound is not particularly limited, but includes, for example, olefin monomers such as ethylene, propylene, and butylene, diene monomers such as butadiene, aromatic group-containing monomers such as styrene, and (meth)acrylic acid ester monomers.
  • olefin monomers such as ethylene, propylene, and butylene
  • diene monomers such as butadiene
  • aromatic group-containing monomers such as styrene
  • (meth)acrylic acid ester monomers ethyrene
  • a copolymer of one or more of these monomers and a monomer having a carboxylic acid group [-COOH] such as (meth)acrylic acid; having a carboxylic acid group such as (meth)acrylic acid examples include homopolymers of monomers; amino acids;
  • the sulfonic acid group-containing compound is not particularly limited, but includes, for example, a copolymer of styrene and acrylamide-2-methylpropanesulfonic acid (salt); styrene, n-butyl acrylate, and acrylamide-2-methylpropanesulfonic acid (salt); and a terpolymer of styrene, 2-ethylhexyl acrylate, and acrylamide-2-methylpropanesulfonic acid (salt).
  • the ether group-containing compound is not particularly limited, but includes, for example, polyethylene glycol and derivatives thereof, fluororesins having an ether group, polyurethane resins having an ether group, and polyphenylene resins having an ether group.
  • the epoxy group-containing compound is not particularly limited, but includes, for example, epoxy resins, modified epoxy resins, (meth)acrylic (co)polymers having epoxy groups, polybutadiene resins having epoxy groups, polyurethane resins having epoxy groups, Examples include adducts or condensates of these resins.
  • the amide group-containing compound is not particularly limited, but includes, for example, poly(N-isopropyl(meth)acrylamide) and poly(N-vinyl-2-pyrrolidone).
  • the weight average molecular weight of the hydrophilic compound is not particularly limited, but is preferably 100 to 1,000,000.
  • the time period for which the filter is immersed in the hydrophilic compound solution is not particularly limited as long as the filter can be coated with the hydrophilic compound, and it depends on the concentration of the hydrophilic compound in the hydrophilic compound solution used, but is preferably is 1 second to 60 minutes, more preferably 10 seconds to 30 minutes.
  • the immersion temperature and atmosphere are not particularly limited and may be appropriately selected depending on the type of hydrophilic compound and the like.
  • the hydrophilic compound when using an aqueous solution as the solution of the hydrophilic compound, even if an untreated filter is immersed in the aqueous solution of the hydrophilic compound, the hydrophilic compound may not be able to penetrate into the inside of the filter. Therefore, before immersing the filter in a solution of a hydrophilic compound, it is preferable to immerse the filter in a water-compatible solvent such as isopropyl alcohol (to impregnate the filter with a water-compatible solvent). .
  • a water-compatible solvent such as isopropyl alcohol
  • the filter may be easily impregnated with the hydrophilic compound by pressing and rubbing, or by applying reduced or increased pressure using a vacuum pressure impregnation device.
  • the water-compatible solvent is not particularly limited, but it is preferable to use a solvent that easily permeates the filter and evaporates easily.
  • a solvent that easily permeates the filter and evaporates easily Specifically, methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl Alcohols such as alcohol, sec-butyl alcohol, tert-butyl alcohol and isobutyl alcohol; Esters such as methyl acetate, ethyl acetate and butyl acetate; Ketones such as acetone and methyl ethyl ketone; Ethers such as tetrahydrofuran and dioxane; dimethyl sulfoxide , N,N-dimethylformamide, and other aprotic polar solvents.
  • the time for immersing the filter in a water-compatible solvent is not particularly limited, but is, for example, 1 minute to 24 hours. Note that the immersion temperature and atmosphere are not particularly limited.
  • the crosslinking agent used in the chemical crosslinking is not particularly limited and may be appropriately selected depending on the type of hydrophilic compound used, but examples include aldehyde compounds such as formaldehyde, glutaraldehyde, and terephthalaldehyde; diacetyl, and chloropentanedione.
  • aldehyde compounds such as formaldehyde, glutaraldehyde, and terephthalaldehyde
  • diacetyl diacetyl, and chloropentanedione.
  • Ketone compounds such as; compounds with reactive halogens such as bis(2-chloroethylurea)-2-hydroxy-4,6-dichloro-1,3,5-triazine; compounds with reactive olefins such as divinylsulfone Compounds; N-methylol compounds; isocyanates; aziridine compounds; carbodiimide compounds; epoxy compounds; halogencarboxaldehydes such as mucochloric acid; dioxane derivatives such as dihydroxydioxane; chromium alum, zirconium sulfate, boric acid, borate, Examples include inorganic crosslinking agents such as phosphates; diazo compounds such as 1,1-bis(diazoacetyl)-2-phenylethane; compounds containing disuccinimidyl ester; and difunctional maleic acid imides. These crosslinking agents may be used alone or in combination of two or more.
  • the crosslinking agent is preferably used in an amount that is in large excess with respect to the amount of the hydrophilic compound in the solution of the hydrophilic compound used.
  • the hydrophilized filter be washed with water or the like for the purpose of removing unreacted hydrophilic compounds. This step may be performed under heating if necessary.
  • the filter is preferably a filter that is transparent or translucent when wetted with the sample liquid.
  • Being transparent or translucent when wetted with a sample solution means, for example, the average transmittance at a wavelength of 400 to 700 nm of the filter immersed in water at 25° C. for 1 minute (hereinafter also referred to as "water immersion average transmittance"). ) is 20% or more. If the filter is transparent or translucent when wetted with sample liquid, it is possible to visually check the position of the pipette tip, how the sample liquid is sucked and discharged, how the sample liquid and carrier are stirred, etc. from outside the container. It's easy to do. Therefore, it becomes easier to handle the situation with automated equipment.
  • filters that are transparent or translucent when wetted with a sample solution include filters made of nonwoven fabric made of ultrafine PTFE fibers and subjected to hydrophilic treatment. This filter is preferable because it not only has excellent transparency but also excellent liquid permeability, so that the sample liquid and the carrier can be easily diffused and mixed in the container.
  • the water immersion average transmittance is measured by the following method.
  • a test piece made of the filter was placed on the inner wall of a 10 mm square measurement cell made of quartz glass, and the cell was filled with pure water.
  • the transmittance (transmittance of light incident perpendicularly to the wall surface of the test piece) was measured at every 10 nm wavelength using an ultraviolet-visible spectrophotometer (UV-2600, manufactured by Shimadzu Corporation).
  • UV-2600 ultraviolet-visible spectrophotometer
  • the average transmittance of the three test pieces at a wavelength of 400 to 700 nm is calculated.
  • Step 1 is a step of obtaining a container (ii) having a carrier in the first chamber to which the substance ⁇ or ⁇ in the sample liquid is adsorbed.
  • the container (i) has a carrier to which the substance ⁇ or ⁇ in the sample liquid is adsorbed in the first chamber, it becomes the container (ii).
  • the carrier is a substance for adsorbing the substance ⁇ or ⁇ in the sample liquid.
  • the carrier has adsorption properties for the substance ⁇ and preferably also non-adsorption properties for impurities.
  • the carrier when producing a purified solution with increased protein purity from a sample solution containing protein, the carrier has adsorption properties for the protein.
  • “adsorptive” means a property that allows adsorption under specific adsorption conditions
  • non-adsorptive means non-adsorptive under specific conditions.
  • carriers having adsorption properties for proteins include clay, agarose, dextran, hydroxyapatite, silica gel, polystyrene, phenol resin, acrylic resin, polyolefin (e.g. polyethylene, polypropylene), polyvinyl chloride, and derivatives thereof.
  • agarose derivatives include Sepharose
  • dextran derivatives include Sephadex
  • hydroxyapatite derivatives include carbonated hydroxyapatite.
  • hydroxyapatite, sepharose, and agarose are preferred, and hydroxyapatite is more preferred.
  • Examples of carriers that have adsorption properties for nucleic acids include hydroxyapatite, silica, magnetic silica beads, cellulose, and agarose or beads immobilized with complementary strands or substances that have structural affinity for nucleic acids.
  • Examples of carriers having adsorption properties for lipids include agarose or beads on which Tim4, which binds to phosphatidylserine, is immobilized.
  • Examples of carriers having adsorption properties for sugars include ion exchange resins, activated carbon, agarose or beads immobilized with lectins and sugar chain-recognizing antibodies, boronic acids and derivatives thereof.
  • the carrier having adsorption properties for exosomes is, for example, agarose or beads immobilized with a substance that has an affinity for molecules expressed on the surface of exosomes, more specifically, Agarose or beads immobilized with a lipid-binding protein such as Tim4 that binds to phosphatidylserine, a lipid-recognizing antibody or a lipid-recognizing aptamer; an antibody or aptamer that recognizes an exosome-specific protein such as a protein belonging to the tetraspanin family such as CD9
  • agarose or beads on which lectin-recognizing molecules are immobilized agarose or beads on which lectin-recognizing molecules are immobilized.
  • the carrier having adsorption properties for viruses is, for example, agarose or beads immobilized with a substance that has an affinity for molecules expressed on the surface of the virus (e.g., the spike protein of the virus).
  • adsorption may be performed using a combination of substances that have specific affinities. That is, the carrier may have one of a combination of substances having specific affinity immobilized thereon via a chemical modification group.
  • Combinations of substances with specific affinity include, for example, antibodies and antigens, antibodies and antibody-binding proteins, GST (glutathione-S-transferase) tags and glutathione, His tags and divalent cations, biotin and avidin. can be mentioned.
  • the carrier When producing a purified liquid with increased purity of the substance ⁇ from a sample solution containing the substance ⁇ , the carrier has adsorption properties for the substance ⁇ , preferably an encapsulated substance, and has adsorption properties for the substance ⁇ . It may have adsorption properties or it may have non-adsorption properties.
  • the carrier when producing a purified solution with increased nucleic acid purity from a sample solution containing a nucleic acid-containing substance, the carrier is preferably a carrier that has adsorption properties to the nucleic acid-containing substance, preferably an encapsulating substance;
  • the carrier may have non-adsorptive properties for nucleic acids, or may have adsorbing properties for nucleic acids.
  • the carrier and substance ⁇ may be By adjusting the conditions for contacting the carrier, the adsorption of the carrier and substance ⁇ can be made higher than that of the carrier and substance ⁇ , or the carrier and substance ⁇ are adsorbed, and the carrier and substance ⁇ are adsorbed. You may choose not to do so.
  • An example of a carrier that has adsorption properties to a substance containing a nucleic acid, preferably an encapsulating substance, more preferably a protein, and has an adsorption property to a nucleic acid is hydroxyapatite.
  • the carrier When producing a purified solution in which the purity of substance ⁇ , which is at least one selected from proteins, lipids, and sugars, is increased from a sample solution containing extracellular vesicles, the carrier is a protein containing proteins constituting the encapsulated substance. It is preferable to have an adsorption property for at least one selected from , lipids, and sugars, and it is more preferable to have an adsorption property for proteins.
  • the carrier may be a carrier that does not adsorb the substance ⁇ , or may have an adsorption property for the substance ⁇ .
  • the carrier when producing a purified solution with increased purity of the virus, extracellular vesicles, or DDS preparation from a sample solution containing the virus, extracellular vesicles, or DDS preparation , the carrier preferably has adsorption properties for at least one selected from proteins, lipids, and sugars constituting the encapsulating substance of the virus, extracellular vesicles, or DDS preparation, and has adsorption properties for lipids. It is more preferable to have adsorption to phospholipids, and it is particularly preferable to have adsorption to phosphatidylserine.
  • agarose or beads immobilized with a substance that has an affinity for phosphatidylserine e.g., a lipid-binding protein such as Tim4 that binds to phosphatidylserine
  • a lipid-binding protein such as Tim4 that binds to phosphatidylserine
  • agarose or beads immobilized with lipid-recognizing antibodies, or lipid-recognizing aptamers can be used as a carrier.
  • agarose or beads immobilized with an antibody or aptamer that recognizes an exosome-specific protein such as a protein belonging to the tetraspanin family such as CD9 can be used as a carrier that has adsorption properties for proteins that constitute the encapsulating substance of exosomes.
  • carriers that have adsorption properties for sugars constituting the encapsulating substance of exosomes include agarose or beads on which lectin-recognizing molecules are immobilized.
  • the carrier When producing a purified solution with increased purity of substance ⁇ , which is at least one selected from nucleic acids, proteins, lipids, and sugars, from a sample solution containing liposomes, the carrier is It is preferable that the material has adsorption properties and non-adsorption properties for the substance ⁇ .
  • Examples of carriers that have non-adsorbing properties for nucleic acids and adsorbing properties for proteins include agarose or beads on which protein-recognizing antibodies are immobilized.
  • Examples of carriers that do not adsorb nucleic acids and adsorb lipids include agarose or beads on which lipid-binding proteins such as Tim4 or lipid-recognizing antibodies are immobilized.
  • Examples of carriers that are non-adsorbent to nucleic acids and adsorbable to sugars include agarose or beads on which lectins and sugar chain-recognizing antibodies are immobilized.
  • Examples of carriers that do not adsorb lipids and adsorb nucleic acids include silica and hydroxyapatite.
  • Examples of carriers that have non-adsorbing properties for lipids and adsorbing properties for proteins include agarose or beads on which antibodies are immobilized, and hydroxyapatite.
  • Examples of carriers that do not adsorb lipids and adsorb sugars include agarose or beads on which lectins and sugar chain-recognizing antibodies are immobilized.
  • Examples of carriers that do not adsorb sugars and adsorb nucleic acids include silica and hydroxyapatite.
  • Examples of carriers that do not adsorb sugars and adsorb proteins include agarose or beads on which antibodies are immobilized, and hydroxyapatite.
  • Examples of carriers that do not adsorb sugars and adsorb lipids include agarose or beads on which lipid-binding proteins such as Tim4 or lipid-recognizing antibodies are immobilized.
  • the above carriers may be used alone or in combination of two or more.
  • the shape of the carrier is not particularly limited, and examples include spherical, particulate, fibrous, rod-like, and plate-like shapes, but spherical and particulate shapes are preferred from the viewpoint of ease of solid-liquid separation and washing.
  • the size of the carrier is not particularly limited as long as it is larger than the pores of the filter, and varies depending on the type of substance ⁇ , but when the carrier is spherical or particulate, the average particle diameter is preferably 0.
  • the diameter is 5 ⁇ m to 2 mm, more preferably 1 ⁇ m to 1.5 mm, even more preferably 1 ⁇ m to 1 mm.
  • the average particle diameter of the carrier is within this range, the carrier and the sample liquid can be easily separated. Note that the average particle diameter can be measured by a light scattering method.
  • the amount of the carrier used in the production method (X) is not particularly limited, and may be appropriately selected depending on the type of carrier, the type of sample liquid, the amount of substance ⁇ and substance ⁇ in the sample liquid, etc. good.
  • Step c After putting the sample liquid into a container (iii) having an opening at the top, the first chamber having a carrier capable of adsorbing substance ⁇ or substance ⁇ and the second chamber are placed in the container (iii). a step in which a substance containing the substance ⁇ or the substance ⁇ is adsorbed onto the carrier by dispersing the substance ⁇ and the substance ⁇ in the sample solution (however, the filter does not contain the substance ⁇ and the substance ⁇ ; is passable)
  • Step d By mixing the sample liquid and a carrier capable of adsorbing substance ⁇ or substance ⁇ and diffusing substance ⁇ and substance ⁇ in the sample liquid, the substance containing substance ⁇ or substance ⁇ is adsorbed. a step of placing the carrier on which the substance ⁇ or substance ⁇ has been adsorbed into the first chamber after obtaining the carrier.
  • Step a the substance ⁇ or the substance ⁇ in the sample liquid placed in the second chamber diffuses in the sample liquid, passes through the filter A, reaches the first chamber, and enters the first chamber. Adsorbs to the carrier in the room. This series of flows is repeated not once but multiple times, so the substance ⁇ or ⁇ is Easy to adsorb onto carriers. Therefore, it is possible to easily produce a purified liquid containing the substance ⁇ with higher purity.
  • step c the sample solution is put into a container (iii) having an opening at the top, and then a carrier capable of adsorbing substance ⁇ or substance ⁇ is provided in the container (iii) and an opening is opened at the top. and a second chamber partitioned from the first chamber by a filter and having an opening at the top.
  • the method of providing the first chamber and the second chamber in the container (iii) is not particularly limited, but for example, the container (iii) may be simply partitioned with one or more of the filters.
  • one chamber in the partitioned container (iii) is designated as the first chamber, and the other part in the container (iii) is designated as the second chamber, and at least one room in the container (iii) is A bag-shaped, cylindrical, etc. member having an opening at the top, the wall of which is composed of the filter, is placed therein, the space inside the bag-shaped, cylindrical, etc. member is defined as a first chamber, and the container (iii)
  • a method (II) in which a space other than the bag-shaped, cylindrical, etc. member inside is used as the second chamber can be mentioned.
  • the method (II) is preferable because the desired first and second chambers can be easily provided.
  • the carrier is placed in the first chamber.
  • a bag-shaped, cylindrical member containing the carrier may be placed in the container (iii), or a bag-shaped, cylindrical member containing the carrier may be placed in the container (iii). After placing a member such as a shape, the carrier may be placed inside the member.
  • step c the substance ⁇ or substance ⁇ in the sample liquid placed in the container (iii) diffuses in the sample liquid and is adsorbed on the carrier in the first chamber.
  • This series of flows is repeated not once but multiple times, so the substance ⁇ or ⁇ is more likely to be adsorbed to the carrier than in conventional methods such as filtration or centrifugation, which have only one opportunity to adsorb to the carrier.
  • Cheap Therefore, it is possible to easily produce a purified liquid containing the substance ⁇ with higher purity.
  • step d the sample solution and the carrier are mixed first, and the substance ⁇ or substance ⁇ can be sufficiently adsorbed to the carrier, so that the substance ⁇ or ⁇ can be adsorbed only once to the carrier, such as filtration or centrifugation.
  • the substance ⁇ or substance ⁇ is more easily adsorbed onto the carrier than in the conventional method according to the method. Therefore, it is possible to easily produce a purified liquid containing the substance ⁇ with higher purity.
  • the substance ⁇ When the substance ⁇ is adsorbed on the carrier, the substance ⁇ can be released from the carrier by changing the adsorption property of the carrier to the substance ⁇ .
  • the liquid that can release the protein from hydroxyapatite is a metal chelating agent such as EDTA with a concentration of 100mM or more, a surfactant such as SDS, Triton Examples include aqueous solutions such as. These may be used alone or in combination of two or more.
  • the sample solution containing the virus, extracellular vesicles, or DDS preparation is separated from the sample solution containing the virus, extracellular vesicles, or DDS preparation in an intact state, and containing the virus, extracellular vesicles, or DDS preparation. , extracellular vesicles, or a purified solution with increased purity of the DDS preparation can be produced.
  • the amount of the cleaning liquid is not particularly limited. Although it may be determined as appropriate depending on the type and amount of impurities, the volume of the cleaning liquid is preferably 20 times or more, more preferably 100 times or more, and still more preferably 200 times or more the volume of the carrier. be.
  • the purification kit (Y) may include the above-mentioned free liquid in addition to a container, a carrier capable of adsorbing substance ⁇ or substance ⁇ , and a filter that does not allow the carrier to pass through.
  • the purification kit (Y) may include the washing liquid in addition to a container, a carrier capable of adsorbing substance ⁇ or substance ⁇ , and a filter that does not allow the carrier to pass through.
  • the purification kit (Y) may also include other reagents or laboratory equipment for sample collection, storage, etc.
  • Another aspect of the present invention is a sample solution containing a substance to be separated (substance ⁇ ) or a substance containing substance ⁇ (substance ⁇ ), which includes any of the following steps a to c. This is a method of adsorbing on a carrier.
  • the first chamber and the second chamber having a carrier capable of adsorbing the substance ⁇ or substance ⁇ are provided in the container (iii), and the sample liquid is placed in the container (iii).
  • Another aspect of the present invention is an object to be separated, which includes any of the following steps a to d and the step of removing the liquid present in the container (ii) after any of the steps a to d.
  • This is a method for separating a substance ⁇ or a substance ⁇ from a sample liquid containing a substance (substance ⁇ ) or a substance containing the substance ⁇ (substance ⁇ ).
  • the PTFE filter bag taken out from the PVA aqueous solution was added to 500 mL of a 5% by mass glutaraldehyde solution (a solution prepared by diluting a 25% glutaraldehyde solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. with pure water to a concentration of 5% by mass). and 5 mL of a 36% aqueous hydrochloric acid solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 30 minutes at room temperature. Next, the PTFE filter bag taken out from the mixed solution was placed in pure water to dissolve unreacted IPA, PVA, and glutaraldehyde.
  • a 5% by mass glutaraldehyde solution a solution prepared by diluting a 25% glutaraldehyde solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. with pure water to a concentration of 5% by mass.
  • a 36% aqueous hydrochloric acid solution manufactured by Fujifilm Wako Pure
  • HT carrier particles Bio-Gel HT Hydroxyapatite manufactured by Bio-Rad, model number: 130-0150
  • PVA hydrophilic
  • Lysis buffer-1 250 mM EDTA aqueous solution, pH 8.0, manufactured by Nippon Gene Co., Ltd., model number: 311-90075 diluted with water
  • the liquid that came out was again added to the inside of the hydrophilic (PVA) treated PTFE filter bag.
  • This operation was repeated five times to release bovine serum albumin labeled with the Cy3 fluorescent dye adsorbed onto the HT carrier particles from the HT carrier particles. Thereafter, the liquid present in the tube was collected from the outside of the hydrophilized (PVA) treated PTFE filter bag, and the resulting solution was designated as Elute-1.
  • bovine serum albumin solution (sample solution containing substance ⁇ ).
  • Example 2 A hydrophilized PVDF porous membrane (manufactured by Merck, Durapore SVLP09050, pore diameter 5 ⁇ m) was molded into a cylinder with an inner diameter of 11 mm and a height of 95 mm (cylindrical shape with a bottom and an opening at the top) using a heat sealer (As One Co., Ltd.).
  • a PVDF filter bag 1 was produced by molding using a PVDF filter bag 1 manufactured by A.
  • a spiral-shaped spacer made of stainless steel (manufactured by DyDo Hunt, number: 30) was inserted inside the PVDF filter bag 1 in order to maintain its shape.
  • the PVDF filter bag 1 containing a spacer was placed inside a tube with a capacity of 14 mL (manufactured by FALCON, model number: 352059), and the upper part of the PVDF filter bag 1 was fixed to the upper part of the tube with a clip. At this time, the PVDF filter bag 1 was fixed so as to have an opening at the top.
  • a first chamber PVDF filter bag 1 with a spacer and an opening at the top
  • a second chamber PVDF filter bag 1 having an opening at the top and having an opening at the top
  • a container having an opening at the top, inside the tube, and outside the PVDF filter bag 1 was obtained.
  • 50 ⁇ L of HT carrier particles Bio-Gel HT Hydroxyapatite manufactured by Bio-Rad, model number: 130-0150 was added into the PVDF filter bag 1 using a micropipette.
  • the cultured influenza A virus (10 9.0 TCID 50 /mL) was diluted 500,000 times with PBS (manufactured by FUJIFILM, model number: 045-29795) to prepare an influenza A virus solution (2000 TCID 50 /mL).
  • this container (tube) and automatic pipetter imitates an automated device that is equipped with only a pipetting means for aspirating and discharging the solution and a means for moving the pipetting means up and down and back and forth. It is.
  • 6.5 mL of the solution was aspirated once at speed 2 and then waited for 2 seconds, then 2 mL of the solution was aspirated once at speed 2 and then waited for 2 seconds.
  • 8.5 mL of the solution was discharged once at speed 3, and then waited for 5 seconds.
  • Lysis buffer (5M guanidine thiocyanate, 100mM EDTA (pH 8.0), 100mM Tris-HCl (pH 6.8), 3% (w/v) Triton X-100, 1% (v/v) 2-mercaptoethanol
  • the results are shown in FIG. 1.
  • the vertical axis of the graph shows ⁇ Rn (intensity of fluorescent signal generated under predetermined PCR conditions), and the horizontal axis shows the number of cycles in quantitative PCR.
  • ⁇ Rn intensity of fluorescent signal generated under predetermined PCR conditions
  • the horizontal axis shows the number of cycles in quantitative PCR.
  • ideal values at the time of concentration are shown.
  • the ideal concentration value is obtained by preparing an influenza A virus solution 10 times more concentrated than Input (i.e., 20,000 TCID 50 /mL), and extracting the nucleic acid containing viral RNA from the solution in the same manner as described above. The calculated solution was used as a sample for PCR.
  • Example 3 Using a heat sealer (As One A PVDF filter bag 2 was produced by molding using PVDF filter bag 2 (manufactured by AS-200).
  • a handmade stainless steel spiral-shaped spacer manufactured by DyDo Hunt, size: 30, diameter: 5 mm, length: 4 cm
  • the PVDF filter bag 2 containing a spacer was placed inside a 2 mL tube (manufactured by Eppendorf, model number: 022431102), and the upper part of the PVDF filter bag 2 was fixed to the upper part of the tube with a clip. At this time, the PVDF filter bag 2 was fixed so as to have an opening at the top.
  • this container (tube) and automatic pipetter imitates an automated device that is equipped with only a pipetting means for aspirating and discharging the solution and a means for moving the pipetting means up and down and back and forth. It is. Human serum-derived exosomes were adsorbed onto the HT carrier particles by suctioning and discharging 0.7 mL of the solution at speed 1 for 1 hour using the programs mode of the automatic pipettor. The subsequent steps of adding and collecting the liquid were performed using a micropipette. After suctioning and discharging the solution for 1 hour, the liquid present in the tube was collected from the outside of the PVDF filter bag 2, and the obtained solution was designated as Through.
  • QIAzol Lysis included in miRNeasy kit 500 ⁇ L of Reagent was added to the inside of PVDF filter bag 2 to release nucleic acids from the HT carrier particles. After that, the liquid present in the tube was collected from the outside of the PVDF filter bag 2, and the process of adding QIAzol Lysis Reagent to the inside of the PVDF filter bag 2 was repeated 5 times to sufficiently release the nucleic acid from the HT carrier particles. I let it happen. Thereafter, the liquid present in the tube was collected from the outside of the PVDF filter bag 2, and a microRNA fraction was obtained according to the miRNeasy kit protocol. The obtained solution was designated as Elute.
  • Example 4 (Preparation of hydrophilic (hydroxyacrylate) treated PTFE filter bag) A nonwoven fabric made of PTFE ultrafine fibers (fabric weight: 16 g/m 2 , 300 mm x 20 mm, average pore diameter 1 ⁇ m) was heated at room temperature (25°C) with a 99.7% isopropyl alcohol (IPA) solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ) for 1 minute.
  • IPA isopropyl alcohol
  • hydroxypropyl methacrylate a mixture of 2-hydroxypropyl ester and 2-hydroxy-1-methylethyl ester
  • HPA hydroxypropyl methacrylate
  • the PTFE nonwoven fabric taken out from the aqueous solution was placed in pure water and boiled at 85° C. for 6 hours to dissolve unreacted IPA, HPA, TEGDA, and AmPS. After boiling, the PTFE nonwoven fabric taken out from the liquid was naturally dried, and the nonwoven fabric was wrapped in a cylindrical shape (with a bottom and an opening at the top) with an inner diameter of 6 mm and a height of 90 mm using double-sided tape (manufactured by Nichiban, Nicetack).
  • a hydrophilic (hydroxy acrylate) treated PTFE filter bag was produced by molding using a strong type (#NW-K10).
  • the PTFE nonwoven fabric sheet taken out from the IPA solution was treated with polyvinyl alcohol (PVA) (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., 160-11485, degree of polymerization: 1500, degree of saponification: 98) adjusted to a concentration of 0.1% by mass. %) aqueous solution for 30 minutes at room temperature.
  • PVA polyvinyl alcohol
  • the PTFE filter taken out from the PVA aqueous solution was added to 500 mL of a 5% by mass glutaraldehyde solution (a solution prepared by diluting a 25% solution of glutaraldehyde manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. with pure water and adjusting the concentration to 5% by mass). and 5 mL of a 36% aqueous hydrochloric acid solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 30 minutes at room temperature.
  • the PTFE nonwoven fabric taken out from the mixed solution was placed in pure water to dissolve unreacted IPA, PVA, and glutaraldehyde. Thereafter, the PTFE nonwoven fabric taken out from the liquid was naturally dried to obtain a hydrophilic (PVA) treated PTFE filter.
  • a 5% by mass glutaraldehyde solution a solution prepared by diluting a 25% solution of glutaraldehyde manufactured by Fujifilm Wako Pure Chemical Industries, Ltd
  • hydrophilic (PVA) treated PTFE filter was immersed in an ethanol solution of 2-methacryloyloxyethylphosphorylcholine (MPC) polymer (manufactured by Intelligent Surface Co., Ltd., MP011 L1) for 30 minutes at room temperature (25°C). I let it happen. Thereafter, the filter taken out from the ethanol solution was air-dried, then washed with pure water and air-dried again.
  • a PTFE filter bag treated with hydrophilicity (PVA-MPC) was prepared by molding it into a cylindrical shape with an opening using double-sided tape (manufactured by Nichiban, Nicetack strong type, #NW-K10).
  • Lysis buffer (5M guanidine thiocyanate, 100mM EDTA (pH 8.0), 100mM Tris-HCl (pH 6.8), 3% (w/v) Triton X-100, 1% (v/v) 2-mercaptoethanol 1 mL was added to the inside of the filter bag, and then the liquid present in the tube was collected from the outside of the filter bag and added to the inside of the filter bag again. This operation was repeated for 5 minutes to absorb the liquid into the HT carrier particles. Influenza A virus was destroyed and nucleic acids containing viral RNA were released from the HT carrier particles.
  • FIG. 3 hydrophilic (hydroxyacrylate) treated PTFE filter bag
  • FIG. 4 hydrophilic (PVA-MPC) treated PTFE filter bag
  • the vertical axis of the graph shows ⁇ Rn (intensity of fluorescent signal generated under predetermined PCR conditions), and the horizontal axis shows the number of cycles in quantitative PCR.
  • ⁇ Rn intensity of fluorescent signal generated under predetermined PCR conditions
  • the horizontal axis shows the number of cycles in quantitative PCR.
  • ideal values at the time of concentration are shown.
  • the ideal concentration value was obtained by preparing an influenza A virus solution 10 times more concentrated than Input (i.e., 20,000 TCID50/mL), and extracting the nucleic acid containing viral RNA from the solution in the same manner as described above. Calculations were made using the solution as a sample for PCR.
  • Example 6 A PTFE nonwoven fabric treated with hydrophilicity (hydroxyacrylate) in the same manner as in Example 4 was wrapped in a cylindrical shape (with a bottom and an opening at the top) with an inner diameter of 16 mm and a height of 40 mm using double-sided tape (manufactured by Nichiban Co., Ltd.).
  • a hydrophilic (hydroxy acrylate) treated PTFE filter bag was produced by molding using Nystack (strong type, #NW-K10). An exosome concentration experiment was conducted using this hydrophilized (hydroxyacrylate) treated PTFE filter bag.
  • a handmade stainless steel spiral-shaped spacer (manufactured by DyDo Hunt, size: 30, diameter: 5 mm, length: 40 mm) was inserted inside the filter bag to maintain its shape.
  • a filter bag containing a spacer was placed inside a 2 mL tube (manufactured by Eppendorf, model number: 022431102), and the top of the filter bag was fixed to the top of the tube with a clip. At this time, the filter bag was fixed so as to have an opening at the top.
  • the first chamber has an opening at the top (a hydrophilic (hydroxyacrylate) treated PTFE filter bag with a spacer and an opening at the top), and the first chamber is partitioned by a hydrophilic (hydroxyacrylate) treated PTFE filter bag.
  • a container (tube) having a second chamber having an opening at the top (a part inside the tube having an opening at the top and outside the hydrophilic (hydroxyacrylate) treated PTFE filter bag) was obtained. . Thereafter, 100 ⁇ L of exosome capture resin (PS Capture Exosome Isolation Resin Kit manufactured by FUJIFILM, #290-80301, average particle diameter of about 34 ⁇ m) was added into the filter bag using a micropipette.
  • exosome capture resin PS Capture Exosome Isolation Resin Kit manufactured by FUJIFILM, #290-80301, average particle diameter of about 34 ⁇ m
  • Freeze-dried exosomes derived from human urine from a healthy individual were dissolved in water to prepare a 1 ⁇ g/ ⁇ L exosome solution.
  • This exosomine solution is 0.1 % (V / v) catfisher albumin (BSA) / PBS solution, Binding Encer (FUJIFILM PS Capture EXOSOME ISOLATION RESIN KIT, # 290 -The diluted with 80301) and 5 ⁇ g / ml (4)
  • An exosome solution of .7 ⁇ 10 9 cells/mL) was prepared. Since this exosome solution contains bovine serum albumin as a contaminant protein, it imitates biological samples (eg, blood, urine, etc.) that contain not only exosomes but also contaminant proteins.
  • the results are shown in FIG.
  • the vertical axis of the graph indicates ⁇ Rn (intensity of fluorescent signal generated under predetermined PCR conditions) and the number of cycles in quantitative PCR.
  • ⁇ Rn intensity of fluorescent signal generated under predetermined PCR conditions
  • ideal values at the time of concentration are shown.
  • the ideal concentration value is to prepare an exosome solution that is 10 times more concentrated than Input (i.e., 50 ⁇ g/mL), extract the microRNA fraction from the solution in the same manner as described above, and use the resulting solution as a sample for PCR. It was calculated using
  • Example 7, 8 The same concentration experiment as in Example 6 was conducted using the same hydrophilic (PVA-MPC) treated PTFE filter bag as in Example 5, except that the amount of Binding Enhancer added was increased by 1 and 10 times.
  • Example 7 is a case in which the amount of Binding Enhancer is increased by 1 times
  • Example 8 is a case in which the amount of Binding Enhancer is increased by 10 times.
  • the results are shown in FIG. 6 (Example 7) and FIG. 7 (Example 8).
  • the amount of Binding Enhancer added As the amount of Binding Enhancer added increased, the amount of concentrated exosomes increased, and quantitative PCR showed an amplification curve closer to the ideal value.
  • Example 9 Using Capan2, a pancreatic cancer-derived cultured cell, as an exosome-supplying cell, an experiment was conducted to concentrate exosomes from cell culture supernatant. It has long been known that the culture supernatant of adipocytes contains exosomes, and adipocytes are widely used as exosome-supplying cells, but this time we investigated cultured cells derived from pancreatic cancer. A certain Capan2 was used as an exosome source cell. An outline of the experiment is shown in FIG. 8(a).
  • Fetal Bovine Serum used for normal cell culture contains bovine-derived exosomes.
  • Capan2 cells were incubated with serum-free medium (D-MEM medium (manufactured by FUJIFILM, #044-29765), 1% Penicillin-Streptomycin ( The cells were cultured using Thermo Fisher Scientific (manufactured by Gibco, #15140122)).
  • the culture supernatant was removed from a flask of Capan2 cells grown to confluence in a serum-containing medium, followed by a cell washing operation (after adding 10 mL of serum-free medium to the flask and washing the cells, immediately The operation of removing the medium) was performed 5 times. Subsequently, 10 mL of serum-free medium was added to the flask and cultured at 37° C. and 5% CO 2 concentration for 48 hours.
  • the prepared spiral-shaped spacer was inserted.
  • a micropipette add 60 ⁇ L of exosome capture resin (PS Capture Exosome Isolation Resin Kit, #290-80301, manufactured by FUJIFILM) into a hydrophilic (PVA-MPC) treated PTFE filter bag with a spacer, and insert it into the bottom end of the filter bag. It was fixed in the flask with a clip so that 1 cm was submerged in the medium.
  • exosome capture resin PS Capture Exosome Isolation Resin Kit, #290-80301, manufactured by FUJIFILM
  • the filter bag was taken out from the flask and placed inside a 14 mL tube (manufactured by FALCON, model number: 352059), and the top of the filter bag was fixed to the top of the tube with a clip. At this time, the filter bag was fixed so as to have an opening at the top.
  • the subsequent steps of adding and collecting the liquid were performed using a 1 mL measuring pipette (manufactured by FALCON, model number: 4485).
  • Exosomes concentrated from the culture supernatant of Capan2 cells were confirmed by Western blotting of CD9, which is an exosome marker.
  • 15 ⁇ L of the eluate was mixed with a sample buffer containing no reducing agent (manufactured by FUJIFILM, #198-13282), treated at 100° C. for 5 minutes, and developed on SDS-PAGE.
  • 15 ⁇ L of an unconcentrated Input sample was subjected to the same treatment.
  • SuperSep (TM) Ace, 10-20% (manufactured by FUJIFILM, #191-15031) was used as the gel for SDS-PAGE.
  • the developed sample was transferred from the gel to a PVDF membrane (Trans-Blot Turbo Mini 0.2 ⁇ m PVDF Transfer Packs, manufactured by Bio-Rad, #1704156), and PVDF Blocking Reagent for PVDF was applied to the PVDF membrane.
  • Can Get Blocking was performed at room temperature for 1 hour using Signal (manufactured by TOYOBO, #NYPBR01).
  • the detection antibodies were anti-Human CD9 (COSMO BIO, #SHI-EXO-M01) as the primary antibody, anti-Mouse IgG-HRP (GE, #NA931) as the secondary antibody, and Can Get Signal Solution (manufactured by TOYOBO).

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