Device for purifying and concentrating liquid containing minute useful substance, and method for producing purified and concentrated liquid of minute useful substance using said device

Abstract

Provided is a purification and concentration device for a liquid containing a minute useful substance, said purification and concentration device being capable of producing a liquid containing a minute useful substance at a high purity and a high concentration (a purified concentrated solution of a minute useful substance), and being capable of more efficiently producing the purified concentrated solution. And a method for producing a purified concentrate of fine useful substances using the device. The purification and concentration device for the liquid containing the tiny useful substances comprises a raw material supply part for supplying the liquid containing the tiny useful substances, a removal part for removing impurities in the liquid, and a purification and concentration part for purifying and concentrating the liquid supplied from the raw material supply part or the removal part by using a hollow fiber membrane, and a first control unit that controls the timing at which the liquid flows from the raw material supply unit to the purification and concentration unit or the liquid flows from the removal unit to the purification and concentration unit.

Description

Technical Field

[0001] The present disclosure relates to a device for purifying and concentrating a liquid containing minute useful substances, and a method for producing a purified concentrated liquid of minute useful substances using the device. Background Art

[0002] As a method for separating and purifying minute useful substances such as extracellular vesicles, antibodies, viruses, proteins, and nucleic acids from a liquid containing the minute useful substances, for example, a method using a separation membrane is known.

[0003] Patent document 1 describes an invention of a method for recovering useful substances, which includes: an effusion step of draining the culture fluid from a cell culture tank and adding a fresh culture medium equal to the drained culture fluid to the culture tank; and a filtration step of filtering the culture fluid extracted from the culture tank using a porous membrane that does not substantially have a dense layer, wherein the filtration in the filtration step is tangential flow filtration, and the permeate velocity in the filtration step is 1.0 LMH or less. Patent document 1 describes that the above-mentioned tiny useful substances can be selected from proteins, viruses, exosomes, and nucleic acids.

[0004] In addition, Patent Document 2 describes an exosome extraction device and an exosome extraction method that can efficiently and massively collect exosomes, which are a type of extracellular vesicles. The exosome extraction device described in Patent Document 2 delivers a raw material liquid containing exosomes supplied from a raw material supply unit to a first storage unit, and then circulates the raw material liquid from the first storage unit through a circulation path including a first filter and a second filter having different functions, thereby concentrating the exosomes in the liquid.

[0005] Patent Document 1: Japanese Patent Application Publication No. 2018-76291

[0006] Patent Document 2: Japanese Patent Application Publication No. 2021-145649 Summary of the invention

[0007] Usually, the raw material liquid containing tiny useful substances often contains impurities. Therefore, a purification and concentration device capable of preparing a liquid containing tiny useful substances at high purity and high concentration (purified concentrated liquid of tiny useful substances) is required. In addition, a purification and concentration device capable of preparing the purified concentrated liquid more efficiently is also required.

[0008] The present invention has been completed in view of the above situation, and its purpose is to provide a purification and concentration device for liquid containing tiny useful substances that can produce a liquid containing tiny useful substances with high purity and high concentration (purified concentrated liquid of tiny useful substances) and can more efficiently prepare the above-mentioned purified concentrated liquid, as well as a method for producing a purified concentrated liquid of tiny useful substances using the device.

[0009] In order to solve the above-mentioned problems, the present disclosure can adopt the following aspects.

[0010] [1] A purification and concentration device for a liquid containing minute amounts of useful substances, comprising: a raw material supply unit for supplying the liquid containing minute amounts of useful substances, a removal unit for removing impurities in the liquid, a purification and concentration unit for purifying and concentrating the liquid supplied from the raw material supply unit or the removal unit using a hollow fiber membrane, and a first control unit for controlling the timing of the flow of the liquid from the raw material supply unit to the purification and concentration unit or the flow of the liquid from the removal unit to the purification and concentration unit.

[0011] [2] The purification and concentrating device according to [1], wherein the removal section includes a carrier column for removing impurities in the liquid, and the carrier column is connected to a flow path for supplying the liquid after the impurities are removed to the purification and concentrating section.

[0012] [3] The purification and concentrating device according to [1] or [2], wherein the removal section includes a filter element for removing impurities in the liquid, and the filter element is inserted in a flow path that supplies the liquid from the raw material supply section to the removal section.

[0013] [4] A purification and concentrating device according to [2] or [3], wherein the removal section comprises a first tank for storing the liquid processed by the carrier column, the first tank is arranged in a flow path for supplying the liquid from the carrier column to the purification and concentrating section, and is connected to a flow path for supplying the liquid in the tank to the purification and concentrating section, and the first control section controls the timing for supplying the liquid in the tank to the purification and concentrating section.

[0014] [5] The purification and concentration device according to any one of [1] to [4], wherein the average inner diameter of the hollow fiber membrane is 0.2 to 1.4 mm and the molecular weight cutoff is 10,000 to 1,000,000.

[0015] [6] A purification and concentrating device according to any one of [1] to [5], wherein the purification and concentrating section comprises a hollow fiber membrane assembly in which a plurality of the hollow fiber membranes are accommodated in a container having an inlet and outlet for the liquid, and the container has at least an inlet and outlet for a first liquid and an inlet and outlet for a second liquid for supplying the liquid to the hollow fiber membranes, and an inlet and outlet for a third liquid for discharging the liquid after passing through the hollow fiber membranes.

[0016] [7] The purification and concentration device according to [6], wherein the hollow fiber membrane module is of an internal pressure type.

[0017] [8] A purification and concentrating device according to [6] or [7], wherein the purification and concentrating section comprises: a second tank storing the liquid supplied from the raw material supply section or the removal section, and connected to a flow path for supplying the liquid to the inlet and outlet of the first liquid of the hollow fiber membrane module; a third tank storing the liquid supplied from the raw material supply section or the removal section, and connected to a flow path for supplying the liquid to the inlet and outlet of the second liquid of the hollow fiber membrane module; a pressurizing device for selectively pressurizing either the second tank or the third tank; and a second control section for controlling the liquid in the second tank and the third tank to be alternately supplied to the hollow fiber membrane module by selectively pressurizing the second tank or the third tank through the pressurizing device.

[0018] [9] A purification and concentration device according to [8], wherein the second control unit alternately pressurizes the second tank and the third tank through the pressurizing device in such a manner as to set the liquid volume in the second tank and the third tank so that the liquid volume in the second tank and the third tank reaches the set value.

[0019]

[10] The purification and concentrating device according to [9], wherein the second tank and the third tank are respectively provided with a tank liquid amount monitoring device including a light sensor at the inlet and outlet of the liquid at the bottom of the tank, and the tank liquid amount monitoring device is configured to irradiate light into the inlet and outlet of the liquid through the light sensor and detect the transmittance of the light, and monitor the liquid amount in the tank based on the change of the transmittance, and the second control unit determines the liquid amount in the second tank and the third tank through the tank liquid amount monitoring device and determines the tank to be pressurized by the pressurizing device.

[0020]

[11] A purification and concentrating device according to [9] or

[10] , wherein the purification and concentrating section includes a camera for monitoring the liquid level positions in the second tank and the third tank, and the second control section determines the tank to be pressurized by the pressurizing device based on information on the liquid level positions in the second tank and the third tank output from the camera.

[0021]

[12] A purification and concentrating device according to any one of [8] to

[11] , wherein the purification and concentrating section comprises: a fourth tank storing a diluent, connected to a flow path for supplying the diluent to the inlet and outlet of the third liquid of the hollow fiber membrane module; and a third control unit controlling the timing of supplying the diluent in the fourth tank to the inlet and outlet of the third liquid of the hollow fiber membrane module, wherein when the total amount of liquid in the second tank and the third tank reaches or falls below the value set by the second control unit, the third control unit supplies the diluent in the fourth tank from the inlet and outlet of the third liquid of the hollow fiber membrane module.

[0022]

[13] A purification and concentrating device according to any one of [8] to

[12] , wherein the purification and concentrating section comprises: a fifth tank connected to the inlet and outlet of the first liquid or the inlet and outlet of the second liquid of the hollow fiber membrane module through a flow path to recover the liquid in the second tank and the third tank; and a fourth control section that controls the recovery of the liquid to the fifth tank.

[0023]

[14] The purification and concentration device according to

[13] , wherein the second tank and the third tank are respectively provided with a tank liquid amount monitoring device including a light sensor at the inlet and outlet of the liquid at the bottom of the tank, and the tank liquid amount monitoring device is constructed in such a manner that the light sensor irradiates the inlet and outlet of the liquid with light and detects the transmittance of the light, and monitors the liquid amount in the tank based on the change of the transmittance, and the fourth control unit selectively pressurizes the second tank or the third tank through the pressurizing device, recovers the liquid in the second tank and the third tank to the fifth tank, determines the liquid amount in the second tank and the third tank through the tank liquid amount monitoring device, and stops the pressurizing device.

[0024]

[15] A purification and concentrating device according to

[13] or

[14] , wherein the purification and concentrating section includes a camera for monitoring the liquid level positions in the second tank and the third tank, the fourth control section selectively pressurizes the second tank or the third tank through the pressurizing device, recovers the liquid in the second tank and the third tank to the fifth tank, and stops the pressurizing device based on the information on the liquid level positions in the second tank and the third tank output from the camera.

[0025]

[16] According to any one of [1] to

[15] , the purification and concentrating device is configured such that the elements of the raw material supply section, the removal section and the purification and concentrating section are arranged in a cover, and has a management section for managing the cleanliness of the cover, and the management section has a fan filter device for supplying filtered air to the cover.

[0026]

[17] The purification and concentration device according to

[16] , wherein the management unit further includes a UV lamp.

[0027]

[18] The purification and concentration device according to any one of [2] to

[17] , wherein the removal section comprises: a sixth tank storing a cleaning solution for cleaning the carrier column; and a seventh tank storing an equilibration solution for equilibrating the carrier column.

[0028]

[19] A purification and concentration device according to any one of [3] to

[18] , wherein the filter element is arranged at a lower portion of the raw material supply section, and the liquid is supplied from the raw material supply section to the filter element by the liquid's own weight.

[0029]

[20] The purification and concentrating device according to any one of [8] to

[19] , wherein the second tank and the third tank are arranged such that the side surfaces in the longitudinal direction are parallel to the longitudinal direction of the hollow fiber membrane module.

[0030]

[21] According to the purification and concentration device described in

[20] , the removal section comprises: a carrier column, which removes impurities in the liquid and is connected to a flow path for supplying the liquid after the impurities are removed to the purification and concentration section; and a first tank, which stores the liquid treated by the carrier column, the second tank and the third tank are connected to the carrier column and the first tank via a flow path, and are arranged above the carrier column and the first tank.

[0031]

[22] The purification and concentration device according to any one of [1] to

[21] , wherein the useful microscopic substance is selected from the group consisting of extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.

[0032]

[23] A method for producing a purified concentrated liquid of a tiny useful substance, which is a method for producing the above-mentioned liquid obtained by purifying and concentrating the tiny useful substance using the purification and concentrating device described in any one of [1] to

[22] , the method comprising: supplying a raw liquid containing the above-mentioned tiny useful substance to the above-mentioned purification and concentrating device, and removing impurities from the above-mentioned liquid; and / or, purifying and concentrating the above-mentioned liquid by a hollow fiber membrane.

[0033]

[24] The method for producing a purified concentrated liquid of minute useful substances according to

[23] , wherein the step of removing impurities from the liquid comprises: removing the impurities from the liquid by adsorbing them through a carrier column.

[0034]

[25] A method for producing a purified concentrated liquid of tiny useful substances according to

[23] or

[24] , wherein the step of purifying and concentrating the liquid through a hollow fiber membrane comprises: performing alternating tangential flow filtration on the liquid through a hollow fiber membrane module.

[0035]

[26] A method for producing a purified concentrated liquid of tiny useful substances according to any one of

[23] to

[25] , wherein the step of purifying and concentrating the liquid through the hollow fiber membrane comprises: recovering the tiny useful substances deposited on the surface of the hollow fiber membrane while performing alternating tangential flow filtration on the liquid through a hollow fiber membrane assembly.

[0036]

[27] A method for producing a purified concentrated liquid of a tiny useful substance according to any one of

[23] to

[26] , wherein the step of purifying and concentrating the liquid by means of the hollow fiber membrane comprises: supplying a diluent to the concentrated liquid for further purification and concentration, and supplying the diluent to the liquid when the amount (B) of the concentrated liquid relative to the amount (A) of the raw liquid (B / A) reaches 1 / 2 to 1 / 200.

[0037]

[28] A method for producing a purified concentrated liquid of a minute useful substance according to any one of

[24] to

[27] , wherein the step of removing impurities in the liquid by the carrier column and the step of purifying and concentrating the liquid by the hollow fiber membrane are performed in parallel.

[0038]

[29] A method for producing a purified concentrated solution of a tiny useful substance according to any one of

[23] to

[28] , wherein the tiny useful substance is at least one selected from an extracellular vesicle, an antibody, a virus, a protein, an enzyme, and a nucleic acid.

[0039] [30-1] A purification and concentration device for a liquid containing minute useful substances, comprising: a raw material supply section for supplying the liquid containing minute useful substances, and the purification and concentration section for purifying and concentrating the liquid supplied from the raw material supply section through a hollow fiber membrane assembly, the hollow fiber membrane assembly having a first liquid inlet and a second liquid inlet for supplying the liquid to the hollow fiber membrane, and a third liquid inlet and outlet for discharging the liquid after passing through the hollow fiber membrane, the purification and concentration section comprising: a second tank for storing the liquid supplied from the raw material supply section, the flow path for supplying the liquid to the first liquid inlet and outlet of the hollow fiber membrane assembly; a third tank for storing the liquid supplied from the raw material supply section, the flow path for supplying the liquid to the first liquid inlet and outlet of the hollow fiber membrane assembly; The flow path of the inlet and outlet of the above-mentioned second liquid of the hollow fiber membrane component; a pressurizing device, which selectively pressurizes any tank of the above-mentioned second tank and the above-mentioned third tank; a second control unit, which controls in a manner of selectively pressurizing the second tank or the above-mentioned third tank through the above-mentioned pressurizing device and alternately supplying the above-mentioned liquid in the above-mentioned second tank and the above-mentioned third tank to the above-mentioned hollow fiber membrane component; and a monitoring device, which detects the amount of the above-mentioned liquid in the above-mentioned second tank and the above-mentioned third tank, the above-mentioned second control unit alternately pressurizes the above-mentioned second tank and the above-mentioned third tank through the above-mentioned pressurizing device in a manner of setting the liquid amount in the above-mentioned second tank and the above-mentioned third tank so that the liquid amount in the above-mentioned second tank and the above-mentioned third tank reaches a set value, and the above-mentioned liquid amount in the above-mentioned second tank and the above-mentioned third tank is determined based on the information of the above-mentioned monitoring device.

[0040] [30-2] A device for purifying and concentrating a liquid containing minute amounts of useful substances, comprising:

[0041] A raw material supply unit for supplying a liquid containing a tiny amount of useful substances, and

[0042] a purification and concentration section for purifying and concentrating the liquid supplied from the raw material supply section by means of a hollow fiber membrane assembly, the hollow fiber membrane assembly comprising: a first liquid inlet and outlet and a second liquid inlet and outlet for supplying the liquid to the hollow fiber membrane, and a third liquid inlet and outlet for discharging the liquid after passing through the hollow fiber membrane, the purification and concentration section comprising: a second tank for storing the liquid supplied from the raw material supply section, the flow path of which is connected to the first liquid inlet and outlet for supplying the liquid to the hollow fiber membrane assembly; a third tank for storing the liquid supplied from the raw material supply section, the flow path of which is connected to the first liquid inlet and outlet for supplying the liquid to the hollow fiber membrane assembly a flow path for the inlet and outlet of two liquids; a liquid feeding device, which selectively feeds any of the above-mentioned liquids in the second tank and the third tank to the above-mentioned hollow fiber membrane module; a second control unit, which controls the above-mentioned liquid feeding device in a manner of selectively supplying the above-mentioned liquids in the second tank and the third tank to the above-mentioned hollow fiber membrane module; and a monitoring device, which monitors the amount of the above-mentioned liquid in the second tank and the third tank, the above-mentioned second control unit sets the liquid amount in the above-mentioned second tank and the third tank, and when the liquid amount in the above-mentioned second tank and the third tank reaches the set value, the above-mentioned liquid feeding device is controlled in a manner of selectively feeding the liquid from the second tank or the third tank to the above-mentioned hollow fiber membrane module,

[0043] The liquid amounts in the second tank and the third tank are determined based on information from the monitoring device.

[0044]

[31] The purification and concentration device according to [30-1] or [30-2], wherein the average inner diameter of the hollow fiber membrane is 0.2 to 2.0 mm and the molecular weight cutoff is 5,000 to 3,000,000.

[0045]

[32] The purification and concentration device according to any one of [30-1] to

[31] , wherein the monitoring device includes a camera for monitoring the liquid level positions in the second tank and the third tank.

[0046]

[33] A purification and concentration device according to any one of [30-1] to

[32] , wherein the monitoring device comprises a light sensor, which is respectively arranged at the inlet and outlet of the liquid at the bottom of the second tank and the third tank, irradiates light into the inlet and outlet of the liquid and detects the transmittance of the light, and monitors the amount of the liquid in the tank based on the change of the transmittance.

[0047]

[34] The purification and concentration device according to any one of [30-1] to

[33] , wherein the hollow fiber membrane module is of an internal pressure type.

[0048]

[35] A purification and concentrating device according to any one of [30-1] to

[34] , wherein the purification and concentrating section comprises: a fourth tank storing a diluent, connected to a flow path for supplying the diluent to the inlet and outlet of the third liquid of the hollow fiber membrane module; and a third control unit for controlling the timing of supplying the diluent in the fourth tank to the inlet and outlet of the third liquid of the hollow fiber membrane module, wherein when the total amount of the liquid in the second tank and the third tank reaches or falls below the value set by the first control unit, the third control unit supplies the diluent in the fourth tank from the inlet and outlet of the third liquid of the hollow fiber membrane module.

[0049]

[36] A purification and concentrating device according to any one of [30-1] to

[35] , wherein the purification and concentrating section comprises: a fifth tank, which is connected to the inlet and outlet of the first liquid or the inlet and outlet of the second liquid of the hollow fiber membrane module through a flow path to recover the liquid in the second tank and the third tank; and a fourth control section, which controls the recovery of the liquid to the fifth tank.

[0050]

[37] According to the purification and concentration device described in

[36] , the fourth control unit selectively pressurizes the second tank or the third tank through the pressurizing device to recover the liquid in the second tank and the third tank to the fifth tank, and determines the amount of the liquid in the second tank and the third tank through the monitoring device to stop the pressurizing device.

[0051]

[38] A purification and concentration device according to any one of [30-1] to

[37] , wherein the above-mentioned tiny useful substances are selected from electroactive polymers, rare metal colloidal particles, pigments, dyes, inorganic or organic nanoparticles, proteins, hormones, cytokines, growth factors, angiogenic factors, growth factors, enzymes, antibodies, plasma proteins, viruses, extracellular vesicles, yeast, yeast, cells, polysaccharides and microalgae.

[0052]

[39] The purification and concentration device according to any one of [1] to

[38] , wherein biomolecules of a different type from the microscopic useful substance are attached to at least a portion of the hollow fiber membrane.

[0053]

[40] A purification and concentration device according to any one of

[12] to

[22] or

[35] to

[39] , wherein the hollow fiber membrane module has at least two inlets and outlets for a third liquid, and the third control unit supplies the diluent in the fourth tank from one of the at least two inlets and outlets for the third liquid of the hollow fiber membrane module while stopping the flow of the liquid through the inlet and outlet for the first liquid and the inlet and outlet for the second liquid of the hollow fiber membrane module, and then supplies the diluent in the fourth tank from at least one of the at least two inlets and outlets for the third liquid of the hollow fiber membrane module while making the inlet and outlet for the first liquid and the inlet and outlet for the second liquid in a state where the flow of the liquid can be carried out.

[0054]

[41] A method for producing a purified concentrated liquid of a minute useful substance, which comprises removing impurities from a liquid of a raw material containing the minute useful substance and impurities, and then purifying and concentrating the liquid to produce a purified concentrated liquid of the minute useful substance, wherein the step of purifying and concentrating the liquid comprises: purifying and concentrating the liquid using a hollow fiber membrane module, and comprising: during the purification and concentration process, pressurizing a diluent from the permeate side of the hollow fiber membrane module to perform a reverse cleaning treatment on the hollow fiber membrane.

[0055]

[42] The method for producing a purified concentrated solution of minute useful substances according to

[41] , wherein the hollow fiber membrane contained in the hollow fiber membrane module is composed of a resin material containing cellulose acetate.

[0056]

[43] The method for producing a purified concentrated solution of minute useful substances according to

[42] , wherein the molecular weight cutoff of the hollow fiber membrane contained in the hollow fiber membrane module is 100,000 to 1,000,000.

[0057]

[44] A method for producing a purified concentrated solution of a minute useful substance according to any one of

[41] to

[43] , wherein the step of removing impurities from the liquid comprises: removing the impurities from the liquid by adsorption through a carrier column.

[0058]

[45] A method for producing a purified concentrated liquid of minute useful substances according to any one of

[41] to

[44] , wherein the step of purifying and concentrating the liquid using the hollow fiber membrane module comprises: performing alternating tangential flow filtration on the liquid.

[0059]

[46] A method for producing a purified concentrated liquid of a tiny useful substance according to any one of

[41] to

[45] , wherein the step of purifying and concentrating the liquid using the hollow fiber membrane assembly comprises: supplying a diluent to the purified and concentrated liquid and further purifying and concentrating the liquid, and supplying the diluent to the liquid when the amount (B) of the purified and concentrated liquid relative to the amount (A) of the raw material liquid (B / A) reaches 1 / 2 to 1 / 200.

[0060]

[47] A method for producing a purified concentrated liquid of a tiny useful substance according to any one of

[44] to

[46] , wherein a step of removing impurities in the liquid by means of the carrier column and a step of purifying and concentrating the liquid using the hollow fiber membrane module are performed in parallel.

[0061]

[48] ​​A method for producing a purified concentrated solution of a tiny useful substance according to any one of

[41] to

[47] , wherein the tiny useful substance is at least one selected from an extracellular vesicle, an antibody, a virus, a protein, an enzyme, and a nucleic acid.

[0062]

[49] The method for producing a purified concentrated solution of a minute useful substance according to any one of

[41] to

[48] , wherein a biomolecule of a different type from that of the minute useful substance is attached to at least a portion of the hollow fiber membrane.

[0063]

[50] The method for producing a purified concentrated solution of minute useful substances according to any one of

[41] to

[49] , comprising: flushing the hollow fiber membrane module before performing the backwashing treatment.

[0064] According to the present disclosure, a purification and concentration device for liquid containing microscopic useful substances (purified concentrated liquid of microscopic useful substances) can be provided, which can produce a liquid containing microscopic useful substances at high purity and high concentration and can more efficiently produce the purified concentrated liquid, and a method for producing a purified concentrated liquid of microscopic useful substances using the device. In addition, the purification and concentration device of the present disclosure is also useful as a device capable of purifying and concentrating microscopic useful substances by automatic control. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a schematic configuration diagram showing an example of the purification and concentrating device according to the first embodiment.

[0066] Figure 2 This is a schematic configuration diagram showing an example of a removal portion according to the first embodiment.

[0067] Figure 3 This is a schematic configuration diagram showing an example of a removal portion according to the first embodiment.

[0068] Figure 4 This is a schematic configuration diagram showing an example of a removal portion according to the first embodiment.

[0069] Figure 5 This is a schematic configuration diagram showing an example of a removal portion according to the first embodiment.

[0070] Figure 6 This is a schematic configuration diagram showing an example of a removal portion according to the first embodiment.

[0071] Figure 7 This is a schematic cross-sectional view showing an example of a hollow fiber membrane module in the purification and concentrating device of the present disclosure.

[0072] Figure 8 This is a schematic configuration diagram showing an example of the purification and concentration section of the first embodiment.

[0073] Fig. 9 This is a schematic configuration diagram showing an example of the purification and concentration section of the first embodiment.

[0074] Fig.10 This is a schematic configuration diagram showing an example of the purification and concentration section of the first embodiment.

[0075] Fig.11 This is a schematic configuration diagram showing an example of the purification and concentration section according to the first embodiment.

[0076] Fig.12 This is a schematic configuration diagram showing an example of the purification and concentrating device according to the first embodiment.

[0077] Fig.13 This is a schematic configuration diagram showing an example of the overall configuration of the purification and concentrating device according to the first embodiment.

[0078] Fig.14 This is a schematic configuration diagram showing an example of the overall configuration of a purification and concentrating device according to the second embodiment.

[0079] Fig.15 This is an example of an apparatus for measuring the γ-globulin permeability of a hollow fiber membrane.

[0080] Fig.16 This is a flowchart showing an example of purification and concentration processing using the purification and concentration device according to the first embodiment.

[0081] Fig.17 This is a graph showing the results of measuring the concentrated solutions obtained in Example 4 and Comparative Example 3 by BCA assay.

[0082] Fig.18 It is the absorbance curve graph of the concentrated solution obtained in Example 4 and Comparative Example 3.

[0083] Explanation of symbols

[0084] 1, 2 purification and concentration device, 10 raw material supply unit, 11 container, 20 removal unit, 21 carrier column, 22 filtering element, 23 first tank (crude purified liquid storage tank), 24 tank (pretreatment liquid storage tank), 25 sixth tank (cleaning liquid storage tank), 26 seventh tank (equilibrium liquid storage tank), 30 purification and concentration unit, 31 second tank, 32 third tank, 33 hollow fiber membrane module, 33a inlet and outlet of first liquid, 33b inlet and outlet of second liquid, 33c, 33d inlet and outlet of third liquid, 34 pressurizing device, 35, 36 tank liquid amount monitoring device, 41-43 flow path, 80 fourth tank (dilution liquid storage tank), 81 fifth tank (recovery tank), 91 fan filter device, 92 UV lamp, 110 first control unit DETAILED DESCRIPTION

[0085] Hereinafter, an example of the present disclosure is described using the accompanying drawings. Each configuration in each embodiment and their combination are examples, and the addition, omission, replacement and other changes of the configuration can be appropriately performed without departing from the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the claims. In addition, for a specific parameter, when multiple upper limits and lower limits are recorded, any upper limit and lower limit can be combined among these upper limits and lower limits to set a preferred numerical range.

[0086] [Device for purifying and concentrating liquid containing minute useful substances]

[0087] The present disclosure relates to a purification and concentration device for a liquid containing minute useful substances. The purification and concentration device of the present disclosure has a first embodiment and a second embodiment. The first embodiment of the purification and concentration device of the present disclosure is described below using the drawings.

[0088] [First embodiment]

[0089] Figure 11 is a schematic diagram showing a purification and concentrating device 1 (hereinafter referred to as "purification and concentrating device 1") of an example of the first embodiment of the present disclosure. The purification and concentrating device 1 of the present disclosure includes: a raw material supply unit 10 for supplying a raw material containing a minute useful substance, a removal unit 20 for removing impurities in a liquid, a purification and concentrating unit 30 for purifying and concentrating the liquid supplied from the raw material supply unit 10 or the removal unit 20 using a hollow fiber membrane, and a first control unit 110 for controlling the timing of the flow of the liquid from the raw material supply unit 10 to the purification and concentrating unit 30 or the flow of the liquid from the removal unit 20 to the purification and concentrating unit 30. The liquid of the raw material supply unit 10 is supplied to the removal unit 20 via a flow path 41, or supplied to the purification and concentrating unit 30 via a flow path 43. In addition, the liquid of the removal unit 20 is supplied to the purification and concentrating unit 30 via a flow path 42. The purification and concentrating device 1 of the present disclosure can produce a liquid containing minute useful substances at a high purity and high concentration (purified concentrated liquid of minute useful substances), and can more efficiently prepare the above-mentioned purified concentrated liquid. In addition, it is also useful as an apparatus capable of purifying and concentrating minute useful substances by automatic control. It should be noted that in the present disclosure, "flow path" refers to a pipeline where a liquid flows.

[0090] <Raw Material Supply Department>

[0091] The raw material supply unit 10 stores a raw material liquid (hereinafter referred to as "raw material liquid") containing a tiny amount of useful substances. The raw material liquid in the raw material supply unit 10 is supplied to the removal unit 20 or the purification and concentration unit 30 according to its purity, that is, the concentration of impurities in the raw material liquid. When the concentration of impurities in the raw material liquid is high, the raw material liquid can be supplied to the removal unit 20, and when the concentration of impurities in the raw material liquid is low, the raw material liquid can be supplied to the purification and concentration unit 30. The raw material supply unit 10 is connected to a flow path 41 for supplying the raw material liquid to the removal unit 20 and a flow path 43 for supplying the raw material liquid to the purification and concentration unit 30.

[0092] The raw material supply unit 10 has a container for storing the raw material liquid. The container may be fixed to the purification and concentration device 1, or may be provided in a manner that allows easy removal. When the container is provided in a manner that allows easy removal, the raw material supply unit 10 may include various devices such as a fixing member for fixing the container.

[0093] The shape and volume of the container for storing the raw material solution are not particularly limited and can be determined according to the type of the micro-useful substance, the amount of the raw material solution, etc. In one embodiment, when purifying and concentrating a raw material solution containing micro-useful substances selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, the volume of the above-mentioned container can be 50 to 5000 mL, or 200 to 2000 mL.

[0094] <Removal section>

[0095] The removal unit 20 removes impurities in the raw material liquid supplied from the raw material supply unit 10 via the flow path 41. According to this configuration, a purified concentrated liquid of minute useful substances can be efficiently prepared. Hereinafter, the liquid from which impurities have been removed by the removal unit 20 is referred to as a "raw purified liquid."

[0096] Figures 2 to 6 Some representative schematic configuration examples of the removal portion 20 are shown.

[0097] Figure 2 The removal section 20 shown is provided with a carrier column 21 for removing impurities in the raw liquid. One end of the carrier column 21 is connected to a flow path 41, and the other end is connected to a flow path 42 for supplying the crude purified liquid to the purification concentration section 30. The flow path 42 from the carrier column 21 can be connected to a flow path (described later) for discharging the liquid to a waste liquid tank (described later).

[0098] The removal unit 20 may also be provided with a device having a container filled with an adsorbent material capable of adsorbing and removing impurities in the raw material liquid instead of the carrier column 21. In these containers, impurities in the raw material liquid can be adsorbed by the adsorbent material through stirring, vibration / oscillation, etc. to remove / reduce impurities. As such a device, a rotator may also be used. The filler adsorbed with impurities can be removed by any filtering unit.

[0099] Figure 3 The removal unit 20 shown in the figure is provided with a carrier column 21 and a first tank 23 for storing a crude purified liquid processed by the carrier column 21. The first tank 23 is arranged in a flow path 42. That is, the flow path 42 is divided into a flow path 42A located on the carrier column 21 side and a flow path 42B located on the purification and concentration section side, sandwiching the first tank 23. The crude purified liquid is stored in the first tank 23 via the flow path 42A from the carrier column 21, and the crude purified liquid stored in the first tank 23 is supplied to the purification and concentration section 30 via the flow path 42B. The flow path 42B is provided with a three-way valve 77. The first control unit 110 opens the three-way valve 77 in the flow path 42B to the purification and concentration section 30 side according to the processing status of the crude purified liquid in the purification and concentration section 30, and supplies the crude purified liquid in the first tank 23 to the purification and concentration section 30. According to this configuration, the crude purified liquid can be temporarily stored in the first tank 23 and then supplied to the purification and concentrating section 30, so that the impurity removal process of the removal section 20 and the purification and concentrating process of the purification and concentrating section 30 can be performed in parallel. As a result, the purification and concentrating process can be performed efficiently. Figures 4 to 6 The structure of the removal portion 20 shown will be described later.

[0100] (Carrier column)

[0101] The support column 21 is filled with a material capable of removing impurities other than minute useful substances in the raw material liquid. The material is preferably an adsorbent material capable of adsorbing and removing impurities in the raw material liquid.

[0102] As the adsorbent, a porous granular material that can adsorb and retain the above-mentioned impurities is preferred. The porous granular material refers to a granular material having a large number of pores on its surface and inside, and can be a porous granular material of a polymer or a porous granular material of an inorganic material. When the tiny useful substances contained in the raw material liquid treated in the purification and concentration device 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, as the above-mentioned porous granular material, it is preferred that the pores inside the pores are positively charged.

[0103] These porous granular materials may be used alone or in combination of a plurality of porous granular materials having different adsorption properties.

[0104] Examples of various porous granules with different adsorption properties include: porous granules with different pore diameters, porous granules with different specific surface areas, porous granules with different average particle sizes, porous granules with different types of substrates constituting the porous granules, porous granules with different types of functional groups existing on the internal surface of the pores of the porous granules, porous granules with different densities, etc.

[0105] As such porous granular material, for example, porous granular materials using polysaccharides such as cellulose, agarose, starch, amylose, dextran, pullulan, glucomannan, polyacrylic acid or its derivatives, polyvinyl alcohol, nylon, polysulfone, polyacrylonitrile, polyethylene, polypropylene, polystyrene, synthetic polymers, glass, porous glass, silica gel, hydroxyapatite, etc. as substrates can be listed. These can be used alone or in combination with more than two kinds. In addition, it can be a mixed mode carrier with ion exchange and size exclusion, preferably a mixed mode carrier with cation exchange and size exclusion.

[0106] The porous granular material can be selected from porous granular materials having an average pore diameter in the range of 0.1 to 1000 nm, depending on the types of impurities contained in the raw material solution, etc. The average pore diameter can be measured by mercury porosimetry or reverse size exclusion chromatography.

[0107] The porous granular material can be selected from porous granular materials having an average particle size in the range of 1 to 1000 μm, depending on the type of impurities contained in the raw material liquid, etc. The average particle size can be measured by a laser diffraction scattering method.

[0108] In one embodiment, when the tiny useful substances contained in the raw material liquid treated in the purification and concentration device 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids, the carrier column 21 may include an antibody column. "Antibody column" refers to a column filled with a carrier fixed with antibodies that can bind to antigens (impurities). In the case where the raw material liquid contains the above-mentioned biological substances, an antibody column filled with a carrier fixed with antibodies that can bind (adsorb) to impurities other than the above-mentioned biological substances can be used. It should be noted that in the raw material liquid containing the above-mentioned biological molecules as tiny useful substances, since it contains multiple impurities in many cases, multiple antibody columns corresponding to the types of impurities can be used.

[0109] In addition, a device (column assembly) that combines multiple carrier columns into a module can also be used to replace the carrier column 21. When using a column assembly, the types of adsorbent materials contained in each carrier column can be the same or different. In addition, the inner diameter and length of the carrier column can be the same or different.

[0110] Figure 4 The removal unit 20 shown in the figure includes: a sixth tank 25 (cleaning liquid storage tank) storing a cleaning liquid for cleaning the carrier column 21, and a seventh tank 26 (equilibrating liquid storage tank) storing an equilibration liquid for equilibrating the carrier column 21. Figure 4 In the removal section 20, the sixth tank 25 is configured to supply the cleaning liquid to the flow path 41 via the flow path 50A. In addition, the seventh tank 26 is configured to supply the equilibration liquid to the flow path 41 via the flow path 50B. The flow paths 50A and 50B are provided with switch valves 71A and 71B. The flow path 42 for supplying the crude purified liquid from the carrier column 21 to the purification and concentration section 30 is provided with a three-way valve 72.

[0111] In one embodiment, it is preferred to wash and balance the carrier column 21 that has been treated with a batch of raw material liquid to reach adsorption saturation, and then further treat the next batch of raw material liquid. The washing and balancing treatment of the carrier column 21 can be implemented by opening the switch valves 71A and 71B provided in the flow paths 50A and 50B, and supplying the washing liquid and the balancing liquid from the sixth tank 25 and the seventh tank 26 to the carrier column 21. It is preferably configured in such a way that the washing liquid and the balancing liquid after the treatment of the carrier column 21 are discharged from the three-way valve 72 provided in the flow path 42 to the waste liquid tank (described later).

[0112] (Cleaning solution and balancing solution)

[0113] As a cleaning liquid for cleaning the carrier column 21, for example, sodium chloride aqueous solution, hydrochloric acid, citric acid, sodium hypochlorite aqueous solution, sodium hydroxide aqueous solution, etc. can be used. From the viewpoint of safety, sodium chloride aqueous solution is preferably used. When the cleaning liquid flows into the carrier column 21, impurities attached to the adsorbent material in the carrier column 21 are removed. After removal with the cleaning liquid, the carrier column 21 is equilibrated. As the equilibration liquid, for example, physiological saline, phosphate buffer (PBS), Tris hydrochloric acid buffer, sodium citrate buffer, citrate phosphate buffer, acetate buffer, boric acid buffer, etc. can be used.

[0114] It should be noted that the material of the sixth tank 25 and the seventh tank 26 for storing the cleaning solution and the balancing solution is not particularly limited, and for example, the sixth tank 25 and the seventh tank 26 may be the same as the second tank 31 and the third tank 32 described later. From the viewpoint of making it easy to visually check the amount of the stored solution in the tank, the sixth tank 25 and the seventh tank 26 are preferably transparent.

[0115] Figure 5 The removal unit 20 shown has a filter member for removing impurities in the liquid before supplying the liquid to the carrier column 21 . Figure 5 The removal section 20 shown is a structure in which a filter element 22 is inserted in a flow path 41. The raw material liquid that has been filtered by the filter element 22 is supplied to the carrier column 21 from the flow path 41A located on the side of the carrier column 21 through the filter element 22 in the flow path 41. According to this structure, the raw material liquid that has been pre-treated by the filter element 22 can be supplied to the carrier column 21. Hereinafter, the raw material liquid that has been treated by the filter element 22 will be described as "pre-treatment liquid". It should be noted that Figure 5 The removal portion 20 in FIG. 1 is configured to include the filter member 22 and the carrier column 21 , but may be configured to include only the filter member 22 .

[0116] Figure 6 The removal unit 20 shown is provided with a tank 24 (pretreatment liquid storage tank) for storing the pretreatment liquid from the filter element 22. The pretreatment liquid from the filter element 22 is stored in the tank 24 via the flow path 41C. The pretreatment liquid stored in the tank 24 is supplied to the carrier column 21 via the flow path 41D. The first control unit 110 (not shown) can open the switch valve 73 in the flow path 41D to supply the pretreatment liquid in the tank 24 to the carrier column 21 according to the progress of the impurity removal treatment of the carrier column 21. According to this configuration, the pretreatment liquid can be temporarily stored in the tank 24 and then supplied to the carrier column 21, so that the filtration treatment of the filter element 22 and the impurity removal treatment of the carrier column 21 can be performed in parallel, and the purification and concentration treatment can be effectively performed, which is preferred.

[0117] exist Figure 6In the embodiment, the filter element 22 is arranged at the lower part of the raw material supply part 10. According to this structure, the raw material liquid is supplied from the raw material supply part 10 to the filter element 22 by the dead weight of the raw material liquid, so the liquid supply member (pump, etc.) for supplying the raw material liquid from the raw material supply part 10 to the filter element 22 can be omitted. Therefore, a more compact purification and concentration device can be made, which is preferred.

[0118] (Filter component)

[0119] The filter component 22 is a component for filtering the raw material liquid. In the case where the tiny useful substances contained in the raw material liquid include extracellular vesicles, the filter component 22 is preferably a filter having a precision filtration membrane (precision filtration membrane assembly). From the viewpoint of effectively filtering and removing impurities such as biological molecules larger than the target extracellular vesicles such as cells in the raw material liquid, the average pore size of the precision filtration membrane (precision filtration membrane assembly) is preferably 0.10 to 0.50 μm, and more preferably 0.15 to 0.30 μm. In addition, as a material for the precision filtration membrane, polyethersulfone, polyvinylidene fluoride, etc. are preferred. In addition, commercially available products composed of these materials can be used for precision filtration membranes.

[0120] (First Control Unit)

[0121] Next, use Fig.16 The flowchart of will explain the details of the first control unit.

[0122] Fig.16 is used Fig.13 The basic flow chart of the purification and concentration device 1 shown in FIG. 1 shows a case where the purification and concentration process is performed. Fig.16 An example of the flow control of the liquid in each control unit is shown in the flowchart.

[0123] The first control unit 110 supplies the raw material liquid to the removal unit 20 or the purification and concentration unit 30 based on the result set according to the impurity concentration of the raw material liquid in the raw material supply unit 10. When the impurity concentration of the raw material liquid is high, the raw material liquid in the raw material supply unit 10 is set to be supplied to the removal unit 20. In this case, the first control unit 110 sets the Fig.13 The three-way valve 82 at the bottom of the container 11 is opened to the flow path 41 side, and the raw material liquid is supplied to the removal part 20 ( Fig.13In the figure, the filter element 22 is the filter element 22). When the impurity concentration of the raw material liquid is low, the raw material liquid in the raw material supply part 10 is set to be supplied to the purification and concentration part 30. In this case, the first control part 110 can open the above-mentioned three-way valve 82 to one side of the flow path 43 to send the raw material liquid to the purification and concentration part 30. The liquid (pretreatment liquid) treated by the filter element 22 is supplied to the tank 24. The first control part 110 can supply the liquid from the tank 24 to the carrier column 21 until the amount of the crude purified liquid in the first tank 23 (crude purified liquid storage tank) reaches the set value. Specifically, the switch valve 73 arranged at the lower part of the tank 24 can be opened to supply the pretreatment liquid to the carrier column 21.

[0124] When the amount of the crude purified liquid in the first tank 23 reaches a preset value, it is sent to the second tank 31. At this time, the first control unit 110 can also confirm the liquid amount in the second tank 31 and the third tank 32. When the first control unit 110 determines that the liquid amount in the second tank 31 and the third tank 32 is the preset liquid amount based on the liquid level monitoring using the tank liquid amount monitoring device 35, 36 and / or the camera described later, the three-way valve 77 connected to the flow path 42B can be opened to the second tank 31 side to supply the crude purified liquid in the first tank 23 to the second tank 31.

[0125] Connect to Fig.13 The flow path 42B of the first tank 23 is provided with a level sensor (not shown) for judging the amount of liquid in the tank. The first control unit 110 can judge whether the crude purified liquid remains in the first tank 23 based on the information of the level sensor. When it is judged that the crude purified liquid in the first tank 23 is all supplied to the second tank 31, the first control unit 110 can open the switch valve 73 to supply the pretreatment liquid in the tank 24 to the carrier column 21 again. By controlling the flow of the liquid in this way, the carrier column 21 can be cleaned and balanced at the end of each batch of processing.

[0126] <Purification and Concentration Department>

[0127] The purification and concentration section 30 purifies and concentrates the liquid supplied from the raw material supply section 10 or the removal section 20 by means of a hollow fiber membrane. Since the purification and concentration are performed using the hollow fiber membrane, a liquid containing a small amount of useful substances at a high purity and a high concentration can be prepared.

[0128] (Hollow Fiber Membrane)

[0129] The material of the hollow fiber membrane of the present disclosure can be appropriately designed according to the purpose of use of the present disclosure, the properties of the liquid containing minute useful substances, and the like.

[0130] Specifically, organic polymer materials such as polyvinylidene fluoride (PVDF), polyethersulfone (PES), polysulfone (PSf), polyolefins (PE, PP, PTFE, etc.), polyamide, polyacrylonitrile, cellulose acetate (CA), and their modified products can be used.

[0131] Furthermore, a composite hollow fiber membrane formed of a plurality of components selected from the above may also be used.

[0132] Among them, in the case where the tiny useful substance is a biological molecule such as an exosome, it is preferred to use a hollow fiber membrane made of cellulose acetate (CA) (i.e., preferably composed of a resin material containing cellulose acetate). By using a hollow fiber membrane made of cellulose acetate, the reduction in filtration speed during purification and concentration treatment can be suppressed, and the recovery effect of tiny useful substances deposited on the membrane surface of the hollow fiber membrane can be further improved. As reasons for this, the following reasons can be cited: the surface charge of the biomolecule is a weak negative charge, and the cellulose acetate membrane is also a weak negative charge. Therefore, the biomolecule and the cellulose acetate membrane will not attract each other with a strong Coulomb force, and the shear force of the tangential flow filtration can be easily made to function by appropriately controlling the circulation linear velocity. In addition, by using a cellulose acetate membrane, the tiny useful substances deposited on the membrane surface of the hollow fiber membrane can be more effectively recovered using the method described later.

[0133] The material of the hollow fiber membrane disclosed in the present invention can be appropriately designed according to the purpose of use of the present invention, the properties of the raw material liquid containing tiny useful substances, etc. On this basis, the surface of the hollow fiber membrane can also be coated with appropriate substances in order to improve the dirt resistance of the surface of the hollow fiber membrane according to the properties of the raw material liquid containing tiny useful substances.

[0134] The coating treatment can be performed, for example, by the same method as the method described in Japanese Patent No. 6081290 for bonding a cationic polymer to the surface of a hollow fiber membrane composed of sulfonated polyethersulfone and polyethersulfone.

[0135] That is, the following methods can be used: a method of immersing the hollow fiber membrane in an aqueous solution (aqueous dispersion) of a coating material; a method of passing an aqueous solution (aqueous dispersion) of a coating material through the hollow fiber membrane in the state of the hollow fiber membrane or a hollow fiber membrane assembly so that the coating material is adsorbed and bonded to the surface of the hollow fiber membrane, and then, the coating material deposited only on a thin layer of the coating material is removed by washing with water.

[0136] When concentrating and purifying a certain extracellular vesicle in a certain culture supernatant, it is sometimes preferable to coat a cellulose acetate hollow fiber membrane with a certain biomolecule from the viewpoint of improving the purification and concentration efficiency and the recovery rate of useful microscopic substances such as extracellular vesicles.

[0137] As an example, the following method can be used: a 1.3 mg / mL skim milk powder solution dissolved in phosphate buffer (PBS) is passed through a cellulose acetate hollow fiber membrane module (molecular weight cutoff 300,000, membrane area 0.062 m2) at a flow rate of 200 mL / min. 2 ) was passed through the hollow fiber membrane for 10 minutes to apply a coating treatment for allowing biomolecules such as casein in the skimmed milk powder to be adsorbed and bound to the surface of the hollow fiber membrane in the form of a thin layer. Then, in order to remove only the excess components deposited on the thin layer, the membrane was washed with 250 mL of phosphate buffered saline (PBS).

[0138] In one embodiment, from the viewpoint of improving the fouling resistance of the hollow fiber membrane, it is preferred that biomolecules of a different type from the above-mentioned tiny useful substance are attached to at least a portion of the hollow fiber membrane. The biomolecules are more preferably attached to an area of ​​more than half of the surface of the hollow fiber membrane, and are preferably attached to the entire area. In one embodiment, it is preferred that a layer containing biomolecules of a different type from the above-mentioned tiny useful substance is formed on at least a portion of the surface of the hollow fiber membrane. "Layer containing biomolecules" means that a substance containing biomolecules is attached to at least a portion of the hollow fiber membrane in a diffused manner. The layer containing biomolecules is preferably formed in an area of ​​more than half of the surface of the hollow fiber membrane, and is more preferably formed in the entire area. In one embodiment, it is preferred that at least a portion of the surface of the hollow fiber membrane is coated with biomolecules of a different type from the above-mentioned tiny useful substance.

[0139] As biological molecules of a type different from the tiny useful substances, there are biological molecules obtained from animal cell tissues such as blood, emulsions, cartilage, and other biological cell tissues. Representative examples include: biological molecules contained in skim milk, skim milk powder, etc. (for example, casein); biological molecules contained in serum, etc. (for example, serum globulin, serum albumin); gelatin (for example, fish gelatin), etc., and one or more of them can be selected from them.

[0140] (Average inner diameter of hollow fiber membrane)

[0141] The average inner diameter of the hollow fiber membrane disclosed in the present invention can be appropriately designed according to the purpose of use of the present invention, the properties of the liquid containing tiny useful substances, etc. In one embodiment, a hollow fiber membrane with an average inner diameter of 0.2mm to 1.4mm can be used. In another embodiment, a hollow fiber membrane with an average inner diameter of 0.2mm to 2.0mm can be used. It should be noted that the "average inner diameter of the hollow fiber membrane" in the present disclosure is a value calculated based on the diameter of the smallest circle inscribed in the hollow portion of a cut surface when a hollow fiber membrane is cut at an arbitrary surface perpendicular to the length direction. In the present disclosure, the above diameter is measured at any 10 or more locations and no more than 100 locations in the above-mentioned cut surface, and the average value thereof is taken as the "average inner diameter".

[0142] It should be noted that the average outer diameter of the hollow fiber membrane of the present disclosure may be 0.3 mm to 2.0 mm. In one embodiment, the average outer diameter of the hollow fiber membrane may be 0.3 mm to 1.4 mm when the average inner diameter is 0.2 mm to 1.0 mm. In addition, in other embodiments, the average outer diameter of the hollow fiber membrane may be 1.5 mm to 2.0 mm when the average inner diameter is 1.0 mm to 1.4 mm. It should be noted that the "average outer diameter of the hollow fiber membrane" is a value calculated based on the diameter of the smallest circle containing the outer edge of the cut surface of the hollow fiber membrane cut by the same method as the average inner diameter of the above-mentioned hollow fiber membrane. In the present disclosure, the above-mentioned diameter is measured at any 10 or more locations and no more than 100 locations in the above-mentioned cut surface, and the average value thereof is taken as the "average outer diameter".

[0143] For the purpose of use of the present disclosure, the average inner diameter of the hollow fiber membrane may be 0.6 mm to 1.4 mm from the viewpoint of easily ensuring a membrane thickness that does not make the hollow fiber membrane too soft and easily improving the recovery efficiency of minute useful substances deposited on the membrane surface of the hollow fiber membrane.

[0144] In addition, the average inner diameter can be adjusted according to the viscosity of the liquid containing the microscopic useful substance during operation. -1 When the liquid viscosity is 10 to 30 mPa·s, it is preferred to use a large-diameter hollow fiber membrane with an average inner diameter of 1.0 mm to 1.4 mm. In addition, when the liquid viscosity during operation of a liquid containing tiny useful substances is low (for example, a liquid temperature of 20°C and a shear rate of 25 s -1When the viscosity of the liquid is less than 10mPa·s, it is preferred to use a hollow fiber membrane with an average inner diameter of 0.6mm to 1.0mm. It should be noted that when the average inner diameter of the hollow fiber membrane increases, the effective membrane area tends to decrease when the above hollow fiber membrane is used as a hollow fiber membrane module. Therefore, from the viewpoint of easily suppressing the decrease in the effective membrane area when a hollow fiber membrane module is made, it is preferred to use a hollow fiber membrane with an average inner diameter of 0.2mm to 1.0mm.

[0145] (Molecular weight cut-off of hollow fiber membrane)

[0146] The molecular weight cutoff of the hollow fiber membrane of the present invention can be appropriately designed according to the purpose of use of the present invention, the properties of the raw material solution containing the tiny useful substance, etc. In one embodiment, the above-mentioned molecular weight cutoff can be 5,000 to 3,000,000, or 10,000 to 1,000,000. In the case where the tiny useful substance is a specific protein, it is preferred to use a hollow fiber membrane with a molecular weight cutoff slightly lower than the molecular weight of the above-mentioned protein (for example, a hollow fiber membrane having a molecular weight cutoff of about 10 to 50% lower than the molecular weight of the protein, etc.). In one embodiment, when the tiny useful substance is an exosome, the molecular weight cutoff of the hollow fiber membrane is preferably 50,000 to 1,000,000, more preferably 70,000 to 800,000, and further preferably 100,000 to 500,000.

[0147] In the case where the tiny useful substance is an extracellular vesicle, from the viewpoint of the efficiency of purification and concentration, the γ-globulin permeability of the hollow fiber membrane assembly disclosed in the present invention is preferably 5 to 90%, more preferably 10 to 80%. In the present disclosure, the method for measuring the permeability of impurities in the liquid containing the above-mentioned γ-globulin containing tiny useful substances is not particularly limited, and existing known methods can be used. Specifically, a method for measuring the permeability of a standard substance having a molecular structure similar to the above-mentioned impurities and having the same degree of molecular weight can be used. In the case of measuring the above-mentioned permeability, a hollow fiber membrane assembly is sometimes used, or sometimes a hollow fiber membrane used in the above-mentioned hollow fiber membrane assembly is directly used. In addition, the above-mentioned permeability of the internal pressure type hollow fiber membrane assembly can also be replaced by the permeability obtained by measuring the method of applying pressure from the outside of the hollow fiber membrane to the inside with a hollow fiber membrane.

[0148] (Hollow fiber membrane module)

[0149] The purification and concentration section 30 in the present disclosure may be a structure having a hollow fiber membrane assembly, wherein the hollow fiber membrane assembly contains a plurality of the above-mentioned hollow fiber membranes in a container having an inlet and outlet for a liquid. In addition, the above-mentioned container at least has: an inlet and outlet 33a for supplying the above-mentioned hollow fiber membrane to the first liquid and an inlet and outlet 33b for the second liquid, and an inlet and outlet 33c, 33d for discharging the third liquid after passing through the above-mentioned hollow fiber membrane.

[0150] The number of hollow fiber membranes used in the hollow fiber membrane module of the present disclosure is not particularly limited, and can be appropriately designed according to the purpose of use of the present disclosure and the weight of the tiny useful substances to be purified and concentrated, so as to obtain the required effective membrane area.

[0151] The hollow fiber membrane is preferably bundled and contained in the above-mentioned container by an adhesive or the like. In the present disclosure, when a liquid containing a tiny useful substance is supplied from the inlet and outlet 33a of the first liquid and / or the inlet and outlet 33b of the second liquid of the hollow fiber membrane module, the liquid flows into one side of the hollow fiber membrane bundle in the container (the inside or outside of the hollow fiber membrane bundle) and is purified and concentrated in the hollow fiber membrane bundle. The liquid that has passed through the hollow fiber membrane bundle flows out to the other side of the hollow fiber membrane bundle (the outside or inside of the hollow fiber membrane bundle) and is discharged from the inlet and outlet 33c, 33d of the third liquid. At this time, the liquid that has not passed through and is retained on the inside or outside of the hollow fiber membrane bundle can be discharged (recovered) from the inlet and outlet of the first liquid and / or the inlet and outlet of the second liquid to the outside of the hollow fiber membrane module. Hereinafter, in the present disclosure, the liquid that has passed through the hollow fiber membrane is recorded as "permeated liquid", and the liquid that has not passed through the hollow fiber membrane and is retained in the hollow fiber membrane is recorded as "concentrated liquid". It should be noted that the "concentrated liquid" refers to the entire liquid that has been purified and concentrated by the hollow fiber membrane.

[0152] A hollow fiber membrane module that supplies liquid containing tiny useful substances to the inner side of a hollow fiber membrane bundle is an internal pressure type hollow fiber membrane module, and a hollow fiber membrane module that supplies liquid to the outer side of a hollow fiber membrane bundle is an external pressure type hollow fiber membrane module. Hereinafter, as an embodiment of a hollow fiber membrane module in the present disclosure, an example of an internal pressure type hollow fiber membrane module is described.

[0153] Figure 7: This is a schematic cross-sectional view showing an example of an internal pressure type hollow fiber membrane assembly 33. The hollow fiber membrane assembly 33 contains a hollow fiber membrane bundle 60 formed by bundling a plurality of hollow fiber membranes in a cylindrical container 61. The above-mentioned plurality of hollow fiber membranes are bundled by a biocompatible sealing adhesive (for example, a polyurethane sealing adhesive, etc.) to form a hollow fiber membrane bundle 60, and are bonded and fixed to the two ends of the length direction in the cylindrical container 61 by adhesive layers 62a and 62b formed by the above-mentioned sealing adhesive. From the viewpoint of transparency, the cylindrical container 61 can be composed of materials such as polycarbonate, poly (meth) acrylate, and polysulfone. It should be noted that the two end faces 61a and 61b of the cylindrical container 61 are in a state where a plurality of hollow fiber membranes are opened. An inlet and outlet 33a for the first liquid and an inlet and outlet 33b for the second liquid are provided at both ends in the length direction of the hollow fiber membrane assembly 33. In addition, the inlet and outlet 33c and 33d of the third liquid are provided on the side portion in the longitudinal direction of the hollow fiber membrane module 33. The liquid supplied from the inlet and outlet 33a of the first liquid and / or the inlet and outlet 33b of the second liquid is sent to the inner side of the hollow fiber membrane bundle 60 to be purified and concentrated. In addition, the permeated liquid that permeates to the outside of the hollow fiber membrane bundle 60 is discharged from the inlet and outlet 33c and 33d of the third liquid provided on the side of the cylindrical container 61. It should be noted that the inlet and outlet of the third liquid are preferably provided at at least two locations on the upper side and the lower side of the cylindrical container 34. Figure 7 In the figure, the inlet and outlet 33c and 33d of the third liquid are provided only on one side of the hollow fiber membrane module 33, but the present invention is not limited thereto. The inlet and outlet 33c and 33d of the third liquid may be provided separately on one side and the other side.

[0154] Figure 7 Although the hollow fiber membrane module of the internal pressure type is exemplified, the hollow fiber membrane module of the present disclosure may also be of the external pressure type as described above. In the case of the external pressure type, the installation position and installation method of the first to third liquid inlets and outlets need to be appropriately designed.

[0155] (Filling rate of hollow fiber membrane module)

[0156] The filling rate in the hollow fiber membrane module disclosed in the present invention is preferably 5 to 55%, more preferably 20 to 55%, and further preferably 25 to 55%. The above filling rate refers to the percentage of the sum of the cross-sectional areas of the outer diameter of the hollow fiber membrane bundle to the cross-sectional area of ​​the short side direction of the generally cylindrical container of the hollow fiber membrane module. When the above filling rate is 5 to 55%, the size of the hollow fiber membrane module can be optimized, and the tiny useful substances deposited on the membrane surface of the hollow fiber membrane can be more effectively recovered.

[0157] Hereinafter, an embodiment in which the purification and concentrating section 30 of the present disclosure includes the hollow fiber membrane module 33 will be described using the drawings.

[0158] Figures 8 to 11 2 shows some representative schematic diagrams of the purification and concentration section 30. It should be noted that the purification and concentration device 1 of the present disclosure sometimes includes valves, automatic valves, backflow prevention valves, orifices, flow meters, pressure gauges, liquid level gauges, rupture plates and other safety devices that are omitted in the schematic diagrams.

[0159] Figure 8 The purification and concentration section 30 shown includes a second tank 31 and a third tank 32, a hollow fiber membrane module 33 connected to the second tank 31 and the third tank 32, and a pressurizing device 34 for pressurizing the second tank 31 and the third tank 32. The pressurizing device 34 is connected to the three-way valve 76 via the gas supply path 52.

[0160] The second tank 31 is a tank for storing the liquid supplied from the raw material supply unit 10 or the removal unit 20, that is, the raw material liquid or the crude purified liquid, and is connected to the inlet and outlet 33a of the first liquid of the hollow fiber membrane module 33 via the flow path 51A. In addition, the third tank 32 is a tank for storing the raw material liquid or the crude purified liquid in the same manner as the second tank 31, and is connected to the inlet and outlet 33b of the second liquid of the hollow fiber membrane module 33 via the flow path 51B. The upper parts of the second tank 31 and the third tank 32 are connected to the pressurizing device 34. The second control unit 120 (not shown) controls in the following manner: by operating the pressurizing device 34, the three-way valve 76 is switched, thereby selectively pressurizing either the second tank 31 or the third tank 32, and the raw material liquid or the crude purified liquid in the second tank 31 or the third tank 32 is alternately (selectively) supplied to the hollow fiber membrane module 33. The liquid discharged from the second tank 31 or the third tank 32 is purified and concentrated by the hollow fiber membrane module 33 and then sent to the other tank. Hereinafter, the liquid stored in the second tank 31 before the purification and concentration process is completed is referred to as the "second stored liquid", and the liquid stored in the third tank 32 before the purification and concentration process is completed is referred to as the "third stored liquid". Figure 8 In the embodiment, the flow paths 42 and 43 are connected to the second tank 31, but the flow paths 42 and 43 may be connected to the third tank 32. The details of the second control unit 120 will be described later.

[0161] (Pressure device)

[0162] The pressurizing device 34 of the present disclosure supplies gas from a gas supply source to the second tank 31 and the third tank 32. The gas for pressurization is supplied to the second tank 31 or the third tank 32 from the gas supply paths 52A and 52B equipped with a pressure gauge (not shown) by switching the three-way valve 76 by the second control unit 120 (not shown). The gas can be selected from inert gases such as nitrogen, argon, and helium, carbon dioxide, filtered air, and the like.

[0163] When the second tank 31 is pressurized by the pressurizing device 34, the second control unit 120 (not shown) supplies gas from a gas supply source (not shown) to the gas supply path 52, and switches the three-way valve 76 to send it to the gas supply path 52A. At this time, the switch valve 74 connected to the exhaust path 53A and the three-way valve 77 of the flow paths 42 and 43 are closed, and the switch valve 75 of the exhaust path 53B is opened. On the other hand, when the third tank 32 is pressurized by the pressurizing device 34, the second control unit 120 supplies gas from a gas supply source (not shown) to the gas supply path 52, and switches the three-way valve 76 to send it to the gas supply path 52B. At this time, the switch valve 75 and the three-way valve 77 of the exhaust path 53B are closed, and the switch valve 74 of the exhaust path 53A is opened. Through these operations, the second tank 31 and the third tank 32 can be selectively pressurized by the pressurizing device 34.

[0164] (Second and third cans)

[0165] In the purification and concentration device 1 of the present disclosure, the second tank 31 and the third tank 32 are preferably transparent so that the liquid level positions of the second storage liquid and the third storage liquid can be observed by the naked eye or the camera described later. In addition, from the viewpoint of preventing the second storage liquid or the third storage liquid from adhering to and remaining on the inner wall surface of the tank, it is more preferably composed of a water-repellent material. Specifically, the second tank 31 and the third tank 32 are preferably composed of a material containing polyacrylonitrile, poly(meth)acrylate and other poly(meth)acrylate, polycarbonate, fluororesin and the like. It should be noted that when the liquid level of the transparent part of the inlet and outlet of the liquid of the second tank 31 or the third tank 32 is managed by the tank liquid amount monitoring device described later, the raw materials of the second tank 31 and the third tank 32 can use opaque materials, such as metal or fiber-reinforced resin tanks.

[0166] exist Figure 8 In the purification and concentration section 30 shown, the second stored liquid enters the hollow fiber membrane module 33 from the inlet and outlet 33a of the first liquid via the flow path 51A, is purified and concentrated by the hollow fiber membrane bundle 60, and is discharged from the inlet and outlet 33b of the second liquid to be sent to the third tank 32. In this case, the second stored liquid is purified and concentrated by tangential flow filtration in which the liquid flows parallel to the membrane surface in the longitudinal direction of the hollow fiber membrane bundle 60. In addition, the permeate after passing through the hollow fiber membrane bundle 60 is discharged from the flow path 54 connected to the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33 to the waste liquid tank (described later).

[0167] Similarly, the third storage liquid enters the hollow fiber membrane assembly 33 from the inlet and outlet 33b of the second liquid via the flow path 51B, is purified and concentrated by the hollow fiber membrane bundle 60, and is discharged from the inlet and outlet 33a of the first liquid and sent to the second tank 31. In this case, the third storage liquid is purified and concentrated by tangential flow filtration in which the liquid flows parallel to the membrane surface in the length direction of the hollow fiber membrane bundle 60. By repeating this operation alternately, tiny useful substances and impurities are not easily deposited on the surface of the hollow fiber membrane. As a result, the reduction in the permeation rate of the liquid can be suppressed, and a high processing efficiency can be maintained. That is, by performing alternating tangential flow filtration on the liquid, a liquid containing tiny useful substances at a higher purity and a higher concentration can be more effectively manufactured.

[0168] The second control unit 120 can set the amount of the second stored liquid and the third stored liquid, and control the pressurizing device 34 and the switch valve so that the second and third stored liquids reach the set values, thereby alternately pressurizing the second tank 31 or the third tank 32 .

[0169] Figure 8 The purification and concentration section 30 may be provided with a device for monitoring the liquid amounts in the second tank 31 and the third tank 32. According to this configuration, the liquid amounts in the second tank 31 and the third tank 32 can be grasped, so that the alternating tangential flow filtration can be performed more efficiently.

[0170] Fig. 9 The purification and concentration section 30 shown has Figure 8 The second tank 31 and the third tank 32 are respectively provided with a tank liquid amount monitoring device 35 and 36 including a light sensor (not shown) at the inlet and outlet of the liquid at the bottom of the tank. The tank liquid amount monitoring device 35 and 36 are configured as follows: the light is irradiated to the inlet and outlet of the liquid of the second tank 31 and the third tank 32 by the light sensor, the transmittance of the light at the inlet and outlet of the liquid is detected, and the liquid amount in the tank is monitored based on the change of the transmittance. The second control unit 120 (not shown) determines the liquid amount of the second stored liquid and the third stored liquid in the second tank 31 and the third tank 32 through the tank liquid amount monitoring device 35 and 36, and determines the tank to be pressurized by the pressurizing device 34.

[0171] (Liquid volume monitoring device in tank)

[0172] The tank liquid amount monitoring device 35, 36 disclosed in the present invention is equipped with a light sensor. The light sensor is equipped with a light-emitting element and a light-receiving element. The light-emitting element irradiates light to the inlet and outlet of the liquid at the bottom of the tank, and the light-receiving element detects the intensity (transmittance) of the transmitted light. It should be noted that the portion of the inlet and outlet of the liquid at the bottom of the tank that is irradiated with light is made of transparent raw materials. The detected transmittance is further sent to the detection unit. When the detection unit detects a change in the transmittance, the second control unit 120 changes the tank that is pressurized by the pressurizing device 34.

[0173] The relationship between the tank liquid amount monitoring device and the second control unit will be described in more detail.

[0174] When the second tank 31 is pressurized by the pressurizing device 34 and the second stored liquid is supplied to the third tank 32, if the amount of the second stored liquid in the second tank 31 decreases, the gas supplied from the pressurizing device 34 is discharged from the inlet and outlet of the liquid of the second tank 31, and the transmittance of the light in the inlet and outlet of the liquid changes. When the tank liquid amount monitoring device 35 detects a change in transmittance, the second control unit 120 (not shown) switches the three-way valve 76 to change the tank pressurized by the pressurizing device 34 from the second tank 31 to the third tank 32. Similarly, when the tank liquid amount monitoring device 36 provided with the inlet and outlet of the liquid of the third tank 32 detects a change in transmittance, the second control unit 120 switches the three-way valve 76 to change the tank pressurized by the pressurizing device 34 from the third tank 32 to the second tank 31. The tank liquid amount monitoring devices 35 and 36 may be fiber sensors. In this way, the second control unit 120 can control the liquids in the second tank 31 and the third tank 32 to be alternately supplied to the hollow fiber membrane module.

[0175] In another embodiment, the amount of liquid in the tank can be monitored based on the image of the camera arranged in the purification and concentration section 30. That is, the purification and concentration section 30 is equipped with a camera for monitoring the liquid level position in the second tank 31 and the third tank 32, and the second control section 120 can grasp the amount of liquid in the tank based on the information of the liquid level position in the second tank 31 and the third tank 32 output by the camera, and determine the tank to be pressurized by the pressurizing device 34. Specifically, the purification and concentration section 30 is equipped with a camera at a position where the liquid level of the second tank 31 and the third tank 32 can be observed, and the image of the liquid level position output by the camera is transmitted to the detection section. The detection section determines the amount of liquid in the second tank 31 and the third tank 32 based on the information of the liquid level position. For example, when the detection section determines that the amount of the second storage liquid in the second tank 31 is less than a preset amount based on the information of the liquid level position of the camera, the second control section 120 switches the three-way valve 76 to change the tank to be pressurized by the pressurizing device 34 from the second tank 31 to the third tank 32. Similarly, when the detection unit determines that the amount of the third stored liquid in the third tank 32 is less than a preset amount, the second control unit 120 switches the three-way valve 76 to change the tank pressurized by the pressurizing device 34 from the third tank 32 to the second tank 31 .

[0176] (Second Control Unit)

[0177] Fig.16 The flowchart of FIG. 1 shows an example of the determination in the liquid circulation control of the second control unit 120. Fig.16 As shown, in order to make the liquid (crude purified liquid or raw material liquid) circulate mainly between the second tank 31 and the third tank 32, the second control unit 120 can operate the switch valve and the pressurizing device. In addition, the second control unit 120 can determine the liquid amount in the second tank 31 and the third tank 32 based on the information obtained by the tank liquid amount monitoring device 35, 36 and / or the camera, and make the liquid in the tank circulate. In addition, the second control unit 120 can also set the liquid amount in the second tank 31 and / or the third tank 32. An example of the method for setting the liquid amount in the tank (setting of the management liquid level position C) will be described later.

[0178] Fig.10 The purification and concentration section 30 includes a cleaning tank that supplies a diluent to the hollow fiber membrane module 33 to clean the plurality of hollow fiber membranes in the hollow fiber membrane bundle 60 . Fig.10The purification and concentration section 30 shown in the figure comprises: a fourth tank 80 storing a diluent, connected to a flow path 55A for supplying the diluent to the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33; and a third control unit 130 (not shown) for controlling the timing of supplying the diluent in the fourth tank 80 to the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33. The fourth tank 80 is connected to a flow path 55B for directly supplying the diluent in the tank to the third tank 32 (or the second tank 31).

[0179] (Third Control Unit)

[0180] The third control unit 130 controls the flow of the diluent in the fourth tank 80 . Fig.16 The flowchart of FIG. 1 shows an example of the judgment in the flow control of the liquid by the third control unit 130. The third control unit 130 can supply the diluent from the fourth tank 80 to either the third tank 32 or the hollow fiber membrane module 33 during the purification and concentration process of the liquid. Hereinafter, the step of supplying the diluent to the third tank 32 is referred to as "dilution process", and the step of supplying the diluent to the hollow fiber membrane module 33 is referred to as "reverse cleaning process".

[0181] (Diluent)

[0182] The diluent may be, for example, pure water, tap water, groundwater, filtered water such as seawater or river water, various aqueous solutions, etc., depending on the purpose of the present disclosure and the type of the micro-useful substance. When the micro-useful substance is an exosome, physiological saline, a buffer for medical or biochemical use, etc. are preferred. As the above-mentioned buffer, for example, phosphate buffer (PBS), Tris hydrochloric acid buffer, sodium citrate buffer, citrate phosphate buffer, acetate buffer, borate buffer, etc. can be used.

[0183] According to the purpose of the present disclosure, the diluent preferably does not contain impurities, but in the case where it is difficult to obtain a diluent without impurities, a diluent with a low impurity concentration (for example, an impurity concentration of less than 500 ppm) may be used.

[0184] When the amount (B) of the concentrated liquid in the second tank 31 (or the third tank 32) is 1 / 2 to 1 / 200 of the amount (B / A) of the raw material liquid containing the minute useful substance, the diluent may be supplied to the concentrated liquid in the second tank 31 (or the third tank 32). At this time, the amount (B) of the concentrated liquid may be equal to the amount of liquid at the management liquid level position C set by the method described later.

[0185] In addition, when the crude purified liquid is supplied from the first tank 23 to the second tank 31 (or the raw liquid is supplied to the second tank 31 via the flow path 43), the third control unit 130 can perform a dilution process. The third control unit 130 can determine that the crude purified liquid or the raw liquid is supplied to the second tank 31 based on the liquid level position information of the tank liquid amount monitoring device 35 and / or the camera. In one embodiment, the third control unit 130 can perform a dilution process (or a reverse cleaning process) until the set liquid volume is reached after performing alternating tangential flow filtration and concentration purification to make the amount of the crude purified liquid reach 1 / 2 to 1 / 200 of the liquid volume, and supply the diluent. In the case of performing a dilution process, the third control unit 130 opens the three-way valve 77A to the side of the flow path 55B, and supplies the diluent in the fourth tank 80 to the third tank 32.

[0186] When the third control unit 130 determines that the total amount of liquid in the second tank 31 and the third tank 32 has reached a predetermined liquid amount or less (for example, below the management liquid level position C set by the method described later), a reverse cleaning process can be performed. When performing a reverse cleaning process, the third control unit 130 opens the three-way valve 77A to the side of the flow path 55A. The diluent in the fourth tank 80 is supplied to the assembly from the inlet and outlet 33c and 33d of the third liquid of the hollow fiber membrane assembly 33 via the flow path 55A. Then, the diluent flows from the permeate side of the hollow fiber membrane bundle 60 toward the concentrate side. When the hollow fiber membrane assembly 33 is an internal pressure type, the diluent supplied to the assembly from the inlet and outlet 33c and 33d of the third liquid flows from the outside to the inside of the hollow fiber membrane bundle 60. Thus, tiny useful substances deposited on the membrane surface of the hollow fiber membrane, especially on the membrane surface inside the hollow fiber membrane, can be recovered from the inlet and outlet 33a of the first liquid and / or the inlet and outlet 33b of the second liquid to the second tank 31 and / or the third tank 32. Through this treatment, tiny useful substances and impurities deposited on the surface of the hollow fiber membrane are stripped off, and the permeation rate of the liquid is basically restored to the original value. In addition, by diluting the liquid with a diluent, the impurities are evenly dispersed and the removal efficiency is improved. As a result, the efficiency of purification and concentration of the liquid can be further improved.

[0187] It should be noted that before implementing reverse cleaning, in order to prevent the residual permeate in the hollow fiber membrane module from mixing with the reverse cleaning liquid and flowing into the concentrated liquid side, it can be rinsed with a cleaning liquid (diluent) in advance, and the residual permeate in the hollow fiber membrane module can be replaced with the cleaning liquid (diluent) optionally.

[0188] At this time, the amount of the cleaning solution (diluent) used is preferably 1 to 8 times, more preferably 2 to 6 times the volume of the permeate remaining in the hollow fiber membrane module.

[0189] When concentrating and purifying a certain extracellular vesicle in a certain culture supernatant, the amount of residual impurity proteins can sometimes be reduced to less than half when a flushing operation is performed before backwashing, compared with a case where a flushing operation is not performed.

[0190] When the hollow fiber membrane module 33 is an internal pressure type, the following Figure 7 An operation of replacing the permeate remaining in the hollow fiber membrane module with the cleaning liquid (diluent) by using either the inlet and outlet 33c or 33d of the third liquid as the inlet and the other as the outlet.

[0191] Since the hollow fiber membrane has liquid flow resistance, if the diluent is supplied from either the third liquid inlet and outlet 33c or 33d, the permeate remaining in the hollow fiber membrane module 33 can be replaced with the diluent with little leakage to the membrane side.

[0192] On the other hand, for example, by providing an on-off valve (not shown) on the flow path near the outlet of the first liquid 33a and the outlet of the second liquid 33b and closing the on-off valve, the flow of the first liquid 33a and the outlet of the second liquid 33b can be stopped. In this state, by supplying a diluent from any one of the outlets 33c and 33d of the third liquid, the diluent is discharged from the other outlet 33c or 33d of the third liquid. In this way, the permeate remaining in the hollow fiber membrane module 33 can be replaced with the diluent.

[0193] The timing of the flushing treatment is controlled by the third control unit. For example, a case where a switch valve not shown is provided in the flow path near the outlet of the first liquid inlet and outlet 33a and the outlet and outlet of the second liquid 33b is described. For the third control unit, when the total amount of the liquid in the second tank 31 and the third tank 32 reaches or less than the value set by the second control unit, the diluent in the fourth tank 80 can be supplied from any one of the at least two outlets and outlets 33c and 33d of the third liquid of the hollow fiber membrane module 33 by closing the switch valves provided near the outlet and outlet of the first liquid 33a and the outlet and outlet of the second liquid 33b to stop the circulation of the liquid. At this time, the switch valve 78 provided in the flow path 54 connected to the waste liquid tank is opened. Then, the switch valves arranged near the inlet and outlet 33a of the first liquid and the inlet and outlet 33b of the second liquid can be opened to make the inlet and outlet 33a of the first liquid and the inlet and outlet 33b of the second liquid in a flowable state, and the dilution liquid in the fourth tank 80 is supplied from any one of at least two inlets and outlets 33c and 33d of the third liquid possessed by the hollow fiber membrane module 33, thereby performing back cleaning.

[0194] At this time, the on-off valve 78 provided in the flow path 54 connected to the waste liquid tank is closed.

[0195] Fig.11 The purification and concentration section 30 shown includes a recovery tank for recovering the concentrated liquid purified and concentrated by the hollow fiber membrane module 33 . Fig.11 The purification and concentration section 30 comprises: a fifth tank 81, which is connected to the inlet and outlet 33a of the first liquid of the hollow fiber membrane module 33 through the flow path 56; and a fourth control unit 140 (not shown), which controls the recovery of the liquid in the second tank 31 and the third tank 32 to the fifth tank 81. It should be noted that the fifth tank 81 can be connected to the inlet and outlet 33b of the second liquid of the hollow fiber membrane module 33. Fig.11 , the flow path 56 is connected to the hollow fiber membrane module 33 via the flow path 51A. During the purification and concentration process based on the alternating tangential flow filtration by the second tank 31 and the third tank 32, the on-off valve 79 is closed. After the purification and concentration process, the on-off valve 79 is opened and the pressurizing device 34 is operated, so that the concentrated liquid in the second tank 31 and the third tank 32 is recovered to the fifth tank 81. It should be noted that the tank for recovering the concentrated liquid may be configured not in the purification and concentration section 30 but outside the purification and concentration device.

[0196] (Fourth Control Unit)

[0197] The fourth control unit 140 controls the timing of recovering the concentrated liquid in the second tank 31 and the third tank 32 . Fig.16 An example of determination in the liquid flow control by the fourth control unit 140 is shown in the flowchart of FIG.

[0198] The fourth control unit determines the amount of the concentrated liquid in the second tank 31 and the third tank 32 through the tank liquid amount monitoring device 35, 36 and / or the camera. The amount of the concentrated liquid can be determined based on the management liquid level position C described later. When it is determined that the purification and concentration process is completed, the fourth control unit 140 pressurizes the second tank 31 and the third tank 32 through the pressurizing device 34. At this time, the on-off valve 74 of the exhaust path 53A, the on-off valve 75 of the exhaust path 53B, the on-off valve 78, and the three-way valves 77 and 77A are closed in advance, and the on-off valve 79 is opened. When the tank liquid amount monitoring device 35, 36 detects a change in transmittance, or when it is determined that the amount of the concentrated liquid in the second tank 31 and the third tank 32 is zero based on the image of the above-mentioned camera, the fourth control unit 140 stops the pressurizing device 34 and ends the recovery of the concentrated liquid to the fifth tank 81. Specifically, the fourth control unit 140 can monitor the amount of concentrated liquid in the flow path 56 through a fiber sensor (not shown) provided at the liquid inlet and outlet of the upper part of the fifth tank 81, and terminate the recovery of the concentrated liquid when it is determined that the amount of liquid is zero. Alternatively, the concentrated liquid recovery operation inside the flow path 56 can be manually operated by a person. In addition, the flow path 56 can be fixed and held by a pneumatic clamp or the like.

[0199] Fig.12 Detailed description of the drawings is given in FIG. 1 . Fig.12 In the purification and concentration device 1, the elements including the raw material supply unit 10, the removal unit 20 and the purification and concentration unit 30 are arranged in a cover. Fig.12 In the purification and concentration device 1, the size of the cover is not particularly limited. In addition, the purification and concentration device 1 is provided with a management unit (not shown) for managing the cleanliness of the inside of the cover. According to this structure, the inside of the purification and concentration device 1 can be kept clean, and contamination of the liquid containing tiny useful substances can be prevented. In addition, a fan filter device 91 for supplying filtered air to the inside of the cover is arranged on the upper part (top) of the cover 100, and a UV lamp 92 is arranged in the cover 100. Through these combinations, the cleanliness of the inside of the cover 100 can be further improved.

[0200] (Fan filter unit)

[0201] The fan filter device 91 has a structure capable of supplying filtered air into the cover. The fan filter device 91 can be set as a filter unit with a fan having a pre-filter built in. As the pre-filter, a HEPA filter, a ULPA filter, a chemical filter, a fiber activated carbon filter, etc. can be used according to the cleanliness in the cover 100.

[0202] (UV Lamp)

[0203] The UV lamp 92 is not particularly limited as long as it can irradiate UV (ultraviolet rays) to sterilize the inside of the cover 100. In addition, the number of UV lamps 92 arranged in the cover 100 is not particularly limited, and two or more UV lamps may be arranged according to the cleanliness of the inside of the cover 100.

[0204] (Cleanliness)

[0205] When the purification and concentration device 1 is configured such that each element is arranged in the cover 100, the cleanliness in the cover 100 can be improved by the above-mentioned management unit. In one embodiment, the cleanliness in the cover 100 can be managed to reach level 8 or below, or level 5 or below, in the cleanliness level specified in ISO14644-1:2015.

[0206] (tiny useful substances)

[0207] There is no particular limitation on the micro-useful substance. In one embodiment, it can be a micro-useful substance with an average particle size of less than 100 μm. In another embodiment, it can be a micro-useful substance with an average molecular weight of 5000 or more. Among them, all substances useful as medicines, cosmetics, and health foods can be used.

[0208] Specific examples of useful microscopic substances include proteins (e.g., heat shock proteins, cytoskeletal proteins, membrane transport proteins, transmembrane proteins, collagen, hyaluronic acid, chondroitin sulfate, etc.), hormones, cytokines, growth factors, angiogenesis factors, growth factors, enzymes, antibodies, plasma proteins, viruses (including virus-like particles), extracellular vesicles (e.g., exosomes, microvesicles, apoptotic bodies, etc.), biological molecules such as yeast, yeast cells and microalgae, polysaccharides such as fucoidan, etc., etc.

[0209] In addition, specific microorganisms that are a type of biomolecule and can be used for biofuels and the like can also be mentioned.

[0210] Among them, tiny useful substances can be selected from extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids.

[0211] [Method for producing a liquid obtained by purifying and concentrating minute useful substances: First embodiment]

[0212] Below, use Fig.13A method for producing a liquid obtained by purifying and concentrating a tiny useful substance using the purification and concentrating device 1 of the present disclosure is described. The production method of the first embodiment of the present disclosure is a method for producing the above-mentioned liquid obtained by purifying and concentrating a tiny useful substance using the purification and concentrating device 1, the method comprising: supplying a raw material liquid containing the above-mentioned tiny useful substance to the above-mentioned purification and concentrating device to remove impurities in the above-mentioned liquid; and / or, purifying and concentrating the above-mentioned liquid using a hollow fiber membrane.

[0213] Fig.13 1 is a structural diagram showing an example of the overall structure of the purification and concentration device 1 of the present disclosure. The raw material supply unit 10 is a structure in which a container 11 is provided for storing a raw material liquid containing a small amount of useful substances. When the present device is started, the three-way valve 82 is opened, and the raw material liquid in the container 11 is supplied to the flow path 59 by falling based on its own weight and a pump (not shown) provided in the flow path 41A. By operating the three-way valve 82, the raw material liquid is supplied to the flow path 41 or the flow path 43. The first control unit 110 supplies the raw material liquid to the removal unit 20 or the purification and concentration unit 30 based on information set by the concentration of impurities in the raw material liquid. The first control unit 110 opens the three-way valve 82 to the side of the flow path 43, and sends the above-mentioned culture solution from the flow path 43 to the purification and concentration unit 30.

[0214] The raw material liquid supplied to the flow path 41 is pre-treated by the filter element 22 and then supplied to the tank 24 via the flow path 41C. The amount of liquid in the tank 24 can be controlled by monitoring the liquid level position based on a level sensor (not shown). When the switch valve 73 is opened, the pre-treatment liquid in the tank 24 is supplied from the flow path 41D to the flow path 41A.

[0215] (Impurity removal treatment)

[0216] The pretreatment liquid supplied to the carrier column 21 is adsorbed and removed by the adsorbent material in the carrier column 21 to form a crude purified liquid, and then supplied to the first tank 23 via the flow path 42A. The impurity removal treatment based on the carrier column 21 can reduce the concentration of impurities in the raw material liquid or the pretreatment liquid to less than 1000 ppm, preferably less than 500 ppm.

[0217] In one embodiment, the impurity removal process may repeat the following steps: after cleaning and balancing the carrier column 21 that has reached adsorption saturation through treatment with a batch of raw material solution, further treating the next batch of raw material solution.

[0218] Fig.13The purification and concentration device 1 is a structure having a sixth tank 25 and a seventh tank 26. When the carrier column 21 that has reached adsorption saturation is cleaned and balanced, first, the switch valve 71A (solenoid valve, etc.) is opened, and the cleaning liquid in the sixth tank 25 is supplied to the filter member 22A via the flow path 50A. After the impurities in the cleaning liquid are removed by the filter member 22A, the cleaning liquid is supplied to the carrier column 21 from the flow path 41A. The cleaning liquid after cleaning the carrier column 21 can be discharged to the waste liquid tank by opening the three-way valve 72. It should be noted that the filter member 22A can adopt the same filter member as the filter member 22. After the cleaning process, the switch valve 71B (solenoid valve, etc.) is opened, and the balancing liquid in the seventh tank 26 is supplied to the filter member 22B. After the impurities in the balancing liquid are removed by the filter member 22B, the balancing liquid is supplied to the carrier column 21 from the flow path 41A. The balancing liquid after the carrier column 21 is balanced can be discharged to the waste liquid tank by opening the three-way valve 72. It should be noted that the filter member 22B may also be the same member as the filter member 22 .

[0219] The cleaning and equilibration of the support column 21 can be arbitrarily set by the amount of the raw material liquid treated by the support column 21. In one embodiment, the cleaning and equilibration of the support column 21 can be performed when 5 to 100 times the volume of the raw material liquid is treated. For example, when 1000 mL of the raw material liquid is treated, the cleaning and equilibration of the support column 21 can be performed every time 20 to 150 mL of the raw material liquid is treated by the support column 21, or the cleaning and equilibration can be performed every time 50 to 100 mL of the raw material liquid is treated.

[0220] In the production method disclosed herein, it is preferred to perform in parallel the step of removing impurities in the raw material liquid by the carrier column 21 and the step of purifying and concentrating the liquid by the hollow fiber membrane of the purification and concentration section 30. In this case, a liquid in which the minute useful substances are purified and concentrated can be produced more efficiently.

[0221] exist Fig.13 In the embodiment of the present invention, the crude purified liquid is stored in the first tank 23 via the flow path 42A. When it is determined that the amount of the second storage liquid and the third storage liquid in the second tank 31 and the third tank 32 reaches the preset liquid amount, the first control unit 110 sends the crude purified liquid stored in the first tank 23 to the flow path 42B. By switching the direction of the three-way valve 77, the crude purified liquid is supplied from the flow path 42B to the second tank 31. When the crude purified liquid is supplied to the second tank 31 (or the third tank 32), the second control unit 120 (not shown) operates the pressurizing device 34 to start the purification and concentration process based on the alternating tangential flow filtration.

[0222] As described above, the purification and concentrating device 1 of the first embodiment of the present disclosure is configured so that the impurity removal process of the removal unit 20 and the purification and concentrating process of the purification and concentrating unit 30 are performed in parallel. In this case, it is preferable to configure a structure with a plurality of tanks for storing liquids processed in each process. From the perspective of making the purification and concentrating device 1 more compact, the configuration position of each tank can be adjusted. For example, Fig.13 In the purification and concentration device 1, the first tank 23 storing the crude purified liquid is arranged above the second tank 31 and the third tank 32, but the second tank 31 and the third tank 32 may be arranged above the first tank 23. Fig.13 As shown, the second tank 31 and the third tank 32 may be arranged so that the side surfaces in the longitudinal direction are parallel to the longitudinal direction of the hollow fiber membrane module 33 .

[0223] The following is a description of the preferred operation when the crude purified liquid (or raw liquid) is subjected to alternating tangential flow filtration by the purification and concentration section 30. In one embodiment, the purification and concentration treatment of the crude purified liquid is preferably divided into the first treatment, the second treatment, and the third treatment. It should be noted that before the first treatment is performed, the liquid volume in the tank is set. The following is a description of an example in which the first treatment is performed after setting the management liquid level position C.

[0224] (Setting of management liquid level position C)

[0225] Before the first process is performed, the amount of managing the liquid level position C is set.

[0226] The first tank 23 stores a crude purified liquid that has been processed by the antibody column 21. The liquid volume at the management liquid level position C is preset to be equal to the liquid volume to be started for the reverse cleaning process and to be 1 / 2 to 1 / 200 of the maximum capacity of the crude purified liquid stored in the first tank 23. When the crude purified liquid in the second tank 31 reaches the volume of the preset management liquid level position C, the crude purified liquid in the first tank 23 can be supplied to the second tank 31. Furthermore, after the crude purified liquid is supplied to the second tank 31, the third control unit 130 opens the three-way valve 77A to the side of the flow path 55B, and supplies the diluent from the fourth tank 80 to the second tank 31.

[0227] After setting the management liquid level position C, the liquid level control positions of the second tank 31 and the third tank 32 are set. The second tank 31 is pressurized by the pressurizing device 34 to send the diluent in the third tank 32 to the second tank 31. At this time, the second control unit 120 sets the low liquid level position (L3) of the third tank 32, and supplies the diluent to the second tank 31 until the diluent in the third tank 32 reaches L3. In addition, the second control unit 120 sets the liquid level position of the second storage liquid (a mixed liquid of the crude purified liquid and the diluent) in the second tank 31 when the diluent in the third tank 32 reaches L3 to the high liquid level position (H2). In addition, from the viewpoint of preventing the second storage liquid from overflowing from the upper part of the second tank 31, the high-high liquid level position (HH2) is set above the high liquid level position. Next, the second control unit 120 pressurizes the second tank 31 by the pressurizing device 34 to send the second storage liquid to the third tank 32. At this time, the second control unit 120 sets the low liquid level position (L2) of the second tank 31, and supplies the second storage liquid to the third tank 32 until the second storage liquid in the second tank 31 reaches L2. In addition, the second control unit 120 sets the liquid level position of the third tank 32 when the second tank 31 reaches L2 as the high liquid level position (H3). From the viewpoint of preventing leakage from the third tank 32, HH3 is set. The second control unit 120 sets the management liquid level position C based on the total liquid volume of H2 and L3, H3 and L2, and moves the liquid within the range of the management liquid level position C to perform alternating tangential flow filtration between the second tank 31 and the third tank 32. It should be noted that L2 and L3 can be set based on the change in the transmittance of the liquid inlet and outlet of the tank obtained by the above-mentioned tank liquid volume monitoring device 35, 36, and can be set based on the image of the liquid level position obtained by the camera.

[0228] In addition, the positions of L2, L3, H2, H3, HH2 and HH3 can be appropriately set according to the capacities of the second tank 31 and the third tank 32 and the amount of the crude purified liquid.

[0229] (Purification and concentration treatment: first treatment)

[0230] Next, the first treatment in the purification and concentration treatment is described. The second control unit 120 alternately pressurizes the second tank 31 and the third tank 32 through the pressurizing device 34, and alternately supplies the second storage liquid and the third storage liquid to the hollow fiber membrane module 33 so that the liquid volume of the management liquid level position C does not decrease. By performing alternating tangential flow filtration in this way, a concentration and purification operation of the liquid containing tiny useful substances is implemented. The permeate is discarded to the waste liquid tank from the inlet and outlet 33c, 33d of the third liquid via the flow path 54.

[0231] It is possible to confirm that the liquid volume in the second tank 31 or the third tank 32 has reached the set value, and then supply an appropriate amount of the crude purified liquid to the second tank 31 or the third tank 32 .

[0232] In addition, it is preferable to perform the backwashing process when the liquid amount in the second tank 31 or the third tank 32 is equal to the liquid amount at the controlled liquid level position C.

[0233] The amount of diluent used for the reverse cleaning treatment can be set to 2 to 200 times the amount of liquid at the management liquid level position C. As the above-mentioned amount of diluent, from the viewpoint that the volume of the second tank 31 or the third tank 32 is not greater than the required amount, it is preferably 2 to 20 times the amount of liquid at the management liquid level position C. In addition, the amount of diluent used for the reverse cleaning treatment can also be appropriately changed during each reverse cleaning operation. In addition, as the timing for implementing the reverse cleaning treatment, it can also be appropriately implemented other than the case where the amount of liquid in the second tank 31 or the third tank 32 is equal to the amount of liquid at the management liquid level position C. As an example, there can be cited: a situation where there is a hidden danger of reduced permeation rate due to pressure changes and flow changes; or a situation where the tendency of reduced permeation rate can be judged, and a situation where a safe operation is implemented to prevent clogging of the hollow fiber membrane in advance, etc.

[0234] In addition, as a dilution treatment, a part of the dilution liquid may be directly sent to the second tank 31 or the third tank 32 .

[0235] The amount of the diluent used in the dilution treatment may be appropriately changed for each dilution operation.

[0236] In one embodiment, the second tank 31 and the third tank 32 are preferably pressurized by the pressurizing device 34 so that the linear velocity of the membrane surface of the hollow fiber membrane module 33 reaches 0.3 m / sec to 2 m / sec. It should be noted that the “linear velocity of the membrane surface” refers to the linear velocity of the membrane surface relative to 1 m / sec. 2 The flow rate per second of the liquid containing tiny useful substances flowing on the membrane surface (m 3 ), in m / s. By pressurizing the tank using the pressurizing device 34 so that the linear velocity of the membrane surface reaches 0.3 to 2.0 m / s, it is easy to improve the purification and concentration efficiency. In addition, the pressure level for increasing the membrane surface velocity will not become too high, and the shear force applied to the tiny useful substances will not be too large, which can easily prevent the tiny useful substances from being deformed. The pressurization in the tank using the pressurizing device 34 can be performed in the range of the linear velocity of the membrane surface reaching 0.5 to 1.5 m / s.

[0237] The operating pressure of the hollow fiber membrane module 33 is preferably adjusted so that the inlet pressure of the liquid is 0.01 MPa to 0.2 MPa, more preferably 0.02 MPa to 0.15 MPa, and further preferably 0.03 MPa to 0.12 MPa.

[0238] In addition, the liquid viscosity of the liquid containing the microscopic useful substance during operation is 20°C at a shear rate of 25 s -1When the viscosity of the liquid is 10 to 30 mPa·s, a thick hollow fiber membrane with an average inner diameter of 1.0 mm to 1.4 mm can be used so that the linear velocity of the membrane surface can be 0.7 to 1.4 m / s. For the operating pressure at this time, it is preferred to adjust the inlet pressure of the liquid to 0.08 MPa to 0.2 MPa.

[0239] In order to improve the fouling resistance of the hollow fiber membrane surface and / or to improve the recovery rate of useful minute substances, the hollow fiber membrane may be coated with an appropriate substance. The coating method is as described above.

[0240] In the first treatment, it is preferred to perform purification and concentration treatment so that the amount of the crude purified liquid reaches a liquid amount equal to the amount of the managed liquid level position C preset by the above method. After the purification and concentration treatment, the third control unit 130 supplies the diluent from the fourth tank 80. The diluent may be supplied to a value of 2 to 200 times the amount of the liquid reaching the managed liquid level position C. That is, in the first treatment, a step of diluting the concentrated liquid by 2 to 20 times may be included.

[0241] The diluent may be directly supplied to the third tank 32 (dilution treatment), or may be supplied from the hollow fiber membrane module 33 to the second tank 31 and the third tank 32 (reverse cleaning treatment). Fig.13 In the purification and concentration device 1, the direction of the three-way valve 77A is opened to the side of the flow path 55A, and the diluent is supplied from the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33. The diluent supplied from the hollow fiber membrane module 33 is supplied to the second tank 31 and the third tank 32. By performing the reverse cleaning treatment, the concentrated liquid can be diluted while recovering the tiny useful substances deposited on the membrane surface of the hollow fiber membrane, so it is preferred. Hereinafter, the liquid obtained by the first treatment is recorded as "the first treatment liquid".

[0242] (Purification and concentration treatment: second treatment)

[0243] The first treated liquid is further subjected to a second treatment to obtain a liquid in which the minute useful substances are further purified and concentrated.

[0244] Since the impurity concentration in the first treatment liquid is reduced by the above-mentioned first treatment, the second treatment is preferably carried out in a manner to further reduce the amount of impurities in the first treatment liquid (i.e., to improve the purity of the tiny useful substance). In one embodiment, the second treatment preferably repeats the following purification and concentration operation: for the amount of the concentrated liquid, relative to the value set by the management liquid level position C, after adding a diluent of 2 to 200 times the amount of the liquid at the management liquid level position C through the reverse cleaning treatment and the dilution treatment, it is further made to reach the liquid amount of the management liquid level position C. That is, in the second treatment, a step of diluting the concentrated liquid by 2 to 20 times may be included. As in the first treatment, the diluent can be directly supplied to the third tank 32 (dilution treatment), or it can be supplied from the hollow fiber membrane module 33 to the second tank 31 and the third tank 32 (reverse cleaning treatment). The second treatment consisting of the purification and concentration treatment and the dilution treatment is preferably carried out for 2 to 20 groups, and more preferably for 2 to 10 groups. Hereinafter, the concentrated liquid obtained by the above-mentioned second treatment is recorded as the "second treatment liquid".

[0245] (Purification and concentration treatment: third treatment)

[0246] The second treatment liquid may be further subjected to a third treatment to obtain a liquid in which the micro-useful substances are further purified and concentrated. In the third treatment, the alternating tangential flow filtration operation may be performed only without using a diluent as required. Hereinafter, the concentrated liquid obtained by the third treatment is recorded as the "third treatment liquid".

[0247] (Recycling)

[0248] The manufacturing method disclosed herein may include a step of recovering the second treatment liquid or the third treatment liquid. The second treatment liquid in the second tank 31, the third tank 32 and the hollow fiber membrane assembly 33 can be recovered to the fifth tank 81 via the flow path 56 by pressurizing with the pressurizing device 34. The fourth control unit 140 opens the switch valve 79 in advance when pressurizing by the pressurizing device 34, and closes the switch valves 74, 75, 78, and the three-way valves 77 and 77A. As described above, the fourth control unit 140 can monitor the amount of concentrated liquid in the flow path 56 by a fiber sensor provided at the inlet and outlet of the liquid on the upper part of the fifth tank 81, and when it is judged that the above-mentioned liquid amount is zero, the recovery of the concentrated liquid can be terminated. Alternatively, the concentrated liquid recovery operation inside the flow path 56 can also be manually operated by a person. In addition, the flow path 56 can be fixed and maintained by a pneumatic clamp or the like.

[0249] (Rinse treatment)

[0250] Before the reverse cleaning is performed, in order to prevent the permeate remaining in the hollow fiber membrane module from mixing with the reverse cleaning liquid and flowing into the concentrated liquid side, a flushing treatment may be performed in advance with a cleaning liquid (diluent), and an operation of replacing the permeate remaining in the hollow fiber membrane module with the cleaning liquid (diluent) may be performed. The flushing treatment method is as described above.

[0251] (Washing out of tiny useful substances deposited on the hollow fiber membrane surface before recycling)

[0252] The manufacturing method of the present disclosure may include an operation of washing out minute useful substances deposited on the hollow fiber membrane surface immediately before the recovery process.

[0253] As an example, in the case of concentrating and purifying a certain extracellular vesicle in a certain culture supernatant, the extracellular vesicles deposited relatively firmly on the hollow fiber membrane surface will not be dispersed in the concentrated solution, so the recovery rate is sometimes reduced in the above-mentioned recovery process. Therefore, an operation process in which the extracellular vesicles deposited on the hollow fiber membrane surface are redispersed in the concentrated solution by a small amount of reverse cleaning solution (diluent) can be implemented at a time just before the recovery process is about to be performed. At this time, it is preferred to fully implement the flushing operation, and the reverse cleaning solution (diluent) used is set to a small amount. As an example, the reverse cleaning solution (diluent) used is preferably half to twice the capacity of the first flow path.

[0254] The liquid passing rate at this time can be set to be equivalent to the liquid passing rate in the purification and concentration step.

[0255] [Other embodiments: Stepwise purification and concentration of a liquid containing minute useful substances]

[0256] Other embodiments of the manufacturing method of the first embodiment of the present disclosure are a method for manufacturing a purified concentrated liquid, which includes the step-by-step purification and concentration of a liquid containing tiny useful substances. As a method for implementing the purification and concentration of a liquid containing tiny useful substances in steps, an example can be given of a case where two or more steps of purification and concentration treatment are performed using hollow fiber membrane modules with two or more average inner diameters. Specifically, the following method can be cited: using the hollow fiber membrane module 33 of the present disclosure with an average inner diameter of 0.6 mm to 1.0 mm to obtain the purified concentrated liquid of the first step, and then using a hollow fiber membrane module device with an average inner diameter of 1.0 mm to 1.4 mm to obtain the final purified concentrated liquid.

[0257] [Second embodiment]

[0258] Next, a second embodiment of the purification and concentration device of the present disclosure will be described using the drawings. In the following description, the same reference numerals are used for components having the same configuration as the first embodiment, and detailed description thereof will be omitted.

[0259] Fig.14 The present invention is a schematic structural diagram showing an example of a purification and concentrating device 2 (hereinafter referred to as "purification and concentrating device 2") according to the second embodiment of the present invention. The purification and concentrating device 2 of the present invention includes: a raw material supply unit 10, and a purification and concentrating unit 30 for purifying and concentrating a liquid supplied from the raw material supply unit 10 through a hollow fiber membrane module 33, wherein the hollow fiber membrane module 33 has a first liquid inlet and outlet 33a and a second liquid inlet and outlet 33b for supplying the liquid to the hollow fiber membrane, and a third liquid inlet and outlet 33c and 33d for discharging the liquid after passing through the hollow fiber membrane, and the purification and concentrating unit 30 includes: a second tank 31 for storing the liquid supplied from the raw material supply unit 10, and connected to a flow path of the first liquid inlet and outlet 33a for supplying the liquid to the hollow fiber membrane module 33; a third tank 32 for storing the liquid supplied from the raw material supply unit 10, and connected to a second liquid inlet and outlet 33a for supplying the liquid to the hollow fiber membrane module 33; a flow path of the inlet and outlet 33b of the body; a pressurizing device 34, which selectively pressurizes any one of the second tank 31 and the third tank 32; a second control unit (not shown) which controls the second tank 31 or the third tank 32 by selectively pressurizing the second tank 31 or the third tank 32 through the pressurizing device 34, and alternately supplies the above-mentioned liquid in the second tank 31 and the third tank 32 to the hollow fiber membrane module 33; a monitoring device (not shown) which monitors the amount of the above-mentioned liquid in the second tank 31 and the third tank 32, the above-mentioned second control unit alternately pressurizes the second tank 31 and the third tank 32 through the pressurizing device 34 in a manner of setting the liquid amount in the second tank 31 and the second tank 31 and making the liquid amount in the second tank 31 and the third tank 32 reach the set value, and the above-mentioned liquid amount in the second tank 31 and the third tank 32 is determined based on the information of the above-mentioned monitoring device.

[0260] The purification and concentration device 2 disclosed in the present invention sometimes includes valves, automatic valves, backflow prevention valves, orifices, flow meters, pressure gauges, liquid level gauges, rupture plates and other safety devices, harmful substance treatment equipment, pressure adjustment equipment, intermediate tanks, and cut-off equipment that can be used for sampling, multi-step molecular weight separation operations, etc., which are omitted in the schematic configuration diagram.

[0261] The monitoring device in the purification and concentration device 2 may appropriately use a known liquid level gauge (liquid level gauge) such as a mechanical or reflective type, a weight monitoring device such as a load cell, an integrating flow meter device, or a combination of multiple devices, depending on the device size (scale).

[0262] Fig.14The purification and concentrating device 2 is a structure that does not have the removal unit 20 of the purification and concentrating device 1. The purification and concentrating device 2 is also useful as a device that can purify and concentrate minute useful substances by automatic control. According to this structure, the raw material liquid in the raw material supply unit 10 is directly supplied to the second tank 31 (or the third tank 32) of the purification and concentrating unit 30 via the flow path 201. Fig.14 The purification and concentration device 2 is useful as a purification and concentration device for a raw material liquid containing high-purity minute useful substances and having a very low impurity concentration in the raw material liquid. Figures 1 to 13 The purification and concentrating device 1 shown has a removal unit 20 and a purification and concentrating unit 30, and can perform the impurity removal process of the removal unit 20 and the purification and concentrating process of the purification and concentrating unit 30 in parallel. That is, the purification and concentrating device 1 is a device that can more efficiently prepare a concentrated liquid obtained by purifying and concentrating minute useful substances. On the other hand, the purification and concentrating device 2 does not have the removal unit 20, and can directly supply the raw material liquid from the raw material supply unit 10 to the purification and concentrating unit 30 to perform the purification and concentrating process of the raw material liquid. Therefore, the purification and concentrating device 2 is also useful as a device for further purifying and concentrating the purified concentrated liquid prepared by the purification and concentrating device 1.

[0263] In addition, by connecting two purification and concentration devices 2, three molecular weight fractionations can be performed. "Three molecular weight fractionations can be performed" means that the permeate of the first membrane and the concentrate of the first membrane can be sent to the second membrane and fractionated into permeate and concentrate. The permeate of the second membrane can be concentrated by the first membrane, and the permeate of the first membrane can be concentrated by another membrane and another concentration device. In one embodiment, the purification and concentration device 2 can be used as a device for fractionating the purified concentrate prepared by the purification and concentration device 1 into low molecular weight (for example, a tiny useful substance with a molecular weight cutoff of 10,000 to 100,000) and high molecular weight (a tiny useful substance with a molecular weight cutoff greater than 100,000).

[0264] exist Fig.14 In the purification and concentrating device 2 of the present invention, the second tank 31 and the third tank 32 may be configured in the same manner as the purification and concentrating device 1. For the purification and concentrating device 2, for example, from the perspective of using the concentrated liquid prepared by the purification and concentrating device 1 as the raw liquid, the volumes of the second tank 31 and the third tank 32 may be smaller than those of the purification and concentrating device 1. In addition, the above tanks may be larger than those of the purification and concentrating device 1. In addition, the first tank 23 may be omitted. In addition, when the liquid level of the transparent portion of the inlet and outlet of the liquid of the second tank 31 or the third tank 32 is managed by a fiber sensor or the like as in the purification and concentrating device 1, the raw material of the second tank 31 and the third tank 32 may be an opaque material, such as a metal or a fiber-reinforced resin tank.

[0265] exist Fig.14 In the purification and concentration device 2, the liquid transfer device is not limited to the pressurizing device 34, and may be performed by a liquid feeding device using a pump or a liquid feeding device using potential energy alone or in combination.

[0266] In order to use potential energy in liquid transfer, the height of the liquid surface in the second tank 31 and the third tank 32 may be controlled by a lifting device or the like, and the height position of the liquid inlet and outlet may be appropriately changed.

[0267] Fig.14 In the purification and concentration device 2, in order to suppress the foaming of the liquid in the second tank 31 or the third tank 32, when the height of the liquid inlet is higher than the liquid surface, the pipe diameter and the liquid delivery flow rate can be appropriately adjusted in a manner not to involve the gas in the upper part of the tank, or a device that can automatically adjust the height of the pipe outlet from the liquid surface, a defoaming / degassing device, etc. can be provided, and an appropriate defoaming agent can also be added according to the situation.

[0268] exist Fig.14 In the purification and concentration device 2, in order to improve the fouling resistance of the hollow fiber membrane surface, and / or improve the purification and concentration efficiency and the recovery rate of extracellular vesicles, the hollow fiber membrane in the hollow fiber membrane assembly 33 can be coated with an appropriate substance by the same method as the purification and concentration device 1.

[0269] exist Fig.14 In the purification and concentration device 2, the average inner diameter of the hollow fiber membrane in the hollow fiber membrane module 33 can be appropriately adjusted by the viscosity of the liquid. In one embodiment, the average inner diameter of the hollow fiber membrane can be 0.2 to 2.0 mm. As in the purification and concentration device 1 described above, in the case of high liquid viscosity (for example, liquid temperature 20°C, shear rate 25s -1 In the case of a liquid viscosity of 10 to 30 mPa·s under the condition of a temperature of 20°C and a shear rate of 25 s / min, it is preferred to use a large-diameter hollow fiber membrane with an average inner diameter of 1.0 mm to 1.4 mm. In addition, in the case of a liquid containing a small amount of useful substances, the liquid viscosity is low during operation (for example, a liquid temperature of 20°C and a shear rate of 25 s / min). -1 In the case where the viscosity of the liquid under the condition is lower than 10mPa·s, it is preferred to use a hollow fiber membrane with an average inner diameter of 0.2mm to 1.0mm. It should be noted that if the average inner diameter of the hollow fiber membrane is large, the effective membrane area is easily reduced when the above hollow fiber membrane is used as a hollow fiber membrane module. Therefore, from the viewpoint of easily suppressing the reduction of the effective membrane area when making a hollow fiber membrane module, it is preferred to use a hollow fiber membrane with an average inner diameter of 0.2mm to 1.0mm.

[0270] exist Fig.14 In the purification and concentration device 2, in addition to the elements shown in the figure, there may be Figures 1 to 13The same elements as the purification and concentration device 1. For example, Fig.14 In the purification and concentrating device 2 of the present invention, the monitoring device for monitoring the liquid amount in the second tank 31 and the third tank 32 may include the above-mentioned tank liquid amount monitoring device 35, 36 and / or camera. The second control unit of the purification and concentrating device 2 may also be configured in the same manner as the second control unit 120 of the purification and concentrating device 1. That is, the second control unit may grasp the liquid amount in the tank based on the change in the transmittance of light detected by the optical sensor included in the tank liquid amount monitoring device 35, 36, and determine the tank to be pressurized by the pressurizing device 34. In addition, the second control unit may grasp the liquid amount in the tank based on the information on the liquid level position output by the camera, and determine the tank to be pressurized by the pressurizing device 34. In the purification and concentrating device 2, the above-mentioned monitoring device preferably includes the tank liquid amount monitoring device 35, 36.

[0271] As described above, in the purification and concentration device 2, the liquid transfer device is not limited to the pressurizing device, and can also be used alone or in combination to transfer liquid using pumping or potential energy. In this case, the second control unit can be configured to control the height of the liquid level in the second tank 31 and the third tank 32 via a lifting device, etc., or to control the height position of the liquid inlet and outlet. With such a configuration, the second control unit can control the liquid transfer device in a manner that the liquid in the second tank and the third tank is selectively supplied to the hollow fiber membrane module.

[0272] Fig.14 The purification and concentrating section 30 in the purification and concentrating device 2 may be provided with a cleaning tank that supplies the diluent to the hollow fiber membrane module 33 to clean the plurality of hollow fiber membranes in the hollow fiber membrane bundle 60. That is, the purification and concentrating section 30 of the purification and concentrating device 2 may be provided with a fourth tank ( Fig.10 Symbol 80), which stores the diluent, is connected to a flow path for supplying the above-mentioned diluent from the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33. In addition, it can be equipped with a third control unit 130 that is the same as the purification and concentration device 1. That is, when the total amount of the liquid in the second tank 31 and the third tank 32 is less than the value set by the second control unit, the third control unit in the purification and concentration device 2 can supply the diluent of the fourth tank from the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33. According to this structure, it is possible to purify and concentrate the raw material liquid while recovering the tiny useful substances deposited on the membrane surface inside the hollow fiber membrane. In addition, the fourth tank can be connected to the second tank 31 or the third tank 32.

[0273] In the purification and concentrating device 2, similarly to the purification and concentrating device 1, before the diluent is supplied (backwashed) to the hollow fiber membrane module 33, in order to prevent the permeate remaining in the hollow fiber membrane module 33 from mixing with the backwashing liquid and flowing into the concentrated liquid side, an operation of pre-rinsing with a cleaning liquid (diluent) and replacing the permeate remaining in the hollow fiber membrane module with the cleaning liquid (diluent) can be arbitrarily performed.

[0274] Fig.14 The purification and concentrating section 30 in the purification and concentrating device 2 may include a fifth tank 81 for recovering the concentrated liquid in the second tank 31, the third tank 32, and the hollow fiber membrane module 33. In addition, a fourth control unit for controlling the recovery of the concentrated liquid to the fifth tank 81 may be provided. The configuration of the fifth tank 81 and the fourth control unit may be the same as that of the purification and concentrating device 1.

[0275] The tiny useful substances purified and concentrated by the purification and concentration device of the second embodiment may be the same as or different from the substances described in the purification and concentration device 1. The tiny useful substances purified and concentrated by the purification and concentration device of the second embodiment may include tiny useful substances for industrial use such as electroactive polymers, rare metal colloidal particles, pigments, dyes, inorganic or organic nanoparticles, etc. Generally speaking, when a liquid containing these tiny useful substances for industrial use is purified and concentrated, it is not necessary to remove impurities in the liquid. Therefore, the device of the second embodiment of the present disclosure can be used appropriately.

[0276] [Method for producing a liquid obtained by purifying and concentrating minute useful substances: Second embodiment]

[0277] As a method for producing a liquid obtained by purifying and concentrating minute useful substances using the purification and concentrating device 2 of the present disclosure, there can be exemplified a method in which the impurity removal process is omitted in the production method using the purification and concentrating device 1 described above. It should be noted that the purification and concentrating device 2 is a configuration without the removal unit 20, but sometimes includes pre-treatment processes such as pre-filtration using a microfiltration membrane or the like, and coagulation and precipitation treatment.

[0278] exist Fig.14 In the purification and concentration device 2, after the raw material liquid is put into the container in the raw material supply part 10, when the switch valve 210 is opened, the raw material liquid is supplied to the second tank 31 (or the third tank 32) through the flow path 201. The raw material liquid supplied to the second tank 31 (or the third tank 32) is purified and concentrated by alternating tangential flow filtration. After the purification and concentration process, the concentrated liquid in the tank and the hollow fiber membrane module is recovered to the fifth tank 81.

[0279] [Other embodiments: Molecular weight fractionation and purification and concentration of minute useful substances]

[0280] Another embodiment of the manufacturing method of the second embodiment of the present disclosure is a method for manufacturing a purified concentrated liquid, which includes molecular weight fractionation of a tiny useful substance and purification and concentration treatment. As a method for implementing molecular weight fractionation of a tiny useful substance into two or more components and concentrating and purifying each liquid, a method of connecting the purification and concentration device 2 of the present disclosure having two or more hollow fiber membrane modules with two or more molecular weight cutoffs, or a method of performing purification and concentration treatment in two or more steps can be exemplified.

[0281] Specifically, in the case of producing a purified concentrated solution of a liquid containing chondroitin sulfate having a wide molecular weight distribution, a purified (molecular weight purified) concentrated solution of chondroitin sulfate having three molecular weight distributions can be obtained by connecting a device having a molecular weight cutoff of 10,000 and a device having a molecular weight cutoff of 70,000, or performing a purification and concentration operation in two or more steps. In this case, the concentration of components having a molecular weight cutoff of less than 10,000 can be performed by concentrating the permeate of the device having a molecular weight cutoff of 10,000 using a device other than a membrane separation device, or by using a NF membrane concentration device (e.g., a device using a membrane described in Japanese Patent No. 7021400).

[0282] Example

[0283] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following description.

[0284] <Production of hollow fiber membrane module 33>

[0285] A hollow fiber membrane bundle 60 consisting of 75 cellulose acetate (CA) hollow fiber membranes (manufactured by DAICEN MEMBRANE-SYSTEMS) with an average inner diameter of 0.8 mm, an average outer diameter of 1.3 mm, and a molecular weight cutoff of 300,000 was placed in a polycarbonate cylindrical container 61 to prepare a Figure 7 The internal pressure hollow fiber membrane module described in . The pure water permeation rate of the hollow fiber membrane module 33 at 0.1 MPa is 28.8 L / Hr. In addition, the γ-globulin permeability measured by the following measurement method is 54%, the effective length of the hollow fiber membrane is 17 cm, and the effective membrane area is 0.0312 m 2 , the filling rate of the hollow fiber membrane is 30%.

[0286] (γ-globulin permeability measurement)

[0287] use Fig.15 The γ-globulin permeability measuring device 300 was used to measure the γ-globulin permeability by the following operation.

[0288] 1) A γ-globulin aqueous solution (γ-globulin concentration: 100 ppm) is placed in the tank 302 .

[0289] 2) Connect the hollow fiber membrane 303 to the flow path 305 .

[0290] 3) Nitrogen gas was injected from valve V-1 to apply a pressure of 0.1 MPa to tank 302. The aqueous solution was filtered from the inner surface to the outer surface of hollow fiber membrane 303, the filtrate was discarded to container 307, and the permeate was recovered to container 306. It should be noted that the initial permeate (about 100 mL) was discarded, and the permeate discharged later was used as a sample for permeability measurement.

[0291] After the permeate sample was prepared by the apparatus 300, the γ-globulin permeability was measured by the following method.

[0292] 4) The absorbance at 280 nm (A1) of the γ-globulin aqueous solution (100 ppm) and the absorbance at 280 nm (A2) of the permeate were measured using a spectrophotometer (manufactured by Shimadzu Corporation, product name "UV2450"), and the γ-globulin transmittance (%) was calculated using the calculation formula (A2 / A1×100).

[0293] <Purification and concentration device 1>

[0294] The following uses Fig.13 The specific operation of the purification and concentration device 1 will be described.

[0295] The elements including the raw material supply unit 10 , the removal unit 20 , and the purification and concentration unit 30 are arranged in a cover 100 (not shown) having a width of 800 mm, a depth of 350 mm, and a height of 1745 mm.

[0296] A fan filter device 91 (manufactured by AS ONE, product name "PURESPACE 01", with ULPA filter unit, air volume 1m 3 / min) (not shown), and the filtered air is configured to flow downward into the cover 100.

[0297] Furthermore, four UV lamps 92 (manufactured by Yamato Electric Industry Co., Ltd., glow type sterilizing lamps) are installed in the cover 100 (not shown).

[0298] A movable caster (not shown) with a free stopper is installed at the bottom of the purification and concentration device 1 .

[0299] A door (not shown) is disposed in front of the purification and concentrating device 1 , and a touch panel (manufactured by KEYENCE, product name “Touch Panel Display Model: VT-W4M”) for managing the process of the purification and concentrating device 1 is attached to the door (not shown).

[0300] The purification and concentration device 1 is provided with a compressed air inlet (not shown) and a DC power supply (not shown). The compressed air is depressurized to 0.05 MPa by a regulator, and the supply destination of the liquid is switched and the supply is controlled through a plurality of switch valves (two-way solenoid valves), three-way valves (three-way solenoid valves), and solenoid valve manifolds in order to perform cleaning, balancing, impurity removal, purification and concentration, and reverse cleaning of the carrier column 21. In addition, the compressed air depressurized to 0.3 MPa by a regulator is also used for the pneumatic clamp when recovering the concentrated liquid.

[0301] (Raw material supply unit 10)

[0302] A container 11 for storing the raw material liquid is provided.

[0303] (Removal portion 20)

[0304] The carrier column 21 was configured such that 5 mL of an adsorbent carrier (manufactured by Cytiva, product name “Capto™ Core 700” (17548101)) was sealed in a column having an inner diameter of 25 mmφ.

[0305] As the filter member 22, a microfiltration membrane (manufactured by Millipore Corporation, product name "Millex-GP", material: polyethersulfone, pore size: 0.22 μm) was used.

[0306] A 2M sodium chloride aqueous solution is put into the sixth tank 25 as a cleaning liquid.

[0307] A 50 mM Tris-HCl (pH 7.2) solution as an equilibration solution was added to the seventh tank 26 .

[0308] like Fig.13 As shown, the liquid is supplied from the tank 24, the sixth tank 25, and the seventh tank 26 to the carrier column 21 through the flow path 41A via a plurality of on-off valves (solenoid valves). A tube pump (not shown) is disposed in the flow path 41A.

[0309] (Purification and Concentration Section 30)

[0310] As the hollow fiber membrane, the hollow fiber membrane module 33 produced as described above was used.

[0311] Transparent containers made of poly(meth)acrylate (capacity 120 mL) were used for the second tank 31 and the third tank 32. Tank liquid level monitoring devices 35 and 36 (Keyence, pipeline-mounted liquid level detection fiber unit (model: FU-95S)) including optical sensors were arranged at the second tank 31 and the liquid inlet and outlet at the bottom of the second tank 31.

[0312] Phosphate buffered saline (PBS) is added to the fourth tank 80 as a diluent.

[0313] [Example 1]

[0314] As a raw material liquid, 800 mL of a culture supernatant of mesenchymal stem cells containing exosomes prepared by the following method was placed in the container 11 .

[0315] <Preparation of culture supernatant of mesenchymal stem cells containing exosomes>

[0316] Human adipose tissue-derived mesenchymal stem cells were cultured using Ultra ExoMCulture Medium for Extracellular Vesicles (FK-K0204024, Santeja).

[0317] When the cells covered about 80% of the wall surface of the culture vessel, the medium was replaced with Ultra ExoMCultureMedium containing no phenol red and culture was continued for 48 hours.

[0318] Then, the culture supernatant was centrifuged at 2000×g for 10 minutes at 4° C. to remove cell debris and prepare a culture supernatant.

[0319] The raw material liquid in the container 11 is supplied to the filtering member 22 to be pretreated in its entirety, and then stored in the tank 24 (pretreatment liquid storage tank).

[0320] Next, 50 mL of the cleaning solution is supplied from the sixth tank 25 (cleaning solution storage tank) to the carrier column 21 at 5 mL / min, and the cleaning solution is discharged to the waste liquid tank after the carrier column 21 is cleaned. Next, 50 mL of the equilibration solution is supplied from the seventh tank 26 (equilibration solution storage tank) to the carrier column 21 at 5 mL / min, and the equilibration solution is discharged to the waste liquid tank after the carrier column 21 is equilibrated.

[0321] <Impurity Removal Process>

[0322] 50 mL of the pretreatment liquid in the tank 24 is supplied to the carrier column 21, and host-derived proteins (HCPs), DNA, etc. in the pretreatment liquid are removed by carrier adsorption. The obtained crude purified liquid is supplied to the first tank 23 via the flow path 42A. Then, the second cleaning and balancing treatment of the carrier column 21 is carried out under the same conditions as above. After the second cleaning and balancing treatment, 50 mL of the pretreatment liquid in the tank 24 is supplied to the carrier column 21 for the second impurity removal treatment. Then, the third cleaning, balancing treatment and third impurity removal treatment of the carrier column 21 are carried out in the same manner. Next, the 4th to 16th cleaning, balancing treatment and 4th to 16th impurity removal treatment of the carrier column 21 are carried out.

[0323] <Purification and concentration treatment>

[0324] The impurity removal process and the purification and concentration process are performed in parallel.

[0325] First, the crude purified liquid (about 200 mL) in the first tank 23 stored by the 1st to 4th impurity removal treatment is sent to the second tank 31 by compressed air of 0.05 MPa. At this time, after the crude purified liquid (about 50 mL) of the first time is sent to the second tank 31, the second tank 31 is pressurized by the pressurizing device 34, and the liquid volume of the management liquid level position C is set to 10 mL, so that the liquid volume of the second tank 31 is not less than 10 mL, and the purification and concentration treatment based on alternating tangential flow filtration (TFF) is implemented. Then, the crude purified liquid of the 2nd to 4th times is sent to the second tank 31 in sequence, and the purification and concentration treatment based on alternating tangential flow filtration (TFF) is continued in a manner that the liquid volume of the second tank 31 is not less than 10 mL.

[0326] Next, a backwash treatment is performed. A 90 mL diluent is used for the backwash treatment. In addition, a phosphate buffer solution (PSB) is used as the diluent. After the backwash treatment is performed using the diluent, a purification and concentration treatment based on alternating tangential flow filtration (TFF) is performed in a manner that the liquid volume of the second tank 31 is not less than 10 mL. Then, the crude purified liquid obtained by the 5th to 8th impurity removal treatment, the crude purified liquid of the 9th to 12th times, and the crude purified liquid of the 13th to 16th times are respectively filled into the second tank 31, and a purification and concentration treatment and a backwash treatment are performed. It should be noted that a backwash treatment is performed whenever the liquid delivery volume of the crude purified liquid after the above-mentioned backwash treatment reaches 200 mL, and the purification and concentration treatment is performed by alternating tangential flow filtration (TFF) in a manner that the liquid volume of the second tank 31 is not less than 10 mL. After a total of 800 mL of the crude purified liquid was purified and concentrated and backwashed and the liquid volume in the second tank 31 reached 10 mL, the backwashing and purification concentration treatments were repeated three times with the dilution volume set to 90 mL (10-fold dilution) to obtain a purified concentrated liquid of exosomes.

[0327] <Recycling>

[0328] The obtained purified concentrated solution of exosomes is recovered in its entirety to the fifth tank 81 via the flow path 56. The recovery process of the concentrated solution is performed by monitoring the amount of concentrated solution in the flow path 56 through a fiber sensor provided at the inlet and outlet of the liquid at the upper part of the fifth tank 81, and the concentrated solution in the two tanks is recovered to the fifth tank 81 before the fiber sensor detects bubbles. The yield of the purified concentrated solution of exosomes obtained at this time is 0.8 to 80 mL, and the recovery rate of exosomes is 50 to 100%. In addition, it is assumed that the protein removal rate is 80% or more.

[0329] [Example 2]

[0330] As the raw material liquid, 800 mL of culture supernatant of mesenchymal stem cells derived from human adipose tissue was used. The protein concentration and CD9 / CD63 positive extracellular vesicles in the culture supernatant were quantified by Bradford method and ELISA method, respectively, and the results showed that the protein concentration was 0.49 mg / mL and 152 pg / mL.

[0331] To the above culture supernatant, 80 mL of a mixed mode carrier with cation exchange and size exclusion balanced with a 50 mM Tris-HCl (pH 7.2) solution was added, and after being dispensed into a 50 mL tube, the mixture was poured and mixed at room temperature at a rotation speed of 10 rpm / min using a rotator for 10 minutes. Then, the supernatant was centrifuged at room temperature for 5 minutes at 10,000 x g, and further filtered using a 0.22 μm filter unit to remove the carrier. The protein concentration in the culture supernatant 790 mL after the carrier treatment was obtained was 0.06 mg / mL, and the exosome concentration was 144 pg / mL.

[0332] Next, the culture supernatant after carrier treatment was supplied from the container 11 to the second tank 31 via the flow path 43, and the pressurization condition of the pressurizing device 34 was set to a nitrogen pressure of 0.04 MPa, and alternating tangential flow filtration was performed. When the residual amount reached 10 mL, 90 mL of phosphate buffer (PBS) was supplied from the fourth tank 80 from one side of the hollow fiber membrane assembly 33 for backwashing. Then, after further repeating the alternating tangential flow filtration and the supply of 90 mL of phosphate buffer twice, 12.9 mL of purified concentrated solution was obtained by alternating tangential flow filtration. The protein concentration of the purified concentrated solution obtained was 0.09 mg / mL, and the exosome concentration was 7149 pg / mL. The protein removal rate calculated based on the concentration and liquid volume was more than 99%, and the recovery rate was 76%.

[0333] [Comparative Example 1]

[0334] As Comparative Example 1, 800 mL of culture supernatant with a protein concentration of 0.52 mg / mL and an exosome concentration of 130 pg / mL was used as the raw material solution, and the raw material solution was supplied from the container 11 to the second tank 31 via the flow path 43, and a purification and concentration treatment based on alternating tangential flow filtration was performed. It should be noted that in Comparative Example 1, the cleaning process in the process was performed by forward cleaning instead of reverse cleaning, and the purification and concentration treatment was performed under the same conditions as Example 2. Forward cleaning was implemented by supplying the dilution in the fourth tank 80 to the third tank 32 via the flow path 55B and then supplying the dilution to the hollow fiber membrane module 33 to clean the hollow fiber membrane. Finally, 11 mL of purified concentrated solution was obtained. In addition, the protein concentration in the purified concentrated solution was less than 0.02 mg / mL, and the exosome concentration was 2595 pg / mL. The protein removal rate calculated based on the concentration and liquid volume was more than 99%, and the recovery rate was 27%. According to the results, it can be seen that the recovery rate can be improved by performing reverse cleaning.

[0335] [Example 3]

[0336] Here, a method of further improving the recovery rate of minute useful substances by coating the surface of a hollow fiber membrane with biomolecules in the purification and concentration step was studied.

[0337] As the raw material solution, 790 mL of culture supernatant of mesenchymal stem cells having a protein concentration of 0.49 mg / mL and an exosome concentration of 166.4 pg / mL was used.

[0338] The concentration of exosomes is equivalent to the amount of CD9 / CD63 fusion standard protein used to prepare a calibration curve when performing quantitative analysis using the CD9 / CD63 ELISA method (EXH0102EL, Cosmo Bio).

[0339] 79 mL of a carrier (a porous granular material with positively charged pores inside, capable of adsorbing and retaining negatively charged unnecessary substances other than extracellular vesicles) equilibrated with 50 mM Tris-HCl (pH 7.2) solution was added to 790 mL of the culture supernatant, and mixed inverted at room temperature for 10 minutes using a rotator. Then, the carrier was removed from the carrier-cell supernatant mixture using a 0.22 μm filter unit (S2GPU11RE, Merck Millipore) to obtain a culture supernatant treated with the carrier.

[0340] Next, the culture supernatant treated with the carrier was filtered, purified, and concentrated by an alternating tangential flow method at a filtration pressure of 30 to 40 kPa. The filtration membrane used was a cellulose acetate hollow fiber membrane module (molecular weight cutoff 300,000, membrane area 0.062 m 2). The filter membrane used was a filter membrane with a coating treatment on the membrane surface with biomolecules. The coating treatment was performed by passing a 1.3 mg / mL skim milk solution dissolved in PBS through the filter membrane at a flow rate of 200 mL / min for 10 minutes. Thus, a coating treatment membrane was obtained in which a certain biomolecule in the skim milk was adsorbed and bound in the form of a thin layer on the membrane surface. Then, the filter membrane was washed with 250 mL of PBS to remove the excess skim milk deposited only on the thin layer.

[0341] Here, the binding state between the filtration membrane and the certain biomolecules in the skim milk is so strong that the filtration membrane does not fall off under water washing and operation conditions, and the filtration performance is hardly reduced even after long-term operation.

[0342] Repeat alternating tangential flow filtration until the volume of the concentrate reaches 10 mL, and then 90 mL of PBS is supplied to the hollow fiber membrane assembly to dilute the concentrate. After further repeating the concentration filtration / dilution process twice, 11.9 mL of purified concentrate (extracellular vesicle purification) was obtained by alternating tangential flow filtration. The extracellular vesicles in the final exosome concentrate were quantitatively analyzed by CD9 / CD63 ELISA (EXH0102EL, Cosmo Bio), and the result was 3746.7 pg / mL, equivalent to the amount of CD9 / CD63 fusion standard protein used to make the calibration curve, with a recovery rate of 33.9%.

[0343] [Comparative Example 2]

[0344] The same operation was carried out using a hollow fiber membrane that had not been coated with skim milk, and the recovery rate was 7.4%.

[0345] The results of Example 3 and Comparative Example 2 show that the recovery rate of useful micro-substances is improved by using a hollow fiber membrane coated with biomolecules (i.e., a hollow fiber membrane having biomolecules of a different type from that of useful micro-substances attached to at least a portion of the membrane).

[0346] [Example 4]

[0347] Here, a method for further improving the purity of the minute useful substances in the purified concentrated liquid obtained in the purification and concentration step was studied.

[0348] As the raw material solution, 40 mL of culture supernatant of amniotic mesenchymal stem cells with a protein concentration of 1.49 mg / mL and an exosome concentration of 196 pg / mL was used. A cellulose acetate hollow fiber membrane module 33 (molecular weight cutoff 300,000, membrane area 0.01 m 2, a purification and concentration device with a permeate side volume (i.e., 10 mL of the hollow fiber external space (ECS: Extracapillary Space)), separated, purified and concentrated the exosomes in the culture supernatant by an alternating tangential flow method with a filtration pressure of 30 to 40 kPa. After the culture supernatant was concentrated to about 10 mL, 40 mL of a diluent (PBS) was injected from the inlet and outlet 33c of the third liquid of the hollow fiber membrane module and discharged to the inlet and outlet 33d of the third liquid. Thus, the impurities in the permeate retained in the ECS were rinsed by PBS. Then, 90 mL of the same diluent (PBS) was pressed from the permeate side of the hollow fiber membrane module 33 into the concentration side, and reverse pressure cleaning (backwashing) was performed. The separation and purification operation was performed again under the same conditions, and the same flushing and backwashing operations were further performed twice at the moment when the culture supernatant reached about 10 mL. After three backwashings, the culture supernatant was concentrated to 5.5 mL. The amount of protein remaining in the purified concentrate was measured by BCA assay, and the result was a protein concentration of 0.16 mg / mL. Fig.17 In addition, the absorbance of the purified concentrate was measured using the TECAN absorbance microplate reader Infinite M Nano+. The results are shown in Fig.18 .

[0349] [Comparative Example 3]

[0350] In the same separation and purification operation as in Example 4, the culture supernatant was concentrated to 5.2 mL without the rinsing operation in the ECS using PBS. The amount of protein remaining in the purified concentrate obtained was measured by BCA assay, and the result was a protein concentration of 0.36 mg / mL. The results are shown in Fig.17 In addition, the absorbance of the obtained purified concentrated solution was measured in the same manner as in Example 4. The results are shown in Fig.18 .

[0351] exist Fig.17 , 18 In the table, "Before" refers to the sample before purification and concentration, "with ECF" refers to the purified concentrated solution obtained by the method of Example 4, and "without ECS" refers to the purified concentrated solution obtained by the method of Comparative Example 3. Fig.17 It is clear that according to the method of Example 4, the concentration of protein as an impurity is further reduced, and the purity of exosomes can be further improved. Fig.18 The peak at a wavelength of 557 nm is derived from phenol red contained in the culture medium as a pH indicator, which corresponds to the impurities in the purified concentrate. Fig.18As shown, according to the method of Example 4, the concentration of phenol red as an impurity is further reduced, and the purity of exosomes can be further improved.

[0352] According to these results, by washing (flushing) the ECS with a diluent such as PBS or saline before the backwashing process of the hollow fiber membrane module to remove impurities remaining in the ECS, the concentration of impurities in the obtained purified concentrated solution can be further reduced, and as a result, the purity of the tiny useful substances can be further improved. As a non-limiting reason, it can be considered that the reason is that by flushing, the impurities in the ECS can be prevented from being mixed into the purified concentrated solution in the subsequent backwashing process using a diluent.

[0353] [Example 5]

[0354] As the raw material solution, 200 mL of culture supernatant of amniotic mesenchymal stem cells with a protein concentration of 1.25 mg / mL and an exosome concentration of 138 pg / mL was used. A cellulose acetate hollow fiber membrane module (molecular weight cutoff 300,000, membrane area 0.01 m 2 , ESC10mL), the exosomes in the culture supernatant were separated, purified and concentrated by alternating tangential flow. After the culture supernatant was concentrated to about 17mL, 40mL of diluent (PBS) was injected from the inlet and outlet 33c of the third liquid of the hollow fiber membrane component and discharged to the inlet and outlet 33d of the third liquid. Thus, the impurities in the permeate retained in the ECS were rinsed by PBS. Then, 160mL of the same diluent (PBS) was pressed from the permeate side of the hollow fiber membrane component 33 into the concentration side, and reverse pressure cleaning (backwashing) was performed. The separation and purification operation was performed again under the same conditions, and the same flushing and backwashing operations were further implemented twice at the moment when the culture supernatant reached about 17mL. The culture supernatant after backwashing was concentrated to 17mL. Backwashing was performed again to wash out the tiny useful substances attached to the surface of the hollow fiber membrane in the purified concentrated liquid, and 50mL of the purified concentrated liquid was recovered. The extracellular vesicles in the purified concentrate were quantitatively analyzed by CD9 / CD63 ELISA (EXH0102EL, CosmoBio), and the result was 486pg / mL, which was equivalent to the amount of CD9 / CD63 fusion standard protein used to prepare the calibration curve, and the recovery rate was 88%. According to this result, by further supplying a diluent to the hollow fiber membrane module before recovering the purified concentrate and performing an elution operation of tiny useful substances, the recovery rate of tiny useful substances can reach more than 80%.

Claims

1. A purification and concentration device for a liquid containing minute useful substances, comprising: A raw material supply unit that supplies a liquid containing tiny useful substances, a removal unit for removing impurities in the liquid, a purification and concentration section for purifying and concentrating the liquid supplied from the raw material supply section or the removal section using a hollow fiber membrane, and A first control unit controls a timing of the flow of the liquid from the raw material supply unit to the purification and concentration unit or a timing of the flow of the liquid from the removal unit to the purification and concentration unit.

2. The purification and concentration device according to claim 1, wherein: The removal unit includes a carrier column for removing impurities in the liquid, and the carrier column is connected to a flow path for supplying the liquid from which impurities have been removed to the purification and concentration unit.

3. The purification and concentration device according to claim 1 or 2, wherein: The removal unit includes a filter member for removing impurities in the liquid. The filter member is inserted in a flow path that supplies the liquid from the raw material supply unit to the removal unit.

4. The purification and concentration device according to claim 2, wherein: The removal unit includes a first tank for storing the liquid processed by the carrier column. The first tank is arranged in the flow path for supplying the liquid from the carrier column to the purification and concentration section, and is connected to the flow path for supplying the liquid in the tank to the purification and concentration section. The first control unit controls a timing for supplying the liquid in the tank to the purification and concentrating unit.

5. The purification and concentration device according to claim 1 or 2, wherein: The average inner diameter of the hollow fiber membrane is 0.2-1.4 mm, and the molecular weight cut-off is 10,000-1,000,000.

6. The purification and concentration device according to claim 1, wherein: The purification and concentration section includes a hollow fiber membrane module in which a plurality of the hollow fiber membranes are housed in a container having an inlet and an outlet for the liquid. The container has at least a first liquid inlet and outlet and a second liquid inlet and outlet for supplying the liquid to the hollow fiber membrane, and a third liquid inlet and outlet for discharging the liquid having passed through the hollow fiber membrane.

7. The purification and concentration device according to claim 6, wherein: The hollow fiber membrane module is of internal pressure type.

8. The purification and concentration device according to claim 6, wherein: The purification and concentration section comprises: a second tank storing the liquid supplied from the raw material supply unit or the removal unit, and connected to a flow path for supplying the liquid to an inlet and outlet of the first liquid of the hollow fiber membrane module; a third tank storing the liquid supplied from the raw material supply unit or the removal unit, and connected to a flow path for supplying the liquid to the inlet and outlet of the second liquid of the hollow fiber membrane module; a pressurizing device for selectively pressurizing any of the second tank or the third tank; and The second control unit controls the second tank or the third tank to be pressurized selectively by the pressurizing device so as to alternately supply the liquid in the second tank and the third tank to the hollow fiber membrane module.

9. The purification and concentration device according to claim 8, wherein: The second control unit sets the liquid amounts in the second tank and the third tank, and alternately pressurizes the second tank and the third tank using the pressurizing device so that the liquid amounts in the second tank and the third tank reach the set values.

10. The purification and concentration device according to claim 9, wherein: The second tank and the third tank are respectively provided with a tank liquid amount monitoring device including an optical sensor at the inlet and outlet of the liquid at the bottom of the tank. The tank liquid amount monitoring device is configured to irradiate light into the liquid inlet and outlet through the optical sensor and detect the transmittance of the light, and monitor the liquid amount in the tank based on changes in the transmittance. The second control unit determines the liquid amounts in the second tank and the third tank by the tank liquid amount monitoring device, and determines the tank to be pressurized by the pressurizing device.

11. The purification and concentration device according to claim 9, wherein: The purification and concentration section includes a camera for monitoring the liquid level positions in the second tank and the third tank. The second control unit determines the tank to be pressurized by the pressurizing device based on the information on the positions of the liquid levels in the second tank and the third tank output from the camera.

12. The purification and concentration device according to claim 8, wherein: The purification and concentration section comprises: a fourth tank storing a diluent, the fourth tank being connected to a flow path for supplying the diluent to the inlet and outlet of the third liquid of the hollow fiber membrane module; and a third control unit for controlling a timing of supplying the diluent in the fourth tank to the inlet and outlet of the third liquid of the hollow fiber membrane module, When the total amount of liquid in the second tank and the third tank becomes equal to or less than a value set by the second control unit, the third control unit supplies the diluent in the fourth tank from the inlet and outlet of the third liquid of the hollow fiber membrane module.

13. The purification and concentration device according to claim 8, wherein: The purification and concentration section comprises: a fifth tank connected to the inlet and outlet of the first liquid or the inlet and outlet of the second liquid of the hollow fiber membrane module through a flow path, and recovering the liquid in the second tank and the third tank; and The fourth control unit controls the recovery of the liquid into the fifth tank.

14. The purification and concentration device according to claim 13, wherein: The second tank and the third tank are respectively provided with a tank liquid amount monitoring device including an optical sensor at the inlet and outlet of the liquid at the bottom of the tank. The tank liquid amount monitoring device is configured to irradiate light into the liquid inlet and outlet through the optical sensor and detect the transmittance of the light, and monitor the liquid amount in the tank based on changes in the transmittance. The fourth control unit selectively pressurizes the second tank or the third tank through the pressurizing device, recovers the liquid in the second tank and the third tank to the fifth tank, determines the liquid amount in the second tank and the third tank through the tank liquid amount monitoring device, and stops the pressurizing device.

15. The purification and concentration device according to claim 13, wherein: The purification and concentration section includes a camera for monitoring the liquid level positions in the second tank and the third tank. The fourth control unit selectively pressurizes the second tank or the third tank through the pressurizing device, recovers the liquid in the second tank and the third tank to the fifth tank, and stops the pressurizing device based on the information of the liquid level position in the second tank and the third tank output from the camera.

16. The purification and concentrating device according to claim 1 or 2, wherein the elements including the raw material supply unit, the removal unit, and the purification and concentrating unit are arranged in a cover, and a management unit for managing the cleanliness in the cover is provided. The management unit includes a fan filter device that supplies filtered air into the cover.

17. The purification and concentration device according to claim 16, wherein: The management unit further includes a UV lamp.

18. The purification and concentration device according to claim 2, wherein: The removing portion comprises: a sixth tank storing a cleaning solution for cleaning the carrier column; and The seventh tank stores an equilibration liquid for equilibrating the support column.

19. The purification and concentration device according to claim 3, wherein: The filter member is disposed at a lower portion of the raw material supply portion, and the liquid is supplied from the raw material supply portion to the filter member by the liquid's own weight. 20 . The purification and concentrating device according to claim 8 , wherein the second tank and the third tank are arranged such that the side surfaces in the longitudinal direction are parallel to the longitudinal direction of the hollow fiber membrane module.

21. The purification and concentration device according to claim 20, wherein: The removing portion comprises: a carrier column for removing impurities from the liquid, and connected to a flow path for supplying the liquid from which the impurities have been removed to the purification and concentration section; and a first tank storing the liquid processed by the carrier column, The second tank and the third tank are connected to the carrier column and the first tank through a flow path, and are arranged above the carrier column and the first tank.

22. The purification and concentration device according to claim 1 or 2, wherein: The tiny useful substances are selected from extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids.

23. A method for producing a purified concentrated liquid of a minute useful substance, which is a method for producing the liquid obtained by purifying and concentrating the minute useful substance using the purification and concentrating device according to claim 1 or 2, the method comprising: The step of supplying the raw material liquid containing the minute useful substance to the purification and concentration device to remove impurities in the liquid; and / or, a step of purifying and concentrating the liquid by means of a hollow fiber membrane.

24. The method for producing a purified concentrated solution of minute useful substances according to claim 23, wherein: The step of removing impurities from the liquid includes: removing the impurities from the liquid by adsorption through a carrier column.

25. The method for producing a purified concentrated solution of minute useful substances according to claim 23, wherein: The step of purifying and concentrating the liquid through the hollow fiber membrane comprises: performing alternating tangential flow filtration on the liquid through the hollow fiber membrane module.

26. The method for producing a purified concentrated solution of minute useful substances according to claim 23, wherein: The step of purifying and concentrating the liquid through the hollow fiber membrane includes: recovering the tiny useful substances deposited on the surface of the hollow fiber membrane while performing alternating tangential flow filtration on the liquid through the hollow fiber membrane module.

27. The method for producing a purified concentrated solution of minute useful substances according to claim 23, wherein: The step of purifying and concentrating the liquid by the hollow fiber membrane comprises: supplying a diluent to the concentrated liquid to further purify and concentrate the liquid; When the amount (B) of the concentrated liquid relative to the amount (A) of the raw material liquid (B / A) reaches 1 / 2 to 1 / 200, the diluent is supplied to the liquid.

28. The method for producing a purified concentrated solution of minute useful substances according to claim 24, wherein: The step of removing impurities from the liquid by the support column and the step of purifying and concentrating the liquid by the hollow fiber membrane are performed in parallel.

29. The method for producing a purified concentrated solution of minute useful substances according to claim 23, wherein: The tiny useful substance is at least one selected from extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids.

30. A device for purifying and concentrating a liquid containing minute useful substances, comprising: A raw material supply unit for supplying a liquid containing a tiny amount of useful substances, and a purification and concentration section for purifying and concentrating the liquid supplied from the raw material supply section by means of a hollow fiber membrane module; The hollow fiber membrane module has a first liquid inlet and a second liquid inlet and an outlet for supplying the liquid to the hollow fiber membrane, and a third liquid inlet and an outlet for discharging the liquid after passing through the hollow fiber membrane. The purification and concentration section comprises: a second tank storing the liquid supplied from the raw material supply unit, the second tank being connected to a flow path for supplying the liquid to the inlet and outlet of the first liquid of the hollow fiber membrane module; a third tank storing the liquid supplied from the raw material supply unit and connected to a flow path for supplying the liquid to the inlet and outlet of the second liquid of the hollow fiber membrane module; A liquid feeding device, which selectively feeds any of the liquids in the second tank and the third tank to the hollow fiber membrane module; a second control unit configured to control the liquid feeding device so as to selectively supply the liquid in the second tank and the third tank to the hollow fiber membrane module; as well as a monitoring device for monitoring the amount of the liquid in the second tank and the third tank, The second control unit sets the liquid amounts in the second tank and the third tank, and controls the liquid delivery device to selectively deliver liquid from the second tank or the third tank to the hollow fiber membrane module when the liquid amounts in the second tank and the third tank reach the set values. The liquid amounts in the second tank and the third tank are determined based on information from the monitoring device.

31. The purification and concentration device according to claim 30, wherein: The average inner diameter of the hollow fiber membrane is 0.2-2.0 mm, and the molecular weight cut-off is 5,000-3,000,000.

32. The purification and concentration device according to claim 30 or 31, wherein: The monitoring device includes a camera that monitors the positions of the liquid levels in the second tank and the third tank.

33. The purification and concentration device according to claim 30 or 31, wherein: The monitoring device includes optical sensors, which are respectively arranged at the inlets and outlets of the liquid at the bottom of the second tank and the third tank, irradiate light into the inlets and outlets of the liquid and detect the transmittance of the light, and monitor the amount of the liquid in the tank based on the change of the transmittance.

34. The purification and concentration device according to claim 30 or 31, wherein: The hollow fiber membrane module is of internal pressure type.

35. The purification and concentration device according to claim 30, wherein: The purification and concentration section comprises: a fourth tank storing a diluent, the fourth tank being connected to a flow path for supplying the diluent to the inlet and outlet of the third liquid of the hollow fiber membrane module; and a third control unit for controlling a timing of supplying the diluent in the fourth tank to the inlet and outlet of the third liquid of the hollow fiber membrane module, When the total amount of the liquid in the second tank and the third tank becomes equal to or less than a value set by the first control unit, the third control unit supplies the diluent in the fourth tank from the inlet and outlet of the third liquid of the hollow fiber membrane module.

36. The purification and concentration device according to claim 30 or 31, wherein: The purification and concentration section comprises: a fifth tank connected to the inlet and outlet of the first liquid or the inlet and outlet of the second liquid of the hollow fiber membrane module through a flow path, and recovering the liquid in the second tank and the third tank; and The fourth control unit controls the recovery of the liquid into the fifth tank.

37. The purification and concentration device according to claim 36, wherein: The liquid delivery device includes a pressurizing device, The fourth control unit selectively pressurizes the second tank or the third tank through the pressurizing device to recover the liquid in the second tank and the third tank to the fifth tank, and determines the liquid amount in the second tank and the third tank through the monitoring device to stop the pressurizing device.

38. The purification and concentration device according to claim 30 or 31, wherein: The tiny useful substances are selected from electroactive polymers, rare metal colloidal particles, pigments, dyes, inorganic or organic nanoparticles, proteins, hormones, cytokines, proliferation factors, angiogenesis factors, growth factors, enzymes, antibodies, plasma proteins, viruses, extracellular vesicles, yeast, yeast, cells, polysaccharides and microalgae.

39. The purification and concentration device according to claim 1, 2, 30 or 31, wherein: Biomolecules of a different type from the minute useful substance are attached to at least a portion of the hollow fiber membrane.

40. The purification and concentration device according to claim 12 or 35, wherein: The hollow fiber membrane module has at least two inlets and outlets for the third liquid. The third control unit supplies the diluent in the fourth tank from one of at least two inlets and outlets of the third liquid of the hollow fiber membrane module while stopping the flow of the liquid at the inlet and outlet of the first liquid and the inlet and outlet of the second liquid of the hollow fiber membrane module, and then supplies the diluent in the fourth tank from at least one of at least two inlets and outlets of the third liquid of the hollow fiber membrane module while making the inlet and outlet of the first liquid and the inlet and outlet of the second liquid in a state where the liquid can flow.

41. A method for producing a purified concentrated solution of a minute useful substance, the method comprising removing impurities from a liquid of a raw material containing the minute useful substance and impurities, and then purifying and concentrating the liquid to produce a purified concentrated solution of the minute useful substance, wherein: The step of purifying and concentrating the liquid includes: purifying and concentrating the liquid using a hollow fiber membrane module, and includes: during the purification and concentration process, pressurizing a diluent from the permeate side of the hollow fiber membrane module to perform a reverse cleaning treatment on the hollow fiber membrane.

42. The method for producing a purified concentrated solution of minute useful substances according to claim 41, wherein: The hollow fiber membrane accommodated in the hollow fiber membrane module is composed of a resin material containing cellulose acetate.

43. The method for producing a purified concentrated solution of minute useful substances according to claim 41, wherein: The molecular weight cutoff of the hollow fiber membrane accommodated in the hollow fiber membrane module is 100,000 to 1,000,000.

44. The method for producing a purified concentrated solution of a minute useful substance according to claim 41 or 42, wherein: The step of removing impurities from the liquid includes: removing the impurities from the liquid by adsorption through a carrier column.

45. The method for producing a purified concentrated solution of a minute useful substance according to claim 41 or 42, wherein: The step of purifying and concentrating the liquid using the hollow fiber membrane module comprises: performing alternating tangential flow filtration on the liquid.

46. ​​The method for producing a purified concentrated solution of a minute useful substance according to claim 41 or 42, wherein: The step of purifying and concentrating the liquid using the hollow fiber membrane module includes: supplying a diluent to the purified and concentrated liquid and further purifying and concentrating it, and supplying the diluent to the liquid when the amount (B) of the purified and concentrated liquid reaches 1 / 2 to 1 / 200 relative to the amount (A) of the raw material liquid (B / A).

47. The method for producing a purified concentrated solution of minute useful substances according to claim 44, wherein: The step of removing impurities in the liquid by the support column and the step of purifying and concentrating the liquid using the hollow fiber membrane module are performed in parallel.

48. The method for producing a purified concentrated solution of a minute useful substance according to claim 41 or 42, wherein: The tiny useful substance is at least one selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.

49. The method for producing a purified concentrated solution of a minute useful substance according to claim 41 or 42, wherein: Biomolecules of a different type from the minute useful substance are attached to at least a portion of the hollow fiber membrane.

50. A method for producing a purified concentrated solution of minute useful substances according to claim 41 or 42, the method comprising: The hollow fiber membrane module is flushed before the backwashing treatment.

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