Purifying and concentrating device for liquid comprising minute useful substance and method for producing purified and concentrated liquid of minute useful substance using same

Abstract

Provided are: a purifying and concentrating device for a liquid comprising a minute useful substance, the device being capable of producing a liquid (purified concentrated liquid of a minute useful substance) comprising a minute useful substance having high purity and high concentration and capable of preparing said purified and concentrated liquid more efficiently; and a method for producing a purified and concentrated liquid of a minute useful substance using said device.　The purifying and concentrating device for the liquid comprising the minute useful substance, the device comprising: a starting material supply part for supplying liquid comprising the minute useful substance; a removing part for removing impurities in the liquid; a purifying and concentrating part for purifying the liquid supplied from the starting material supply part or the removing part using a hollow fiber membrane and concentrating the liquid; and a first control part for controlling the timing of flow of the liquid from the starting material supply part to the purifying and concentrating part or flow of the liquid from the removing part to the purifying and concentrating part.

Description

Apparatus for purifying and concentrating liquid containing useful micro-substances and method for producing purified and concentrated liquid of useful micro-substances using the same

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

[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 substances, for example, a method using a separation membrane is known.

[0003] Patent Document 1 describes a method for recovering useful substances, which includes a bleeding step of discharging a culture medium from a cell culture tank and adding an equal amount of fresh medium to the culture tank as the discharged culture medium, and a filtration step of filtering the culture medium extracted from the culture tank using a porous membrane that is substantially free of a dense layer, wherein the filtration in the filtration step is tangential flow filtration and the velocity of the permeate in the filtration step is 1.0 LMH or less. Patent Document 1 also describes that the minute useful substances may be selected from the group consisting of proteins, viruses, exosomes, and nucleic acids.

[0004] Furthermore, Patent Document 2 describes an exosome extraction device and an exosome extraction method that can efficiently collect large amounts of exosomes, a type of extracellular vesicle. The exosome extraction device described in Patent Document 2 sends a raw material liquid containing exosomes supplied from a raw material supply unit to a first reservoir, and then circulates the liquid from the first reservoir through a circulation path that includes a first filter and a second filter, each of which has a different function, to concentrate the exosomes in the liquid.

[0005] JP 2018-76291 A JP 2021-145649 A

[0006] Generally, raw liquids containing useful micro-substances often contain impurities. Therefore, there is a demand for a purification and concentration apparatus that can prepare a liquid containing useful micro-substances at high purity and high concentration (a purified and concentrated solution of useful micro-substances). There is also a demand for a purification and concentration apparatus that can prepare the purified and concentrated solution more efficiently.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a purification and concentration device for liquids containing micro-useful substances, which is capable of producing a liquid containing micro-useful substances at high purity and high concentration (a purified and concentrated liquid of micro-useful substances) and which can prepare the purified and concentrated liquid more efficiently, and a method for producing a purified and concentrated liquid of micro-useful substances using the same.

[0008] To solve the above problems, the present disclosure can employ the following means. [1] An apparatus for purifying and concentrating a liquid containing useful micro substances, comprising: a raw material supply unit that supplies a liquid containing useful micro substances; a removal unit that removes impurities from the liquid; a purification and concentration unit that purifies and concentrates the liquid supplied from the raw material supply unit or the removal unit using a hollow fiber membrane; and a first control unit that controls the timing of flow of the liquid from the raw material supply unit to the purification and concentration unit or the timing of flow of the liquid from the removal unit to the purification and concentration unit. [2] The apparatus for purifying and concentrating described in [1], wherein the removal unit comprises a carrier column connected to a flow path that removes impurities from the liquid and supplies the liquid after impurity removal to the purification and concentration unit. [3] The apparatus for purifying and concentrating described in [1] or [2], wherein the removal unit comprises a filter member that removes impurities from the liquid, and the filter member is interposed in the flow path that supplies the liquid from the raw material supply unit to the removal unit. [4] The purification and concentration apparatus according to [2] or [3], wherein the removal unit comprises a first tank for storing the liquid treated in the carrier column, the first tank being disposed in a flow path for supplying the liquid from the carrier column to the purification and concentration unit, and a flow path for supplying the liquid in the tank to the purification and concentration unit being connected thereto, and the first control unit controls the timing for supplying the liquid in the tank to the purification and concentration unit. [5] The purification and concentration apparatus according to any of [1] to [4], wherein the hollow fiber membranes have an average inner diameter of 0.2 to 1.4 mm and a molecular weight cutoff of 10,000 to 1,000,000. [6] The purification and concentration apparatus according to any of [1] to [5], wherein the purification and concentration unit comprises a hollow fiber membrane module in which a plurality of the hollow fiber membranes are housed in a container having an inlet and outlet for the liquid, and the container has at least a first inlet and an outlet for a liquid and a second inlet and an outlet for a liquid for supplying the liquid to the hollow fiber membranes, and a third inlet and an outlet for discharging the liquid that has permeated the hollow fiber membranes. [7] The purification and concentration apparatus described in [6], wherein the hollow fiber membrane module is an internal pressure type.[8] The purification and concentration unit comprises: a second tank for storing the liquid supplied from the raw material supply unit or the removal unit, the second tank having a flow path connected to an inlet / outlet for the first liquid of the hollow fiber membrane module for supplying the liquid; a third tank for storing the liquid supplied from the raw material supply unit or the removal unit, the third tank having a flow path connected to an inlet / outlet for the second liquid of the hollow fiber membrane module for supplying the liquid; a pressurizing device for selectively pressurizing either the second tank or the third tank; and a second control unit for controlling the pressurizing device to selectively pressurize the second tank or the third tank so that the liquid in the second tank and the third tank are alternately supplied to the hollow fiber membrane module. [9] The purification and concentration unit according to [8], wherein the second control unit sets the liquid volumes in the second tank and the third tank, and alternately pressurizes the second tank and the third tank with the pressurizing device so that the liquid volumes in the second tank and the third tank become the set values.

[10] The second tank and the third tank are each equipped with a tank liquid level monitoring device including an optical sensor at the liquid inlet / outlet port at the bottom of the tank, the tank liquid level monitoring device is configured to irradiate light into the liquid inlet / outlet port with the optical sensor to detect light transmittance and monitor the liquid level in the tank based on changes in the transmittance, the second control unit determines the liquid levels in the second tank and the third tank using the tank liquid level monitoring device and decides which tank to pressurize with the pressurizing device.

[11] The purification and concentration device according to [9] or

[10] , wherein the purification and concentration unit is equipped with a camera for monitoring liquid level positions in the second tank and the third tank, and the second control unit decides which tank to pressurize with the pressurizing device based on information on the liquid level positions in the second tank and the third tank output from the camera.

[12] The purification and concentration apparatus according to any one of [8] to

[11] , wherein the purification and concentration unit comprises: a fourth tank for storing a dilution liquid, the fourth tank having a flow path connected thereto for supplying the dilution liquid to an inlet / outlet for the third liquid of the hollow fiber membrane module; and a third control unit for controlling timing for supplying the dilution liquid in the fourth tank to the inlet / outlet for the third liquid of the hollow fiber membrane module, wherein the third control unit supplies the dilution liquid in the fourth tank from the inlet / outlet for the third liquid of the hollow fiber membrane module when the total amount of liquid in the second tank and the third tank is equal to or less than a value set by the second control unit.

[13] The purification and concentration apparatus according to any one of [8] to

[12] , wherein the purification and concentration unit comprises: a fifth tank connected to the inlet / outlet for the first liquid or the inlet / outlet for the second liquid of the hollow fiber membrane module by a flow path, and for recovering the liquid in the second tank and the third tank; and a fourth control unit for controlling recovery of the liquid into the fifth tank.

[14] The second tank and the third tank are each equipped with a tank liquid level monitoring device including an optical sensor at the liquid inlet / outlet at the bottom of the tank, and the tank liquid level monitoring device is configured to irradiate light into the liquid inlet / outlet with the optical sensor to detect the light transmittance and monitor the liquid level in the tank based on changes in the transmittance, and the fourth control unit selectively pressurizes the second tank or the third tank with the pressurizing device to recover the liquid in the second tank and the third tank into the fifth tank, and determines the liquid levels in the second tank and the third tank with the tank liquid level monitoring device, and stops the pressurizing device.

[15] The purification and concentration unit is equipped with a camera that monitors the liquid level position in the second tank and the third tank, and the fourth control unit selectively pressurizes the second tank or the third tank using the pressure device to recover the liquid in the second tank and the third tank into the fifth tank, and stops the pressure device based on information on the liquid level position in the second tank and the third tank output from the camera. The purification and concentration apparatus described in

[13] or

[14] .

[16] The purification and concentration apparatus according to any one of [1] to

[15] , wherein elements including the raw material supply unit, the removal unit, and the purification and concentration unit are arranged in a housing, and further comprising a management unit that manages the cleanliness level within the housing, the management unit comprising a fan filter device that supplies filtered air into the housing.

[17] The purification and concentration apparatus according to

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

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

[17] , wherein the removal unit comprises a sixth tank that stores a cleaning solution for washing the carrier column and a seventh tank that stores an equilibration solution for equilibrating the carrier column.

[19] The purification and concentration apparatus according to any one of [3] to

[18] , wherein the filtering element is arranged below the raw material supply unit, and the liquid is supplied from the raw material supply unit to the filtering element by the weight of the liquid.

[20] The purification and concentration apparatus according to any one of [8] to

[19] , wherein the longitudinal sides of the second tank and the third tank are arranged parallel to the longitudinal direction of the hollow fiber membrane module.

[21] The purification and concentration apparatus according to

[20] , wherein the removal section comprises a carrier column connected to a flow path for removing impurities from the liquid and supplying the liquid after impurity removal to the purification and concentration section, and a first tank for storing the liquid treated in the carrier column, the second tank and the third tank being connected to the carrier column and the first tank by flow paths and being arranged above the carrier column and the first tank.

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

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

[23] A method for producing a liquid in which useful micro substances have been purified and concentrated using the purification and concentration device described in any one of [1] to

[22] , comprising supplying a raw material liquid containing the useful micro substances to the purification and concentration device and removing impurities from the liquid, and / or purifying and concentrating the liquid with a hollow fiber membrane.

[24] A method for producing a purified and concentrated liquid of useful micro substances described in

[23] , wherein removing impurities from the liquid comprises adsorbing and removing impurities from the liquid using a carrier column.

[25] The method for producing a purified and concentrated solution of useful micro substances according to

[23] or

[24] , wherein purifying and concentrating the solution with the hollow fiber membranes comprises subjecting the solution to alternating tangential flow filtration in a hollow fiber membrane module.

[26] The method for producing a purified and concentrated solution of useful micro substances according to any of

[23] to

[25] , wherein purifying and concentrating the solution with the hollow fiber membranes comprises subjecting the solution to alternating tangential flow filtration in a hollow fiber membrane module while recovering the useful micro substances deposited on the surfaces of the hollow fiber membranes.

[27] The method for producing a purified and concentrated solution of useful micro substances according to any of

[23] to

[26] , wherein purifying and concentrating the solution with the hollow fiber membranes comprises supplying a diluent to the concentrated solution to further purify and concentrate it, and the diluent is supplied to the solution when the ratio of the amount (B) of the concentrated solution to the amount (A) of the raw material solution (B) (B / A) becomes 1 / 2 to 1 / 200.

[28] A method for producing a purified and concentrated solution of a useful micro substance according to any one of

[24] to

[27] , wherein the removal of impurities in the solution using the carrier column and the purification and concentration using the hollow fiber membrane are carried out in parallel.

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

[23] to

[28] , wherein the useful micro substance is at least one selected from the group consisting of extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.[30-1] A purification and concentration unit includes a raw material supply unit that supplies a liquid containing a useful micro substance, and a purification and concentration unit that purifies and concentrates the liquid supplied from the raw material supply unit using a hollow fiber membrane module, wherein the hollow fiber membrane module has a first liquid inlet / outlet and a second liquid inlet / outlet for supplying the liquid to the hollow fiber membrane, and a third liquid inlet / outlet for discharging the liquid that has permeated the hollow fiber membrane, and the purification and concentration unit includes a second tank that stores the liquid supplied from the raw material supply unit and has a flow path for supplying the liquid connected to the first liquid inlet / outlet of the hollow fiber membrane module, a third tank that stores the liquid supplied from the raw material supply unit and has a flow path for supplying the liquid connected to the second liquid inlet / outlet of the hollow fiber membrane module, and a second control unit that controls the pressurization device to selectively pressurize the second tank or the third tank, and supply the liquid in the second tank and the third tank alternately to the hollow fiber membrane module; and a monitoring device that monitors the amount of the liquid in the second tank and the third tank, wherein the second control unit sets the amount of liquid in the second tank and the third tank, and causes the pressurization device to alternately pressurize the second tank and the third tank so that the amount of liquid in the second tank and the third tank becomes a set value, and the amount of liquid in the second tank and the third tank is determined based on information from the monitoring device.[30-2] A raw material supply unit that supplies a liquid containing useful micro substances; and a purification and concentration unit that purifies and concentrates the liquid supplied from the raw material supply unit using a hollow fiber membrane module, wherein the hollow fiber membrane module has a first liquid inlet / outlet and a second liquid inlet / outlet for supplying the liquid to the hollow fiber membranes, and a third liquid inlet / outlet for discharging the liquid that has permeated the hollow fiber membranes, and the purification and concentration unit comprises: a second tank that stores the liquid supplied from the raw material supply unit and has a flow path that supplies the liquid connected to the first liquid inlet / outlet of the hollow fiber membrane module; and a third tank that stores the liquid supplied from the raw material supply unit and has a flow path that supplies the liquid connected to the second liquid inlet / outlet of the hollow fiber membrane module. a liquid delivery device that selectively delivers the liquid in either the second tank or the third tank to the hollow fiber membrane module, a second control unit that controls the liquid delivery device so as to selectively supply the liquid in the second tank or the third tank to the hollow fiber membrane module, and a monitoring device that monitors the amount of the liquid in the second tank and the third tank, wherein the second control unit sets the amount of liquid in the second tank and the third tank, and when the amount of liquid in the second tank and the third tank reaches a set value, controls the liquid delivery device to selectively deliver the liquid from the second tank or the third tank to the hollow fiber membrane module, and the amount of liquid in the second tank and the third tank is determined based on information from the monitoring device.

[31] An apparatus for purifying and concentrating a liquid containing a useful micro substance according to [30-1] or [30-2], wherein the hollow fiber membrane has an average inner diameter of 0.2 to 2.0 mm and a molecular weight cutoff of 5,000 to 3,000,000.

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

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

[33] The purification and concentration apparatus according to any one of [30-1] to

[32] , wherein the monitoring device is equipped with an optical sensor provided at the liquid inlet and outlet at the bottom of the second tank and the third tank, respectively, which irradiates light into the liquid inlet and outlet to detect light transmittance and monitors the amount of the liquid in the tank based on changes in the transmittance.

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

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

[35] The purification and concentration apparatus according to any one of [30-1] to

[34] , wherein the purification and concentration unit comprises: a fourth tank for storing a dilution liquid, the fourth tank being connected to a flow path for supplying the dilution liquid to an inlet / outlet for the third liquid of the hollow fiber membrane module; and a third control unit for controlling timing for supplying the dilution liquid in the fourth tank to the inlet / outlet for the third liquid of the hollow fiber membrane module, wherein the third control unit supplies the dilution liquid in the fourth tank from the inlet / outlet for the third liquid of the hollow fiber membrane module when the sum of the liquid amounts in the second tank and the third tank is equal to or less than a value set by the first control unit.

[36] The purification and concentration apparatus according to any one of [30-1] to

[35] , wherein the purification and concentration unit comprises: a fifth tank connected to the inlet / outlet for the first liquid or the inlet / outlet for the second liquid of the hollow fiber membrane module by a flow path, and for recovering the liquid in the second tank and the third tank; and a fourth control unit for controlling recovery of the liquid into the fifth tank.

[37] The purification and concentration apparatus according to

[36] , wherein the fourth control unit selectively pressurizes the second tank or the third tank using the pressurizing device to recover the liquid in the second tank and the third tank into the fifth tank, and stops the pressurizing device by determining the liquid volume in the second tank and the third tank using the monitoring device.

[38] The purification and concentration apparatus according to any one of [30-1] to

[37] , wherein the useful minute substance is selected from electroactive polymers, rare metal colloid particles, pigments, dyes, inorganic or organic nanoparticles, proteins, hormones, cytokines, growth factors, angiogenesis factors, growth factors, enzymes, antibodies, plasma proteins, viruses, extracellular vesicles, yeast, cells, polysaccharides, and microalgae.

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

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

[40] The purification and concentration apparatus described in any of

[12] to

[22] or any of

[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 diluted liquid in the fourth tank from one of the at least two inlets and outlets for the third liquid that the hollow fiber membrane module has 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 diluted liquid in the fourth tank from at least one of the at least two inlets and outlets for the third liquid that the hollow fiber membrane module has while allowing the flow of the liquid through the inlet and outlet for the first liquid and the inlet and outlet for the second liquid.

[41] A method for producing a purified and concentrated solution of useful micro substances by removing impurities from a raw material liquid containing useful micro substances and impurities, and then purifying and concentrating the liquid, wherein purifying and concentrating the liquid includes purifying and concentrating the liquid using a hollow fiber membrane module, and during the purification and concentration process, includes backwashing the hollow fiber membranes by pressurizing a diluent into the permeate side of the hollow fiber membrane module.

[42] A method for producing a purified and concentrated solution of useful micro substances according to

[41] , wherein the hollow fiber membranes housed in the hollow fiber membrane module are made of a resin material containing cellulose acetate.

[43] A method for producing a purified and concentrated solution of useful micro substances according to

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

[44] A method for producing a purified and concentrated solution of useful micro substances according to any of

[41] to

[43] , wherein removing impurities from the liquid includes adsorbing and removing impurities from the liquid using a carrier column.

[45] A method for producing a purified and concentrated solution of a useful micro substance according to any one of

[41] to

[44] , wherein purifying and concentrating the liquid using the hollow fiber membrane module comprises subjecting the liquid to alternating tangential flow filtration.

[46] The method for producing a purified and concentrated solution of a useful micro substance according to any one of

[41] to

[45] , wherein purifying and concentrating the liquid using the hollow fiber membrane module includes supplying a diluent to the purified and concentrated liquid to further purify and concentrate the liquid, and the diluent is supplied to the liquid when the amount (B) of the purified and concentrated liquid relative to the amount (A) of the raw liquid (B) (B / A) becomes 1 / 2 to 1 / 200.

[47] The method for producing a purified and concentrated solution of a useful micro substance according to any one of

[44] to

[46] , wherein removing impurities from the liquid using the carrier column and purifying and concentrating the liquid using the hollow fiber membrane module are carried out in parallel.

[48] The method for producing a purified and concentrated solution of a useful micro substance according to any one of

[41] to

[47] , wherein the useful micro substance is at least one selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.

[49] A method for producing a purified and concentrated solution of a useful micro substance according to any one of

[41] to

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

[50] A method for producing a purified and concentrated solution of a useful micro substance according to any one of

[41] to

[49] , comprising flushing the hollow fiber membrane module prior to the backwashing treatment.

[0009] According to the present disclosure, it is possible to provide a device for purifying and concentrating a liquid containing useful micro substances, which is capable of producing a liquid containing useful micro substances at high purity and high concentration (a purified and concentrated liquid of useful micro substances) and which can more efficiently prepare the purified and concentrated liquid, and a method for producing a purified and concentrated liquid of useful micro substances using the same. In addition, the purifying and concentrating device of the present disclosure is also useful as a device that can purify and concentrate useful micro substances by automatic control.

[0010] FIG. 1 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to a first embodiment. FIG. 2 is a schematic configuration diagram showing an example of a removal section according to the first embodiment. FIG. 3 is a schematic configuration diagram showing an example of a removal section according to the first embodiment. FIG. 4 is a schematic configuration diagram showing an example of a removal section according to the first embodiment. FIG. 5 is a schematic cross-sectional view showing an example of a hollow fiber membrane module in the purification and concentration apparatus of the present disclosure. FIG. 6 is a schematic configuration diagram showing an example of a purification and concentration section according to the first embodiment. FIG. 7 is a schematic configuration diagram showing an example of a purification and concentration section according to the first embodiment. FIG. 8 is a schematic configuration diagram showing an example of a purification and concentration section according to the first embodiment. FIG. 9 is a schematic configuration diagram showing an example of a purification and concentration section according to the first embodiment. FIG. 10 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the first embodiment. FIG. 11 is a schematic configuration diagram showing an example of an overall configuration of the purification and concentration apparatus according to the first embodiment. FIG. 12 is a schematic configuration diagram showing an example of the overall configuration of the purification and concentration apparatus according to the second embodiment. FIG. 13 is an example of an apparatus for measuring γ-globulin permeability of a hollow fiber membrane. FIG. 14 is a flowchart showing an example of a purification and concentration process using the purification and concentration apparatus according to the first embodiment. FIG. 15 is a graph showing the results of BCA assay of the concentrates obtained in Example 4 and Comparative Example 3. 1 is an absorbance chart of the concentrates obtained in Example 4 and Comparative Example 3.

[0011] An example of the present disclosure will be described below using the drawings. However, each configuration and their combinations in each embodiment are merely examples, and addition, omission, substitution, and other modifications of configurations are possible as appropriate within the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the scope of the claims. Furthermore, when multiple upper and lower limit values ​​are listed for a specific parameter, any upper and lower limit values ​​can be combined to form a suitable numerical range.

[0012] [Apparatus for purifying and concentrating a liquid containing useful micro-substances] The present disclosure relates to an apparatus for purifying and concentrating a liquid containing useful micro-substances. The apparatus for purifying and concentrating the liquid of the present disclosure has a first embodiment and a second embodiment. The first embodiment of the apparatus for purifying and concentrating the liquid of the present disclosure will be described below with reference to the drawings.

[0013] First Embodiment FIG. 1 is a schematic diagram showing a purification and concentration apparatus 1 (hereinafter referred to as "purification and concentration apparatus 1") according to a first embodiment of the present disclosure. The purification and concentration apparatus 1 of the present disclosure includes a raw material supply unit 10 that supplies a raw material containing useful micro substances, a removal unit 20 that removes impurities from the liquid, a purification and concentration unit 30 that purifies and concentrates 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 that controls the timing of the flow of the liquid from the raw material supply unit 10 to the purification and concentration unit 30 or the flow of the liquid from the removal unit 20 to the purification and concentration unit 30. The liquid from the raw material supply unit 10 is supplied to the removal unit 20 via a flow path 41 or to the purification and concentration unit 30 via a flow path 43. The liquid from the removal unit 20 is supplied to the purification and concentration unit 30 via a flow path 42. The purification and concentration apparatus 1 of the present disclosure is capable of producing a liquid containing useful micro substances at high purity and high concentration (a purified and concentrated liquid of useful micro substances) and can more efficiently prepare the purified and concentrated liquid. The present invention is also useful as a device capable of purifying and concentrating minute useful substances through automatic control.In the present disclosure, the term "flow path" refers to a pipe through which a liquid flows.

[0014] <Raw material supply section> A raw material liquid (hereinafter referred to as "raw material liquid") containing minute useful substances is stored in the raw material supply section 10. The raw material liquid in the raw material supply section 10 is supplied to the removal section 20 or the purification and concentration section 30 depending on its purity, i.e., 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 set to be supplied to the removal section 20, and when the concentration of impurities in the raw material liquid is low, the raw material liquid can be set to be supplied to the purification and concentration section 30. A flow path 41 for supplying the raw material liquid to the removal section 20 and a flow path 43 for supplying the raw material liquid to the purification and concentration section 30 are connected to the raw material supply section 10.

[0015] 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 apparatus 1, or may be installed so as to be easily removable. If the container is installed so as to be easily removable, the raw material supply unit 10 may include various equipment such as fixing members for fixing the container.

[0016] The shape and volume of the container for storing the raw material liquid are not particularly limited and can be determined depending on the type of useful minute substance, the amount of the raw material liquid, etc. In one embodiment, when purifying and concentrating a raw material liquid containing a useful minute substance selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, nucleic acids, etc., the volume of the container may be 50 to 5,000 mL, or may be 200 to 2,000 mL.

[0017] <Removal section> The removal section 20 removes impurities from the raw material liquid supplied from the raw material supply section 10 via the flow path 41. With this configuration, a purified and concentrated solution of useful minute substances can be efficiently prepared. Hereinafter, the liquid from which impurities have been removed in the removal section 20 will be referred to as a "roughly purified solution."

[0018] 2 to 6 show several representative schematic configuration examples of the removal section 20. The removal section 20 shown in FIG. 2 is provided with a carrier column 21 for removing impurities from the raw material 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 partially purified liquid to the purification and concentration section 30. A flow path (described below) for discharging the liquid to a waste liquid tank (described below) may be connected to the flow path 42 from the carrier column 21. Instead of the carrier column 21, the removal section 20 may be provided with a device equipped with a container filled with an adsorbent capable of adsorbing and removing impurities from the raw material liquid. In such a container, impurities in the raw material liquid can be adsorbed onto the adsorbent by stirring, vibration, shaking, etc., thereby removing or reducing the impurities. A rotator can also be used as such a device. The packing material to which the impurities have been adsorbed can be removed by an optional filtration unit.

[0019] The removal unit 20 shown in FIG. 3 includes a carrier column 21 and a first tank 23 that stores the crudely purified liquid treated in the carrier column 21. The first tank 23 is disposed 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 unit side, with the first tank 23 in between. The crudely purified liquid is stored in the first tank 23 from the carrier column 21 via the flow path 42A, and the crudely purified liquid stored in the first tank 23 is supplied to the purification and concentration unit 30 via the flow path 42B. A three-way valve 77 is provided in the flow path 42B. Depending on the treatment status of the crudely purified liquid in the purification and concentration unit 30, the first control unit 110 opens the three-way valve 77 in the flow path 42B to the purification and concentration unit 30 side, and supplies the crudely purified liquid in the first tank 23 to the purification and concentration unit 30. According to this configuration, the partially purified liquid can be temporarily stored in the first tank 23 and then supplied to the purification and concentration section 30, so that the impurity removal process in the removal section 20 and the purification and concentration process in the purification and concentration section 30 can be performed in parallel. As a result, the purification and concentration processes can be performed efficiently. The configuration of the removal section 20 shown in Figures 4 to 6 will be described later.

[0020] (Carrier Column) The carrier column 21 is filled with a material capable of removing impurities other than useful micro-substances from the raw liquid. The material is preferably an adsorbent capable of adsorbing and removing impurities from the raw liquid. The adsorbent is preferably a porous granular material capable of adsorbing and retaining the impurities. Porous granular materials refer to granular substances with numerous pores on their surface and inside, and polymeric porous granular materials or inorganic porous granular materials can be used. When the useful micro-substances contained in the raw liquid to be treated in the purification and concentration apparatus 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, the porous granular materials preferably have positively charged pores inside. These porous granular materials can be used singly or in combination with multiple materials having different adsorption capabilities. Examples of multiple porous granular materials with different adsorption capabilities include those with different pore sizes, specific surface areas, average particle sizes, types of substrates constituting the porous granular materials, and types or densities of functional groups present on the inner surfaces of the pores of the porous granular materials. Examples of such porous granular materials include those based on polysaccharides such as cellulose, agarose, starch, amylose, dextran, pullulan, and glucomannan; synthetic polymers such as polyacrylic acid or derivatives thereof, polyvinyl alcohol, nylon, polysulfone, polyacrylnitrile, polyethylene, polypropylene, and polystyrene; glass, porous glass, silica gel, and hydroxyapatite. These may be used alone or in combination of two or more. They may also be mixed-mode carriers having ion exchange and size exclusion, preferably mixed-mode carriers having cation exchange and size exclusion.

[0021] The porous granular material can be selected from those having an average pore diameter in the range of 0.1 to 1,000 nm depending on the type of impurities contained in the raw material liquid. The average pore diameter can be measured by mercury intrusion porosimetry or inverse size exclusion chromatography. The porous granular material may also be selected from those having an average particle diameter in the range of 1 to 1,000 μm depending on the type of impurities contained in the raw material liquid. The average particle diameter can be measured by laser diffraction scattering.

[0022] In one embodiment, when the useful micro-substances contained in the raw material solution to be treated by the purification and concentration apparatus 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, the carrier column 21 may include an antibody column. An "antibody column" refers to a column packed with carriers onto which antibodies capable of binding to antigens (impurities) are immobilized. When the raw material solution contains the aforementioned biological substances, an antibody column packed with carriers onto which antibodies capable of binding (adsorbing) impurities other than the biological substances can be used. Note that, because raw material solutions containing the aforementioned biological molecules as useful micro-substances often contain multiple types of impurities, multiple antibody columns may be used depending on the types of impurities.

[0023] Alternatively, a column module formed by combining multiple carrier columns may be used instead of the carrier column 21. When a column module is used, the types of adsorbents contained in the carrier columns may be the same or different. The inner diameters and lengths of the carrier columns may also be the same or different.

[0024] The removal section 20 shown in Figure 4 includes a sixth tank 25 (washing solution storage tank) that stores a washing solution for washing the carrier column 21, and a seventh tank 26 (equilibration solution storage tank) that stores an equilibration solution for equilibrating the carrier column 21. In the removal section 20 of Figure 4, the sixth tank 25 is configured to be able to supply the washing solution to the flow path 41 via flow path 50A. Furthermore, the seventh tank 26 is configured to be able to supply the equilibration solution to the flow path 41 via flow path 50B. On-off valves 71A and 71B are disposed in the flow paths 50A and 50B. A three-way valve 72 is provided in the flow path 42 that supplies the partially purified solution from the carrier column 21 to the purification and concentration section 30.

[0025] In one embodiment, it is preferable that the carrier column 21, which has been treated with one batch of raw material solution and reached adsorption saturation, is washed and equilibrated, and then the next batch of raw material solution is treated. The washing and equilibration treatment of the carrier column 21 can be performed by opening the on-off valves 71A and 71B provided in the flow paths 50A and 50B and supplying the washing solution and equilibration solution from the sixth tank 25 and the seventh tank 26 to the carrier column 21. It is preferable that the washing solution and equilibration solution after treating the carrier column 21 are discharged from the three-way valve 72 provided in the flow path 42 to a waste liquid tank (described later).

[0026] (Washing Solution and Equilibration Solution) Examples of the washing solution used to wash the carrier column 21 include aqueous sodium chloride solutions, hydrochloric acid, citric acid, aqueous sodium hypochlorite solutions, and aqueous sodium hydroxide solutions. From the viewpoint of safety, it is preferable to use aqueous sodium chloride solutions. When the washing solution flows into the carrier column 21, impurities adhering to the adsorbent in the carrier column 21 are removed. After removal with the washing solution, the carrier column 21 is equilibrated. Examples of the equilibration solution that can be used include physiological saline, phosphate buffer solution (PBS), Tris-HCl buffer solution, sodium citrate buffer solution, citrate phosphate buffer solution, acetate buffer solution, and borate buffer solution. The materials of the sixth tank 25 and the seventh tank 26 that store the washing solution and the equilibration solution are not particularly limited, and may have the same structure as the second tank 31 and the third tank 32 described below. From the viewpoint of easily visually checking the amount of liquid stored in the tanks, it is preferable that the sixth tank 25 and the seventh tank 26 be transparent.

[0027] The removal unit 20 shown in FIG. 5 includes a filter member that removes impurities from the liquid before supplying it to the carrier column 21. The removal unit 20 shown in FIG. 5 is configured with a filter member 22 interposed in a flow path 41. The raw material liquid filtered by the filter member 22 is supplied to the carrier column 21 from a flow path 41A that is located on the carrier column 21 side of the filter member 22 within the flow path 41. With this configuration, the raw material liquid that has been pretreated by the filter member 22 can be supplied to the carrier column 21. Hereinafter, the raw material liquid treated by the filter member 22 will be referred to as the "pretreated liquid." Note that although the removal unit 20 in FIG. 5 includes the filter member 22 and the carrier column 21, it may also be configured with only the filter member 22.

[0028] The removal unit 20 shown in FIG. 6 includes a tank 24 (pretreatment liquid storage tank) that stores the pretreatment liquid from the filtration member 22. The pretreatment liquid from the filtration member 22 is stored in the tank 24 via a flow path 41C. The pretreatment liquid stored in the tank 24 is supplied to the carrier column 21 via a flow path 41D. The first control unit 110 (not shown) can open the on-off 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 process in the carrier column 21. This configuration is preferable because the pretreatment liquid can be temporarily stored in the tank 24 and then supplied to the carrier column 21, allowing the filtration process by the filtration member 22 and the impurity removal process by the carrier column 21 to be performed in parallel, thereby enabling efficient purification and concentration processes.

[0029] 6, the filtering member 22 is configured to be disposed below the raw material supply unit 10. With this configuration, the supply of the raw material liquid from the raw material supply unit 10 to the filtering member 22 proceeds due to the weight of the raw material liquid, so that a liquid delivery member (such as a pump) for supplying the raw material liquid from the raw material supply unit 10 to the filtering member 22 can be omitted. This is preferable because it allows for a more compact purification and concentration apparatus.

[0030] (Filtering Member) The filtering member 22 is a member that filters the raw material liquid. When the useful microsubstances contained in the raw material liquid include extracellular vesicles, the filtering member 22 is preferably a filter equipped with a microfiltration membrane (microfiltration membrane module). From the viewpoint of efficiently filtering out impurities such as cells in the raw material liquid and biomolecules larger than the target extracellular vesicles, the average pore size of the microfiltration membrane (microfiltration membrane module) is preferably 0.10 to 0.50 μm, and more preferably 0.15 to 0.30 μm. Furthermore, preferred materials for the microfiltration membrane include polyethersulfone and polyvinylidene fluoride. Furthermore, commercially available microfiltration membranes made of these materials can be used.

[0031] (First Control Unit) Next, details of the first control unit will be described using the flowchart of FIG. 16 . FIG. 16 is a basic flowchart for performing a purification and concentration process using the purification and concentration apparatus 1 shown in FIG. 13 . The flowchart of FIG. 16 shows an example of liquid flow control in each control unit. The first control unit 110 can supply the raw material liquid to the removal unit 20 or the purification and concentration unit 30 based on the result set by 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 opens the three-way valve 82 located at the bottom of the container 11 in FIG. 13 to the flow path 41 side, and supplies the raw material liquid to the removal unit 20 (filtering member 22 in FIG. 13 ). When the impurity concentration of the raw material liquid is low, the raw material liquid in the raw material supply unit 10 is set to be supplied to the purification and concentration unit 30. In this case, the first control unit 110 opens the three-way valve 82 to the flow path 43 side, and can send the raw material liquid toward the purification and concentration unit 30. The liquid (pretreatment liquid) treated by the filtration member 22 is supplied to the tank 24. The first control unit 110 can supply the liquid from the tank 24 to the carrier column 21 until the amount of the roughly purified liquid in the first tank 23 (roughly purified liquid storage tank) reaches a set value. Specifically, the pretreatment liquid can be supplied to the carrier column 21 by opening an on-off valve 73 provided at the bottom of the tank 24.

[0032] When the amount of the partially purified liquid in the first tank 23 reaches a preset value, the liquid is sent to the second tank 31. At this time, the first control unit 110 can also check the liquid amounts in the second tank 31 and the third tank 32. When the first control unit 110 determines that the liquid amounts in the second tank 31 and the third tank 32 are the preset liquid amounts based on liquid level monitoring by tank liquid amount monitoring devices 35, 36 and / or a camera, which will be described later, the first control unit 110 opens the three-way valve 77 connected to the flow path 42B toward the second tank 31, and can supply the partially purified liquid in the first tank 23 to the second tank 31.

[0033] 13 is provided with a level sensor (not shown) that determines the amount of liquid in the tank. Based on information from the level sensor, the first control unit 110 can determine whether or not a partially purified liquid remains in the first tank 23. When it determines that all of the partially purified liquid in the first tank 23 has been supplied to the second tank 31, the first control unit 110 opens the on-off valve 73 to supply the pretreatment liquid in the tank 24 again to the carrier column 21. By controlling the flow of liquid in this manner, the carrier column 21 can be washed and equilibrated each time one batch of processing is completed.

[0034] <Purification and concentration section> The purification and concentration section 30 uses hollow fiber membranes to purify and concentrate the liquid supplied from the raw material supply section 10 or the removal section 20. Since the liquid is purified and concentrated using hollow fiber membranes, it is possible to prepare a liquid containing useful micro substances at high purity and high concentration.

[0035] (Hollow Fiber Membrane) The material of the hollow fiber membrane of the present disclosure can be appropriately designed depending on the intended use of the present disclosure and the properties of the liquid containing the useful micro-substance. 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 forms can be used. Furthermore, composite hollow fiber membranes composed of multiple components selected from the above can also be used. Among these, when the useful micro-substance is a biomolecule such as an exosome, it is preferable to use a hollow fiber membrane made of cellulose acetate (CA) (i.e., it is preferable to use a resin material containing cellulose acetate). Using a hollow fiber membrane made of cellulose acetate can suppress a decrease in filtration rate during the purification and concentration process and further improve the recovery effect of useful micro-substances deposited on the membrane surface of the hollow fiber membrane. The reason for this is that the surface charge of biomolecules is weakly negative, and the cellulose acetate membrane is also weakly negative, so there is no strong Coulomb force between the biomolecules and the cellulose acetate membrane, and by appropriately controlling the circulation linear velocity, the shear force of cross-flow filtration can function easily.In addition, by using a cellulose acetate membrane, useful micro-substances deposited on the membrane surface of the hollow fiber membrane can be recovered more efficiently using the method described below.

[0036] The material of the hollow fiber membrane of the present disclosure can be designed appropriately depending on the intended use of the present disclosure and the properties of the raw material liquid containing the useful micro-substances. In addition, depending on the properties of the raw material liquid containing the useful micro-substances, the hollow fiber membrane surface can also be coated with an appropriate substance to improve the fouling resistance.

[0037] The coating treatment can be carried out by a method similar to the method of bonding a cationic polymer to the surface of a hollow fiber membrane made of sulfonated polyethersulfone and polyethersulfone described in Japanese Patent No. 6081290. That is, a method can be used in which the hollow fiber membrane is immersed in an aqueous solution (aqueous dispersion) of the coating substance, or a method can be used in which an aqueous solution (aqueous dispersion) of the coating substance is passed through the hollow fiber membrane in the state of a hollow fiber membrane or hollow fiber membrane module to adsorb and bond the coating substance to the surface of the hollow fiber membrane, and then the coating substance that is merely deposited on the thin layer of the coating substance is removed by washing with water.

[0038] When concentrating and purifying certain extracellular vesicles from certain culture supernatants, coating a cellulose acetate hollow fiber membrane with certain biomolecules may be preferable in terms of increasing the efficiency of purification and concentration and the recovery rate of useful micro-substances such as extracellular vesicles.

[0039] As an example, a hollow fiber membrane module made of cellulose acetate (molecular weight cutoff: 300,000, membrane area: 0.062 m) ２ ) is passed through a hollow fiber membrane at a flow rate of 200 mL / min for 10 minutes, and a coating treatment is performed to adsorb and bind biomolecules such as casein in the skim milk powder to the surface of the hollow fiber membrane as a thin layer. After that, the membrane is washed with 250 mL of phosphate buffer solution (PBS) to remove excess components that have simply accumulated on the thin layer.

[0040] In one embodiment, from the viewpoint of improving the fouling resistance of the hollow fiber membrane, it is preferable that a type of biomolecule different from the useful microsubstance is attached to at least a portion of the hollow fiber membrane. It is more preferable that the biomolecule is attached over more than half of the surface area of ​​the hollow fiber membrane, and it is preferable that the biomolecule is attached over the entire area. In one embodiment, it is preferable that a layer containing a type of biomolecule different from the useful microsubstance is formed on at least a portion of the surface of the hollow fiber membrane. The "layer containing biomolecules" means that a substance containing biomolecules is attached with a certain spread to at least a portion of the surface of the hollow fiber membrane. It is preferable that the layer containing biomolecules is formed over more than half of the surface area of ​​the hollow fiber membrane, and it is more preferable that it is formed over the entire area. In one embodiment, it is preferable that at least a portion of the surface of the hollow fiber membrane is coated with a type of biomolecule different from the useful microsubstance.

[0041] Biomolecules of a type different from minute useful substances are those obtained from animal cell tissues such as blood, milk, cartilage, and other biological cell tissues, and representative examples include biomolecules contained in skim milk and non-fat powdered milk (e.g., casein); biomolecules contained in serum (e.g., serum globulin, serum albumin); gelatin (e.g., fish gelatin); and the like, and may be one or more selected from these.

[0042] (Average Inner Diameter of Hollow Fiber Membrane) The average inner diameter of the hollow fiber membrane of the present disclosure can be designed appropriately depending on the intended use of the present disclosure, the properties of the liquid containing the useful minute substance, and the like. In one embodiment, a hollow fiber membrane having an average inner diameter of 0.2 mm to 1.4 mm can be used. In another embodiment, a hollow fiber membrane having an average inner diameter of 0.2 mm to 2.0 mm can be used. Note that the "average inner diameter of the hollow fiber membrane" in the present disclosure is a value calculated by cutting a single hollow fiber membrane along an arbitrary plane perpendicular to the longitudinal direction and calculating the diameter of the smallest circle inscribed in the hollow portion of the cut surface. In the present disclosure, the diameters are measured at any 10 to 100 locations on the above cut surface, and the average value is defined as the "average inner diameter."

[0043] The hollow fiber membranes of the present disclosure may have an average outer diameter of 0.3 mm to 2.0 mm. In one embodiment, when the average inner diameter of the hollow fiber membrane is 0.2 mm to 1.0 mm, the average outer diameter may be 0.3 mm to 1.4 mm. In another embodiment, when the average inner diameter of the hollow fiber membrane is 1.0 mm to 1.4 mm, the average outer diameter may be 1.5 mm to 2.0 mm. The "average outer diameter of the hollow fiber membrane" is a value calculated from the diameter of the smallest circle that encompasses the outer edge of the cut surface of a hollow fiber membrane cut in the same manner as the aforementioned average inner diameter of the hollow fiber membrane. In the present disclosure, the diameters are measured at any 10 to 100 locations on the cut surface, and the average value is defined as the "average outer diameter."

[0044] In the intended use of the present disclosure, the hollow fiber membrane may have an average inner diameter of 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 flexible and easily improving the recovery efficiency of useful micro substances deposited on the membrane surface of the hollow fiber membrane. Furthermore, the average inner diameter may be adjusted depending on the liquid viscosity of the liquid containing useful micro substances during operation. When the liquid viscosity is high (for example, when the liquid temperature is 20°C and the shear rate is 25 s －１ In addition, when the viscosity of the liquid containing the minute useful substance is low during operation (for example, when the liquid temperature is 20°C and the shear rate is 25 s), it is preferable to use a large-diameter hollow fiber membrane having an average inner diameter of 1.0 mm to 1.4 mm. －１ is less than 10 mPa s), it is preferable to use hollow fiber membranes with an average inner diameter of 0.6 mm to 1.0 mm. Note that if the average inner diameter of the hollow fiber membrane is large, the effective membrane area is likely to decrease when the hollow fiber membrane is used as a hollow fiber membrane module. Therefore, from the viewpoint of easily suppressing a decrease in the effective membrane area when used as a hollow fiber membrane module, it is preferable to use hollow fiber membranes with an average inner diameter of 0.2 mm to 1.0 mm.

[0045] (Molecular Weight Cutoff of Hollow Fiber Membrane) The molecular weight cutoff of the hollow fiber membrane of the present disclosure can be appropriately designed depending on the intended use of the present disclosure, the properties of the raw material solution containing the minute useful substance, etc. In one embodiment, the molecular weight cutoff may be 5,000 to 3,000,000, or may be 10,000 to 1,000,000. When the minute useful substance is a specific protein, it is preferable to use a hollow fiber membrane with a molecular weight cutoff slightly lower than the molecular weight of the protein (for example, a hollow fiber membrane having a molecular weight cutoff approximately 10 to 50% lower than the molecular weight of the protein). In one embodiment, when the minute 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 even more preferably 100,000 to 500,000.

[0046] When the useful minute substance is an extracellular vesicle, the γ-globulin permeability of the hollow fiber membrane module of the present disclosure is preferably 5 to 90%, more preferably 10 to 80%, from the viewpoint of purification and concentration efficiency. In the present disclosure, the method for measuring the permeability of impurities in a solution containing the useful minute substance, including the aforementioned γ-globulin, is not particularly limited, and conventionally known methods can be employed. Specifically, a method for measuring the permeability of a standard substance having a molecular structure similar to that of the impurity and a molecular weight comparable to that of the impurity can be employed. When measuring the permeability, a hollow fiber membrane module may be used, or a single hollow fiber membrane may be directly used in the hollow fiber membrane module. Furthermore, the permeability of an internal pressure hollow fiber membrane module may be substituted for the permeability measured by applying pressure from the outside of a single hollow fiber membrane to permeate the impurity to the inside.

[0047] (Hollow Fiber Membrane Module) The purification and concentration section 30 of the present disclosure may be configured to include a hollow fiber membrane module in which a plurality of hollow fiber membranes are housed in a container having a liquid inlet / outlet. The container has at least a first liquid inlet / outlet 33a and a second liquid inlet / outlet 33b for supplying liquid to the hollow fiber membranes, and third liquid inlets / outlets 33c and 33d for discharging liquid that has permeated the hollow fiber membranes. The number of hollow fiber membranes used in the hollow fiber membrane module of the present disclosure is not particularly limited. The number of hollow fiber membranes to be incorporated into the module can be designed appropriately to obtain the required effective membrane area, depending on the intended use of the present disclosure and the weight of the useful minute substance to be purified and concentrated.

[0048] The hollow fiber membranes are preferably bundled together with adhesive or the like and housed in the container. In the present disclosure, when a liquid containing useful micro-substances is supplied from the first liquid inlet / outlet 33a and / or the second liquid inlet / outlet 33b of the hollow fiber membrane module, the liquid flows into one side of the hollow fiber membrane bundle in the container (inside or outside the hollow fiber membrane bundle) and is purified and concentrated by the hollow fiber membrane bundle. The liquid that permeates the hollow fiber membrane bundle flows out the other side of the hollow fiber membrane bundle (inside or outside the hollow fiber membrane bundle) and is discharged from the third liquid inlet / outlet 33c, 33d. At this time, the liquid that remains inside or outside the hollow fiber membrane bundle without permeating can be discharged (recovered) from the hollow fiber membrane module through the first liquid inlet / outlet and / or the second liquid inlet / outlet. Hereinafter, in the present disclosure, the liquid that permeates the hollow fiber membranes will be referred to as the "permeate," and the liquid that does not permeate the hollow fiber membranes and remains in the hollow fiber membranes will be referred to as the "concentrate." The term "concentrated liquid" refers to all liquids purified and concentrated using hollow fiber membranes.

[0049] A hollow fiber membrane module in which a liquid containing useful micro-substances is supplied to the inside of a hollow fiber membrane bundle is an internal pressure type hollow fiber membrane module, and a hollow fiber membrane module in which a liquid is supplied to the outside of a hollow fiber membrane bundle is an external pressure type hollow fiber membrane module. Hereinafter, an example of an internal pressure type hollow fiber membrane module will be described as one embodiment of the hollow fiber membrane module in the present disclosure.

[0050] FIG. 7 is a schematic cross-sectional view showing an example of an internal pressure type hollow fiber membrane module 33. The hollow fiber membrane module 33 contains a hollow fiber membrane bundle 60, which is a bundle of multiple hollow fiber membranes, housed within a cylindrical container 61. The multiple hollow fiber membranes are bound together with a biocompatible sealing adhesive (e.g., a polyurethane-based sealing adhesive) to form the hollow fiber membrane bundle 60, and are bonded and fixed to both longitudinal ends of the cylindrical container 61 with adhesive layers 62a, 62b made of the sealing adhesive. From the perspective of transparency, the cylindrical container 61 may be made of a material such as polycarbonate, poly(meth)acrylate, or polysulfone. Note that both end faces 61a, 61b of the cylindrical container 61 have multiple hollow fiber membranes open. An inlet / outlet 33a for a first liquid and an inlet / outlet 33b for a second liquid are provided at both longitudinal ends of the hollow fiber membrane module 33. In addition, third liquid inlets and outlets 33c and 33d are provided on the longitudinal side of the hollow fiber membrane module 33. The liquid supplied from the first liquid inlet and outlet 33a and / or the second liquid inlet and outlet 33b is sent to the inside of the hollow fiber membrane bundle 60 and purified and concentrated. The permeated liquid that has permeated to the outside of the hollow fiber membrane bundle 60 is discharged from the third liquid inlets and outlets 33c and 33d provided on the side of the cylindrical container 61. It is preferable that the third liquid inlets and outlets be provided in at least two locations, one at the upper side and one at the lower side of the cylindrical container 61. In FIG. 7, the third liquid inlets and outlets 33c and 33d are provided on only one side of the hollow fiber membrane module 33, but this is not a limitation. The third liquid inlets and outlets 33c and 33d may be provided separately on one side and the other side.

[0051] 7 illustrates an internal pressure type hollow fiber membrane module, but the hollow fiber membrane module of the present disclosure may be an external pressure type as described above. In the case of an external pressure type, it is necessary to appropriately design the installation locations and installation method of the inlets and outlets for the first to third liquids.

[0052] (Filling rate of hollow fiber membrane module) The filling rate of the hollow fiber membrane module of the present disclosure is preferably 5 to 55%, more preferably 20 to 55%, and even more preferably 25 to 55%. The filling rate refers to the percentage of the total cross-sectional area of ​​the outer diameter portion of the hollow fiber membrane bundle relative to the cross-sectional area of ​​the hollow fiber membrane module, generally a cylindrical container, in the short direction. If the filling rate is 5 to 55%, useful minute substances deposited on the membrane surfaces of the hollow fiber membranes can be more efficiently recovered while optimizing the size of the hollow fiber membrane module.

[0053] Hereinafter, an embodiment in which the purification and concentration section 30 of the present disclosure includes a hollow fiber membrane module 33 will be described with reference to the drawings.

[0054] 8 to 11 show some representative schematic configuration diagrams of the purification and concentration section 30. Note that the purification and concentration apparatus 1 of the present disclosure may include safety devices such as valves, automatic valves, check valves, orifices, flow meters, pressure gauges, level gauges, and rupture discs, which are omitted from the schematic configuration diagrams.

[0055] The purification and concentration section 30 shown in Figure 8 includes a second tank 31, 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 that pressurizes the second tank 31 and the third tank 32. The pressurizing device 34 is connected to a three-way valve 76 via a gas supply line 52. The second tank 31 is a tank that stores the liquid supplied from the raw material supply unit 10 or the removal unit 20, i.e., the raw material liquid or the roughly purified liquid, and is connected to a first liquid inlet / outlet 33a of the hollow fiber membrane module 33 via a flow path 51A. Similarly to the second tank 31, the third tank 32 is a tank that stores the raw material liquid or the roughly purified liquid and is connected to a second liquid inlet / outlet 33b of the hollow fiber membrane module 33 via a flow path 51B. The upper portions of the second tank 31 and the third tank 32 are connected to the pressurizing device 34. The second control unit 120 (not shown) operates the pressurizing device 34 and switches the three-way valve 76 to selectively pressurize either the second tank 31 or the third tank 32, thereby controlling the raw material liquid or partially purified liquid in the second tank 31 or the third tank 32 to be 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 in the hollow fiber membrane module 33 and then sent to the other tank. Hereinafter, the liquid stored in the second tank 31 before the completion of the purification and concentration process will be referred to as the "second stored liquid," and the liquid stored in the third tank 32 before the completion of the purification and concentration process will be referred to as the "third stored liquid." Note that, although FIG. 8 shows a configuration in which the flow paths 42 and 43 are connected to the second tank 31, the flow paths 42 and 43 may also be connected to the third tank 32. The second control unit 120 will be described in detail later.

[0056] (Pressurization Device) The pressurization device 34 of the present disclosure supplies gas from a gas supply source into 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 gas supply paths 52A, 52B equipped with a pressure gauge (not shown) by switching a three-way valve 76 by a second control unit 120 (not shown). The gas can be selected from an inert gas such as nitrogen, argon, or helium, carbon dioxide, filtered air, or the like.

[0057] 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 line 52 and switches the three-way valve 76 to send the gas to the gas supply line 52A. At this time, the second control unit 120 closes the on-off valve 74 of the gas vent line 53A and the three-way valve 77 connecting the flow paths 42 and 43, and opens the on-off valve 75 of the gas vent line 53B. On the other hand, when the third tank 32 is pressurized by the pressurizing device 34, the second control unit 120 supplies gas from the gas supply source (not shown) to the gas supply line 52 and switches the three-way valve 76 to send the gas to the gas supply line 52B. At this time, the second control unit 120 closes the on-off valve 75 and the three-way valve 77 of the gas vent line 53B, and opens the on-off valve 74 of the gas vent line 53A. By these operations, the pressurizing device 34 can selectively pressurize the second tank 31 and the third tank 32.

[0058] (Second Tank and Third Tank) In the purification and concentration apparatus 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 and third stored liquids can be observed visually or with a camera described below. Furthermore, from the viewpoint of preventing the second or third stored liquid from adhering and remaining on the inner wall surface of the tank, it is more preferable that the second tank 31 and the third tank 32 be made of a water-repellent material. Specifically, the second tank 31 and the third tank 32 are preferably made of a material containing polyacrylonitrile; poly(meth)acrylate such as poly(meth)acrylic acid ester; polycarbonate; fluororesin, or the like. Note that, when the liquid level in the transparent portion of the liquid inlet / outlet of the second tank 31 or the third tank 32 is managed using a tank liquid level monitoring device or the like described below, the second tank 31 and the third tank 32 can be made of an opaque material, such as a metal or fiber-reinforced resin tank.

[0059] In the purification and concentration section 30 shown in Figure 8, the second pooled liquid enters the hollow fiber membrane module 33 from the first liquid inlet / outlet 33a via the flow path 51A, is purified and concentrated in the hollow fiber membrane bundle 60, and is then discharged from the second liquid inlet / outlet 33b and sent to the third tank 32. In this case, the second pooled liquid is purified and concentrated by tangential flow filtration, in which the liquid flows parallel to the membrane surfaces in the longitudinal direction of the hollow fiber membrane bundle 60. The permeated liquid that has permeated the hollow fiber membrane bundle 60 is discharged to a waste liquid tank (described below) from a flow path 54 connected to the third liquid inlets 33c and 33d of the hollow fiber membrane module 33.

[0060] Similarly, the third pooled liquid enters the hollow fiber membrane module 33 through the second liquid inlet / outlet 33b via the flow path 51B, is purified and concentrated in the hollow fiber membrane bundle 60, and is then discharged through the first liquid inlet / outlet 33a and sent to the second tank 31. In this case, too, the third pooled liquid is purified and concentrated by tangential flow filtration, in which the liquid flows parallel to the membrane surfaces in the longitudinal direction of the hollow fiber membrane bundle 60. By alternately repeating this operation, it becomes difficult for useful micro-substances and impurities to accumulate on the hollow fiber membrane surfaces. As a result, a decrease in the liquid permeation rate can be suppressed, and high treatment efficiency can be maintained. In other words, by subjecting the liquid to alternating tangential flow filtration, a liquid containing useful micro-substances at higher purity and higher concentrations can be more efficiently produced.

[0061] The second control unit 120 sets the amount of the second stored liquid and the third stored liquid, and controls the pressure device 34 and the on-off 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.

[0062] The purification and concentration section 30 in Fig. 8 may be configured to include a device for monitoring the liquid volume in the second tank 31 and the third tank 32. With this configuration, the liquid volumes in the second tank 31 and the third tank 32 can be monitored, allowing for more efficient alternating tangential flow filtration.

[0063] The purification and concentration unit 30 shown in Figure 9 has the same configuration as the purification and concentration unit 30 shown in Figure 8, and further includes tank liquid level monitors 35, 36, each of which includes an optical sensor (not shown) at the liquid inlet and outlet ports at the bottom of the tank, for monitoring liquid levels in the second tank 31 and the third tank 32. The tank liquid level monitors 35, 36 are configured to irradiate the liquid inlet and outlet ports of the second tank 31 and the third tank 32 with light using the optical sensor to detect the light transmittance at the liquid inlet and outlet ports, and monitor the liquid levels in the tanks based on changes in the transmittance. A second control unit 120 (not shown) determines the liquid levels of the second and third stored liquid in the second tank 31 and the third tank 32 using the tank liquid level monitors 35, 36, and determines which tank to pressurize using the pressurizing device 34.

[0064] (Tank liquid level monitoring device) The tank liquid level monitoring devices 35, 36 of the present disclosure are equipped with an optical sensor. The optical sensor has a light-emitting element and a light-receiving element, and the light-emitting element emits light toward the liquid inlet / outlet at the bottom of the tank, and the light-receiving element detects the intensity of the transmitted light (transmittance). The portion of the tank bottom that is illuminated by light from the liquid inlet / outlet is made of a transparent material. The detected transmittance is then sent to the detection unit. When the detection unit detects a change in transmittance, the second control unit 120 changes the tank to be pressurized by the pressurizing device 34.

[0065] The relationship between the tank liquid level monitor and the second control unit will be described in more detail. 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 liquid inlet / outlet of the second tank 31, causing a change in the light transmittance through the liquid inlet / outlet. When the tank liquid level monitor 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 level monitor 36 provided at the liquid inlet / outlet 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 level monitors 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.

[0066] In other embodiments, the liquid levels in the tanks may be monitored based on images captured by a camera disposed in the purification and concentration unit 30. That is, the purification and concentration unit 30 may include a camera for monitoring the liquid level positions in the second tank 31 and the third tank 32, and the second control unit 120 may determine the liquid levels in the tanks from information on the liquid level positions in the second tank 31 and the third tank 32 output from the camera, and determine which tank to pressurize with the pressurizing device 34. Specifically, the purification and concentration unit 30 includes a camera positioned to observe the liquid levels in the second tank 31 and the third tank 32, and the image of the liquid level position output from the camera is transmitted to a detection unit. The detection unit determines the liquid levels in the second tank 31 and the third tank 32 from the information on the liquid level position. For example, when the detection unit determines from the information on the liquid level position captured by the camera that the amount of the second stored liquid in the second tank 31 has fallen below a predetermined amount, the second control unit 120 switches the three-way valve 76 to change the tank to be pressurized with the pressurizing device 34 from the second tank 31 to the third tank 32. Similarly, if the detection unit determines that the amount of the third stored liquid in the third tank 32 has fallen below a predetermined amount, the second control unit 120 switches the three-way valve 76 to change the tank pressurized by the pressure device 34 from the third tank 32 to the second tank 31.

[0067] (Second Control Unit) An example of a determination made by the second control unit 120 in controlling the flow of liquid is shown in the flowchart of FIG. 16. As shown in FIG. 16, the second control unit 120 can operate an on-off valve and a pressurizing device to mainly circulate the liquid (crudely purified liquid or raw material liquid) between the second tank 31 and the third tank 32. The second control unit 120 can also determine the liquid levels in the second tank 31 and the third tank 32 based on information from the in-tank liquid level monitoring devices 35, 36 and / or cameras, and can circulate the liquid in the tanks. Furthermore, the second control unit 120 can also set the liquid levels in the second tank 31 and / or the third tank 32. An example of a method for setting the liquid levels in the tanks (setting the controlled liquid level position C) will be described later.

[0068] The purification and concentration unit 30 in Fig. 10 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. The purification and concentration unit 30 shown in Fig. 10 includes a fourth tank 80 that stores the diluent and is connected to a flow path 55A that supplies the diluent to the third liquid inlets and outlets 33c and 33d of the hollow fiber membrane module 33, and a third control unit 130 (not shown) that controls the timing at which the diluent in the fourth tank 80 is supplied to the third liquid inlets and outlets 33c and 33d of the hollow fiber membrane module 33. A flow path 55B that directly supplies the diluent in the tank to the third tank 32 (or the second tank 31) is connected to the fourth tank 80.

[0069] (Third Control Unit) The third control unit 130 controls the flow of the diluted solution in the fourth tank 80. An example of the determination made by the third control unit 130 in controlling the flow of the solution is shown in the flowchart of Figure 16. The third control unit 130 can supply the diluted solution 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 solution. Hereinafter, supplying the diluted solution to the third tank 32 will be referred to as the "dilution process," and supplying the diluted solution to the hollow fiber membrane module 33 will be referred to as the "backwashing process."

[0070] (Diluted Solution) Depending on the purpose of the present disclosure and the type of useful micro-substance, the diluted solution may be, for example, pure water; filtered water such as tap water, groundwater, seawater, or river water; or various aqueous solutions. When the useful micro-substance is exosomes, physiological saline or a medical or biochemical buffer solution is preferred. Examples of the buffer solution that can be used include phosphate buffer (PBS), Tris-HCl buffer, sodium citrate buffer, citrate phosphate buffer, acetate buffer, and borate buffer. Depending on the purpose of the present disclosure, the diluted solution is preferably impurity-free; however, if it is difficult to obtain an impurity-free diluted solution, a diluted solution with a low impurity concentration (e.g., an impurity concentration of less than 500 ppm) can be used.

[0071] The diluted liquid can be supplied to the concentrated liquid in the second tank 31 (or the third tank 32) when the ratio (B) of the amount of concentrated liquid in the second tank 31 (or the third tank 32) to the amount (A) of the raw liquid containing the minute useful substances (B / A) becomes 1 / 2 to 1 / 200. In this case, the amount (B) of the concentrated liquid may be equal to the liquid volume at the controlled liquid level position C set by the method described below. Furthermore, when the roughly purified liquid is supplied from the first tank 23 to the second tank 31 (or when 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 roughly purified liquid or the raw liquid has been supplied to the second tank 31 based on the liquid level position information from the tank liquid level monitoring device 35 and / or the camera. In one embodiment, the third control unit 130 performs alternating tangential flow filtration to concentrate and purify the crude liquid until the volume of the crude liquid is reduced to 1 / 2 to 1 / 200 of the original volume, and then performs a dilution process (or backwashing process) until the volume of the crude liquid reaches a set volume, thereby supplying the diluted liquid. When performing the dilution process, the third control unit 130 opens the three-way valve 77A to the flow path 55B side, and supplies the diluted liquid in the fourth tank 80 to the third tank 32.

[0072] The third control unit 130 can perform backwashing when it determines that the total amount of liquid in the second tank 31 and the third tank 32 has fallen below a predetermined liquid amount (for example, below the controlled liquid level C set by a method described below). To perform backwashing, the third control unit 130 opens the three-way valve 77A to the flow path 55A. The diluted liquid in the fourth tank 80 is supplied into the hollow fiber membrane module 33 through the third liquid inlets 33c and 33d via the flow path 55A. The diluted liquid then flows from the permeate side to the concentrate side of the hollow fiber membrane bundle 60. If the hollow fiber membrane module 33 is an internal pressure type, the diluted liquid supplied into the module through the third liquid inlets 33c and 33d flows from the outside to the inside of the hollow fiber membrane bundle 60. This allows the minute useful substances deposited on the membrane surface of the hollow fiber membrane, particularly on the membrane surface inside the hollow fiber membrane, to be recovered into the second tank 31 and / or the third tank 32 through the first liquid inlet / outlet 33a and / or the second liquid inlet / outlet 33b. This process peels off the minute useful substances and impurities deposited on the hollow fiber membrane surface, restoring the liquid permeation rate to almost its original value. Furthermore, by diluting the liquid with the diluent, the impurities are uniformly dispersed, increasing the removal efficiency. As a result, the efficiency of liquid purification and concentration can be further improved.

[0073] Before backwashing, a flushing treatment can be carried out in advance with a cleaning liquid (diluted liquid) to prevent the permeated liquid remaining in the hollow fiber membrane module from mixing with the backwashing liquid and flowing into the concentrated liquid side, and the permeated liquid remaining in the hollow fiber membrane module can be replaced with the cleaning liquid (diluted liquid).

[0074] In this case, the amount of the washing liquid (diluted liquid) used is preferably 1 to 8 times, more preferably 2 to 6 times, the volume of the permeated liquid remaining in the hollow fiber membrane module.

[0075] When concentrating and purifying certain extracellular vesicles from certain culture supernatants, performing a flushing operation before backwashing can sometimes reduce the amount of remaining impurity proteins to less than half of that when no flushing operation is performed.

[0076] When the hollow fiber membrane module 33 is an internal pressure type, the permeate remaining in the hollow fiber membrane module can be replaced with a cleaning liquid (diluted liquid) using either the third liquid inlet / outlet 33c or 33d in Figure 7 as an inlet and the other as an outlet. Because the hollow fiber membranes have resistance to liquid flow, supplying the diluted liquid through either the third liquid inlet / outlet 33c or 33d allows the permeate remaining in the hollow fiber membrane module 33 to be replaced with the diluted liquid with minimal leakage into the membrane. Alternatively, for example, an on-off valve (not shown) can be provided in the flow path near the outlets of the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b, and closing this on-off valve can stop the flow of the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b. In this state, supplying the diluted liquid through either the third liquid inlet / outlet 33c or 33d causes the diluted liquid to be discharged from the other third liquid inlet / outlet 33c or 33d. This allows the permeated liquid remaining in the hollow fiber membrane module 33 to be replaced with the diluent.

[0077] The timing of the flushing process is controlled by the third control unit. For example, in a case where on-off valves (not shown) are provided on the flow paths near the outlets of the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b, when the total amount of the liquid in the second tank 31 and the third tank 32 falls below the value set by the second control unit, the third control unit can supply the dilution liquid in the fourth tank 80 from one of the at least two third liquid inlets 33c and 33d of the hollow fiber membrane module 33 by closing the on-off valves provided near the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b to stop the flow of the liquid. At this time, the on-off valve 78 provided on the flow path 54 connected to the waste liquid tank is open. Thereafter, the on-off valves installed near the inlet / outlet 33a for the first liquid and the inlet / outlet 33b for the second liquid are opened to allow flow through the inlet / outlet 33a for the first liquid and the inlet / outlet 33b for the second liquid, and backwashing can be performed by supplying the dilution liquid in the fourth tank 80 from one of the at least two inlets / outlets 33c and 33d for the third liquid that the hollow fiber membrane module 33 has. At this time, the on-off valve 78 installed in the flow path 54 connected to the waste liquid tank is closed.

[0078] The purification and concentration unit 30 shown in FIG. 11 includes a recovery tank for recovering the concentrated liquid purified and concentrated by the hollow fiber membrane module 33. The purification and concentration unit 30 in FIG. 11 includes a fifth tank 81 connected to the first liquid inlet / outlet 33a of the hollow fiber membrane module 33 via a flow path 56, and a fourth control unit 140 (not shown) that controls the recovery of the liquid from the second tank 31 and the third tank 32 into the fifth tank 81. The fifth tank 81 may also be connected to the second liquid inlet / outlet 33b of the hollow fiber membrane module 33. In FIG. 11 , the flow path 56 is connected to the hollow fiber membrane module 33 via a flow path 51A. While the purification and concentration process by alternating tangential flow filtration is being performed in 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, allowing the concentrated liquid from the second tank 31 and the third tank 32 to be recovered in the fifth tank 81. The tank for collecting the concentrated liquid may be arranged outside the purification and concentration device, rather than inside the purification and concentration section 30.

[0079] (Fourth Control Unit) The fourth control unit 140 controls the timing of recovering the concentrated liquid from the second tank 31 and the third tank 32. An example of the determination made by the fourth control unit 140 in controlling the flow of liquid is shown in the flowchart of FIG.

[0080] The fourth control unit 140 determines the volume of the concentrated liquid in the second tank 31 and the third tank 32 using the tank liquid volume monitoring devices 35, 36 and / or the camera. The volume of the concentrated liquid may be determined from the controlled liquid level position C described below. When it is determined that the purification and concentration process is complete, the fourth control unit 140 pressurizes the second tank 31 and the third tank 32 using the pressurizing device 34. At this time, the on-off valve 74 of the gas vent path 53A, the on-off valve 75 of the gas vent path 53B, the on-off valve 78, and the three-way valves 77 and 77A are closed, and the on-off valve 79 is opened. When the tank liquid volume monitoring devices 35, 36 detect a change in transmittance, or when it is determined from the camera images that the volume of the concentrated liquid in the second tank 31 and the third tank 32 has reached zero, the fourth control unit 140 stops the pressurizing device 34 and terminates the collection of the concentrated liquid into the fifth tank 81. Specifically, the fourth control unit 140 may monitor the amount of concentrated liquid in the flow path 56 using a fiber sensor (not shown) installed at the liquid inlet / outlet at the top of the fifth tank 81, and may terminate collection of the concentrated liquid when it determines that the liquid amount has reached zero. Alternatively, the operation of collecting the concentrated liquid inside the flow path 56 may be manually performed by a person. Furthermore, the flow path 56 may be fixed and held using an air gripper or the like.

[0081] FIG. 12 shows an example of the configuration of a purification and concentration apparatus according to the first embodiment. In the purification and concentration apparatus 1 of FIG. 12, elements including a raw material supply unit 10, a removal unit 20, and a purification and concentration unit 30 are arranged within a housing. The size of the housing of the purification and concentration apparatus 1 of FIG. 12 is not particularly limited. The purification and concentration apparatus 1 also includes a control unit (not shown) that manages the cleanliness level within the housing. This configuration allows the interior of the purification and concentration apparatus 1 to be kept clean and prevents contamination of the liquid containing minute useful substances. A fan filter device 91 that supplies filtered air into the housing is arranged at the top (top) of the housing 100, and a UV lamp 92 is arranged within the housing 100. This combination further increases the cleanliness level within the housing 100.

[0082] (Fan Filter Device) The fan filter device 91 has a configuration that allows filtered air to be supplied into the housing. The fan filter device 91 can be a filter unit with a fan that has a built-in pre-filter. As the pre-filter, a HEPA filter, ULPA filter, chemical filter, fiber activated carbon filter, etc. can be used depending on the cleanliness level inside the housing 100.

[0083] (UV Lamp) The UV lamp 92 is not particularly limited as long as it can irradiate UV (ultraviolet rays) to sterilize the inside of the housing 100. In addition, the number of UV lamps 92 arranged inside the housing 100 is also not particularly limited, and two or more UV lamps may be arranged depending on the cleanliness level inside the housing 100.

[0084] (Cleanliness) When the purification and concentration apparatus 1 is configured such that each element is disposed inside the housing 100, the above-described management unit can increase the cleanliness inside the housing 100. In one embodiment, the cleanliness inside the housing 100 may be managed to be class 8 or less, or class 5 or less, according to the cleanliness classes defined in ISO 14644-1:2015.

[0085] (Useful Micro Substances) The useful micro substance is not particularly limited. In one embodiment, it may be a useful micro substance with an average particle size of less than 100 μm. In another embodiment, it may be a useful micro substance with an average molecular weight of 5,000 or more. Among these, any substance useful as a pharmaceutical, cosmetic, or health food product may be used. Specific examples of useful micro 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, enzymes, antibodies, plasma proteins, viruses (including virus-like particles), extracellular vesicles (e.g., exosomes, microvesicles, apoptotic bodies, etc.), biomolecules such as yeast, yeast cells, and microalgae, and polysaccharides such as fucoidan. Other examples include specific microorganisms, which are a type of biomolecule and are used for biofuels, etc. Among these, useful micro substances may be selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.

[0086] [Method for producing a liquid in which useful micro substances are purified and concentrated: First embodiment] A method for producing a liquid in which useful micro substances are purified and concentrated using the purification and concentration device 1 of the present disclosure will be described below with reference to Fig. 13. The production method according to the first embodiment of the present disclosure is a method for producing a liquid in which useful micro substances are purified and concentrated using the purification and concentration device 1, and includes supplying a raw material liquid containing the useful micro substances to the purification and concentration device and removing impurities in the liquid, and / or purifying and concentrating the liquid using a hollow fiber membrane.

[0087] FIG. 13 is a diagram showing an example of the overall configuration of the purification and concentration apparatus 1 of the present disclosure. The raw material supply unit 10 is configured to include a container 11 for storing a raw material liquid containing useful micro-substances. When the apparatus is started, the three-way valve 82 opens, and the raw material liquid in the container 11 is supplied to the flow path 59 by falling under its own weight and by a pump (not shown) provided in the flow path 41A. The raw material liquid is supplied to the flow path 41 or the flow path 43 by operating the three-way valve 82. 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 according to the concentration of impurities in the raw material liquid. The first control unit 110 opens the three-way valve 82 to the flow path 43 side, and sends the culture liquid from the flow path 43 to the purification and concentration unit 30.

[0088] The raw material liquid supplied to flow path 41 is pretreated by filter member 22 and then supplied to tank 24 via flow path 41C. The amount of liquid in tank 24 can be controlled by monitoring the liquid level with a level sensor (not shown). When on-off valve 73 is opened, the pretreatment liquid in tank 24 is supplied from flow path 41D to flow path 41A.

[0089] (Impurity Removal Treatment) The pretreatment liquid supplied to the carrier column 21 has impurities adsorbed and removed by the adsorbent in the carrier column 21 to form a roughly purified liquid, which is then supplied to the first tank 23 via the flow path 42A. The impurity removal treatment using the carrier column 21 may be to reduce the concentration of impurities in the raw material liquid or the pretreatment liquid to 1,000 ppm or less, preferably to 500 ppm or less.

[0090] In one embodiment, the impurity removal process may be repeated by washing and equilibrating the carrier column 21 that has reached adsorption saturation after processing one batch of raw material liquid, and then processing the next batch of raw material liquid. The purification and concentration apparatus 1 in FIG. 13 is configured to include a sixth tank 25 and a seventh tank 26. When washing and equilibrating the carrier column 21 that has reached adsorption saturation, first, the on-off valve 71A (e.g., an electromagnetic valve) is opened, and the washing solution in the sixth tank 25 is supplied to the filtration element 22A via the flow path 50A. After the impurities in the washing solution are removed by the filtration element 22A, the washing solution is supplied to the carrier column 21 via the flow path 41A. After washing the carrier column 21, the washing solution can be discharged to a waste tank by opening the three-way valve 72. The same filtering element as the filtration element 22 can be used for the filtration element 22A. After the washing process, the on-off valve 71B (e.g., an electromagnetic valve) is opened, and the equilibration solution in the seventh tank 26 is supplied to the filtration element 22B. After impurities in the equilibration liquid are removed by the filter member 22B, the equilibration liquid is supplied from the flow path 41A to the carrier column 21. After equilibrating the carrier column 21, the equilibration liquid can be discharged to a waste tank by opening the three-way valve 72. The filter member 22B can be made of the same material as the filter member 22.

[0091] The washing and equilibration of the carrier column 21 can be set arbitrarily depending on the amount of the raw material solution treated in the carrier column 21. In one embodiment, the washing and equilibration of the carrier column 21 may be performed when a raw material solution having a volume 5 to 100 times the volume of the carrier column 21 has been treated. For example, when treating 1,000 mL of raw material solution, the washing and equilibration treatment of the carrier column 21 can be performed every time 20 to 150 mL of the raw material solution is treated in the carrier column 21, and the washing and equilibration treatment can also be performed every time 50 to 100 mL of the raw material solution is treated.

[0092] In the manufacturing method of the present disclosure, it is preferable to simultaneously remove impurities from the raw liquid using the carrier column 21 and purify and concentrate the liquid using the hollow fiber membranes in the purification and concentration section 30. In this case, a liquid in which useful micro-substances are purified and concentrated can be produced more efficiently. In FIG. 13 , the crudely purified liquid is stored in the first tank 23 via flow path 42A. When the first control unit 110 determines that the amounts of the second and third stored liquids in the second and third tanks 31 and 32 are equal to predetermined liquid volumes, it sends the crudely purified liquid stored in the first tank 23 to flow path 42B. By switching the direction of the three-way valve 77, the crudely purified liquid is supplied from flow path 42B to the second tank 31. Once the crudely 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 initiate a purification and concentration process using alternating tangential flow filtration.

[0093] As described above, the purification and concentration apparatus 1 according to the first embodiment of the present disclosure may be configured so that the impurity removal process in the removal section 20 and the purification and concentration process in the purification and concentration section 30 are performed in parallel. In this case, it is preferable to configure the apparatus 1 so that multiple tanks are arranged to store the liquids processed in each step. The positions of the tanks may be adjusted to make the purification and concentration apparatus 1 more compact. For example, in the purification and concentration apparatus 1 shown in FIG. 13 , the first tank 23 for storing the crude liquid is arranged above the second tank 31 and the third tank 32. However, the second tank 31 and the third tank 32 may also be arranged above the first tank 23. Furthermore, as shown in FIG. 13 , the longitudinal sides of the second tank 31 and the third tank 32 may be arranged parallel to the longitudinal direction of the hollow fiber membrane module 33.

[0094] A preferred operation for performing alternating tangential flow filtration of the crude liquid (or raw liquid) in the purification and concentration section 30 will be described below. In one embodiment, the purification and concentration process of the crude liquid is preferably performed in three stages: a first process, a second process, and a third process. Before performing the first process, the liquid volume in the tank is set. An example will be described below in which the controlled liquid level position C is set and then the first process is performed.

[0095] (Setting the Control Liquid Level Position C) Before performing the first treatment, the amount at the control liquid level position C is set. The first tank 23 contains the crudely purified solution treated in the antibody column 21. The liquid volume at the control liquid level position C is equal to the liquid volume at which the backwashing treatment should be started, and is preset to be 1 / 2 to 1 / 200 of the maximum volume of the crudely purified solution stored in the first tank 23. When the amount of the crudely purified solution in the second tank 31 reaches the amount set at the preset control liquid level position C, the crudely purified solution in the first tank 23 can be supplied to the second tank 31. Furthermore, after the crudely purified solution has been supplied to the second tank 31, the third control unit 130 opens the three-way valve 77A to the flow path 55B side to supply the diluent from the fourth tank 80 to the second tank 31.

[0096] After setting the controlled 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, and the diluted liquid in the third tank 32 is sent 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 diluted liquid to the second tank 31 until the diluted liquid in the third tank 32 reaches L3. Furthermore, the second control unit 120 sets the liquid level position of the second stored liquid (a mixture of the partially purified liquid and the diluted liquid) in the second tank 31 when the diluted liquid in the third tank 32 reaches L3 as the high liquid level position (H2). Furthermore, from the viewpoint of preventing the second stored liquid from overflowing from the top of the second tank 31, a 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, and sends the second stored liquid to the third tank 32. At this time, the second control unit 120 sets a low liquid level position (L2) for the second tank 31 and supplies the second stored liquid to the third tank 32 until the second stored liquid in the second tank 31 reaches L2. Furthermore, the second control unit 120 sets the liquid level position of the third tank 32 when the second tank 31 reaches L2 as a high liquid level position (H3). The third tank 32 also sets HH3 to prevent leakage. The second control unit 120 sets a controlled liquid level position C based on the total liquid volume of H2 and L3, and H3 and L2, and performs alternating tangential flow filtration between the second tank 31 and the third tank 32 by moving the liquid within the range of the controlled liquid level position C. Note that L2 and L3 may be set based on changes in the transmittance of the liquid inlets and outlets of the tanks as measured by the tank liquid level monitoring devices 35 and 36, or based on images of the liquid level position captured by a camera. Furthermore, the positions of L2, L3, H2, H3, HH2, and HH3 can be set appropriately depending on the capacity of the second tank 31 and the third tank 32, and the amount of the partially purified liquid.

[0097] (Purification and concentration process: first process) Next, the first process in the purification and concentration process will be described. The second control unit 120 alternately pressurizes the second tank 31 and the third tank 32 using the pressure device 34 so that the liquid volume does not fall below the controlled liquid level position C, and alternately supplies the second and third stored liquids to the hollow fiber membrane module 33. By performing alternate tangential flow filtration in this manner, a concentration and purification operation is carried out on the liquid containing minute useful substances. The permeated liquid is discharged from the third liquid inlets and outlets 33c and 33d via the flow path 54 to a waste liquid tank.

[0098] After confirming that the liquid volume in the second tank 31 or the third tank 32 has reached the set value, an appropriate amount of the roughly purified liquid can be additionally supplied to the second tank 31 or the third tank 32. Furthermore, when the liquid volume in the second tank 31 or the third tank 32 becomes equal to the liquid volume at the controlled liquid level position C, it is preferable to perform a backwashing process.

[0099] The amount of diluent used in the backwashing process can be 2 to 200 times the volume of the liquid at the controlled liquid level position C. The amount of diluent is preferably 2 to 20 times the volume of the liquid at the controlled liquid level position C, so as to prevent the volume of the second tank 31 or the third tank 32 from becoming unnecessarily large. The amount of diluent used in the backwashing process can also be changed as needed with each backwashing operation. Furthermore, the backwashing process can be performed at any time other than when the volume of the liquid in the second tank 31 or the third tank 32 becomes equal to the volume of the liquid at the controlled liquid level position C. Examples of such timing include when there is concern about a decrease in the permeation rate due to pressure fluctuations or flow rate fluctuations, when a tendency for the permeation rate to decrease can be determined, or when safety operations are being performed to prevent blockage of the hollow fiber membrane. A portion of the diluent can also be directly sent to the second tank 31 or the third tank 32 for dilution. The amount of diluent used in the dilution process can also be changed as needed with each dilution operation.

[0100] 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 on the membrane surface of the hollow fiber membrane module 33 is 0.3 m / sec to 2 m / sec. ２The flow rate per second of the liquid containing the micro-useful substance flowing against the membrane surface (m ３ ) and the unit is m / sec. By pressurizing the tank with the pressurizing device 34 so that the linear velocity on the membrane surface is 0.3 to 2.0 m / sec, the purification and concentration efficiency is likely to be improved, and the pressure level for increasing the membrane surface velocity is not too high, which makes it easy to prevent the deformation of the useful micro substance due to excessive shear force applied to the useful micro substance. The pressurization inside the tank by the pressurizing device 34 may be carried out in a range so that the linear velocity on the membrane surface is 0.5 to 1.5 m / sec.

[0101] The operating pressure of the hollow fiber membrane module 33 is preferably adjusted to 0.01 MPa to 0.2 MPa, more preferably 0.02 MPa to 0.15 MPa, and even more preferably 0.03 MPa to 0.12 MPa at the inlet of the liquid. In addition, the liquid viscosity during operation of the liquid containing the useful micro substance is adjusted to 0.01 MPa to 0.2 MPa at a liquid temperature of 20°C and a shear rate of 25 s －１ When the liquid viscosity is 10 to 30 mPa s, a large-diameter hollow fiber membrane having an average inner diameter of 1.0 mm to 1.4 mm can be used so that the linear velocity on the membrane surface can be 0.7 to 1.4 m / s. In this case, the operating pressure is preferably adjusted to 0.08 MPa to 0.2 MPa at the liquid inlet.

[0102] The hollow fiber membrane may be coated with an appropriate substance to improve the fouling resistance of the hollow fiber membrane surface and / or to increase the recovery rate of useful micro-substances. The coating method is as described above.

[0103] In the first process, the purification and concentration process is preferably performed until the volume of the crude liquid reaches the volume equal to the volume of the controlled liquid level position C, which is preset by the above-described method. After the purification and concentration process, the third control unit 130 supplies a dilution liquid from the fourth tank 80. The dilution liquid can be supplied until the volume reaches 2 to 200 times the volume of the controlled liquid level position C. That is, the first process can include diluting the concentrated liquid 2 to 20 times. The dilution liquid may be supplied directly to the third tank 32 (dilution process) or may be supplied from the hollow fiber membrane module 33 to the second tank 31 and the third tank 32 (backwashing process). When performing the backwashing process, in the purification and concentration apparatus 1 of FIG. 13, the three-way valve 77A is opened to the flow path 55A, and the dilution liquid is supplied from the third liquid inlets 33c and 33d of the hollow fiber membrane module 33. The dilution liquid supplied from the hollow fiber membrane module 33 is supplied to the second tank 31 and the third tank 32. The backwashing treatment is preferable because it allows the concentrated liquid to be diluted while recovering minute useful substances deposited on the membrane surface of the hollow fiber membrane. Hereinafter, the liquid obtained by the first treatment will be referred to as the "first treated liquid."

[0104] (Purification and Concentration Treatment: Second Treatment) The first treatment liquid is further subjected to a second treatment to obtain a liquid in which useful micro substances are further purified and concentrated. Because the impurity concentration in the first treatment liquid is reduced by the first treatment described above, the second treatment is preferably performed to further reduce the amount of impurities in the first treatment liquid (i.e., to increase the purity of the useful micro substances). In one embodiment, the second treatment preferably involves adding a diluent in an amount 2 to 200 times the volume of the concentrated liquid set at the controlled liquid level position C through a backwashing or dilution treatment, and then repeating the purification and concentration operation until the volume reaches the controlled liquid level position C. That is, the second treatment may include diluting the concentrated liquid 2 to 20 times. As in the first treatment, the diluent may be supplied directly to the third tank 32 (dilution treatment) or from the hollow fiber membrane module 33 to the second tank 31 and the third tank 32 (backwashing treatment). The second treatment, which combines the purification and concentration treatment and the dilution treatment, is preferably performed 2 to 20 times, more preferably 2 to 10 times. Hereinafter, the concentrated liquid obtained by the above-mentioned second treatment will be referred to as the "second treatment liquid."

[0105] (Purification and concentration treatment: third treatment) The second treatment liquid can be further subjected to a third treatment to obtain a liquid in which useful micro-substances are further purified and concentrated. In the third treatment, no dilution liquid is used, and only alternating tangential flow filtration operation can be carried out to the extent necessary. Hereinafter, the concentrated liquid obtained by the above-mentioned third treatment will be referred to as the "third treatment liquid."

[0106] (Recovery Process) The manufacturing method of the present disclosure may include 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 module 33 can be recovered into the fifth tank 81 via the flow path 56 by pressurizing it with the pressurizing device 34. When pressurizing it with the pressurizing device 34, the fourth control unit 140 opens the on-off valve 79 and closes the on-off valves 74, 75, and 78 and the three-way valves 77 and 77A. As described above, the fourth control unit 140 may monitor the amount of concentrated liquid in the flow path 56 using a fiber sensor installed at the liquid inlet / outlet at the top of the fifth tank 81, and may terminate the recovery of the concentrated liquid when it determines that the liquid amount has reached zero. Alternatively, the operation of recovering the concentrated liquid in the flow path 56 can be manually performed by a person. Furthermore, the flow path 56 can be fixed and held using an air gripper or the like.

[0107] (Flushing Treatment) Before backwashing, a flushing treatment can be optionally performed in advance with a cleaning liquid (diluted liquid) to prevent the permeated liquid remaining in the hollow fiber membrane module from mixing with the backwashing liquid and flowing into the concentrated liquid side, and the permeated liquid remaining in the hollow fiber membrane module can be replaced with the cleaning liquid (diluted liquid). The method of the flushing treatment is as described above.

[0108] (Washing-out Operation of Useful Micro Substances Deposited on the Hollow Fiber Membrane Surface Immediately Before the Recovery Process) The manufacturing method of the present disclosure may include a washing-out operation of useful micro substances deposited on the hollow fiber membrane surface immediately before the recovery process. For example, when concentrating and purifying certain extracellular vesicles from certain culture supernatants, the extracellular vesicles that are relatively firmly deposited on the hollow fiber membrane surface may not be dispersed in the concentrate, resulting in a low recovery rate in the recovery process. Therefore, an operation step of redispersing the extracellular vesicles deposited on the hollow fiber membrane surface into the concentrate using a small amount of backwashing solution (dilution solution) can be performed immediately before the recovery process. In this case, it is preferable to perform a sufficient flushing operation and use a small amount of backwashing solution (dilution solution). For example, the volume of the backwashing solution (dilution solution) used is preferably half to twice the volume of the first flow path. The liquid passage speed in this case can be the same as the liquid passage speed in the purification and concentration step.

[0109] Another embodiment of the production method according to the first embodiment of the present disclosure is a method for producing a purified and concentrated liquid, which includes stepwise purification and concentration of a liquid containing a useful micro substance. An example of a method for stepwise purification and concentration of a liquid containing a useful micro substance is performing two or more stages of purification and concentration treatment using hollow fiber membrane modules with two or more average inner diameters. Specifically, a first-stage purified and concentrated liquid is obtained using a hollow fiber membrane module 33 according to the present disclosure having an average inner diameter of 0.6 mm to 1.0 mm, and then a final purified and concentrated liquid is obtained using a hollow fiber membrane module device having an average inner diameter of 1.0 mm to 1.4 mm.

[0110] [Second Embodiment] Next, a second embodiment of the purification and concentration apparatus of the present disclosure will be described with reference to the drawings. In the following description, the same reference numerals are used for components having the same configuration as those of the first embodiment, and detailed description thereof will be omitted. Figure 14 is a schematic diagram showing an example of the purification and concentration apparatus 2 of the second embodiment of the present disclosure (hereinafter referred to as "purification and concentration apparatus 2"). The purification and concentration apparatus 2 of the present disclosure includes a raw material supply unit 10 and a purification and concentration unit 30 that purifies and concentrates the liquid supplied from the raw material supply unit 10 using a hollow fiber membrane module 33. The hollow fiber membrane module 33 has a first liquid inlet / outlet 33a and a second liquid inlet / outlet 33b for supplying the liquid to the hollow fiber membranes, and third liquid inlets / outlets 33c and 33d for discharging the liquid that has permeated the hollow fiber membranes. The purification and concentration unit 30 includes a second tank 31 that stores the liquid supplied from the raw material supply unit 10 and has a flow path connected to the first liquid inlet / outlet 33a of the hollow fiber membrane module 33 for supplying the liquid, and a third tank 32 that stores the liquid supplied from the raw material supply unit 10 and has a flow path connected to the second liquid inlet / outlet 33b of the hollow fiber membrane module 33 for supplying the liquid. a pressure device 34 that selectively pressurizes either the second tank 31 or the third tank 32; a second control unit (not shown) that controls the pressure device 34 to selectively pressurize the second tank 31 or the third tank 32 and alternately supply the liquid in the second tank 31 and the third tank 32 to the hollow fiber membrane module 33; and a monitoring device (not shown) that monitors the amount of liquid in the second tank 31 and the third tank 32, wherein the second control unit sets the amount of liquid in the second tank 31 and the third tank 32, and causes the pressure device 34 to alternately pressurize the second tank 31 and the third tank 32 so that the amount of liquid in the second tank 31 and the third tank 32 becomes a set value, and the amount of liquid in the second tank 31 and the third tank 32 is determined based on information from the monitoring device. The purification and concentration apparatus 2 of the present disclosure may include valves, automatic valves, check valves, orifices, flow meters, pressure gauges, level gauges, safety devices such as rupture discs, abatement equipment, pressure adjustment equipment, intermediate tanks, and branch removal equipment that can be used for sampling, multistage molecular weight splitting operations, etc., which are omitted from the schematic configuration diagram.Furthermore, the monitoring device in the purification and concentration device 2 can be a known mechanical or reflective type liquid level gauge, a weight monitoring device such as a load cell, or an integrating flow meter, either alone or in combination, depending on the scale of the device.

[0111] The purification and concentration apparatus 2 of FIG. 14 does not have the removal section 20 of the purification and concentration apparatus 1. The purification and concentration apparatus 2 is also useful as an apparatus capable of purifying and concentrating useful micro substances under automatic control. With this configuration, the raw material liquid in the raw material supply section 10 is supplied directly to the second tank 31 (or third tank 32) of the purification and concentration section 30 via the flow path 201. The purification and concentration apparatus 2 of FIG. 14 is useful as an apparatus for purifying and concentrating a raw material liquid containing highly pure useful micro substances, with an extremely low impurity concentration. The purification and concentration apparatus 1 shown in FIGS. 1 to 13 has the removal section 20 and the purification and concentration section 30, and can perform the impurity removal process in the removal section 20 and the purification and concentration process in the purification and concentration section 30 in parallel. In other words, the purification and concentration apparatus 1 is an apparatus that can more efficiently prepare a concentrated solution in which useful micro substances have been purified and concentrated. On the other hand, the purification and concentration apparatus 2 does not have the removal section 20, and the raw material liquid can be directly supplied from the raw material supply section 10 to the purification and concentration section 30, allowing the raw material liquid to be purified and concentrated. Therefore, the purification and concentration apparatus 2 is also useful as an apparatus for further purifying and concentrating the purified and concentrated liquid prepared in the purification and concentration apparatus 1.

[0112] Furthermore, by connecting two purification and concentration apparatuses 2, three types of molecular weight fractions can be obtained. "Three types of molecular weight fractions can be obtained" means that the permeate from the first membrane and the concentrate from the first membrane can be sent to the second membrane and fractionated into a permeate and a concentrate. The permeate from the second membrane can be concentrated using the first membrane, and the permeate from the first membrane can also be concentrated using another membrane or another concentrator. In one embodiment, the purification and concentration apparatus 2 can also be used as an apparatus for fractionating the purified concentrate prepared in the purification and concentration apparatus 1 into low molecular weight fractions (e.g., useful microsubstances with a molecular weight cutoff of 10,000 to 100,000) and high molecular weight fractions (useful microsubstances with a molecular weight cutoff of more than 100,000).

[0113] In the purification and concentration apparatus 2 of FIG. 14 , the second tank 31 and the third tank 32 can have the same configuration as the purification and concentration apparatus 1. Because the purification and concentration apparatus 2 is expected to use a concentrated liquid prepared in an apparatus such as the purification and concentration apparatus 1 as the raw liquid, the volumes of the second tank 31 and the third tank 32 can be smaller than those of the second tank 31 and the third tank 32 of the purification and concentration apparatus 1. These tanks can also be larger than those of the purification and concentration apparatus 1. The first tank 23 can also be omitted. Furthermore, as with the purification and concentration apparatus 1, if the liquid level in the transparent portion of the liquid inlet / outlet of the second tank 31 or the third tank 32 is controlled using a fiber sensor or the like, the second tank 31 and the third tank 32 can be made of an opaque material, such as a metal or fiber-reinforced resin tank. In the purification and concentration apparatus 2 of FIG. 14 , the liquid transfer device is not limited to the pressurizing device 34, and can also be a liquid transfer device that uses a pump or potential energy to transfer liquid, either alone or in combination. In order to use potential energy for the liquid transfer, the liquid levels in the second tank 31 and the third tank 32 can be controlled by an elevator or the like, and the height positions of the liquid inlet and outlet can also be changed as appropriate. In the purification and concentration apparatus 2 of Figure 14, in order to suppress foaming of the liquid in the second tank 31 or the third tank 32, if the height position of the liquid inlet is higher than the liquid level, the pipe diameter and liquid transfer flow rate can be appropriately adjusted so as not to entrain gas from the upper part of the tank, or equipment that can automatically adjust the height from the pipe outlet to the liquid level, foam breaking / defoaming equipment, etc. can be installed, and an appropriate antifoaming agent can also be added as needed.

[0114] In the purification and concentration apparatus 2 of FIG. 14 , the hollow fiber membranes in the hollow fiber membrane module 33 may be coated with an appropriate substance in the same manner as in the purification and concentration apparatus 1 in order to improve the fouling resistance of the hollow fiber membrane surface and / or to increase the purification and concentration efficiency and the recovery rate of extracellular vesicles. In the purification and concentration apparatus 2 of FIG. 14 , the average inner diameter of the hollow fiber membranes in the hollow fiber membrane module 33 can be adjusted appropriately depending on the viscosity of the liquid. In one embodiment, the average inner diameter of the hollow fiber membranes may be 0.2 to 2.0 mm. As with the purification and concentration apparatus 1 described above, when the liquid viscosity is high (for example, when the liquid temperature is 20°C and the shear rate is 25 s－１ In addition, when the viscosity of the liquid containing the minute useful substance is low during operation (for example, when the liquid temperature is 20°C and the shear rate is 25 s), it is preferable to use a large-diameter hollow fiber membrane having an average inner diameter of 1.0 mm to 1.4 mm. －１ In the case where the liquid viscosity in the test piece is less than 10 mPa s, it is preferable to use hollow fiber membranes having an average inner diameter of 0.2 mm to 1.0 mm. Note that if the average inner diameter of the hollow fiber membrane is large, the effective membrane area is likely to decrease when the hollow fiber membrane is used as a hollow fiber membrane module. Therefore, from the viewpoint of easily suppressing a decrease in the effective membrane area when used as a hollow fiber membrane module, it is preferable to use hollow fiber membranes having an average inner diameter of 0.2 mm to 1.0 mm.

[0115] The purification and concentration apparatus 2 of FIG. 14 may have the same elements as the purification and concentration apparatus 1 of FIGS. 1 to 13 in addition to the elements shown in the figure. For example, in the purification and concentration apparatus 2 of FIG. 14, the monitoring device for monitoring the liquid levels in the second tank 31 and the third tank 32 may include the above-mentioned tank liquid level monitoring devices 35, 36 and / or a camera. The second control unit of the purification and concentration apparatus 2 may also have the same configuration as the second control unit 120 of the purification and concentration apparatus 1. That is, the second control unit can determine the liquid levels in the tanks based on changes in light transmittance detected by the optical sensors provided in the tank liquid level monitoring devices 35, 36, and determine which tanks to pressurize with the pressurizing device 34. Furthermore, the second control unit can determine the liquid levels in the tanks based on information on the liquid level position output from the camera, and determine which tanks to pressurize with the pressurizing device 34. In the purification and concentration apparatus 2, the monitoring device preferably includes the tank liquid level monitoring devices 35, 36. As described above, the liquid transfer device in the purification and concentration apparatus 2 is not limited to a pressurizing device, and pumping and liquid transfer utilizing potential energy can be used alone or in combination. In this case, the second control unit can be configured to control the liquid level in the second tank 31 and the third tank 32 via an elevator or the like, and can also be configured to control the height positions of the liquid inlet and outlet ports. With this configuration, the second control unit can control the liquid delivery device to selectively supply the liquid in the second tank and the third tank to the hollow fiber membrane module.

[0116] The purification and concentration section 30 in the purification and concentration apparatus 2 in FIG. 14 may be configured to include a cleaning tank that supplies a diluent to the hollow fiber membrane module 33 and cleans the multiple hollow fiber membranes in the hollow fiber membrane bundle 60. That is, the purification and concentration section 30 of the purification and concentration apparatus 2 may include a fourth tank (reference numeral 80 in FIG. 10 ) that stores the diluent and is connected to a flow path for supplying the diluent from the third liquid inlets 33c and 33d of the hollow fiber membrane module 33. It may also include a third control unit 130 similar to that of the purification and concentration apparatus 1. That is, the third control unit in the purification and concentration apparatus 2 can supply the diluent from the fourth tank from the third liquid inlets 33c and 33d of the hollow fiber membrane module 33 when the total amount of liquid in the second tank 31 and the third tank 32 falls below the value set by the second control unit. This configuration allows the raw material liquid to be purified and concentrated while recovering minute useful substances deposited on the membrane surfaces inside the hollow fiber membranes. The fourth tank may be connected to the second tank 31 or the third tank 32. In the purification and concentration apparatus 2, similarly to the purification and concentration apparatus 1, before supplying (backwashing) the diluent to the hollow fiber membrane module 33, in order to prevent the permeated liquid remaining in the hollow fiber membrane module 33 from mixing with the backwashing liquid and flowing into the concentrated liquid side, an operation of previously flushing with a cleaning liquid (diluent) and replacing the permeated liquid remaining in the hollow fiber membrane module with the cleaning liquid (diluent) can be optionally performed.

[0117] 14 may include a fifth tank 81 for recovering the concentrated liquid from the second tank 31, the third tank 32, and the hollow fiber membrane module 33. It may also include a fourth control unit for controlling the recovery of the concentrated liquid into the fifth tank 81. The configurations of the fifth tank 81 and the fourth control unit may be the same as those of the purification and concentration apparatus 1.

[0118] The useful micro-substances purified and concentrated by the purification and concentration apparatus according to the second embodiment may be the same as those described for the purification and concentration apparatus 1, or may be different. The useful micro-substances purified and concentrated by the purification and concentration apparatus according to the second embodiment may include useful micro-substances for industrial applications, such as electroactive polymers, rare metal colloid particles, pigments, dyes, and inorganic or organic nanoparticles. Generally, when purifying and concentrating a liquid containing such useful micro-substances for industrial applications, no treatment for removing impurities from the liquid is required. Therefore, the apparatus according to the second embodiment of the present disclosure can be suitably used.

[0119] [Method for producing a liquid in which useful micro substances have been purified and concentrated: Second embodiment] An example of a method for producing a liquid in which useful micro substances have been purified and concentrated using the purification and concentration apparatus 2 of the present disclosure is a method that includes a step in which the impurity removal process is omitted from the production method using the above-mentioned purification and concentration apparatus 1. Note that the purification and concentration apparatus 2 does not have a removal unit 20, but may include pretreatment steps such as a pre-filtration process using a microfiltration membrane or a coagulation and sedimentation process.

[0120] In the purification and concentration apparatus 2 shown in Figure 14, after the raw material liquid is charged into a container or the like in the raw material supply unit 10, when the on-off valve 210 is opened, the raw material liquid is supplied to the second tank 31 (or the third tank 32) via 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 tanks and the hollow fiber membrane module is recovered in the fifth tank 81.

[0121] [Other Embodiments: Molecular Weight Fractionation and Purification / Concentration of Useful Micro Substances] Another embodiment of the production method according to the second embodiment of the present disclosure is a method for producing a purified concentrate, including molecular weight fractionation and purification / concentration of useful micro substances. Examples of methods for molecular weight fractionation of useful micro substances into two or more components and concentrating and purifying each of the components include connecting two or more purification / concentration devices 2 of the present disclosure, each equipped with a hollow fiber membrane module having two or more molecular weight cutoffs, or performing two or more stages of purification / concentration processing. Specifically, when producing a purified concentrate of a liquid containing chondroitin sulfate with a wide molecular weight distribution, a device with a molecular weight cutoff of 10,000 can be connected to a device with a molecular weight cutoff of 70,000, or two or more stages of purification / concentration processing can be performed to obtain a purified (molecular weight purified) concentrate of chondroitin sulfate with three molecular weight distributions. In this case, the concentration of components with a molecular weight cutoff of less than 10,000 can be achieved by concentrating the permeate from the device with a molecular weight cutoff of 10,000 using a device other than a membrane separation device, or by using an NF membrane concentrator (e.g., a device using the membrane described in Japanese Patent Publication No. 7,021,400).

[0122] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following description.

[0123] <Preparation of hollow fiber membrane module 33> A hollow fiber membrane bundle 60, which was a bundle of 75 hollow fiber membranes (manufactured by Daisen Membrane Systems Co., Ltd.) made of cellulose acetate (CA) 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 housed in a polycarbonate cylindrical container 61, to prepare the internal pressure type hollow fiber membrane module shown in Figure 7. The pure water permeation flow rate of the hollow fiber membrane module 33 at 0.1 MPa was 28.8 L / Hr. Furthermore, the γ-globulin permeability measured by the following measurement method was 54%, and the effective length of the hollow fiber membrane was 17 cm and the effective membrane area was 0.0312 m. ２ The filling rate of the hollow fiber membrane was 30%.

[0124] (Measurement of γ-globulin permeability) Using the γ-globulin permeability measuring device 300 of FIG. 15, γ-globulin permeability was measured by the following procedure. 1) A γ-globulin aqueous solution (γ-globulin concentration 100 ppm) was charged into the tank 302. 2) The hollow fiber membrane 303 was connected to the flow path 305. 3) Nitrogen gas was injected through the valve V-1 to apply a pressure of 0.1 MPa to the tank 302. The aqueous solution was filtered from the inner surface to the outer surface of the hollow fiber membrane 303, the filtrate was discarded in the container 307, and the permeate was recovered in the container 306. The initial permeate (approximately 100 mL) was discarded, and the permeate discharged thereafter was used as a sample for permeability measurement. After preparing a sample of the permeate using the device 300, the γ-globulin permeability was measured by the following method. 4) Using a spectrophotometer (Shimadzu Corporation, product name "UV2450"), the absorbance (A1) at 280 nm of the γ-globulin aqueous solution (100 ppm) and the absorbance (A2) at 280 nm of the permeated solution were measured, and the γ-globulin transmittance (%) was calculated using the formula (A2 / A1×100).

[0125] <Purification and concentration apparatus 1> A specific operation when using the purification and concentration apparatus 1 of Fig. 13 will be described below. Elements including the raw material supply section 10, removal section 20, and purification and concentration section 30 are arranged in a housing 100 (not shown) with a width of 800 mm, a depth of 350 mm, and a height of 1745 mm. A fan filter device 91 (manufactured by AS ONE Corporation, product name "Pure Space 01", equipped with a ULPA filter unit, air volume 1 m) is installed at the top of the purification and concentration apparatus 1. ３ / min) (not shown) is provided so that filtered air flows down into the housing 100. Four UV lamps 92 (glow-type germicidal lamps, manufactured by Yamato Denki Sangyo Co., Ltd.) (not shown) are also installed inside the housing 100. Casters with free stoppers (not shown) are attached to the bottom of the purification and concentration apparatus 1. A door (not shown) is provided on the front of the purification and concentration apparatus 1, and a touch panel (Keyence Corporation, product name "Touch Panel Display Model: VT-W4M") (not shown) is attached to the door for managing the processes of the purification and concentration apparatus 1. A compressed air intake (not shown) and a DC power supply (not shown) are provided in the purification and concentration apparatus 1. The compressed air is decompressed to 0.05 MPa by a regulator, and the liquid supply destination is switched and the supply is controlled via a plurality of on-off valves (two-port solenoid valves), three-way valves (three-port solenoid valves), and solenoid valve manifolds for cleaning, equilibration treatment, impurity removal treatment, purification and concentration treatment, and backwashing treatment of the carrier column 21. In addition, the compressed air decompressed to 0.3 MPa by a regulator is also used for the air grip when recovering the concentrated liquid.

[0126] (Raw Material Supply Unit 10) A container 11 for storing raw material liquid is provided.

[0127] (Removal section 20) The carrier column 21 is a column with an inner diameter of 25 mm, and contains an adsorbent carrier (manufactured by Cytiva, product name "Capto"). ＴＭ The filter 22 is a microfiltration membrane (Millipore Corporation, product name "Millex-GP", material: polyethersulfone, pore size: 0.22 μm). The sixth tank 25 is charged with a 2 M aqueous solution of sodium chloride as a cleaning solution. The seventh tank 26 is charged with a 50 mM Tris-HCl (pH 7.2) solution as an equilibration solution. As shown in FIG. 13, the filter 22 is configured so that liquid is supplied from the tank 24, the sixth tank 25, and the seventh tank 26 to the carrier column 21 via a flow path 41A via multiple on-off valves (solenoid valves). A tube pump (not shown) is provided in the flow path 41A.

[0128] (Purification and concentration section 30) The hollow fiber membrane module 33 prepared above is used as the hollow fiber membrane. Transparent containers (120 mL capacity) made of poly(meth)acrylate are used for the second tank 31 and the third tank 32. Tank liquid level monitors 35, 36 (pipe-mounted liquid level detection fiber unit (model: FU-95S) manufactured by Keyence Corporation) including optical sensors are placed at the second tank 31 and the liquid inlet / outlet at the bottom of the second tank 31. Phosphate buffer solution (PBS) is charged into the fourth tank 80 as a diluent.

[0129] Example 1: 800 mL of exosome-containing mesenchymal stem cell culture supernatant prepared by the method described below was placed in container 11 as a raw material solution. <Preparation of exosome-containing mesenchymal stem cell culture supernatant> Human adipose tissue-derived mesenchymal stem cells were cultured using Ultra ExoM Culture Medium for Extracellular Vesicles (FK-K0204024, Santeja). With the cells covering approximately 80% of the adhesive surface of the culture vessel, the medium was replaced with phenol red-free Ultra ExoM Culture Medium and cultured for 48 hours. The culture supernatant was then centrifuged at 2000 x g for 10 minutes at 4°C to remove cell debris and prepare a culture supernatant.

[0130] The raw material liquid in the container 11 is supplied to the filtration member 22 to perform pretreatment in its entirety, and then stored in the tank 24 (pretreatment liquid storage tank). Next, 50 mL of cleaning liquid is supplied from the sixth tank 25 (cleaning liquid storage tank) to the carrier column 21 at 5 mL / min to clean the carrier column 21, and then the cleaning liquid is discharged into the waste liquid tank. Then, 50 mL of equilibration liquid is supplied from the seventh tank 26 (equilibration liquid storage tank) to the carrier column 21 at 5 mL / min to equilibrate the carrier column 21, and then the equilibration liquid is discharged into the waste liquid tank.

[0131] <Impurity Removal Treatment> 50 mL of the pretreatment solution in the tank 24 is supplied to the carrier column 21, and host-derived proteins (HCPs), DNA, etc. in the pretreatment solution are adsorbed and removed by the carrier. The resulting roughly purified solution is supplied to the first tank 23 via the flow path 42A. Thereafter, a second washing and equilibration treatment of the carrier column 21 is carried out under the same conditions as above. After the second washing and equilibration treatment, 50 mL of the pretreatment solution in the tank 24 is supplied to the carrier column 21 to carry out a second impurity removal treatment. Thereafter, a third washing and equilibration treatment of the carrier column 21 and a third impurity removal treatment are carried out in the same manner. Subsequently, the fourth to sixteenth washing and equilibration treatment of the carrier column 21 and the fourth to sixteenth impurity removal treatments are carried out.

[0132] <Purification and Concentration Treatment> The impurity removal treatment and the purification and concentration treatment are performed in parallel. First, the crudely purified liquid (approximately 200 mL) stored in the first tank 23 after the first to fourth impurity removal treatments is transferred to the second tank 31 using compressed air at 0.05 MPa. At this time, the first crudely purified liquid (approximately 50 mL) is first transferred to the second tank 31, and then the second tank 31 is pressurized using the pressure device 34 to set the liquid volume at the controlled liquid level position C to 10 mL. The purification and concentration treatment is performed by alternating tangential flow filtration (TFF) so that the liquid volume in the second tank 31 does not fall below 10 mL. Thereafter, while the crudely purified liquids from the second to fourth treatments are sequentially transferred to the second tank 31, the purification and concentration treatment by alternating tangential flow filtration (TFF) is continuously performed so that the liquid volume in the second tank 31 does not fall below 10 mL.

[0133] Next, a backwashing process is performed. 90 mL of diluent is used for the backwashing process. Phosphate buffer solution (PSB) is used as the diluent. After performing the backwashing process with the diluent, a purification and concentration process is performed by alternating tangential flow filtration (TFF) so that the liquid volume in the second tank 31 does not fall below 10 mL. Thereafter, the crudely purified liquid obtained by the fifth to eighth impurity removal processes, the crudely purified liquid obtained by the ninth to twelfth impurity removal processes, and the crudely purified liquid obtained by the thirteenth to sixteenth impurity removal processes are each added to the second tank 31, while a purification and concentration process and a backwashing process are performed. The backwashing process is performed every time the volume of the crudely purified liquid sent after the aforementioned backwashing process reaches 200 mL, and the purification and concentration process is performed by alternating tangential flow filtration (TFF) so that the liquid volume in the second tank 31 does not fall below 10 mL. A total of 800 mL of crudely purified liquid is subjected to purification and concentration processes and backwashing processes. After the liquid volume in the second tank 31 reaches 10 mL, the backwashing process and purification and concentration processes are repeated three times with a diluted liquid volume of 90 mL (10-fold dilution) to obtain a purified and concentrated exosome solution.

[0134] <Recovery Process> The obtained purified exosome concentrate is recovered in its entirety into the fifth tank 81 via the flow path 56. The concentrate recovery process involves monitoring the amount of concentrate in the flow path 56 with a fiber sensor installed at the liquid inlet / outlet at the top of the fifth tank 81, and recovering the concentrate in the two tanks into the fifth tank 81 until the fiber sensor detects air bubbles. The yield of the exosome purified concentrate obtained at this time is 0.8 to 80 mL, and the exosome recovery rate is 50 to 100%. It is also expected that the protein removal rate will be 80% or more.

[0135] Example 2: 800 mL of human adipose tissue-derived mesenchymal stem cell culture supernatant was used as the starting material. The protein concentration and CD9 / CD63-positive extracellular vesicles in the culture supernatant were quantified by the Bradford method and ELISA, respectively, to find that the protein concentrations were 0.49 mg / mL and 152 pg / mL. 80 mL of a mixed-mode carrier with cation exchange and size exclusion equilibrated with 50 mM Tris-HCl (pH 7.2) was added to the culture supernatant, dispensed into a 50 mL tube, and then mixed by end-over-end mixing at 10 rpm / min at room temperature for 10 minutes. The mixture was then centrifuged at 10,000 x g for 5 minutes at room temperature, and the supernatant was further filtered using a 0.22 μm filtration unit to remove the carrier. The protein concentration in the resulting 790 mL of carrier-treated culture supernatant was 0.06 mg / mL, and the exosome concentration was 144 pg / mL.

[0136] Next, the carrier-treated culture supernatant was supplied from container 11 to second tank 31 via flow path 43, and alternating tangential flow filtration was performed under pressure conditions in pressure device 34 with a nitrogen gas pressure of 0.04 MPa. When the remaining volume reached 10 mL, 90 mL of phosphate buffer solution (PBS) was supplied from the fourth tank 80 to the hollow fiber membrane module 33 side for backwashing. Alternating tangential flow filtration and the supply of 90 mL of phosphate buffer solution were then repeated two more times, after which 12.9 mL of purified concentrate was obtained by alternating tangential flow filtration. The protein concentration of the obtained purified concentrate was 0.09 mg / mL, and the exosome concentration was 7149 pg / mL. The protein removal rate calculated from the concentration and liquid volume was over 99%, and the recovery rate was 76%.

[0137] Comparative Example 1: In 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. The raw material solution was supplied from container 11 to second tank 31 via flow path 43, and a purification and concentration process was performed using alternating tangential flow filtration. In Comparative Example 1, the purification and concentration process was performed under the same conditions as in Example 2, except that the intermediate washing step was performed by forward washing instead of backwashing. Forward washing was performed by supplying the diluted solution in fourth tank 80 to third tank 32 via flow path 55B, and then supplying the diluted solution to hollow fiber membrane module 33 to wash the hollow fiber membrane. Finally, 11 mL of purified concentrate was obtained. The protein concentration in the purified concentrate was 0.02 mg / mL or less, and the exosome concentration was 2595 pg / mL. The protein removal rate calculated from the concentration and liquid volume was 99% or more, and the recovery rate was 27%. These results demonstrate that backwashing improves the recovery rate.

[0138] Example 3: Here, we investigated a method for further improving the recovery rate of useful micromaterials by coating the surface of hollow fiber membranes with biomolecules during the purification and concentration process. As the raw material, 790 mL of mesenchymal stem cell culture supernatant with a protein concentration of 0.49 mg / mL and an exosome concentration of 166.4 pg / mL was used. The exosome concentration was quantitatively analyzed using a CD9 / CD63 ELISA (EXH0102EL, Cosmo Bio) and corresponds to the amount of CD9 / CD63 fusion standard protein used to create the calibration curve. To 790 mL of culture supernatant, 79 mL of a carrier (a porous granular material with positively charged pores and capable of adsorbing and retaining negatively charged waste materials, excluding extracellular vesicles) equilibrated with 50 mM Tris-HCl (pH 7.2) solution was added, and the mixture was mixed by inversion at room temperature for 10 minutes using a rotator. Thereafter, the carrier was removed from the carrier-cell supernatant mixture using a 0.22 μm filtration unit (S2GPU11RE, Merck Millipore) to obtain a carrier-treated culture supernatant.

[0139] Next, the carrier-treated culture supernatant was purified and concentrated by filtration using an alternating tangential flow system at a filtration pressure of 30 to 40 kPa. The filtration membrane used was a hollow fiber membrane module made of cellulose acetate (molecular weight cutoff: 300,000, membrane area: 0.062 m). ２ ) was used. The filtration membrane used had its surface coated with biomolecules. The coating treatment was performed by passing a 1.3 mg / mL solution of skim milk dissolved in PBS through the filtration membrane at a flow rate of 200 mL / min for 10 minutes. This resulted in a coated membrane in which certain biomolecules in the skim milk were adsorbed and bound to the membrane surface as a thin layer. The filtration membrane was then washed with 250 mL of PBS to remove excess skim milk that had simply accumulated on the thin layer.

[0140] Here, the binding state between the filtration membrane and certain biomolecules in skim milk is strong enough that they do not come off under washing and operating conditions, and furthermore, there is almost no decrease in filtration performance even after long-term operation.

[0141] Alternating tangential flow filtration was repeated until the volume of the concentrate reached 10 mL, after which the concentrate was diluted by supplying 90 mL of PBS to the hollow fiber membrane module. This concentration, filtration, and dilution process was repeated two more times, and then 11.9 mL of purified concentrate (extracellular vesicle purified product) was obtained by alternating tangential flow filtration. The extracellular vesicles in the final exosome concentrate were quantitatively analyzed using the CD9 / CD63 ELISA method (EXH0102EL, Cosmo Bio), and the CD9 / CD63 fusion standard protein used to create the calibration curve was found to be 3,746.7 pg / mL, with a recovery rate of 33.9%.

[0142] Comparative Example 2 When the same procedure was carried out using a hollow fiber membrane that had not been coated with skim milk, the recovery rate was 7.4%.

[0143] The results of Example 3 and Comparative Example 2 showed that the recovery rate of micro-useful substances can be improved by using a hollow fiber membrane coated with biomolecules (i.e., a hollow fiber membrane having a type of biomolecules different from the micro-useful substances attached to at least a portion of the membrane).

[0144] Example 4 Here, a method for further improving the purity of useful minute substances in the purified concentrate obtained in the purification and concentration step was investigated.

[0145] As a raw material solution, 40 mL of culture supernatant of amniotic membrane-derived mesenchymal stem cells with a protein concentration of 1.49 mg / mL and an exosome concentration of 196 pg / mL was used. The exosomes in the culture supernatant were separated into 33 hollow fiber membrane modules made of cellulose acetate (molecular weight cutoff: 300,000, membrane area: 0.01 m). ２ Using a purification and concentration device equipped with a permeate volume (i.e., 10 mL of hollow fiber extracapillary space (ECS)), separation, purification, and concentration were performed using an alternating tangential flow method with a filtration pressure of 30 to 40 kPa. After concentrating the culture supernatant to approximately 10 mL, 40 mL of diluent (PBS) was injected through the third liquid inlet / outlet 33c of the hollow fiber membrane module and discharged through the third liquid inlet / outlet 33d. This flushed out impurities in the permeate remaining in the ECS with PBS. Then, 90 mL of the same diluent (PBS) was forced from the permeate side to the concentration side of the hollow fiber membrane module 33 to perform backpressure washing (backwashing). The separation and purification operation was performed again under the same conditions, and when the culture supernatant reached approximately 10 mL, the same flushing and backwashing operation was performed two more times. After three backwashings, the culture supernatant was concentrated to 5.5 mL. The amount of remaining protein in the purified concentrate was measured by BCA assay, and the protein concentration was found to be 0.16 mg / mL. The results are shown in Figure 17. The absorbance of the purified concentrate was also measured using an Infinite M Nano+ absorbance microplate reader manufactured by TECAN. The results are shown in Figure 18.

[0146] Comparative Example 3: In the same separation and purification procedure as in Example 4, the culture supernatant was concentrated to 5.2 mL without flushing the ECS with PBS. The amount of residual protein in the resulting purified concentrate was measured by BCA assay, and the protein concentration was found to be 0.36 mg / mL. The results are shown in Figure 17. The absorbance of the resulting purified concentrate was also measured in the same manner as in Example 4. The results are shown in Figure 18.

[0147] In Figures 17 and 18, "Before" refers to the sample before the purification and concentration process, "with ECF" refers to the purified concentrate obtained by the method of Example 4, and "without ECS" refers to the purified concentrate obtained by the method of Comparative Example 3. As is clear from Figure 17, the method of Example 4 further reduces the concentration of protein impurities, thereby further improving the purity of exosomes. In Figure 18, the peak at a wavelength of approximately 557 nm is a peak derived from phenol red, which is contained in the culture medium as a pH indicator and corresponds to an impurity in the purified concentrate. As shown in Figure 18, the method of Example 4 further reduces the concentration of phenol red as an impurity, thereby further improving the purity of exosomes.

[0148] These results indicate that flushing the ECS with a diluent such as PBS or saline to remove impurities remaining in the ECS before the backwashing process of the hollow fiber membrane module can further reduce the concentration of impurities in the resulting purified concentrate, thereby further improving the purity of the useful micro-substances. A non-limiting reason for this is thought to be that flushing prevents impurities in the ECS from being reconstituted in the purified concentrate during the subsequent backwashing process with the diluent.

[0149] [Example 5] As a raw material solution, 200 mL of culture supernatant of amniotic membrane-derived mesenchymal stem cells with a protein concentration of 1.25 mg / mL and an exosome concentration of 138 pg / mL was used. The exosomes in the culture supernatant were separated into 200 mL of a cellulose acetate hollow fiber membrane module (molecular weight cutoff: 300,000, membrane area: 0.01 m). ２Separation, purification, and concentration were performed using an alternating tangential flow method using a purification and concentration device equipped with a 10 mL ECS. After concentrating the culture supernatant to approximately 17 mL, 40 mL of diluent (PBS) was injected through the third liquid inlet / outlet 33c of the hollow fiber membrane module and discharged through the third liquid inlet / outlet 33d. This flushed impurities in the permeate remaining in the ECS with PBS. Then, 160 mL of the same diluent (PBS) was forced from the permeation side to the concentration side of the hollow fiber membrane module 33 to perform backpressure washing (backwashing). The separation and purification operation was performed again under the same conditions, and when the culture supernatant reached approximately 17 mL, the same flushing and backwashing operation was performed two more times. The culture supernatant after backwashing was concentrated to 17 mL. Backwashing was performed again to wash out the useful micro-substances adhering to the hollow fiber membrane surface into the purified concentrate, and 50 mL of purified concentrate was recovered. The extracellular vesicles in the purified concentrate were quantitatively analyzed using the CD9 / CD63 ELISA method (EXH0102EL, Cosmo Bio). The concentration of the CD9 / CD63 fusion standard protein used to create the calibration curve was 486 pg / mL, resulting in a recovery rate of 88%. These results demonstrate that the recovery rate of useful micro-substances can be increased to 80% or higher by further supplying a diluent to the hollow fiber membrane module immediately before collecting the purified concentrate and washing out the useful micro-substances.

[0150] 1, 2 Purification and concentration apparatus, 10 Raw material supply section, 11 Container, 20 Removal section, 21 Carrier column, 22 Filtering member, 23 First tank (roughly purified liquid storage tank), 24 Tank (pretreatment liquid storage tank), 25 Sixth tank (washing liquid storage tank), 26 Seventh tank (equilibration liquid storage tank), 30 Purification and concentration section, 31 Second tank, 32 Third tank, 33 Hollow fiber membrane module, 33a First liquid inlet / outlet, 33b Second liquid inlet / outlet, 33c, 33d Third liquid inlet / outlet, 34 Pressurizing device, 35, 36 Tank liquid level monitoring device, 41 to 43 Flow path, 80 Fourth tank (diluted liquid storage tank), 81 Fifth tank (recovery tank), 91 Fan filter device, 92 UV lamp, 110 First control section

Claims

1. A raw material supply unit that supplies a liquid containing minute useful substances; A removal unit for removing impurities from 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 that controls the timing of flow of the liquid from the raw material supply unit to the purification and concentration unit, or the timing of flow of the liquid from the removal unit to the purification and concentration unit.

2. 2. The purification and concentration apparatus according to claim 1, wherein the removal section comprises a carrier column connected to a flow path for removing impurities from the liquid and supplying the liquid after the impurities have been removed to the purification and concentration section.

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

4. the removal unit includes a first tank configured to store the liquid treated in the carrier column, the first tank is disposed in a flow path for supplying the liquid from the carrier column to the purification and concentration section, and a flow path for supplying the liquid in the tank to the purification and concentration section is connected thereto; The purification and concentration apparatus according to claim 2 , wherein the first control unit controls a timing for supplying the liquid in the tank to the purification and concentration unit.

5. The purification and concentration apparatus according to claim 1 or 2, wherein the hollow fiber membrane has an average inner diameter of 0.2 to 1.4 mm and a molecular weight cutoff of 10,000 to 1,000,000.

6. the purification and concentration section includes a hollow fiber membrane module in which a plurality of the hollow fiber membranes are accommodated in a container having an inlet and outlet for the liquid, 2. The purification and concentration apparatus according to claim 1, wherein the container has at least a first liquid inlet / outlet and a second liquid inlet / outlet for supplying the liquid to the hollow fiber membrane, and a third liquid inlet / outlet for discharging the liquid that has permeated the hollow fiber membrane.

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

8. the purification and concentration section comprises a second tank configured to store the liquid supplied from the raw material supply section or the removal section, and a flow path for supplying the liquid to an inlet / outlet for the first liquid of the hollow fiber membrane module is connected to the second tank; a third tank that stores the liquid supplied from the raw material supply unit or the removal unit and has a flow path connected to an inlet / outlet for the second liquid of the hollow fiber membrane module, the third tank having a flow path that supplies the liquid to the second liquid inlet / outlet; a pressurizing device that selectively pressurizes either the second tank or the third tank; and a second control unit that controls the pressurizing device to selectively pressurize the second tank or the third tank so as to alternately supply the liquid in the second tank and the liquid in the third tank to the hollow fiber membrane module.

9. 9. The purification and concentration apparatus according to claim 8, wherein the second control unit sets the liquid volume in the second tank and the third tank, and alternately pressurizes the second tank and the third tank with the pressurizing device so that the liquid volume in the second tank and the third tank becomes the set value.

10. the second tank and the third tank are each provided with a tank liquid level monitoring device including an optical sensor at an inlet / outlet for the liquid at the bottom of the tank; the tank liquid level monitoring device is configured to detect a light transmittance by irradiating light into the liquid inlet / outlet port with the optical sensor, and monitor the liquid level in the tank based on a change in the light transmittance; 10. The purification and concentration apparatus according to claim 9, wherein the second control unit determines the liquid volumes in the second tank and the third tank using the tank liquid volume monitoring device, and determines which tank is to be pressurized by the pressurizing device.

11. the purification and concentration unit includes a camera for monitoring liquid level positions in the second tank and the third tank, 10. The purification and concentration apparatus according to claim 9, wherein the second control unit determines the tank to be pressurized by the pressurizing device based on information on liquid level positions in the second tank and the third tank output from the camera.

12. the purification and concentration section includes a fourth tank that stores a diluent and is connected to a flow path that supplies the diluent to an inlet / outlet for the third liquid of the hollow fiber membrane module; a third control unit that controls a timing at which the dilution liquid in the fourth tank is supplied to an inlet / outlet for the third liquid of the hollow fiber membrane module, 9. The purification and concentration apparatus according to claim 8, wherein the third control unit supplies the dilution liquid in the fourth tank from an inlet / outlet for the third liquid of the hollow fiber membrane module when a 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.

13. 9. The purification and concentration apparatus according to claim 8, wherein the purification and concentration section comprises a fifth tank connected via a flow path to an inlet / outlet for the first liquid or an inlet / outlet for the second liquid of the hollow fiber membrane module, and which recovers the liquid in the second tank and the third tank, and a fourth control section which controls the recovery of the liquid into the fifth tank.

14. the second tank and the third tank are each provided with a tank liquid level monitoring device including an optical sensor at an inlet / outlet for the liquid at the bottom of the tank; the tank liquid level monitoring device is configured to detect a light transmittance by irradiating light into the liquid inlet / outlet port with the optical sensor, and monitor the liquid level in the tank based on a change in the light transmittance; 14. The purification and concentration apparatus according to claim 13, wherein the fourth control unit selectively pressurizes the second tank or the third tank using the pressurizing device to recover the liquid in the second tank and the third tank into the fifth tank, and determines the liquid levels in the second tank and the third tank using the liquid level monitoring device in the tank, and stops the pressurizing device.

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

16. The raw material supply unit, the removal unit, and the purification and concentration unit are configured to be disposed in a housing, and a management unit is provided for managing a cleanliness level within the housing; The purification and concentration apparatus according to claim 1 or 2, wherein the management section includes a fan filter device that supplies filtered air into the housing.

17. The purification and concentration apparatus according to claim 16 , wherein the management unit further comprises a UV lamp.

18. 3. The purification and concentration apparatus according to claim 2, wherein the removal unit comprises a sixth tank that stores a washing liquid for washing the carrier column, and a seventh tank that stores an equilibration liquid for equilibrating the carrier column.

19. 4. The purification and concentration apparatus according to claim 3, wherein the filtering member is disposed below the raw material supply unit, and the liquid is supplied from the raw material supply unit to the filtering member by the weight of the liquid.

20. The purification and concentration apparatus according to claim 8 , wherein the second tank and the third tank have longitudinal sides arranged parallel to the longitudinal direction of the hollow fiber membrane module.

21. the removal unit includes a carrier column connected to a flow path for removing impurities from the liquid and supplying the liquid after the impurities have been removed to the purification and concentration unit, and a first tank for storing the liquid treated in the carrier column; The purification and concentration apparatus according to claim 20 , wherein the second tank and the third tank are connected to the carrier column and the first tank by a flow path and are disposed above the carrier column and the first tank.

22. The purification and concentration device according to claim 1 or 2, wherein the minute useful substance is selected from the group consisting of extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.

23. A method for producing a liquid in which useful micro substances are purified and concentrated, using the purification and concentration apparatus according to claim 1 or 2, comprising the steps of: A method for producing a purified and concentrated solution of useful micro-substances, comprising: supplying a raw liquid containing the useful micro-substances to the purification and concentration device to remove impurities in the liquid, and / or purifying and concentrating the liquid using a hollow fiber membrane.