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

Abstract

The present invention provides: a liquid containing a minute useful substance at a high purity and a high concentration (a purified concentrated solution of a minute useful substance), the purified concentrated solution being more efficiently prepared; the present invention relates to a device for purifying and concentrating a liquid containing a minute useful substance, which is less likely to cause foreign matter from the outside during the purification and concentration process, and a method for producing a purified and concentrated liquid containing a minute useful substance using the device. This purification and concentration device is provided with: a hollow fiber membrane module in which a plurality of hollow fiber membranes are accommodated in a container; the container has a first liquid inlet / outlet and a second liquid inlet / outlet for supplying a liquid containing a minute useful substance to the hollow fiber membrane, and a third liquid inlet / outlet for discharging the liquid having passed through the hollow fiber membrane; a first flow path which is connected to the inlet / outlet of the first liquid and the inlet / outlet of the second liquid and forms a circulation path including the hollow fiber membrane module; a second flow path that supplies the liquid to the first flow path; and one or more pumps which are inserted in the first flow path and circulate the liquid in a positive and negative possible manner.

Description

Technical Field

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

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

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

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

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

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

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

[0008] In addition, the purified concentrated solution containing minute useful substances is sometimes used for medical purposes, so a device is also required that is unlikely to cause foreign matter to mix in from the outside during the purification and concentration process. In addition, a device that can purify and concentrate minute useful substances by automatic control is also required.

[0009] The present invention has been made in view of the above circumstances, and its purpose is to provide a purification and concentration device for a liquid containing a microscopic useful substance, which is less likely to be mixed with foreign matter from the outside during the purification and concentration process, and a method for producing a purified concentrated liquid containing a microscopic useful substance using the device, wherein the device can produce a liquid containing a microscopic useful substance at a high purity and high concentration (purified concentrated liquid of a microscopic useful substance), and can more efficiently produce the purified concentrated liquid. In addition, the present invention also aims to provide a device capable of purifying and concentrating microscopic useful substances by automatic control.

[0010] In order to solve the above-mentioned problems, the present disclosure can adopt the following aspects.

[0011] [1] A device for purifying and concentrating a liquid containing minute amounts of useful substances, comprising:

[0012] A hollow fiber membrane module containing a plurality of hollow fiber membranes in a container, wherein the container has a first liquid inlet and a second liquid inlet for supplying a liquid containing a tiny useful substance to the hollow fiber membranes, and a third liquid inlet and outlet for discharging the liquid after passing through the hollow fiber membranes;

[0013] A first flow path connected to the inlet and outlet of the first liquid and the inlet and outlet of the second liquid to form a circulation path including the hollow fiber membrane module;

[0014] a second flow path that supplies the liquid to the first flow path; and

[0015] One or more pumps are inserted into the first flow path to circulate the liquid in a forward and reverse manner.

[0016] [2] The purification and concentration device according to [1], wherein the pump is a tube pump.

[0017] [3] The purification and concentration device according to [1] or [2], wherein the capacity of the first flow path is 5 to 100 mL.

[0018] [4] The purification and concentrating device according to any one of [1] to [3], wherein the pump comprises at least: a first pump arranged at the inlet and outlet side of the first liquid in the first flow path, and a second pump arranged at the inlet and outlet side of the second liquid.

[0019] [5] The purification and concentration device according to any one of [1] to [4], wherein the average inner diameter of the hollow fiber membrane is 0.2 to 1.4 mm and the molecular weight cutoff is 10,000 to 1,000,000.

[0020] [6] The purification and concentration device according to any one of [1] to [5], wherein the first flow path is formed in a detachable structure.

[0021] [7] The purification and concentration device according to any one of [1] to [6], wherein the second flow path is connected to a raw material storage unit that stores a raw material liquid of the liquid containing the minute useful substance.

[0022] [8] The purification and concentration device according to [7], wherein a carrier column for removing impurities in the liquid supplied from the raw material storage unit is inserted in the second flow path.

[0023] [9] The purification and concentrating device according to [7] or [8], wherein a filter member for removing impurities in the liquid supplied from the raw material storage unit is inserted in the second flow path.

[0024]

[10] The purification and concentrating device according to [8] or [9], comprising a first tank that stores the liquid from which impurities in the liquid have been removed and the liquid in the first flow path,

[0025] The first tank is connected to the second flow path, and a third flow path for supplying the liquid in the tank to the first flow path is connected to the first tank.

[0026]

[11] The purification and concentrating device according to

[10] , further comprising a second tank storing a diluent, the second tank being connected to a fourth flow path for supplying the diluent to the hollow fiber membrane module.

[0027]

[12] The purification and concentration device according to

[11] , wherein the second tank and the first tank are connected via a fifth flow path.

[0028]

[13] The purification and concentrating device according to

[12] , wherein all of the first to fifth flow paths are detachably configured.

[0029]

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

[11] to

[13] , comprising:

[0030] a liquid level position monitoring device including a light sensor for irradiating light toward the third flow path and detecting the transmittance of the light, and

[0031] a first control unit that controls a timing of supplying the diluent in the second tank to the hollow fiber membrane module based on an output of the liquid level position monitoring device.

[0032]

[15] The purification and concentrating device according to

[14] , wherein the first control unit controls the timing of supplying the diluent in the second tank from the inlet and outlet of the third liquid of the hollow fiber membrane module.

[0033]

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

[15] , comprising a third tank connected to the first flow path and configured to recover the liquid in the first flow path.

[0034]

[17] The purification and concentrating device according to any one of [1] to

[16] , wherein one or more of the first flow path, the second flow path, and the hollow fiber membrane module are arranged in a cover.

[0035]

[18] The purification and concentration device according to

[17] , comprising a management unit for managing the cleanliness inside the cover,

[0036] The management unit is provided with a fan filter device for supplying filtered air.

[0037]

[19] The purification and concentration device according to

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

[0038]

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

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

[0039]

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

[20] , wherein at least the hollow fiber membrane module and the first flow path are housed in a shell of a detachable cartridge.

[0040]

[22] The purification and concentration device according to any one of [8] to

[21] , wherein at least the second flow path and the carrier column are housed in a detachable cartridge shell.

[0041]

[23] The purification and concentration device according to

[22] , wherein the hollow fiber membrane module and the first flow path are accommodated in the shell.

[0042]

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

[15] to

[23] , wherein the hollow fiber membrane has at least two inlets and outlets for the third liquid, and the first control unit supplies the diluent from one of the at least two inlets and outlets for the third liquid while stopping the flow of the liquid through the inlet and outlet of the first liquid and the inlet and outlet of the second liquid of the hollow fiber membrane module, and then supplies the diluent from one of the at least two inlets and outlets for the third liquid while opening the flow of the liquid through the inlet and outlet of the first liquid and the inlet and outlet of the second liquid of the hollow fiber membrane module.

[0043]

[25] A method for producing a purified concentrated liquid containing minute amounts of useful substances, the method comprising:

[0044] The steps of supplying a raw liquid containing minute useful substances to the purification and concentration device, and purifying and concentrating the liquid using the hollow fiber membrane module,

[0045] The step of purifying and concentrating the liquid is performed while circulating the liquid in the circulation path including the hollow fiber membrane module in a forward and reversible manner.

[0046]

[26] The method for producing a purified concentrated liquid according to

[25] , comprising: changing the circulation direction of the liquid in the first flow path according to the purification and concentration time of the liquid or according to the flow rate of the liquid circulating in the circulation path.

[0047]

[27] The method for producing a purified concentrated liquid according to

[25] or

[26] , wherein the liquid is supplied to the hollow fiber membrane module so that the pressure between the hollow fiber membranes in the hollow fiber membrane module reaches 0.02 to 0.2 MPa to perform purification and concentration.

[0048]

[28] According to the method for producing a purified concentrated liquid described in

[27] , the method includes: among the two or more pumps inserted in the above-mentioned first flow path, the liquid delivery direction of the first pump arranged on the inlet and outlet side of the above-mentioned first liquid and the liquid delivery direction of the second pump arranged on the inlet and outlet side of the above-mentioned second liquid are set to be clockwise or counterclockwise relative to each other, and one of the above-mentioned first pump and the above-mentioned second pump is set as a main pump, the output of the above-mentioned main pump is set to be higher than the output of the other above-mentioned pumps, and the above-mentioned liquid in the above-mentioned first flow path is delivered to the above-mentioned hollow fiber membrane module.

[0049]

[29] The method for producing a purified concentrated liquid according to any one of

[25] to

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

[0050]

[30] A method for producing a purified concentrated liquid according to any one of

[25] to

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

[0051]

[31] The method for producing a purified concentrated liquid according to any one of

[25] to

[30] , wherein the step of purifying and concentrating the liquid using the hollow fiber membrane module comprises: diluting the concentrated liquid by 2 to 200 times with a diluent, and then further purifying and concentrating the liquid;

[0052] The timing for supplying the diluent to the concentrated liquid is determined based on the liquid level position of the liquid in the first flow path.

[0053]

[32] The method for producing a purified concentrated liquid according to any one of

[25] to

[31] , comprising: recovering the entire amount of the purified concentrated liquid in the first flow path.

[0054]

[33] The method for producing a purified concentrate according to any one of

[25] to

[32] , wherein the above-mentioned small useful substances are selected from extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids.

[0055]

[34] The method for producing a purified concentrated liquid according to any one of

[25] to

[33] , wherein the step of purifying and concentrating the liquid using the hollow fiber membrane module includes: flushing the hollow fiber membrane module.

[0056] According to the present disclosure, a purification and concentration device for a liquid containing microscopic useful substances which is not easily mixed with foreign matter from the outside during the purification and concentration process and a method for producing a purified concentrated liquid containing microscopic useful substances using the device can be provided, the device can produce a liquid containing microscopic useful substances at a high purity and high concentration (purified concentrated liquid of microscopic useful substances), and can more efficiently prepare the purified concentrated liquid. In addition, the purification and concentration device of the present disclosure is also useful as a device capable of purifying and concentrating microscopic useful substances by automatic control. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0058] Figure 2 This is a schematic cross-sectional view showing an example of a hollow fiber membrane module in the purification and concentrating device of the present disclosure.

[0059] Figure 3 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0060] Figure 4 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0061] Figure 5 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0062] Figure 6 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0063] Figure 7 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0064] Figure 8 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0065] Fig. 9 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0066] Fig.10 This is a flow chart showing an example of purification and concentration processing using the purification and concentration device of the present disclosure.

[0067] Fig.11 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0068] Fig.12 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0069] Fig.13 This is a schematic configuration diagram showing an example of the purification and concentration device of the present disclosure.

[0070] Fig.14 This is a graph showing the results of measuring the concentrated solutions obtained in Example 2 and Comparative Example 2 by BCA assay.

[0071] Fig.15 It is the absorbance curve graph of the concentrated solution obtained in Example 2 and Comparative Example 2.

[0072] Explanation of symbols

[0073] 1 purification and concentration device, 10 first flow path, 11 liquid level position monitoring device, 20 (20A to 20E) second flow path, 21 carrier column, 22 filtering member, 23, 24 waste liquid tank, 30a, 30b circulation path (circulation path 30), 31 first pump, 32 second pump, 33 hollow fiber membrane module, 41 flow meter (clamp-type flow meter), 50 raw material storage part, 51 cleaning liquid storage tank, 52 balancing liquid storage tank, 60 first tank, 61A, 61B flow path, 62a, 62b inflow direction, 63 cumulative flow meter (clamp-type cumulative flow meter), 64 exhaust valve, 65 exhaust pipeline, 80 third tank, 90 second tank, 100 membrane treatment cartridge, 200 pre-treatment cartridge, 300 cover, 301 frame, 400A, 400B pre-treatment and membrane treatment cartridge, 101, 201, 401 shell DETAILED DESCRIPTION

[0074] Hereinafter, an example of the present disclosure is described using the accompanying drawings. Each configuration in each embodiment and their combination are examples, and the addition, omission, replacement and other changes of the configuration can be appropriately performed without departing from the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the claims. In addition, for a specific parameter, when multiple upper limits and lower limits are recorded, any upper limit and lower limit can be combined among these upper limits and lower limits to set a preferred numerical range.

[0075] In the present disclosure, the liquid that has passed through the hollow fiber membrane in the hollow fiber membrane module is recorded as "permeated liquid", and the liquid that has not passed through the hollow fiber membrane but is retained in the hollow fiber membrane is recorded as "concentrated liquid". It should be noted that "concentrated liquid" refers to the entire liquid that has been purified and concentrated by the hollow fiber membrane.

[0076] [Device for purifying and concentrating liquid containing minute useful substances]

[0077] The purification and concentration device for liquid containing tiny useful substances disclosed in the present invention comprises: a hollow fiber membrane assembly containing a plurality of hollow fiber membranes in a container, the container having an inlet and outlet of a first liquid and an inlet and outlet of a second liquid for supplying the liquid containing tiny useful substances to the hollow fiber membranes, and an inlet and outlet of a third liquid for discharging the liquid after passing through the hollow fiber membranes; a first flow path connected to the inlet and outlet of the first liquid and the inlet and outlet of the second liquid to form a circulation path containing the hollow fiber membrane assembly; a second flow path that supplies the liquid to the first flow path; and one or more pumps inserted in the first flow path to circulate the liquid in a forward and reverse manner. The first embodiment of the purification and concentration device disclosed in the present invention is described below using the accompanying drawings.

[0078] Figure 1 This is a schematic configuration diagram showing an example of a purification and concentration device 1 (hereinafter referred to as “device 1 ”) of the present disclosure. Figure 1 The device 1 comprises: a hollow fiber membrane assembly 33 containing a plurality of hollow fiber membranes in a container, the container having a first liquid inlet and outlet 33a and a second liquid inlet and outlet 33b for supplying a liquid containing tiny useful substances to the hollow fiber membranes, and a third liquid inlet and outlet 33c, 33d for discharging the liquid after passing through the hollow fiber membranes; a first flow path 10, which is connected to the first liquid inlet and outlet 33a and the second liquid inlet and outlet 33b to form a circulation path 30 including the hollow fiber membrane assembly 33; a second flow path 20, which supplies the liquid to the first flow path 10; and one or more pumps, which are inserted in the first flow path 10 to circulate the liquid in a forward and reverse manner. It should be noted that Figure 1 In the device 1, the first pump 31 and the second pump 32 are inserted in the first flow path 10. According to this structure, the liquid containing tiny useful substances can be purified and concentrated while circulating in the first flow path 10 connected to the hollow fiber membrane module 33 in a forward and reverse manner. Therefore, the purified concentrated liquid of the tiny useful substances can be produced more efficiently. In addition, since a container for purification and concentration treatment is not required, a compact device can be made. As a result, the device 1 can be adjusted to a size that can be configured in a clean room, etc., and foreign matter can be prevented from mixing from the outside during purification and concentration treatment.

[0079] <First flow path>

[0080] exist Figure 1 In the device 1, the first flow path 10 is connected to the hollow fiber membrane module 33 to form a circulation path 30. One or more pumps are inserted in the first flow path 10. It should be noted that in the present disclosure, "flow path" refers to a pipeline for liquid flow. In addition, a "circulation path" refers to a path constructed in a manner so that the liquid in the flow path circulates once and returns to its original position, and the circulation direction is indicated by an arrow in the accompanying drawing.

[0081] exist Figure 1 In the device 1, the first flow path 10 can be connected and disconnected with one or more pumps and hollow fiber membrane modules 33. Figure 1 In the case of the device 1, the first flow path 10 can be formed by a resin tube, and the resin tube is connected to one or more pumps and hollow fiber membrane modules 33. By setting the first flow path 10 to be detachable from other elements, after the purification and concentration treatment of the liquid containing tiny useful substances, the first flow path 10 can be removed from other elements and discarded. According to such a structure, the first flow path 10 can be discarded and replaced with a new flow path in each batch, so foreign matter is less likely to mix. In addition, the device 1 can also be made into a more compact structure. In addition, by forming the flow path with a resin tube, the device 1 can also be manufactured cheaply. There is no particular limitation on the resin tube constituting the first flow path 10. From the perspective of excellent toughness / chemical resistance / resilience, cleanliness, etc., it is preferably a tube made of thermoplastic elastomer (for example, PharMed tube, etc.), a tube made of ester polyurethane (for example, Tygon tube, etc.), a nylon tube, a silicone tube, and a polyethylene tube.

[0082] Figure 1 The first flow path 10 shown in the figure has a circulation path 30a in which the liquid flows in a clockwise direction and a circulation path 30b in which the liquid flows in a counterclockwise direction. Figure 1In the device 1, the liquid supplied from the second flow path 20 to the first flow path 10 is pumped by the first pump 31 and the second pump 32 so that it circulates in the circulation path 30a or the circulation path 30b. The liquid in the first flow path 10 is purified and concentrated in the hollow fiber membrane module 33 while circulating in the circulation path 30a or the circulation path 30b. It should be noted that the circulation path 30a and the circulation path 30b are collectively referred to as "circulation path 30". The liquid that has passed through the hollow fiber membrane in the hollow fiber membrane module 33 is discharged from the inlet and outlet 33c and 33d of the third liquid, and is discarded to the waste liquid tank (described later) via the flow path 40.

[0083] exist Figure 1 In the device 1 shown, the first flow path 10 is connected to the first liquid inlet 33a and the second liquid inlet 33b of the hollow fiber membrane module 33. Figure 2 As shown, the hollow fiber membrane bundle 35 is arranged in parallel with the length direction of the hollow fiber membrane module 33. Therefore, the liquid entering from the inlet and outlet 33a of the first liquid of the hollow fiber membrane module 33 is purified and concentrated by tangential flow filtration in which the liquid flows parallel to the membrane surface in the length direction of the hollow fiber membrane bundle 35, and then discharged from the inlet and outlet 33b of the second liquid. Similarly, the liquid entering from the inlet and outlet 33b of the second liquid is also filtered by the tangential flow and discharged from the inlet and outlet 33a of the first liquid. That is, in Figure 1 In the device 1, the liquid is purified and concentrated while being subjected to alternating tangential flow filtration. By repeating this operation alternately, tiny useful substances and impurities are not easily deposited on the surface of the hollow fiber membrane. As a result, the reduction in the permeation rate of the liquid can be suppressed, and the processing efficiency can be maintained at a high level. That is, a liquid containing tiny useful substances at a higher purity and a higher concentration can be more effectively produced.

[0084] exist Figure 1 In the device 1, the capacity of the first flow path 10 is preferably 5 to 100 mL, more preferably 8 to 50 mL. It should be noted that the capacity of the first flow path 10 refers to the capacity of the entire flow path. With regard to the capacity of the first flow path 10, for example, when the first flow path 10 is composed of a resin tube, it can be adjusted by changing the length and inner diameter of the above-mentioned tube. It should be noted that the capacity (liquid volume) of the liquid in the first flow path 10 can be determined based on the information of the liquid level position monitoring device 11 described later.

[0085] (Pump)

[0086] The one or more pumps inserted in the first flow path 10 are preferably tube pumps. Figure 1In the device 1, the two pumps in the first flow path 10 are tube pumps. When the first flow path 10 is composed of a resin tube, by setting one or more pumps in the first flow path 10 as tube pumps, it is easier to load and unload the resin tube. In addition, a more compact device can be made. In addition, the device 1 can also be made into a simpler (simple structure and inexpensive) structure.

[0087] exist Figure 1 In the device 1, a first pump 31 is arranged on the first liquid inlet and outlet 33a side of the hollow fiber membrane module 33, and a second pump 32 is arranged on the second liquid inlet and outlet 33b side. By arranging two or more, preferably two pumps in a manner of sandwiching the hollow fiber membrane module 33, the liquid can be circulated more efficiently.

[0088] (Hollow fiber membrane module)

[0089] The device 1 in the present disclosure includes a hollow fiber membrane module 33 in which a plurality of hollow fiber membranes are accommodated in a container having an inlet and an outlet for a liquid.

[0090] (Hollow Fiber Membrane)

[0091] The material of the hollow fiber membrane of the present disclosure can be appropriately designed according to the purpose of use of the present disclosure, the properties of the liquid containing minute useful substances, and the like.

[0092] Specifically, organic polymer materials such as polyvinylidene fluoride (PVDF), polyethersulfone (PES), polysulfone (PSf), polyolefins (PE, PP, PTFE, etc.), polyamide, polyacrylonitrile, cellulose acetate (CA), and their modified products can be used.

[0093] Furthermore, a composite hollow fiber membrane formed of a plurality of components selected from the above may also be used.

[0094] Among them, in the case where the tiny useful substance is a biomolecule such as an exosome, it is preferred to use a hollow fiber membrane made of cellulose acetate (CA). By using a hollow fiber membrane made of CA (hereinafter sometimes also recorded as "CA membrane"), the reduction in filtration speed during purification and concentration treatment can be suppressed, and the recovery effect of tiny useful substances deposited on the membrane surface of the hollow fiber membrane can be further improved. As reasons for this, the following reasons can be cited: the surface charge of the biomolecule is a weak negative charge, and the CA membrane is also a weak negative charge. Therefore, the biomolecule and the CA membrane will not attract each other with a strong Coulomb force, and the shear force of the tangential flow filtration can be easily made to function by appropriately controlling the circulation linear velocity. In addition, by using a CA membrane, the tiny useful substances deposited on the membrane surface of the hollow fiber membrane can be more effectively recovered using the method described later.

[0095] The material of the hollow fiber membrane disclosed in the present invention can be appropriately designed according to the purpose of use of the present invention, the properties of the raw material liquid containing tiny useful substances, etc. On this basis, the surface of the hollow fiber membrane can also be coated with appropriate substances in order to improve the dirt resistance of the surface of the hollow fiber membrane according to the properties of the raw material liquid containing tiny useful substances.

[0096] The coating treatment can be performed, for example, by the same method as the method described in Japanese Patent No. 6081290 for bonding a cationic polymer to the surface of a hollow fiber membrane composed of sulfonated polyethersulfone and polyethersulfone.

[0097] That is, the following methods can be used: a method of immersing the hollow fiber membrane in an aqueous solution (aqueous dispersion) of a coating material; a method of passing an aqueous solution (aqueous dispersion) of a coating material through the hollow fiber membrane in the state of the hollow fiber membrane or a hollow fiber membrane assembly so that the coating material is adsorbed and bonded to the surface of the hollow fiber membrane, and then, the coating material deposited only on a thin layer of the coating material is removed by washing with water.

[0098] When concentrating and purifying a certain extracellular vesicle in a certain culture supernatant, it is sometimes preferable to coat a hollow fiber membrane made of cellulose acetate with a certain biomolecule from the viewpoint of improving the purification and concentration efficiency and the recovery rate of useful microscopic substances such as extracellular vesicles.

[0099] As an example, the following method can be used: a 1.3 mg / mL skim milk powder solution dissolved in phosphate buffer (PBS) is passed through a cellulose acetate hollow fiber membrane module (molecular weight cutoff 300,000, membrane area 0.062 m2) at a flow rate of 200 mL / min. 2 ) was passed through the hollow fiber membrane for 10 minutes to apply a coating treatment for allowing biomolecules such as casein in the skimmed milk powder to be adsorbed and bound to the surface of the hollow fiber membrane in the form of a thin layer. Then, in order to remove only the excess components deposited on the thin layer, the membrane was washed with 250 mL of phosphate buffered saline (PBS).

[0100] In one embodiment, from the viewpoint of improving the fouling resistance of the hollow fiber membrane, it is preferred that biomolecules of a different type from the above-mentioned tiny useful substance are attached to at least a portion of the hollow fiber membrane. The biomolecules are more preferably attached to an area of ​​more than half of the surface of the hollow fiber membrane, and are preferably attached to the entire area. In one embodiment, it is preferred that a layer containing biomolecules of a different type from the above-mentioned tiny useful substance is formed on at least a portion of the surface of the hollow fiber membrane. "Layer containing biomolecules" means that a substance containing biomolecules is attached to at least a portion of the hollow fiber membrane in a diffused manner. The layer containing biomolecules is preferably formed in an area of ​​more than half of the surface of the hollow fiber membrane, and is more preferably formed in the entire area. In one embodiment, it is preferred that at least a portion of the surface of the hollow fiber membrane is coated with biomolecules of a different type from the above-mentioned tiny useful substance.

[0101] As biological molecules of a type different from the tiny useful substances, there are biological molecules obtained from animal cell tissues such as blood, emulsions, cartilage, and other biological cell tissues. Representative examples include: biological molecules contained in skim milk, skim milk powder, etc. (for example, casein); biological molecules contained in serum, etc. (for example, serum globulin, serum albumin); gelatin (for example, fish gelatin), etc., and one or more of them can be selected from them.

[0102] (Average inner diameter of hollow fiber membrane)

[0103] The average inner diameter of the hollow fiber membrane disclosed in the present invention can be appropriately designed according to the purpose of use of the present invention, the properties of the liquid containing tiny useful substances, etc. In one embodiment, a hollow fiber membrane with an average inner diameter of 0.2 mm to 1.4 mm can be used. It should be noted that the "average inner diameter of the hollow fiber membrane" in the present disclosure is a value calculated based on the diameter of the smallest circle inscribed in the hollow portion of a cut surface when a hollow fiber membrane is cut at an arbitrary surface perpendicular to the length direction. In the present disclosure, the above diameter is measured at any 10 or more locations and no more than 100 locations in the above-mentioned cut surface, and the average value thereof is taken as the "average inner diameter".

[0104] It should be noted that the average outer diameter of the hollow fiber membrane of the present disclosure may be 0.3 mm to 2.0 mm. In one embodiment, the average outer diameter of the hollow fiber membrane may be 0.3 mm to 1.4 mm when the average inner diameter is 0.2 mm to 1.0 mm. In addition, in other embodiments, the average outer diameter of the hollow fiber membrane may be 1.5 mm to 2.0 mm when the average inner diameter is 1.0 mm to 1.4 mm. It should be noted that the "average outer diameter of the hollow fiber membrane" is a value calculated based on the diameter of the smallest circle containing the outer edge of the cut surface of the hollow fiber membrane cut by the same method as the average inner diameter of the above-mentioned hollow fiber membrane. In the present disclosure, the above-mentioned diameter is measured at any 10 or more locations and no more than 100 locations in the above-mentioned cut surface, and the average value thereof is taken as the "average outer diameter".

[0105] For the purpose of use of the present disclosure, the average inner diameter of the hollow fiber membrane may be 0.6 mm to 1.4 mm from the viewpoint of easily ensuring a membrane thickness that does not make the hollow fiber membrane too soft and easily improving the recovery efficiency of minute useful substances deposited on the membrane surface of the hollow fiber membrane.

[0106] In addition, the average inner diameter can be adjusted according to the viscosity of the liquid containing the microscopic useful substance during operation. -1 When the liquid viscosity is 10 to 30 mPa·s, it is preferred to use a large-diameter hollow fiber membrane with an average inner diameter of 1.0 mm to 1.4 mm. In addition, when the liquid viscosity during operation of a liquid containing tiny useful substances is low (for example, a liquid temperature of 20°C and a shear rate of 25 s -1 When the viscosity of the liquid is less than 10mPa·s, it is preferred to use a hollow fiber membrane with an average inner diameter of 0.6mm to 1.0mm. It should be noted that when the average inner diameter of the hollow fiber membrane increases, the effective membrane area tends to decrease when the above hollow fiber membrane is used as a hollow fiber membrane module. Therefore, from the viewpoint of easily suppressing the decrease in the effective membrane area when a hollow fiber membrane module is made, it is preferred to use a hollow fiber membrane with an average inner diameter of 0.2mm to 1.0mm.

[0107] (Molecular weight cut-off of hollow fiber membrane)

[0108] The molecular weight cutoff of the hollow fiber membrane of the present invention can be appropriately designed according to the purpose of use of the present invention, the properties of the raw material solution containing the tiny useful substance, etc. In one embodiment, the above-mentioned molecular weight cutoff can be 5,000 to 3,000,000, or 10,000 to 1,000,000. In the case where the tiny useful substance is a specific protein, it is preferred to use a hollow fiber membrane with a molecular weight cutoff slightly lower than the molecular weight of the above-mentioned protein (for example, a hollow fiber membrane having a molecular weight cutoff of about 10 to 50% lower than the molecular weight of the protein, etc.). In one embodiment, when the tiny useful substance is an exosome, the molecular weight cutoff of the hollow fiber membrane is preferably 50,000 to 1,000,000, more preferably 70,000 to 800,000, and further preferably 100,000 to 500,000.

[0109] In the case where the tiny useful substance is an extracellular vesicle, from the viewpoint of the efficiency of purification and concentration, the γ-globulin permeability of the hollow fiber membrane assembly disclosed in the present invention is preferably 5 to 90%, more preferably 10 to 80%. In the present disclosure, the method for measuring the permeability of impurities in the liquid containing the above-mentioned γ-globulin containing tiny useful substances is not particularly limited, and existing known methods can be used. Specifically, a method for measuring the permeability of a standard substance having a molecular structure similar to the above-mentioned impurities and having the same degree of molecular weight can be used. In the case of measuring the above-mentioned permeability, a hollow fiber membrane assembly is sometimes used, or sometimes a hollow fiber membrane used in the above-mentioned hollow fiber membrane assembly is directly used. In addition, the above-mentioned permeability of the internal pressure type hollow fiber membrane assembly can also be replaced by the permeability obtained by measuring the method of applying pressure from the outside of the hollow fiber membrane to the inside with a hollow fiber membrane.

[0110] The number of hollow fiber membranes used in the hollow fiber membrane module of the present disclosure is not particularly limited, and can be appropriately designed according to the purpose of use of the present disclosure and the weight of the tiny useful substances to be purified and concentrated, so as to obtain the required effective membrane area.

[0111] The hollow fiber membrane is preferably bundled and housed in the above-mentioned container by an adhesive or the like. In the present disclosure, when a liquid containing a tiny useful substance is supplied from the inlet and outlet of the first liquid and / or the inlet and outlet of the second liquid of the hollow fiber membrane module, the liquid flows into one side of the hollow fiber membrane bundle in the container (the inside or outside of the hollow fiber membrane bundle) and is purified and concentrated in the hollow fiber membrane bundle. The permeate that has passed through the hollow fiber membrane bundle flows out to the other side of the hollow fiber membrane bundle (the outside or inside of the hollow fiber membrane bundle) and is discharged from the inlet and outlet of the third liquid. At this time, the concentrated liquid that has not passed through and is retained on the inside or outside of the hollow fiber membrane bundle can be discharged (recovered) from the inlet and outlet of the first liquid and / or the inlet and outlet of the second liquid to the outside of the hollow fiber membrane module.

[0112] A hollow fiber membrane module that supplies a liquid containing minute useful substances to the inside of a hollow fiber membrane bundle is an internal 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.

[0113] Figure 2 : is a schematic cross-sectional view showing an example of an internal pressure type hollow fiber membrane assembly 33. The hollow fiber membrane assembly 33 contains a hollow fiber membrane bundle 35 formed by bundling a plurality of hollow fiber membranes in a cylindrical container 34. The above-mentioned plurality of hollow fiber membranes are bundled by a biocompatible sealing adhesive (for example, a polyurethane sealing adhesive, etc.) to form a hollow fiber membrane bundle 35, and are bonded and fixed to the two ends of the length direction in the cylindrical container 34 by adhesive layers 36a and 36b formed by the above-mentioned sealing adhesive. From the viewpoint of transparency, the cylindrical container 34 can be composed of materials such as polycarbonate, poly (meth) acrylate, polysulfone, etc. It should be noted that the two end faces 34a and 34b of the cylindrical container 34 are in a state where a plurality of hollow fiber membranes are opened. An inlet and outlet 33a of the first liquid and an inlet and outlet 33b of the second liquid are provided at both ends in the length direction of the hollow fiber membrane assembly 33. In addition, inlet and outlet 33c, 33d for the third liquid are provided on the side surface in the longitudinal direction of the hollow fiber membrane module 33. The liquid supplied from the inlet and outlet 33a of the first liquid and / or the inlet and outlet 33b of the second liquid is sent to the inner side of the hollow fiber membrane bundle 35 to be purified and concentrated. In addition, the permeated liquid that permeates to the outside of the hollow fiber membrane bundle 35 is discharged from the inlet and outlet 33c, 33d of the third liquid provided on the side surface of the cylindrical container 34. It should be noted that the inlet and outlet of the third liquid are preferably provided at at least two locations on the upper side and the lower side surface of the cylindrical container 34. Figure 2 In the figure, the inlet and outlet 33c and 33d of the third liquid are provided only on one side of the hollow fiber membrane module 33, but the present invention is not limited thereto. The inlet and outlet 33c and 33d of the third liquid may be provided separately on one side and the other side.

[0114] (Filling rate of hollow fiber membrane module)

[0115] The filling rate in the hollow fiber membrane module disclosed in the present invention is preferably 5 to 55%, more preferably 20 to 55%, and further preferably 25 to 55%. The above filling rate refers to the percentage of the sum of the cross-sectional areas of the outer diameter of the hollow fiber membrane bundle to the cross-sectional area of ​​the short side direction of the generally cylindrical container of the hollow fiber membrane module. When the above filling rate is 5 to 45%, the size of the hollow fiber membrane module can be optimized, and the tiny useful substances deposited on the membrane surface of the hollow fiber membrane can be more effectively recovered.

[0116] Figure 3The device 1 is provided with a third tank 80 connected to the first flow path 10 and recovering the liquid in the first flow path 10. The third tank 80 is connected to the flow path 81B, and the flow path 81B is connected to the flow path 81A via the switch valve 76. The flow path 81A is further connected to the first flow path 10. In addition, the switch valve 75 is also provided in the first flow path 10. During the purification and concentration treatment of the liquid, the switch valve 75 is opened and the switch valve 76 is closed. After the purification and concentration treatment is completed, the switch valve 75 is closed and the switch valve 76 is opened. In this way, the entire amount of the concentrated liquid in the first flow path 10 is recovered to the third tank 80.

[0117] The recovery of the concentrated liquid into the third tank 80 can be controlled by the second control unit.

[0118] exist Figure 3 In the device 1, the second control unit may first open the switch valve 76, and use the first pump 31 and the second pump 32 to send the clean gas from the exhaust part. Furthermore, the switch valve 75 may be closed at any time while the above-mentioned gas supply is performed, and the concentrated liquid in the first flow path 10 may be recovered to the third tank 80. Furthermore, the second control unit may operate the first pump 31 and the second pump 32 in such a manner that the concentrated liquid in the first flow path 10 circulates in the circulation path 30a. That is, the first pump 31 and the second pump 32 may be operated in such a manner that the liquid sending directions are clockwise and in the same direction. By closing the switch valve 75, the concentrated liquid flowing through the circulation path 30a can be sent to the third tank 80 via the flow paths 81A and 81B. In addition, the second control unit may start the recovery process after judging that the concentration process in the first flow path 10 is completed through the liquid level position monitoring device 11 described later. Alternatively, the recovery operation of the concentrated liquid in the first flow path 10 may be manually operated by a person.

[0119] <Second flow path>

[0120] The second flow path 20 supplies the liquid containing minute useful substances to the first flow path 10 . Figures 4 to 8 Some representative schematic structures of the second flow path 20 are shown in FIG. It should be noted that the purification and concentration device 1 of the present disclosure sometimes includes valves, automatic valves, backflow prevention valves, orifices, flow meters, pressure gauges, liquid level gauges, rupture plates and other safety devices that are omitted in the schematic structure diagram. It should be noted that from the perspective of easy loading and unloading operations, it is preferred that the sensor parts of the flow meter and the pressure gauge contact the liquid containing tiny useful substances via a tube (that is, a structure that does not directly contact the liquid). In addition, as a flow meter, for example, a clamp-type cumulative flow meter or the like can be preferably used.

[0121] exist Figure 4 In the device 1, the second flow path 20 is connected to a raw material storage unit 50 for storing a raw material liquid containing a tiny amount of useful substances. Figure 4In the device 1, the second flow path 20 is divided into flow paths 20A and 20B via the on-off valve 70, the flow path 20A is connected to the raw material storage unit 50, and the flow path 20B is connected to the first flow path 10. Figure 4 In the device 1, by opening the on-off valve 70, the raw material liquid in the raw material storage part 50 can be supplied to the first flow path 10. It should be noted that the direction of gravity is not reflected in the drawings of the present disclosure.

[0122] (Raw material storage department)

[0123] The raw material storage unit 50 has a container for storing the raw material liquid. The container may be fixed to the device 1 or may be arranged in a manner that allows easy removal. When the container is arranged in a manner that allows easy removal, the raw material storage unit 50 may include various devices such as a fixing member for fixing the container.

[0124] The shape and volume of the container for storing the raw material solution are not particularly limited and can be determined according to the type of the tiny useful substance, the amount of the raw material solution, etc. In one embodiment, when the raw material solution containing tiny useful substances selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids is purified and concentrated, the volume of the above-mentioned container can be 50 to 5000 mL, or 200 to 2000 mL. The above-mentioned container can be composed of an infusion bag so that each batch can be easily disassembled.

[0125] Figure 5 In the device 1, a carrier column 21 is inserted in the second flow path, and the carrier column 21 removes impurities in the raw material liquid supplied from the raw material storage unit 50. Figure 5 In the device 1, the second flow path 20 can be divided into: a flow path 20A connected to the raw material storage part 50, a flow path 20C for supplying the raw material liquid to the carrier column 21, and a flow path 20B for supplying the liquid treated by the carrier column 21 (hereinafter referred to as "crude purified liquid") to the first flow path 10, and a switch valve 70 is arranged between the flow path 20A and the flow path 20C. When the switch valve 70 is opened, the raw material liquid in the raw material storage part 50 is supplied to the carrier column 21. In addition, a three-way valve 71 is inserted in the flow path 20B, and one end of the three-way valve 71 is connected to a flow path 20E for allowing the liquid to flow to the waste liquid tank 23.

[0126] (Carrier column)

[0127] The support column 21 is filled with a material capable of removing impurities other than minute useful substances in the raw material liquid. The material is preferably an adsorbent material capable of adsorbing and removing impurities in the raw material liquid.

[0128] As the adsorbent, a porous granular material that can adsorb and retain the above-mentioned impurities is preferred. The porous granular material refers to a granular material having a large number of pores on its surface and inside, and can be a porous granular material of a polymer or a porous granular material of an inorganic material. When the tiny useful substances contained in the raw material liquid treated in the device 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, as the above-mentioned porous granular material, it is preferred that the pores inside the pores are positively charged.

[0129] These porous granular materials may be used alone or in combination of a plurality of porous granular materials having different adsorption properties.

[0130] Examples of various porous granules with different adsorption properties include: porous granules with different pore diameters, porous granules with different specific surface areas, porous granules with different average particle sizes, porous granules with different types of substrates constituting the porous granules, porous granules with different types of functional groups existing on the internal surface of the pores of the porous granules, porous granules with different densities, etc.

[0131] Examples of such porous granular materials include those based on polysaccharides such as cellulose, agarose, starch, amylose, dextran, pullulan, glucomannan, polyacrylic acid or its derivatives, polyvinyl alcohol, nylon, polysulfone, polyacrylonitrile, polyethylene, polypropylene, polystyrene, glass, porous glass, silica gel, hydroxyapatite, etc. These may be used alone or in combination of two or more.

[0132] The porous granular material can be selected from porous granular materials having an average pore diameter in the range of 0.1 to 1000 nm, depending on the types of impurities contained in the raw material solution, etc. The average pore diameter can be measured by mercury porosimetry or reverse size exclusion chromatography.

[0133] The porous granular material can be selected from porous granular materials having an average particle size in the range of 1 to 1000 μm, depending on the type of impurities contained in the raw material liquid, etc. The average particle size can be measured by a laser diffraction scattering method.

[0134] In one embodiment, when the tiny useful substances contained in the raw material liquid treated in the device 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids, the carrier column 21 may include an antibody column. "Antibody column" refers to a column filled with a carrier fixed with antibodies that can bind to antigens (impurities). In the case where the raw material liquid contains the above-mentioned biological substances, an antibody column filled with a carrier fixed with antibodies that can bind (adsorb) to impurities other than the above-mentioned biological substances can be used. It should be noted that in the raw material liquid containing the above-mentioned biological molecules as tiny useful substances, since it contains multiple impurities in many cases, multiple antibody columns corresponding to the types of impurities can be used.

[0135] In addition, a device (column assembly) that combines multiple carrier columns into a module can also be used to replace the carrier column 21. When using a column assembly, the types of adsorbent materials contained in each carrier column can be the same or different. In addition, the inner diameter and length of the carrier column can be the same or different.

[0136] Figure 6 In the device 1 , a filter member 22 is inserted in the second flow path, and the filter member 22 removes impurities in the raw material liquid supplied from the raw material storage unit 50 . Figure 6 In the device 1, the raw material liquid is treated by the filter element 22 before the liquid is supplied to the carrier column 21. Figure 6 In the device 1, the second flow path 20 can be divided into: a flow path 20A connecting the raw material storage part 50 and the filter element 22, a flow path 20D connected to the switch valve 70 for supplying the liquid treated by the filter element 22 (hereinafter referred to as "pretreatment liquid") to the carrier column 21, a flow path 20C connecting the switch valve 70 and the carrier column 21, and a flow path 20B for supplying the crude purified liquid to the first flow path 10. When the switch valve 70 is opened, the pretreatment liquid is supplied to the carrier column 21 via the flow path 20C. According to this structure, the pretreatment liquid that has been pretreated by the filter element 22 can be supplied to the carrier column 21. It should be noted that Figure 6 The device 1 is formed to have a structure including a filter member 22 and a carrier column 21 , but may also have a structure including only the filter member 22 .

[0137] (Filter component)

[0138] The filter component 22 is a component for filtering the raw material liquid. In the case where the tiny useful substances contained in the raw material liquid include very fine biomolecules such as extracellular vesicles, the filter component 22 is preferably a filter having a precision filtration membrane (precision filtration membrane assembly). From the viewpoint of effectively filtering and removing impurities such as biomolecules larger than the target extracellular vesicles such as cells in the raw material liquid, the average pore size of the precision filtration membrane (precision filtration membrane assembly) is preferably 0.10 to 0.50 μm, and more preferably 0.15 to 0.30 μm. In addition, as a material for the precision filtration membrane, polyethersulfone, polyvinylidene fluoride, etc. are preferred. In addition, commercially available products composed of these materials can be used for precision filtration membranes.

[0139] Figure 7 The device 1 includes a first tank 60, which stores the raw liquid (crude purified liquid) after the impurities are removed and the liquid in the first flow path 10. The first tank 60 is connected to the flow path 61A, and the flow path 61A is connected to the flow path 61B via the switch valve 72. Figure 7 In the device 1, the crude purified liquid treated by the carrier column 21 is transported to the flow path 61B via the flow path 20B, and then flows along the inflow direction 62a to be supplied to the first tank 60. The crude purified liquid stored in the first tank 60 is supplied to the flow path 61A, flows along the inflow direction 62b to be sent to the flow path 61B, and is sent to the first flow path 10. The flow direction of the liquid is indicated by an arrow in the figure. Figure 7 In the device 1, among the flow paths 61A and 61B, the flow path flowing along the inflow direction 62b is described as "third flow path 62b".

[0140] (First Can)

[0141] In the device 1 of the present disclosure, the first tank 60 is a tank for storing the crude purified liquid (or pretreatment liquid) and the liquid in the first flow path 10. In one embodiment, the first tank 60 can be composed of an infusion bag so that each batch can be easily disassembled. Hereinafter, the liquid stored in the first tank 60 is recorded as "first storage liquid".

[0142] The first tank 60 is preferably transparent so that the liquid level of the first storage liquid can be observed. In addition, from the viewpoint of preventing the storage liquid in the tank from adhering to and remaining on the inner wall surface of the tank, it is more preferably formed of a water-repellent material. Specifically, the first tank 60 is preferably formed of a material including polyacrylonitrile, poly(meth)acrylate such as poly(meth)acrylate, polycarbonate, fluororesin, and the like.

[0143] for Figure 7 The flow path when the raw material liquid is processed in the device 1 is specifically described.

[0144] The raw material liquid in the raw material storage section 50 is supplied to the carrier column 21 via the flow paths 20A and 20C by opening the switch valve 70. After the impurities are removed by the carrier column 21, when the three-way valve 71 is opened to the flow path 20B side, the crude purified liquid is supplied from the flow path 20B to the flow path 61B. At this time, if there is no liquid in the first flow path 10, the crude purified liquid supplied to the flow path 61B flows along the inflow direction 62b and is sent to the first flow path 10. On the other hand, when the purification and concentration treatment is being performed in the first flow path 10, the crude purified liquid is sent to the flow path 61A along the inflow direction 62a. When the switch valve 72 is opened, the crude purified liquid is stored in the first tank 60. The crude purified liquid in the first tank 60 can be supplied to the first flow path 10 via the third flow path 62b when the switch valve 72 is opened.

[0145] Figure 8 The device 1 has Figure 7 The structure further includes a second tank 90 for storing the diluent. In the second tank 90, the fourth flow path 71A for supplying the diluent in the tank to the hollow fiber membrane module 33 and the fifth flow path 71B for supplying the diluent to the flow path 61B are connected to the three-way valve 74, respectively. Figure 8 The device 1 is provided with a liquid level position monitoring device 11 in a flow path 61B forming the third flow path 62b. In addition, a cumulative flow meter 63 may be provided in a flow path 61A connected to the first tank 60. In one embodiment, from the viewpoint of making the device 1 more compact and simple in design, the liquid capacity of the first tank 60 is particularly preferably controlled by a flow meter measurement value obtained by a clamp-type flow meter. That is, the cumulative flow meter 63 is particularly preferably a clamp-type cumulative flow meter.

[0146] exist Figure 8 In the device 1, the fourth flow path 71A is connected to the flow path 40 connected to the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33. Figure 8 In the device 1, the flow path 40 is provided with a switch valve 73 and a flow meter 41. When the diluent is supplied to the fourth flow path 71A, the three-way valve 74 is opened to the side of the fourth flow path 71A, and the switch valve 73 is closed. Thus, the diluent is supplied to the inlet and outlet 33c, 33d of the third liquid of the hollow fiber membrane module 33 via the flow path 40. The amount of the diluent supplied to the hollow fiber membrane module 33 can be managed by the flow meter 41. On the other hand, when the diluent is supplied to the fifth flow path 71B, the three-way valve 74 is opened to the side of the fifth flow path 71B, and the switch valve 72 is opened. Thus, the diluent flows along the inflow direction 62a and is supplied to the flow path 61B. That is, the second tank 90 and the first tank 60 are connected together through the fifth flow path 71B. It should be noted that from the perspective of making the device 1 a more compact and simple design, the flow meter 41 is preferably a clamp-type flow meter.

[0147] In addition, the fifth flow path 71B may be connected to the top of the first tank 60. The liquid can be sent by a tube pump, high-pressure gas, potential energy (water head difference), or the like.

[0148] (Diluent)

[0149] The diluent stored in the second tank 90 can be, for example, pure water, tap water, groundwater, filtered water such as river water, various aqueous solutions, etc., depending on the purpose of the present disclosure and the type of the micro-useful substance. In the case where the micro-useful substance is an exosome, physiological saline, a buffer for medical or biochemical use, etc. are preferably used. As the above-mentioned buffer, for example, phosphate buffer (PBS), Tris hydrochloric acid buffer, sodium citrate buffer, citrate phosphate buffer, acetate buffer, borate buffer, etc. can be used.

[0150] It should be noted that, according to the purpose of the present disclosure, the diluent preferably does not contain impurities, but when it is difficult to obtain a diluent without impurities, a diluent with a low impurity concentration (for example, an impurity concentration of less than 500 ppm) can be used.

[0151] As mentioned above, Figure 8 The device 1 has Figure 7 The first control unit may control the timing of supplying the diluent in the second tank 90 to the hollow fiber membrane module 33 based on the output from the liquid level position monitoring device 11 .

[0152] (Liquid level monitoring device)

[0153] Figure 8 The device 1 is provided with a liquid level monitoring device 11. Figure 8 In the device 1, the liquid level position monitoring device 11 is inserted into the third flow path 62b. In one embodiment, the liquid level position monitoring device 11 is preferably inserted near the connection portion between the flow path 61B forming the third flow path 62b and the first flow path 10. In the case of the liquid level position monitoring device 11, the portion of the flow path where the liquid level position monitoring device 11 is inserted and located at the lower part of the device can be included in the capacity of the first flow path 10. That is, in Figure 8 In the device 1, the capacity of the first flow path 10 can be set to the portion forming the circulation path 30 and the portion of the flow path including the lower portion of the liquid level position monitoring device 11. Figure 8In the device 1, the flow path 61B is made of a transparent raw material. The liquid level position monitoring device 11 includes a light sensor that irradiates light toward the flow path 61B and detects the transmittance of the light. The light sensor includes a light-emitting element and a light-receiving element, and irradiates light from the light-emitting element toward the flow path 61A, and detects the transmitted light intensity (transmittance) of the light through the light-receiving element. The detected transmittance is further sent to the detection unit. When the detection unit detects a change in the transmittance, in order to supply the diluent in the second tank 90 to the fourth flow path 71A or the fifth flow path 71B, the first control unit can control the three-way valve 74. It should be noted that the liquid level position monitoring device 11 can be a fiber sensor.

[0154] (First Control Unit)

[0155] The first control unit of the present disclosure can control the timing of supplying the diluent in the second tank 90 to the hollow fiber membrane module 33. An example of the determination in the diluent flow control of the first control unit is shown in FIG. Fig.10 Flowchart of the process. Fig.10 is used Fig. 9 A flow chart showing a situation in which device 1 performs purification and concentration treatment of a liquid.

[0156] The relationship between the liquid level position monitoring device 11 and the first control unit will be described in more detail.

[0157] exist Figure 8 In the device 1, the first stored liquid in the first tank 60 flows in the inflow direction 62b due to the dead weight of the liquid, is supplied to the flow path 61B, and then is sent to the first flow path 10. In the first flow path 10, the first stored liquid is purified and concentrated while circulating in the circulation path 30. As described above, the purification and concentration treatment in the first flow path 10 can be performed by operating one or more pumps inserted in the first flow path 10 to circulate the liquid. In this way, the first stored liquid can be continuously supplied from the first tank 60 to the first flow path 10.

[0158] When the amount of the first stored liquid in the first tank 60 decreases, the gas in the first tank 60 is discharged to the third flow path 62b. At this time, the amount of liquid in the first flow path 10 decreases, and when the bubbles reach the liquid level position monitoring device 11 arranged in the flow path 61B, the transmittance of light in the flow path changes. If the liquid level position monitoring device 11 detects a change in transmittance, the first control unit can determine that the amount of liquid in the first flow path 10 has decreased, and can open the three-way valve 74 to the side of the fourth flow path 71A. Furthermore, the first control unit can operate more than one pump in the first flow path 10 (preferably more than two pumps in the first flow path 10) to make the hollow fiber membrane module 33 have a negative pressure. Figure 8In the case of the device 1, the first control unit operates the first pump 31 in such a way that the liquid in the flow path flows in the counterclockwise direction, and operates the second pump 32 in such a way that the liquid in the flow path flows in the clockwise direction. Further, it is preferred to operate the first pump 31 and the second pump 32 in such a way that the pump outputs of the first pump 31 and the second pump 32 are the same value. Through this operation, the hollow fiber membrane module 33 becomes a negative pressure. When the hollow fiber membrane module 33 is a negative pressure, the diluent in the second tank 90 is supplied to the first flow path 10 from the inlet and outlet 33c, 33d side of the third liquid of the hollow fiber membrane module 33 via the fourth flow path 71A. At this time, the switch valve 73 of the flow path 40 is closed. In addition, the amount of diluent supplied to the hollow fiber membrane module 33 can be managed by the flow meter 41.

[0159] The diluent supplied to the assembly from the inlet and outlet 33c and 33d of the third liquid flows from the outside to the inside of the hollow fiber membrane bundle 35. Thus, while recovering the tiny useful substances deposited on the membrane surface of the hollow fiber membrane, especially the membrane surface inside the hollow fiber membrane, the diluent flows from the inlet and outlet 33a of the first liquid and the inlet and outlet 33b of the second liquid to the first flow path 10. The diluent supplied to the first flow path 10 dilutes the liquid while further flowing along the inflow direction 62a and being sent to the first tank 60. The process of supplying the diluent from the permeate side of the hollow fiber membrane assembly 33 in this way is described as "reverse cleaning process". Through the reverse cleaning process, the tiny useful substances and impurities deposited on the surface of the hollow fiber membrane are peeled off, and it is easy to restore the permeation rate of the liquid to the original value. In addition, by diluting the liquid with the diluent, the impurities are evenly dispersed and the removal efficiency is improved. As a result, the efficiency of purification and concentration of the liquid can be further improved.

[0160] The first control unit may also supply the diluent in the second tank 90 to the first tank 60 via the fifth flow path 71B. For example, when the liquid in the first flow path 10 is diluted without backwashing, the first control unit may open the three-way valve 74 to the fifth flow path 71B side and supply the diluent to the flow path 61B. Such a process is described as "dilution process". When the first control unit determines that the amount of liquid in the first flow path 10 is reduced based on the information of the liquid level position monitoring device 11, the three-way valve 74 may be opened to the fifth flow path 71B side. Further, the first control unit may stop one or more pumps in the first flow path 10. Thus, the diluent supplied to the flow path 61B is supplied to the first flow path 10, and the liquid in the flow path can be diluted. The diluent supplied to the first flow path 10 can flow along the inflow direction 62a while diluting the liquid in the first flow path 10 and be stored in the first tank 60. The amount of the liquid stored in the first tank 60 by dilution can also be managed by the integrating flow meter 63. As described above, the integrating flow meter 63 is preferably a clamp-type integrating flow meter from the viewpoint of making the device 1 more compact and simple in design.

[0161] exist Figure 8 In the device 1, all the flow paths from the first flow path to the fifth flow path can be a detachable structure. For example, the first flow path 10, the second flow path 20 (flow paths 20A, 20B, 20C and 20E), the third flow path 62b (flow paths 61A and 61B), the fourth flow path 71A, and the fifth flow path 71B can be all composed of resin tubes, and the above-mentioned resin tubes are connected to each element. By setting the structure in which all the flow paths from the first flow path to the fifth flow path are detachable from other elements, after the purification and concentration treatment of the liquid containing tiny useful substances is performed, all the flow paths from the first flow path to the fifth flow path can be disassembled from other elements and discarded. According to this structure, the first flow path to the fifth flow path can be discarded and replaced with a new flow path in each batch, so foreign matter is less likely to mix. In addition, the device 1 can also be made into a simpler structure.

[0162] In addition, from the viewpoint of easy installation and removal, it is preferred that the sensor parts of the flow meter and the pressure gauge contact the liquid containing the minute useful substance via the tube (ie, a structure that does not directly contact the liquid). As the flow meter, for example, a clamp-type integrating flow meter can be preferably used.

[0163] In the purification and concentration device of the first embodiment of the present disclosure, at least one of the first flow path, the second flow path, and the hollow fiber membrane module may be arranged in a cover. From the perspective of a device with less mixing of foreign matter, all the elements may be arranged in a cover.

[0164] In addition, a management unit for managing the cleanliness of the hood may also be provided. According to this configuration, the cleanliness of the purification and concentration device disclosed herein can be maintained, and contamination of liquid containing tiny useful substances can be easily prevented. The management unit may include a fan filter device for supplying filtered air to the hood, and a UV lamp. Through these combinations, the cleanliness of the hood can be further improved.

[0165] (Fan filter unit)

[0166] The fan filter device has a structure capable of supplying filtered air into the hood. The fan filter device can be a filter unit with a fan having a pre-filter built in. As the pre-filter, a HEPA filter, a ULPA filter, a chemical filter, a fiber activated carbon filter, etc. can be used depending on the cleanliness of the hood.

[0167] (UV Lamp)

[0168] The UV lamp is not particularly limited as long as it can irradiate UV (ultraviolet rays) to sterilize the inside of the cover. In addition, the number of UV lamps configured in the cover is not particularly limited either, and more than two UV lamps can be configured according to the cleanliness in the cover.

[0169] (Cleanliness)

[0170] When the purification and concentration device of the present disclosure is configured such that each element is arranged in a cover, it is preferred that the cleanliness in the cover is improved by the above-mentioned management unit. In one embodiment, the cleanliness in the cover can be managed to reach level 8 or below, or to reach level 5 or below, in the cleanliness level specified in ISO14644-1:2015.

[0171] Furthermore, since the purification and concentration device 1 of the present disclosure can be compact in size, it can be carried into a facility such as a clean booth or a clean room where cleanliness is controlled, and can be operated more easily.

[0172] (tiny useful substances)

[0173] The useful microscopic substance is preferably selected from the group consisting of extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids.

[0174] <Filter Cartridge>

[0175] In the purification and concentration device 1, each element of the device can be configured in the form of a detachable filter cartridge. According to this configuration, each element can be easily replaced by simply replacing the filter cartridge. Figures 11 to 13 A specific example will be described. Figures 11 to 13 In the figure, the purification and concentration device 1 includes a frame 301 in a cover 300 .

[0176] (Membrane treatment cartridge)

[0177] exist Fig.11 In the purification and concentrating device 1, the hollow fiber membrane module 33 and the first flow path 10 are accommodated in the shell 101 of the membrane treatment cartridge 100 which is freely attached and detached with respect to the frame 301. That is, in detail, the membrane treatment cartridge 100 accommodates the hollow fiber membrane module 33 and the first flow path 10 in the shell 101, and the shell 101 is freely attached and detached with respect to the frame 301 of the purification and concentrating device 1 via a mounting member not shown in the figure. According to this structure, the hollow fiber membrane module 33 and the first flow path 10 can be easily replaced by replacing only the membrane treatment cartridge 100.

[0178] In the purification and concentration device 1, at least a portion of the first flow path 10 is formed of a resin tube. The first pump 31 and the second pump 32 are tube pumps capable of locking a portion of the first flow path 10, and the tube pumps are mounted on a frame 301. The tube pump flows the liquid in the tube constituting a portion of the first flow path 10, and is a device having a roller that presses the tube to cause the liquid inside to flow in one direction. The tube pump is provided with a guide frame in a manner surrounding the roller, and the tube that is a portion of the first flow path 10 is hooked on the roller along the guide frame for use.

[0179] When the membrane treatment cartridge 100 is mounted on the frame 301, the third liquid inlet and outlet 33c and 33d of the hollow fiber membrane module 33 may be connected to the flow path 40 connected to the waste liquid tank via a connecting member as needed.

[0180] The purification and concentration device 1 preferably has a cartridge installation portion on the front side of the device where the membrane treatment cartridge 100 can be placed. By having the cartridge installation portion on the front side of the device, the cartridge can be replaced more easily.

[0181] As a modified example of the above-mentioned structure, the first pump 31 and the second pump 32 may be accommodated in the membrane treatment cartridge 100. In this case, the hollow fiber membrane module 33, the first flow path 10, the first pump 31, and the second pump 32 are accommodated in the shell 101 of the membrane treatment cartridge 100 which can be freely loaded and unloaded. According to this structure, the hollow fiber membrane module 33, the first flow path 10, the first pump 31, and the second pump 32 can be easily replaced by replacing only the membrane treatment cartridge 100. In this case, the first pump 31 and the second pump 32 after use can also be reused after being cleaned or the like.

[0182] (Pre-treatment filter cartridge)

[0183] exist Fig.11In the purification and concentration device 1, the second flow path 20 and the carrier column 21 are accommodated in the pretreatment filter cartridge 200 which is freely loaded and unloaded relative to the frame 301 together with the open valve 70 and the three-way valve 71 provided in the second flow path 20. That is, in detail, the pretreatment filter cartridge 200 accommodates the second flow path 20 (the open valve 70 and the three-way valve 71) and the carrier column 21 in the shell 201, and the shell 201 is freely loaded and unloaded relative to the frame 301 of the purification and concentration device 1 via the mounting member which is not shown in the figure. According to this structure, the second flow path 20 and the carrier column 21 can be easily replaced by replacing only the pretreatment filter cartridge 200. When the pretreatment filter cartridge 200 is installed in the frame 301, the second flow path 20 is connected to the raw material storage part 50 which stores the raw material liquid of the above-mentioned liquid via the connecting member as needed.

[0184] The connection between the pre-treatment cartridge 200 and the membrane treatment cartridge 100 can be performed by connecting the first flow path 10 of the membrane treatment cartridge 100 and the second flow path 20 of the pre-treatment cartridge 200 via a connecting member as needed. The connecting member can be provided on the membrane treatment cartridge 100 and / or the pre-treatment cartridge 200, or on the frame 301.

[0185] The purification and concentration device 1 preferably has a cartridge installation portion on the front side of the device where the pretreatment cartridge 200 can be placed. By having the cartridge installation portion on the front side of the device, the cartridge can be replaced more easily.

[0186] (Pretreatment and membrane treatment cartridge 400A)

[0187] exist Fig.12 In the purification and concentration device 1, the hollow fiber membrane module 33, the first flow path 10, the second flow path 20 and the carrier column 21 are accommodated in the shell 401 of the pre-treatment and membrane treatment cartridge 400A which is freely loaded and unloaded relative to the frame 301 together with the open valve 70 and the three-way valve 71 provided in the second flow path 20. That is, in detail, the pre-treatment and membrane treatment cartridge 400A accommodates the hollow fiber membrane module 33, the first flow path 10, the second flow path 20 (the open valve 70 and the three-way valve 71) and the carrier column 21 in the shell 401, and the shell 401 is freely loaded and unloaded relative to the frame 301 of the purification and concentration device 1 via the mounting member not shown in the figure. According to this structure, when it is installed on the frame 301, the process of connecting the membrane treatment cartridge 100 with the pre-treatment cartridge 200 can be omitted. In addition, the hollow fiber membrane module 33, the first flow path 10, the second flow path 20 and the carrier column 21 can be easily replaced at one time by replacing only one cartridge. The contents described in the membrane treatment cartridge 100 and the pre-treatment cartridge 200 also apply here.

[0188] (Pretreatment and membrane treatment cartridge 400B)

[0189] exist Fig.13In the purification and concentrating device 1, the hollow fiber membrane module 33, the carrier column 21, the filter element 22, the first flow path 10, the second flow path 20, the third flow paths 61A and 61B, the fourth flow path 71A, the fifth flow path 71B, the flow paths 40, 53A, 53B, 54A, 54B, 81A, 81B, the exhaust line 65, the on-off valves 72, 75, 76, 77A, 77B, 78, and the three-way valves 71, 74 are accommodated in the housing 401 of the pre-treatment and membrane treatment cartridge 400B which is freely attached and detached from the frame 301. According to this configuration, in the purification and concentrating device 1, each element to be replaced in each batch can be easily replaced at once by replacing only one cartridge.

[0190] exist Fig.13 In the purification and concentration device 1, the first flow path 10 is at least partially composed of a resin tube as described above, and the first pump 31 and the second pump 32 are tube pumps. As described above, the tube pump includes a tube and a guide frame, and the tube as a part of the first flow path 10 is used along the guide frame and hooked on the roller.

[0191] exist Fig.13 In the purification and concentration device 1, the third flow paths 61A and 61B, and the fourth flow path 71A are also at least partially composed of a resin tube. The liquid level monitoring device 11, the flow meter 41, and the cumulative flow meter 63 are mounted on the frame 301, and have a tube installation portion where the tubes can be installed. Each tube constituting a part of the third flow paths 61A and 61B or the fourth flow path 71A is installed in the tube installation portion for use. It is also preferred that at least a part of the other flow paths is also composed of a resin tube.

[0192] When the pre-treatment and membrane treatment filter cartridge 400 is disposed on the frame 301, the third flow path 61A, the fourth flow path 71A, the flow paths 40 and 81B can be connected to the first tank 60, the second tank 90, the third tank 80 or the waste liquid tank 24 via connecting components as needed. The second flow paths 20, 20E, the flow paths 53A, 54A can be connected to the raw material storage part 50, the cleaning liquid storage tank 51, the balancing liquid storage tank 52 or the waste liquid tank 23 via connecting components as needed. The connecting components can be disposed on the pre-treatment and membrane treatment filter cartridge 400 or on the frame 301.

[0193] As a variation of the above-mentioned structure, one or more other elements selected from the first pump 31, the second pump 32, the liquid level monitoring device 11 and the cumulative flow meter 63 can be accommodated in the membrane treatment and pretreatment filter cartridge, so that the filter cartridge composed of the hollow fiber membrane component 33, the various flow paths and the above-mentioned other elements can be freely loaded and unloaded relative to the above-mentioned frame 301.

[0194] The purification and concentration device 1 preferably has a cartridge installation portion on the front side of the device where the pretreatment and membrane treatment cartridges 400A and 400B can be placed. By having the cartridge installation portion on the front side of the device, the cartridge can be replaced more easily.

[0195] An embodiment of the device 1 of the present disclosure has been described, but the device 1 of the present disclosure is not limited to the combination of the above-mentioned structures. One of the characteristics of the device 1 of the present disclosure is that a compact and simple design can be achieved. Therefore, from the perspective of obtaining a device 1 with a more compact and simple design, a part or all of the above-mentioned preferred structures can also be omitted. That is, from the perspective of easily obtaining a device 1 with a more compact and simple design, it is preferably formed into a structure in which the above-mentioned management unit (including a fan filter device and a UV lamp) is omitted.

[0196] [Method for producing liquid obtained by purifying and concentrating tiny useful substances]

[0197] Below, use Fig. 9 A method for producing a liquid obtained by purifying and concentrating minute useful substances using the device 1 of the present disclosure will be described. Fig. 9 1 is a structural diagram showing an example of the overall structure of the device 1 disclosed in the present invention. The manufacturing method disclosed in the present invention is a method for manufacturing a liquid obtained by purifying and concentrating a tiny useful substance using the device 1, the method comprising: a step of supplying a raw liquid containing the above-mentioned tiny useful substance to the device 1, and a step of purifying and concentrating the above-mentioned liquid using the above-mentioned hollow fiber membrane module, the method being characterized in that: the step of purifying and concentrating the above-mentioned liquid is performed while the above-mentioned liquid is circulated in the above-mentioned circulation path containing the above-mentioned hollow fiber membrane module in a forward and reverse manner.

[0198] The production method disclosed herein includes the step of supplying a raw material solution to the apparatus 1. As the raw material solution, a culture solution, a dispersion solution, etc. containing a microscopic useful substance can be used. In addition, the concentration of the microscopic useful substance in the raw material solution is not particularly limited.

[0199] exist Fig. 9 In the device 1, the raw material liquid is stored in the raw material storage part 50. Then, it is supplied to the filter element 22 through the flow path 20A. After the raw material liquid is pre-treated by the filter element 22, it is supplied to the flow path 20D. When the switch valve 70 is opened, the pre-treatment liquid is supplied to the carrier column 21 through the flow path 20C.

[0200] Fig. 9In the device 1, a tank 51 storing a cleaning liquid (hereinafter referred to as "cleaning liquid storage tank 51") and a tank 52 storing a balancing liquid (hereinafter referred to as "balancing liquid storage tank 52") are respectively connected to the flow path 20C. They are used to clean and balance the carrier column 21. That is, the manufacturing method disclosed in the present invention may include the steps of cleaning and balancing the carrier column 21.

[0201] When the carrier column 21 is cleaned and equilibrated, first, the switch valve 77A (solenoid valve, etc.) is opened to supply the cleaning liquid in the cleaning liquid storage tank 51 to the flow path 20C via the flow paths 53A and 53B. The cleaning liquid supplied to the flow path 20C is discarded to the waste liquid tank 23 via the flow path 20E after cleaning the carrier column 21. After the cleaning process, the switch valve 77B (solenoid valve, etc.) is opened to supply the equilibration liquid in the equilibration liquid storage tank 52 to the flow path 20C via the flow paths 54A and 54B. The equilibration liquid supplied to the flow path 20C is discarded to the waste liquid tank 23 via the flow path 20E after equilibration of the carrier column 21.

[0202] (Cleaning solution and balancing solution)

[0203] As a cleaning liquid for cleaning the carrier column 21, for example, sodium chloride aqueous solution, hydrochloric acid, citric acid, sodium hypochlorite aqueous solution, sodium hydroxide aqueous solution, etc. can be used. From the viewpoint of safety, sodium chloride aqueous solution is preferably used. When the cleaning liquid flows into the carrier column 21, impurities attached to the adsorbent material in the carrier column 21 are removed. After removal with the cleaning liquid, the carrier column 21 is equilibrated. As the equilibration liquid, for example, physiological saline, phosphate buffer (PBS), Tris hydrochloric acid buffer, sodium citrate buffer, citrate phosphate buffer, acetate buffer, boric acid buffer, etc. can be used.

[0204] It should be noted that the materials of the cleaning liquid storage tank 51 and the balancing liquid storage tank 52 for storing the cleaning liquid and the balancing liquid are not particularly limited, and can be, for example, the same configuration as the first tank 60 and the second tank 90 described above. From the viewpoint of easily confirming the amount of the stored liquid in the tank by naked eye observation, the cleaning liquid storage tank 51 and the balancing liquid storage tank 52 are preferably transparent.

[0205] The cleaning and equilibration of the support column 21 can be arbitrarily set according to the amount of the raw material liquid treated by the support column 21. In one embodiment, with respect to the treatment amount of the support column 21 relative to the total mass of the raw material liquid, the support column 21 can be cleaned and equilibrated when 5 to 100 times the capacity of the support column is treated. For example, when 1000 mL of the raw material liquid is treated, it is preferred that the support column 21 be cleaned and equilibrated each time 20 to 150 mL of the raw material liquid, more preferably 50 to 100 mL, is treated with the support column 21.

[0206] (Impurity removal treatment)

[0207] The pretreatment liquid supplied to the carrier column 21 is adsorbed and removed by the adsorbent material in the carrier column 21 to form a crude purified liquid, which is then sent to the flow path 61B via the flow path 20B. In one embodiment, the impurity removal treatment based on the carrier column 21 can reduce the concentration of impurities in the raw material liquid or the pretreatment liquid to less than 1000 ppm, preferably less than 500 ppm.

[0208] (Purification and concentration treatment)

[0209] The crude purified liquid supplied to the flow path 61B can flow along the inflow direction 62b and be stored in the first tank 60. The first storage liquid can be supplied to the first flow path 10 via the third flow path 62b according to the progress of the purification and concentration treatment of the liquid in the first flow path 10. The liquid (first storage liquid) supplied to the first flow path 10 is purified and concentrated while circulating in the circulation paths 30a and 30b.

[0210] By operating one or more pumps inserted in the first flow path 10, the liquid in the first flow path 10 can be circulated through the human circulation path 30. Fig. 9 In the device 1, when the first pump 31 and the second pump 32 are both tube pumps, in order to circulate the liquid through the circulation path 30a, the liquid delivery directions of the two pumps are set to be clockwise relative to each other, and one of the first pump 31 and the second pump 32 is set as the main pump, and the output of the main pump is set higher than the output of the other pumps. In one embodiment, when the hollow fiber membrane module 33 is used as the starting point, the main pump is preferably set as a pump located on the upstream side of the liquid. Fig. 9In the case of the device 1, when the liquid is circulated through the circulation path 30a, the output of the first pump 31 is preferably set higher than the output of the second pump 32. In addition, when the liquid is circulated through the circulation path 30b, the output of the second pump 32 is preferably set higher than the output of the first pump 31. In addition, the output difference of the pumps at this time is preferably 2 to 15%, more preferably 4 to 20%. It should be noted that, when these pumps are tube pumps, the output of the first pump 31 and the second pump 32 can be set by changing the discharge flow rate (mL / min) or the output (rotation speed).

[0211] In the manufacturing method disclosed in the present invention, it is preferred that the liquid in the first flow path 10 is circulated alternately in the circulation paths 30a and 30b. The timing of switching the circulation direction of the liquid can be managed according to the purification and concentration time of the liquid, and can also be managed according to the flow rate of the liquid circulating in the circulation path 30. Specifically, it can be appropriately set according to the contamination of the hollow fiber membrane by the liquid containing tiny useful substances. In one embodiment, when the circulation direction is switched according to the purification and concentration time, it is preferably every 1 second to 10 minutes, and more preferably every 3 seconds to 5 minutes. In addition, when the circulation direction is switched according to the flow rate of the above-mentioned liquid (the cumulative flow rate after switching the circulation direction of the liquid), it is preferably every 5 mL to 300 mL, and more preferably every 10 mL to 200 mL.

[0212] The liquid in the first flow path 10 can be purified and concentrated while being alternately circulated through the circulation path 30. The liquid entering the hollow fiber membrane module 33 is purified and concentrated by alternate tangential flow filtration. The permeated liquid can be discarded to the waste liquid tank 24 from the third liquid inlet and outlet 33c, 33d of the hollow fiber membrane module 33 via the flow path 40.

[0213] In addition, the liquid viscosity of the liquid containing the microscopic useful substance during operation is 20°C at a shear rate of 25 s -1 When the viscosity of the liquid under the pressure 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.

[0214] In one embodiment, the liquid in the first flow path 10 can be circulated in such a manner that the pressure between the hollow fiber membranes of the hollow fiber membrane module 33 is 0.02 to 0.2 MPa. The pressure is preferably 0.02 to 0.15 MPa, more preferably 0.03 to 0.12 MPa. It should be noted that the pressure between the hollow fiber membranes refers to the pressure difference between the pressure applied to the inner surface of the hollow fiber membrane and the pressure applied to the outer surface.

[0215] (Reverse cleaning process)

[0216] The manufacturing method disclosed herein may include: performing reverse cleaning on the hollow fiber membrane, recovering tiny useful substances deposited on the membrane surface of the hollow fiber membrane, and performing alternating tangential flow filtration on the liquid through the hollow fiber membrane module 33. For the reverse cleaning process, when the liquid amount in the first flow path 10 is below the set value, it can be performed by supplying the dilution liquid in the second tank 90 to the inlet and outlet side of the third liquid of the hollow fiber membrane module 33. As a method for setting the liquid amount in the first flow path 10, for example, when using Fig. 9 In the case of the device 1, it can be set to the liquid volume when the liquid level position monitoring device 11 detects a change in permeability. In this case, the first control unit can open the three-way valve 74 to the side of the fourth flow path 71A, thereby implementing a backwash treatment. In addition, the liquid delivery direction of the first pump 31 can be set to a counterclockwise direction, and the liquid delivery direction of the second pump 32 can be set to a clockwise direction in a manner that makes the hollow fiber membrane module 33 negative pressure. In addition, it is preferred to set the outputs of the first pump 31 and the second pump 32 to the same value (the output difference between the first pump 31 and the second pump 32 is in the range of 0 to 4%) for operation. At this time, the outputs of the first pump 31 and the second pump 32 can be set arbitrarily. From the perspective of energy saving, the outputs of the first pump 31 and the second pump 32 can be less than 30% or less than 20%.

[0217] In one embodiment, as a timing for performing the backwash treatment, it can also be appropriately performed when the amount of liquid in the first flow path 10 is below the set value. As an example, there can be listed: a situation where there is a risk of a decrease in the permeation rate due to pressure changes or flow changes, a situation where a tendency of a decrease in the permeation rate can be determined, and a situation where a safety operation for preventing the hollow fiber membrane from being blocked in advance is performed.

[0218] When a reverse cleaning treatment is performed, a diluent having an amount of 2 to 200 times the amount of liquid set by the liquid level position monitoring device 11 is preferably supplied from the hollow fiber membrane module 33 to the first flow path 10. At this time, the excess diluent dilutes the liquid in the first flow path 10, and flows along the inflow direction 62a and is stored in the first tank 60. In addition, the amount of the diluent is preferably managed by a flow meter 41. In one embodiment, the amount of the diluent is more preferably set to 2 to 20 times the amount of the liquid. By performing such a reverse cleaning treatment, it is possible to dilute the concentrated liquid while recovering tiny useful substances deposited on the membrane surface of the hollow fiber membrane, so it is preferred.

[0219] In addition, the amount of the above-mentioned diluent can be controlled to be 2 to 10 times the amount of the above-mentioned liquid in the initial stage of the purification and concentration treatment (for example, the stage of purification and concentration treatment of the crude purified liquid), and the purification and concentration efficiency can be improved by further increasing the amount of the above-mentioned diluent to 2 to 15 times the amount of the diluent supplied in the initial stage in the intermediate and subsequent stages of the purification and concentration treatment (for example, the stage of re-purifying and concentrating the liquid that has been diluted more than once).

[0220] It should be noted that before implementing reverse cleaning, in order to prevent the residual permeate in the hollow fiber membrane module from mixing with the reverse cleaning liquid and flowing into the concentrated liquid side, it can be rinsed with a cleaning liquid (diluent) in advance, and the residual permeate in the hollow fiber membrane module can be replaced with the cleaning liquid (diluent) optionally.

[0221] At this time, the amount of the cleaning solution (diluent) used is preferably 1 to 8 times, more preferably 2 to 6 times the volume of the permeate remaining in the hollow fiber membrane module.

[0222] When concentrating and purifying a certain extracellular vesicle in a certain culture supernatant, when a flushing operation is performed before backwashing, the amount of residual impurity proteins can sometimes be reduced to half or less compared to when no flushing operation is performed.

[0223] When the hollow fiber membrane module 33 is an internal pressure type, the following Figure 2 An operation of replacing the permeate remaining in the hollow fiber membrane module with the cleaning liquid (diluent) by using either the inlet and outlet 33c or 33d of the third liquid as the inlet and the other as the outlet.

[0224] Since the hollow fiber membrane has liquid flow resistance, if the diluent is supplied from either the third liquid inlet and outlet 33c or 33d, the permeate remaining in the hollow fiber membrane module 33 can be replaced with the diluent with little leakage to the membrane side.

[0225] For example, by stopping the flow of the first pump 31 and the second pump 32, the flow of the first liquid inlet and outlet 33a and the second liquid inlet and outlet 33b can be stopped. In this state, by supplying a diluent from one of the third liquid inlet and outlet 33c and 33d using a pump (not shown), the diluent is discharged from the other third liquid inlet and outlet 33c or 33d. Thus, the permeate remaining in the hollow fiber membrane module 33 can be replaced with the diluent.

[0226] In addition, in the state where the flow of the first pump 31 and the second pump 32 is stopped, the flow of the above liquids at the inlet and outlet 33a of the first liquid and the inlet and outlet 33b of the second liquid is stopped by closing the valve not shown. In this state, the diluent is supplied from any one of the inlet and outlet 33c and 33d of the third liquid using a pump not shown, and the diluent is discharged from the other inlet and outlet 33c or 33d of the third liquid. In this way, the permeate remaining in the hollow fiber membrane module 33 can be replaced with the diluent.

[0227] The timing of the flushing treatment can be controlled by the first control unit. That is, the first control unit can supply the diluent in the second tank 90 from any one of the at least two third liquid inlets 33c and 33d of the hollow fiber membrane module 33, based on the output of the liquid level position monitoring device, in a state where the flow of the first pump 31 and the second pump 32 is stopped, and the flow of the first liquid inlet 33a and the second liquid inlet 33b is stopped by closing the valve not shown in the figure. At this time, the switch valve 73 provided in the flow path 40 connected to the waste liquid tank is opened.

[0228] Then, the valve is opened to allow the inlet and outlet 33a of the first liquid and the inlet and outlet 33b of the second liquid to flow, and the diluent in the second tank 90 is supplied from any one of the at least two inlets and outlets 33c and 33d of the third liquid of the hollow fiber membrane module 33 using the first pump 31 and the second pump 32, or using other pumps (not shown) etc., so that backwashing can be performed. At this time, the switch valve 73 provided in the flow path 40 connected to the waste liquid tank is closed.

[0229] (Dilution treatment)

[0230] The manufacturing method disclosed in the present invention can also dilute the purified and concentrated concentrate while further purifying and concentrating it. In one embodiment, the dilution ratio of the concentrate is preferably 2 to 200 times, more preferably 2 to 20 times. The dilution process can be performed when the amount of liquid (concentrated liquid) in the first flow path 10 is below the set value. Fig. 9In the case of the device 1, the liquid volume when the liquid level position monitoring device 11 detects a change in transmittance can be set in the same manner as the above-mentioned backwashing treatment. In the case of a dilution treatment, the first control unit can also open the three-way valve 74 to the side of the fifth flow path 71B. In addition, the first control unit preferably stops the first pump 31 and the second pump 32 when the dilution treatment is performed. In one embodiment, the purification and concentration treatment can also be performed in parallel with the dilution treatment. When the purification and concentration treatment is performed in parallel with the dilution treatment, the fifth flow path 71B is preferably connected to the top of the first tank 60. In addition, the delivery of the dilution liquid to the first tank 60 can be implemented by a tube pump, high-pressure gas, and potential energy (head difference). In addition, when the first pump 31 and the second pump 32 are stopped during the dilution treatment, the dilution liquid supplied to the fifth flow path 71B is supplied from the flow path 61B to the first flow path 10, and then flows along the inflow direction 62a and is supplied to the first tank 60.

[0231] In the manufacturing method disclosed in the present invention, the following steps are preferably performed in parallel: the step of removing impurities in the raw liquid by the carrier column 21, and the step of purifying and concentrating the liquid by the hollow fiber membrane module 33. In this case, the liquid obtained by purifying and concentrating the tiny useful substances can be produced more efficiently. Fig. 9 In the device 1, the crude purified liquid after impurities are removed by the carrier column 21 flows along the inflow direction 62a and is stored in the first tank 60. The first stored liquid in the first tank 60 can be supplied to the first flow path 10 through the third flow path 62b when the purification and concentration treatment is performed.

[0232] (Recycling)

[0233] The manufacturing method disclosed herein may include: recovering the entire amount of the concentrated liquid in the first flow path 10. For the concentrated liquid in the first flow path 10, by opening the switch valve 76 and using the first pump 31 and the second pump 32 to deliver clean gas from the exhaust part, and closing the switch valve 75 at any time, the entire amount can be recovered to the third tank 80 via the flow paths 81A and 81B. Fig. 9 In the device 1, the second control unit opens the switch valve 76, and further opens the exhaust valve 64 and the switch valve 78. The exhaust valve 64 is connected to the flow path 61B via the exhaust pipe 65. In addition, the switch valve 78 is inserted in the exhaust pipe 65. Then, the second control unit can recover the concentrated liquid by closing the switch valve 75 at any time.

[0234] In addition, the first pump 31 and the second pump 32 can be operated in such a manner that the concentrated liquid in the first flow path 10 circulates in the circulation path 30a. That is, the liquid delivery direction of the first pump 31 and the second pump 32 can be made the same clockwise direction. At this time, the output of the first pump 31 and the second pump 32 can be set arbitrarily. In one embodiment, from the perspective of energy saving, the output of the first pump 31 and the second pump 32 is preferably less than 50%, and more preferably less than 30%. In addition, the output difference between the first pump 31 and the second pump 32 is preferably the same degree (for example, in the range of 0 to 4%). The recovery operation of the concentrated liquid in the first flow path 10 can also be manually operated by a person.

[0235] (Washing out of tiny useful substances deposited on the hollow fiber membrane surface before recycling)

[0236] The manufacturing method of the present disclosure may include an operation of washing out minute useful substances deposited on the hollow fiber membrane surface immediately before the recovery process.

[0237] For example, when concentrating and purifying a certain type of extracellular vesicles in a certain culture supernatant, the extracellular vesicles deposited relatively firmly on the hollow fiber membrane surface are not dispersed in the concentrated solution, and thus the recovery rate may be reduced in the above recovery treatment.

[0238] In particular, when the amount of concentrated liquid in the first flow path 10 is small, if the extracellular vesicles deposited relatively firmly on the hollow fiber membrane surface cannot be recovered, the purification concentration efficiency will be reduced. Therefore, an operation step of redispersing the extracellular vesicles deposited on the hollow fiber membrane surface in the concentrated liquid by a small amount of reverse cleaning liquid (diluent) can be performed at a time just before the recovery process. At this time, it is preferred to fully perform the above-mentioned flushing operation and set the reverse cleaning liquid (diluent) used to a small amount. As an example, the volume of the reverse cleaning liquid (diluent) used is preferably half to twice the volume of the first flow path.

[0239] The liquid passing rate at this time can be set to be equivalent to the liquid passing rate in the purification and concentration step.

[0240] [Example 1]

[0241] Here, a method of further improving the recovery rate of minute useful substances by coating the surface of a hollow fiber membrane with biomolecules was studied.

[0242] As the raw material solution, 790 mL of culture supernatant of mesenchymal stem cells having a protein concentration of 0.49 mg / mL and an exosome concentration of 166.4 pg / mL was used.

[0243] The concentration of exosomes is equivalent to the amount of CD9 / CD63 fusion standard protein used to prepare a calibration curve when performing quantitative analysis using the CD9 / CD63 ELISA method (EXH0102EL, Cosmo Bio).

[0244] 79 mL of a carrier (a porous granular material with positively charged pores inside, capable of adsorbing and retaining negatively charged unnecessary substances other than extracellular vesicles) equilibrated with 50 mM Tris-HCl (pH 7.2) solution was added to 790 mL of the culture supernatant, and mixed inverted at room temperature for 10 minutes using a rotator. Then, the carrier was removed from the carrier-cell supernatant mixture using a 0.22 μm filter unit (S2GPU11RE, Merck Millipore) to obtain a culture supernatant treated with the carrier.

[0245] Next, the culture supernatant treated with the carrier was filtered, purified, and concentrated by an alternating tangential flow method at a filtration pressure of 30 to 40 kPa. The filtration membrane used was a cellulose acetate hollow fiber membrane module (molecular weight cutoff 300,000, membrane area 0.062 m 2 ). The filter membrane used was a filter membrane with a coating treatment on the membrane surface with biomolecules. The coating treatment was performed by passing a 1.3 mg / mL skim milk solution dissolved in PBS through the filter membrane at a flow rate of 200 mL / min for 10 minutes. Thus, a coating treatment membrane was obtained in which a certain biomolecule in the skim milk was adsorbed and bound in the form of a thin layer on the membrane surface. Then, the filter membrane was washed with 250 mL of PBS to remove the excess skim milk deposited only on the thin layer.

[0246] Here, the binding state between the filtration membrane and the certain biomolecules in the skim milk is so strong that the filtration membrane does not fall off under water washing and operation conditions, and the filtration performance is hardly reduced even after long-term operation.

[0247] Repeat alternating tangential flow filtration until the volume of the concentrate reaches 10 mL, and then 90 mL of PBS is supplied to the hollow fiber membrane assembly to dilute the concentrate. After further repeating the concentration filtration / dilution process twice, 11.9 mL of purified concentrate (extracellular vesicle purification) was obtained by alternating tangential flow filtration. The extracellular vesicles in the final exosome concentrate were quantitatively analyzed by CD9 / CD63 ELISA (EXH0102EL, Cosmo Bio), and the result was 3746.7 pg / mL, equivalent to the amount of CD9 / CD63 fusion standard protein used to make the calibration curve, with a recovery rate of 33.9%.

[0248] [Comparative Example 1]

[0249] The same operation was carried out using a hollow fiber membrane that had not been coated with skim milk, and the recovery rate was 7.4%.

[0250] The results of Example 1 and Comparative Example 1 show that the recovery rate of useful micro-substances is improved by using a hollow fiber membrane coated with biomolecules (i.e., a hollow fiber membrane having biomolecules of a different type from useful micro-substances attached to at least a portion of the membrane).

[0251] [Example 2]

[0252] Here, a method for further improving the purity of the minute useful substances in the obtained purified concentrated liquid was studied.

[0253] As the raw material solution, 40 mL of culture supernatant of amniotic mesenchymal stem cells with a protein concentration of 1.49 mg / mL and an exosome concentration of 196 pg / mL was used. A cellulose acetate hollow fiber membrane module 33 (molecular weight cutoff 300,000, membrane area 0.01 m 2 , a purification and concentration device with a permeate side volume (i.e., 10 mL of the hollow fiber external space (ECS: Extracapillary Space)), separated, purified and concentrated the exosomes in the culture supernatant by an alternating tangential flow method with a filtration pressure of 30 to 40 kPa. After the culture supernatant was concentrated to about 10 mL, 40 mL of a diluent (PBS) was injected from the inlet and outlet 33c of the third liquid of the hollow fiber membrane module and discharged to the inlet and outlet 33d of the third liquid. Thus, the impurities in the permeate retained in the ECS were rinsed by PBS. Then, 90 mL of the same diluent (PBS) was pressed from the permeate side of the hollow fiber membrane module 33 into the concentration side, and reverse pressure cleaning (backwashing) was performed. The separation and purification operation was performed again under the same conditions, and the same flushing and backwashing operations were further performed twice at the moment when the culture supernatant reached about 10 mL. After three backwashings, the culture supernatant was concentrated to 5.5 mL. The amount of protein remaining in the purified concentrate was measured by BCA assay, and the result was a protein concentration of 0.16 mg / mL. Fig.14 In addition, the absorbance of the purified concentrate was measured using the TECAN absorbance microplate reader Infinite M Nano+. The results are shown in Fig.15 .

[0254] [Comparative Example 2]

[0255] In the same separation and purification operation as in Example 2, the culture supernatant was concentrated to 5.2 mL without the rinsing operation in the ECS using PBS. The amount of protein remaining in the purified concentrate obtained was measured by BCA assay, and the result was a protein concentration of 0.36 mg / mL. The results are shown in Fig.14 In addition, the absorbance of the obtained purified concentrated solution was measured in the same manner as in Example 2. The results are shown in Fig.15 .

[0256] exist Fig.14 , 15 In the table, "Before" refers to the sample before purification and concentration, "with ECF" refers to the purified concentrated solution obtained by the method of Example 2, and "without ECS" refers to the purified concentrated solution obtained by the method of Comparative Example 2. Fig.14 It is clear that according to the method of Example 2, the concentration of protein as an impurity is further reduced, and the purity of exosomes can be further improved. Fig.15 The peak at a wavelength of 557 nm is derived from phenol red contained in the culture medium as a pH indicator, which corresponds to the impurities in the purified concentrate. Fig.15 As shown, according to the method of Example 2, the concentration of phenol red as an impurity is further reduced, and the purity of exosomes can be further improved.

[0257] According to these results, by washing (flushing) the ECS with a diluent such as PBS or saline before the backwashing process of the hollow fiber membrane module to remove impurities remaining in the ECS, the concentration of impurities in the obtained purified concentrated solution can be further reduced, and as a result, the purity of the tiny useful substances can be further improved. As a non-limiting reason, it can be considered that the reason is that by flushing, the impurities in the ECS can be prevented from being mixed into the purified concentrated solution in the subsequent backwashing process using a diluent.

[0258] [Example 3]

[0259] As the raw material solution, 200 mL of culture supernatant of amniotic mesenchymal stem cells with a protein concentration of 1.25 mg / mL and an exosome concentration of 138 pg / mL was used. A cellulose acetate hollow fiber membrane module (molecular weight cutoff 300,000, membrane area 0.01 m 2, ESC10mL), the exosomes in the culture supernatant were separated, purified and concentrated by alternating tangential flow. After the culture supernatant was concentrated to about 17mL, 40mL of diluent (PBS) was injected from the inlet and outlet 33c of the third liquid of the hollow fiber membrane component and discharged to the inlet and outlet 33d of the third liquid. Thus, the impurities in the permeate retained in the ECS were rinsed by PBS. Then, 160mL of the same diluent (PBS) was pressed from the permeate side of the hollow fiber membrane component 33 into the concentration side, and reverse pressure cleaning (backwashing) was performed. The separation and purification operation was performed again under the same conditions, and the same flushing and backwashing operations were further implemented twice at the moment when the culture supernatant reached about 17mL. The culture supernatant after backwashing was concentrated to 50mL. Backwashing was performed again to wash out the tiny useful substances attached to the surface of the hollow fiber membrane in the purified concentrated liquid, and 50mL of purified concentrated liquid was recovered. The extracellular vesicles in the purified concentrate were quantitatively analyzed by CD9 / CD63 ELISA (EXH0102EL, CosmoBio), and the result was 486pg / mL, which was equivalent to the amount of CD9 / CD63 fusion standard protein used to prepare the calibration curve, and the recovery rate was 88%. According to this result, by further supplying a diluent to the hollow fiber membrane module before recovering the purified concentrate and performing an elution operation of tiny useful substances, the recovery rate of tiny useful substances can reach more than 80%.

Claims

1. A device for purifying and concentrating a liquid containing minute useful substances, comprising: A hollow fiber membrane module containing a plurality of hollow fiber membranes in a container, the container having a first liquid inlet and a second liquid inlet for supplying a liquid containing a tiny useful substance to the hollow fiber membranes, and a third liquid inlet and outlet for discharging the liquid after passing through the hollow fiber membranes; A first flow path connected to the inlet and outlet of the first liquid and the inlet and outlet of the second liquid to form a circulation path including the hollow fiber membrane module; a second flow path that supplies the liquid to the first flow path; and One or more pumps are inserted into the first flow path to circulate the liquid in a forward and reverse manner.

2. The purification and concentration device according to claim 1, wherein: The pump is a tube pump.

3. The purification and concentration device according to claim 1 or 2, wherein: The capacity of the first flow channel is 5 to 100 mL.

4. The purification and concentration device according to claim 1 or 2, wherein: The pump includes at least a first pump disposed on the inlet and outlet side of the first liquid in the first flow path and a second pump disposed on the inlet and outlet side of the second liquid.

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

6. The purification and concentration device according to claim 1 or 2, wherein: The first flow channel is formed in a detachable configuration.

7. The purification and concentration device according to claim 1 or 2, wherein: The second flow path is connected to a raw material storage portion storing a raw material liquid of the liquid containing the minute useful substance.

8. The purification and concentration device according to claim 7, wherein: A carrier column for removing impurities in the liquid supplied from the raw material storage unit is inserted in the second flow path.

9. The purification and concentration device according to claim 7, wherein: A filter member for removing impurities in the liquid supplied from the raw material storage unit is inserted in the second flow path.

10. The purification and concentrating device according to claim 8, comprising a first tank for storing the liquid from which impurities in the liquid have been removed and the liquid in the first flow path, The first tank is connected to the second flow path, and the first tank is connected to a third flow path that supplies the liquid in the tank to the first flow path. 11 . The purification and concentration device according to claim 10 , further comprising a second tank storing a diluent, the second tank being connected to a fourth flow path for supplying the diluent to the hollow fiber membrane module.

12. The purification and concentration device according to claim 11, wherein: The second tank and the first tank are connected via a fifth flow path.

13. The purification and concentration device according to claim 12, wherein: All of the first to fifth flow paths are detachably configured.

14. The purification and concentration device according to claim 11, comprising: a liquid level position monitoring device including a light sensor for irradiating light toward the third flow path and detecting the transmittance of the light, and A first control unit controls the timing of supplying the diluent in the second tank to the hollow fiber membrane module based on the output of the liquid level position monitoring device.

15. The purification and concentration device according to claim 14, wherein: The first control unit controls a timing of supplying the diluent in the second tank from the inlet and outlet of the third liquid of the hollow fiber membrane module. 16 . The purification and concentration device according to claim 1 , further comprising a third tank connected to the first flow path and configured to recover the liquid in the first flow path. 17 . The purification and concentrating device according to claim 1 , wherein one or more of the first flow path, the second flow path, and the hollow fiber membrane module are disposed in a cover.

18. The purification and concentration device according to claim 17, further comprising a management unit for managing the cleanliness of the inside of the cover. The management unit includes a fan filter device for supplying filtered air.

19. The purification and concentration device according to claim 18, wherein: The management unit includes a UV lamp.

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

21. The purification and concentration device according to claim 1 or 2, wherein: At least the hollow fiber membrane module and the first flow path are accommodated in a detachable cartridge case.

22. The purification and concentration device according to claim 8, wherein: At least the second flow path and the carrier column are housed in a detachable cartridge housing.

23. The purification and concentration device according to claim 22, wherein: The hollow fiber membrane module and the first flow path are accommodated in the housing.

24. The purification and concentration device according to claim 15, wherein: The hollow fiber membrane has at least two inlets and outlets for the third liquid. The first control unit supplies the diluent from one of the at least two third liquid inlets and outlets while stopping the flow of the liquid at the first liquid inlet and outlet and the second liquid inlet and outlet of the hollow fiber membrane module, and then supplies the diluent from one of the at least two third liquid inlets and outlets while opening the flow of the liquid at the first liquid inlet and outlet and the second liquid inlet and outlet of the hollow fiber membrane module.

25. A method for producing a purified concentrated solution containing minute useful substances, which is a method for producing a purified concentrated solution containing minute useful substances using the purification and concentration device according to claim 1 or 2, the method comprising: The steps of supplying a raw liquid of a liquid containing minute useful substances to the purification and concentration device, and purifying and concentrating the liquid using the hollow fiber membrane module, The step of purifying and concentrating the liquid is performed while the liquid is circulated in the circulation path including the hollow fiber membrane module in a forward and reversible manner.

26. The method for producing a purified concentrate according to claim 25, comprising: The circulation direction of the liquid in the first flow path is changed according to the purification and concentration time of the liquid or according to the flow rate of the liquid circulating in the circulation path.

27. The method for producing a purified concentrated solution according to claim 25, wherein: The liquid is supplied to the hollow fiber membrane module so that the pressure between the hollow fiber membranes in the hollow fiber membrane module reaches 0.02 to 0.2 MPa to be purified and concentrated.

28. The method for producing a purified concentrate according to claim 27, comprising: Among the two or more pumps inserted in the first flow path, the delivery direction of the first pump arranged on the inlet and outlet side of the first liquid and the delivery direction of the second pump arranged on the inlet and outlet side of the second liquid are set to be clockwise or counterclockwise relative to each other, and one of the first pump and the second pump is set as a main pump, and the output of the main pump is set higher than the outputs of the other pumps to deliver the liquid in the first flow path to the hollow fiber membrane module.

29. The method for producing a purified concentrated solution according to claim 25, wherein: The step of purifying and concentrating the liquid by using the hollow fiber membrane assembly comprises: using the hollow fiber membrane assembly to perform alternating tangential flow filtration on the liquid.

30. The method for producing a purified concentrated solution according to claim 25, wherein: The step of purifying and concentrating the liquid by using the hollow fiber membrane module includes: recovering the tiny useful substances deposited on the membrane surface of the hollow fiber membrane while performing alternating tangential flow filtration on the liquid by using the hollow fiber membrane module.

31. The method for producing a purified concentrated solution according to claim 25, wherein: The step of purifying and concentrating the liquid by using the hollow fiber membrane assembly comprises: diluting the concentrated liquid by 2 to 200 times with a diluent, and then further purifying and concentrating the liquid. The timing for supplying the diluent to the concentrated liquid is determined based on the liquid level position of the liquid in the first flow path.

32. The method for producing a purified concentrate according to claim 25, comprising: The entire amount of the purified concentrate in the first flow path is recovered.

33. The method for producing a purified concentrated solution according to claim 25, wherein: The tiny useful substances are selected from extracellular vesicles, antibodies, viruses, proteins, enzymes and nucleic acids.

34. The method for producing a purified concentrated solution according to claim 25, wherein: The step of purifying and concentrating the liquid by using the hollow fiber membrane assembly includes: flushing the hollow fiber membrane assembly.

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