Device for purifying and concentrating liquid that contains minute useful substance, and method for producing purified concentrate that contains minute useful substance using said device

Abstract

Provided are: a device for purifying and concentrating a liquid that contains a minute useful substance, the device being capable of producing a liquid that contains a minute useful substance at high purity and high concentration (a purified concentrate of a minute useful substance), being capable of more efficiently preparing the purified concentrate, and making it less likely for foreign substances to be mixed in from the outside during a purification and concentration process; and a method for producing a purified concentrate that contains a minute useful substance using said device.　A purification and concentration device according to the present disclosure comprises: a hollow fiber membrane module in which a plurality of hollow fiber membranes are contained in a container, the container having a first liquid inlet / outlet and a second liquid inlet / outlet for supplying a liquid that contains a minute useful substance, and a third liquid inlet / outlet for discharging the liquid after the liquid has passed through the hollow fiber membranes; a first flow path that is connected to the first liquid inlet / outlet and the second liquid inlet / outlet, the first flow path forming a circulation path that includes the hollow fiber membrane module; a second flow path that supplies the liquid to the first flow path; and one or more pumps that are interposed in the first flow path, the one or more pumps circulating the liquid in a forward-and-reverse manner.

Description

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

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

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

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

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

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

[0006] In general, raw liquids containing useful micro substances often contain impurities. Therefore, there is a demand for an apparatus that can prepare a liquid containing useful micro substances at high purity and high concentration (a purified and concentrated solution of useful micro substances). There is also a demand for an apparatus that can prepare the purified and concentrated solution more efficiently. Furthermore, since purified and concentrated solutions containing useful micro substances are sometimes used for medical purposes, there is also a demand for an apparatus that is less susceptible to the introduction of foreign matter from the outside during the purification and concentration process. There is also a demand for an apparatus that can purify and concentrate useful micro substances using automatic control.

[0007] The present invention has been made in view of the above circumstances, and aims to provide an apparatus for purifying and concentrating a liquid containing useful micro substances at high purity and high concentration (a purified and concentrated liquid of useful micro substances) that can more efficiently prepare the purified and concentrated liquid and that is less likely to be contaminated with foreign matter from the outside during the purification and concentration process, and a method for producing a purified and concentrated liquid containing useful micro substances using the same. Another aim is to provide an apparatus that can purify and concentrate useful micro substances using automatic control.

[0008] To solve the above problems, the present disclosure can employ the following means. [1] An apparatus for purifying and concentrating a liquid containing useful micro substances, comprising: a hollow fiber membrane module containing a plurality of hollow fiber membranes in a container, the container having an inlet / outlet for a first liquid and an inlet / outlet for a second liquid for supplying a liquid containing useful micro substances to the hollow fiber membranes, and a third inlet / outlet for discharging the liquid that has permeated the hollow fiber membranes; a first flow path connected to the inlet / outlet for the first liquid and the inlet / outlet for the second liquid and forming a circulation path including the hollow fiber membrane module; a second flow path for supplying the liquid to the first flow path; and one or more pumps interposed in the first flow path for circulating the liquid in a forward and reverse direction. [2] The apparatus for purifying and concentrating according to [1], wherein the pump is a tube pump. [3] The apparatus for purifying and concentrating according to [1] or [2], wherein the capacity of the first flow path is 5 to 100 mL. [4] The purification and concentration apparatus according to any one of [1] to [3], wherein the pump comprises at least a first pump arranged on an inlet / outlet side of the first liquid in the first flow path and a second pump arranged on an inlet / outlet side of the second liquid. [5] The purification and concentration apparatus according to any one of [1] to [4], wherein the hollow fiber membrane has an average inner diameter of 0.2 to 1.4 mm and a molecular weight cutoff of 10,000 to 1,000,000. [6] The purification and concentration apparatus according to any one of [1] to [5], wherein the first flow path is detachable. [7] The purification and concentration apparatus according to any one of [1] to [6], wherein the second flow path is connected to a raw material reservoir that stores a raw material liquid of the liquid containing the useful minute substance. [8] The purification and concentration apparatus according to [7], wherein a carrier column that removes impurities from the liquid supplied from the raw material reservoir is disposed in the second flow path. [9] The purification and concentration apparatus according to [7] or [8], wherein a filtration member that removes impurities from the liquid supplied from the raw material reservoir is disposed in the second flow path.

[10] The purification and concentration apparatus according to [8] or [9], further comprising a first tank for storing the liquid after impurities in the liquid have been removed and the liquid in the first flow path, wherein the first tank is connected to the second flow path, and a third flow path is connected to the first tank for supplying the liquid in the tank toward the first flow path.

[11] The purification and concentration apparatus according to

[10] , comprising a second tank for storing a diluted solution and connected to a fourth flow path for supplying the diluted solution to the hollow fiber membrane module.

[12] The purification and concentration apparatus according to

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

[13] The purification and concentration apparatus according to

[12] , wherein all of the flow paths from the first flow path to the fifth flow path are detachable.

[14] The purification and concentration apparatus according to any of

[11] to

[13] , comprising a liquid level position monitoring device including an optical sensor that irradiates light toward the third flow path and detects light transmittance, and a first control unit that controls the timing of supplying the diluted solution 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 apparatus according to

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

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

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

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

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

[18] The purification and concentration apparatus according to

[17] , further comprising a control unit for controlling the cleanliness of the interior of the housing, the control unit comprising a fan filter device for supplying filtered air.

[19] The purification and concentration apparatus according to

[18] , wherein the control unit comprises a UV lamp.

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

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

[21] The purification and concentration apparatus 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 detachable cartridge case.

[22] The purification and concentration apparatus 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 case.

[23] The purification and concentration apparatus according to

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

[24] A purification and concentration apparatus described in any 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 dilution liquid from one of the at least two inlets and outlets for the third liquid while stopping the flow of the liquid through the inlets and outlets for the first liquid and the inlets and outlets for the second liquid of the hollow fiber membrane module, and then supplies the dilution liquid from one of the at least two inlets and outlets for the third liquid while opening the flow of the liquid through the inlets and outlets for the first liquid and the inlets and outlets for the second liquid of the hollow fiber membrane module.

[25] A method for producing a purified concentrate containing useful micro substances using the purification and concentration apparatus described in any one of [1] to

[24] , comprising: supplying a raw material liquid containing useful micro substances to the purification and concentration apparatus; and purifying and concentrating the liquid with the hollow fiber membrane module, wherein the purification and concentration are carried out while the liquid is circulated reversibly through the circulation path including the hollow fiber membrane module.

[26] A method for producing a purified concentrate according to

[25] , comprising changing the circulation direction of the liquid in the first flow path depending on the purification and concentration time of the liquid or depending on the flow rate of the liquid circulated through the circulation path.

[27] A method for producing a purified concentrate according to

[25] or

[26] , wherein the liquid is supplied to the hollow fiber membrane module so that the pressure across the hollow fiber membranes in the hollow fiber membrane module is 0.02 to 0.2 MPa, and then purified and concentrated.

[28] The method for producing a purified concentrate according to

[27] , wherein, of the two or more pumps interposed in the first flow path, a first pump arranged on an inlet / outlet side for the first liquid and a second pump arranged on an inlet / outlet side for the second liquid are mutually configured to send liquid in a clockwise or counterclockwise direction, and one of the first pump and the second pump is used as a main pump, and an output of the main pump is set higher than an output of the other pump, thereby sending the liquid in the first flow path toward the hollow fiber membrane module.

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

[25] to

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

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

[25] to

[29] , wherein purifying and concentrating the liquid using the hollow fiber membrane module comprises subjecting the liquid to alternating tangential flow filtration using the hollow fiber membrane module while recovering the useful micro substance deposited on the membrane surface of the hollow fiber membrane.

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

[25] to

[30] , wherein purifying and concentrating the liquid using the hollow fiber membrane module comprises diluting the concentrated liquid 2 to 200 times with a diluent, followed by further purification and concentration, and the timing for supplying the diluent to the concentrated liquid is determined by the liquid level position of the liquid in the first flow path.

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

[25] to

[31] , wherein the method comprises recovering the entire amount of the purified concentrate in the first flow path.

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

[25] to

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

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

[25] to

[33] , wherein purifying and concentrating the liquid using the hollow fiber membrane module includes subjecting the hollow fiber membrane module to a flushing treatment.

[0009] According to the present disclosure, it is possible to provide a device for purifying and concentrating a liquid containing useful micro substances at high purity and high concentration (a purified and concentrated liquid of useful micro substances), which can more efficiently prepare the purified and concentrated liquid and is less likely to be contaminated with foreign matter from the outside during the purification and concentration process, and a method for producing a purified and concentrated liquid containing useful micro substances using the same. The purification and concentration device of the present disclosure is also useful as a device that can purify and concentrate useful micro substances using automatic control.

[0010] FIG. 1 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 2 is a schematic cross-sectional view showing an example of a hollow fiber membrane module in the purification and concentration apparatus according to the present disclosure. FIG. 3 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 4 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 5 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 6 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 7 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 8 is a flowchart showing an example of a purification and concentration process using the purification and concentration apparatus according to the present disclosure. FIG. 9 is a schematic configuration diagram showing an example of a purification and concentration apparatus according to the present disclosure. FIG. 10 is a graph showing the results of measuring the concentrates obtained in Example 2 and Comparative Example 2 by BCA assay. FIG. 11 is an absorbance chart of the concentrates obtained in Example 2 and Comparative Example 2.

[0011] An example of the present disclosure will be described below using the drawings. However, the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of configurations are possible as appropriate within the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the scope of the claims. Furthermore, when multiple upper and lower limit values ​​are specified for a specific parameter, any of these upper and lower limit values ​​can be combined to form a suitable numerical range. In this disclosure, the liquid that has permeated the hollow fiber membrane in the hollow fiber membrane module is referred to as the "permeate," and the liquid that does not permeate the hollow fiber membrane but remains in the hollow fiber membrane is referred to as the "concentrate." Note that the "concentrate" refers to all liquids that have been purified and concentrated using the hollow fiber membrane.

[0012] [Apparatus for purifying and concentrating a liquid containing a useful micro substance] The apparatus for purifying and concentrating a liquid containing a useful micro substance of the present disclosure comprises a hollow fiber membrane module having a container containing a plurality of hollow fiber membranes, the container having a first liquid inlet / outlet and a second liquid inlet / outlet for supplying the liquid containing the useful micro substance to the hollow fiber membranes, and a third liquid inlet / outlet for discharging the liquid that has permeated the hollow fiber membranes, a first flow path connected to the first liquid inlet / outlet and the second liquid inlet / outlet and forming a circulation path including the hollow fiber membrane module, a second flow path for supplying the liquid to the first flow path, and one or more pumps interposed in the first flow path and for circulating the liquid in a forward and reverse direction. A first embodiment of the apparatus for purifying and concentrating a liquid of the present disclosure will be described below with reference to the drawings.

[0013] FIG. 1 is a schematic diagram showing an example of a purification and concentration apparatus 1 (hereinafter referred to as "apparatus 1") according to the present disclosure. The apparatus 1 of FIG. 1 includes a hollow fiber membrane module 33 having a container containing a plurality of hollow fiber membranes, the container having a first liquid inlet / outlet 33a and a second liquid inlet / outlet 33b for supplying a liquid containing a useful micro substance to the hollow fiber membranes, and third liquid inlets / outlets 33c and 33d for discharging the liquid that has permeated the hollow fiber membranes; a first flow path 10 connected to the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b and forming a circulation path 30 including the hollow fiber membrane module 33; a second flow path 20 for supplying the liquid to the first flow path 10; and one or more pumps interposed within the first flow path 10 for reversibly circulating the liquid. The apparatus 1 of FIG. 1 also includes a first pump 31 and a second pump 32 interposed within the first flow path 10. With this configuration, the liquid containing the useful micro substance can be purified and concentrated while circulating reversibly through the first flow path 10 connected to the hollow fiber membrane module 33. This allows for more efficient production of a purified and concentrated liquid of the useful micro substance. Furthermore, since no container is required for the purification and concentration process, the device can be made compact. As a result, the device 1 can be adjusted to a size that can be placed in a clean booth or the like, and the introduction of foreign matter from outside can be prevented during the purification and concentration process.

[0014] <First flow path> In the device 1 of Fig. 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 installed in the first flow path 10. In this disclosure, the term "flow path" refers to a pipe through which a liquid flows. The term "circulation path" refers to a path configured so that the liquid in the flow path makes a full circle and returns to its original position, and the direction of circulation is indicated by an arrow in the drawing.

[0015] In the device 1 of FIG. 1 , the first flow path 10 is detachable from one or more pumps and hollow fiber membrane modules 33. In the device 1 of FIG. 1 , the first flow path 10 is constructed of a resin tube, and the resin tube can be connected to one or more pumps and hollow fiber membrane modules 33. By configuring the first flow path 10 to be detachable from other elements in this manner, the first flow path 10 can be removed from the other elements and discarded after the purification and concentration process of the liquid containing the useful micro substance. This configuration allows the first flow path 10 to be discarded and replaced with a new one for each batch, making it less likely for foreign matter to be mixed in. This also allows the device 1 to be more compact. Furthermore, by configuring the flow path with a resin tube, the device 1 can be manufactured inexpensively. The resin tube that constitutes the first flow path 10 is not particularly limited, but from the viewpoints of excellent toughness, chemical resistance, recovery elasticity, cleanability, etc., thermoplastic elastomer tubes (e.g., PharMed tubes), ester-based polyurethane tubes (e.g., Tygon tubes), nylon tubes, silicone tubes, polyethylene tubes, etc. are preferred.

[0016] The first flow path 10 shown in Figure 1 is formed with a circulation path 30a in which the liquid flows clockwise and a circulation path 30b in which the liquid flows counterclockwise. In the device 1 shown in Figure 1, the liquid supplied from the second flow path 20 into the first flow path 10 is pumped by a first pump 31 and a second pump 32 to circulate through 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 through the circulation path 30a or the circulation path 30b. The circulation paths 30a and 30b are collectively referred to as "circulation path 30." The liquid that has permeated the hollow fiber membranes in the hollow fiber membrane module 33 is discharged from third liquid inlets and outlets 33c and 33d and disposed of in a waste liquid tank (described below) via a flow path 40.

[0017] In the apparatus 1 shown in FIG. 1 , the first flow path 10 is connected to a first liquid inlet / outlet 33 a and a second liquid inlet / outlet 33 b of the hollow fiber membrane module 33. As shown in FIG. 2 , the hollow fiber membrane bundle 35 is arranged within the hollow fiber membrane module 33 so that it is parallel to the longitudinal direction of the hollow fiber membrane module 33. Therefore, the liquid entering the hollow fiber membrane module 33 through the first liquid inlet / outlet 33 a is purified and concentrated by tangential flow filtration, in which the liquid flows parallel to the membrane surface in the longitudinal direction of the hollow fiber membrane bundle 35, and then discharged through the second liquid inlet / outlet 33 b. Similarly, the liquid entering through the second liquid inlet / outlet 33 b is also tangentially filtered and discharged through the first liquid inlet / outlet 33 a. That is, in the apparatus 1 shown in FIG. 1 , the liquid can be purified and concentrated while undergoing alternate tangential flow filtration. By alternately repeating this operation, it becomes difficult for useful micro-substances and impurities to accumulate on the hollow fiber membrane surface. As a result, the decrease in the permeation rate of the liquid can be suppressed and the treatment efficiency can be maintained at a high level, which means that a liquid containing useful minute substances at a higher purity and higher concentration can be produced more efficiently.

[0018] In the device 1 of FIG. 1 , the capacity of the first flow path 10 is preferably 5 to 100 mL, and more preferably 8 to 50 mL. The capacity of the first flow path 10 refers to the capacity of the entire flow path. For example, when the first flow path 10 is configured with a resin tube, the capacity of the first flow path 10 can be adjusted by changing the length or inner diameter of the tube. The volume (liquid amount) of the liquid in the first flow path 10 may be determined based on information from a liquid level position monitoring device 11, which will be described later.

[0019] (Pump) Preferably, one or more pumps disposed in the first flow path 10 are tube pumps. In the device 1 of FIG. 1 , the two pumps in the first flow path 10 are tube pumps. When the first flow path 10 is configured with a resin tube, using a tube pump for one or more pumps in the first flow path 10 makes it easier to attach and detach the resin tube. Furthermore, a more compact device can be achieved. Furthermore, the device 1 can be configured more simply (with a simple structure and low cost). In the device 1 of FIG. 1 , a first pump 31 is disposed on the side of an inlet / outlet 33a for the first liquid of the hollow fiber membrane module 33, and a second pump 32 is disposed on the side of an inlet / outlet 33b for the second liquid. By disposing two or more pumps, preferably two, on either side of the hollow fiber membrane module 33, the liquid can be circulated more efficiently.

[0020] (Hollow Fiber Membrane Module) The device 1 according to the present disclosure includes a hollow fiber membrane module 33 in which a plurality of hollow fiber membranes are housed in a container having an inlet and outlet for liquid.

[0021] (Hollow Fiber Membrane) The material of the hollow fiber membrane of the present disclosure can be appropriately designed depending on the intended use of the present disclosure and the properties of the liquid containing the useful micro-substance. Specifically, organic polymer materials such as polyvinylidene fluoride (PVDF), polyethersulfone (PES), polysulfone (PSf), polyolefins (PE, PP, PTFE, etc.), polyamide, polyacrylonitrile, cellulose acetate (CA), and their modified forms can be used. Furthermore, composite hollow fiber membranes composed of multiple components selected from the above can also be used. Of these, when the useful micro-substance is a biomolecule such as exosomes, it is preferable to use a hollow fiber membrane made of cellulose acetate (CA). The use of a hollow fiber membrane made of CA (hereinafter sometimes referred to as a "CA membrane") can suppress a decrease in filtration rate during the purification and concentration process and further improve the recovery effect of useful micro-substances deposited on the membrane surface of the hollow fiber membrane. The reason for this is that the surface charge of biomolecules is weakly negative, and the CA membrane is also weakly negative, so there is no strong Coulomb force between the biomolecules and the CA membrane, and by appropriately controlling the circulation linear velocity, the shear force of cross-flow filtration can be easily exerted.In addition, by using a CA membrane, useful micro-substances deposited on the membrane surface of the hollow fiber membrane can be more efficiently recovered using the method described below.

[0022] The material of the hollow fiber membrane of the present disclosure can be designed appropriately depending on the intended use of the present disclosure and the properties of the raw material liquid containing the useful micro-substances. In addition, depending on the properties of the raw material liquid containing the useful micro-substances, the hollow fiber membrane surface can also be coated with an appropriate substance to improve the fouling resistance.

[0023] The coating treatment can be carried out by a method similar to the method of bonding a cationic polymer to the surface of a hollow fiber membrane made of sulfonated polyethersulfone and polyethersulfone described in Japanese Patent No. 6081290. That is, a method can be used in which the hollow fiber membrane is immersed in an aqueous solution (aqueous dispersion) of the coating substance, or a method can be used in which an aqueous solution (aqueous dispersion) of the coating substance is passed through the hollow fiber membrane in the state of a hollow fiber membrane or hollow fiber membrane module to adsorb and bond the coating substance to the surface of the hollow fiber membrane, and then the coating substance that is merely deposited on the thin layer of the coating substance is removed by washing with water.

[0024] When concentrating and purifying certain extracellular vesicles from certain culture supernatants, coating a cellulose acetate hollow fiber membrane with certain biomolecules may be preferable in terms of increasing the efficiency of purification and concentration and the recovery rate of useful micro-substances such as extracellular vesicles.

[0025] As an example, a hollow fiber membrane module made of cellulose acetate (molecular weight cutoff: 300,000, membrane area: 0.062 m) ２ ) is passed through a hollow fiber membrane at a flow rate of 200 mL / min for 10 minutes, and a coating treatment is performed to adsorb and bind biomolecules such as casein in the skim milk powder to the surface of the hollow fiber membrane as a thin layer. After that, the membrane is washed with 250 mL of phosphate buffer solution (PBS) to remove excess components that have simply accumulated on the thin layer.

[0026] In one embodiment, from the viewpoint of improving the fouling resistance of the hollow fiber membrane, it is preferable that a type of biomolecule different from the useful microsubstance is attached to at least a portion of the hollow fiber membrane. It is more preferable that the biomolecule is attached over more than half of the surface area of ​​the hollow fiber membrane, and it is preferable that the biomolecule is attached over the entire area. In one embodiment, it is preferable that a layer containing a type of biomolecule different from the useful microsubstance is formed on at least a portion of the surface of the hollow fiber membrane. The "layer containing biomolecules" means that a substance containing biomolecules is attached with a certain spread to at least a portion of the surface of the hollow fiber membrane. It is preferable that the layer containing biomolecules is formed over more than half of the surface area of ​​the hollow fiber membrane, and it is more preferable that it is formed over the entire area. In one embodiment, it is preferable that at least a portion of the surface of the hollow fiber membrane is coated with a type of biomolecule different from the useful microsubstance.

[0027] Biomolecules of a type different from minute useful substances are those obtained from animal cell tissues such as blood, milk, cartilage, and other biological cell tissues, and representative examples include biomolecules contained in skim milk and non-fat powdered milk (e.g., casein); biomolecules contained in serum (e.g., serum globulin, serum albumin); gelatin (e.g., fish gelatin); and the like, and may be one or more selected from these.

[0028] (Average inner diameter of hollow fiber membrane) The average inner diameter of the hollow fiber membrane of the present disclosure can be designed appropriately depending on the intended use of the present disclosure, the properties of the liquid containing the useful minute substance, and the like. In one embodiment, a hollow fiber membrane having an average inner diameter of 0.2 mm to 1.4 mm can be used. Note that the "average inner diameter of the hollow fiber membrane" in the present disclosure is a value calculated by cutting a single hollow fiber membrane along an arbitrary plane perpendicular to the longitudinal direction and calculating the diameter of the smallest circle inscribed in the hollow portion of the cut surface. In the present disclosure, the diameters are measured at any 10 to 100 locations on the above cut surface, and the average value is defined as the "average inner diameter."

[0029] The hollow fiber membranes of the present disclosure may have an average outer diameter of 0.3 mm to 2.0 mm. In one embodiment, when the average inner diameter of the hollow fiber membrane is 0.2 mm to 1.0 mm, the average outer diameter may be 0.3 mm to 1.4 mm. In another embodiment, when the average inner diameter of the hollow fiber membrane is 1.0 mm to 1.4 mm, the average outer diameter may be 1.5 mm to 2.0 mm. The "average outer diameter of the hollow fiber membrane" is a value calculated from the diameter of the smallest circle that encompasses the outer edge of the cut surface of a hollow fiber membrane cut in the same manner as for the average inner diameter of the hollow fiber membrane described above. In the present disclosure, the diameters are measured at any 10 to 100 locations on the cut surface, and the average value is defined as the "average outer diameter."

[0030] In the intended use of the present disclosure, the hollow fiber membrane may have an average inner diameter of 0.6 mm to 1.4 mm, from the viewpoint of easily ensuring a membrane thickness that does not make the hollow fiber membrane too flexible and easily improving the recovery efficiency of useful micro substances deposited on the membrane surface of the hollow fiber membrane. Furthermore, the average inner diameter may be adjusted depending on the liquid viscosity of the liquid containing useful micro substances during operation. When the liquid viscosity is high (for example, when the liquid temperature is 20°C and the shear rate is 25 s －１ In addition, when the viscosity of the liquid containing the minute useful substance is low during operation (for example, when the liquid temperature is 20°C and the shear rate is 25 s), it is preferable to use a large-diameter hollow fiber membrane having an average inner diameter of 1.0 mm to 1.4 mm. －１ is less than 10 mPa s), it is preferable to use hollow fiber membranes with an average inner diameter of 0.6 mm to 1.0 mm. Note that if the average inner diameter of the hollow fiber membrane is large, the effective membrane area is likely to decrease when the hollow fiber membrane is used as a hollow fiber membrane module. Therefore, from the viewpoint of easily suppressing a decrease in the effective membrane area when used as a hollow fiber membrane module, it is preferable to use hollow fiber membranes with an average inner diameter of 0.2 mm to 1.0 mm.

[0031] (Molecular Weight Cutoff of Hollow Fiber Membrane) The molecular weight cutoff of the hollow fiber membrane of the present disclosure can be appropriately designed depending on the intended use of the present disclosure, the properties of the raw material solution containing the minute useful substance, etc. In one embodiment, the molecular weight cutoff may be 5,000 to 3,000,000, or may be 10,000 to 1,000,000. When the minute useful substance is a specific protein, it is preferable to use a hollow fiber membrane with a molecular weight cutoff slightly lower than the molecular weight of the protein (for example, a hollow fiber membrane having a molecular weight cutoff approximately 10 to 50% lower than the molecular weight of the protein). In one embodiment, when the minute useful substance is an exosome, the molecular weight cutoff of the hollow fiber membrane is preferably 50,000 to 1,000,000, more preferably 70,000 to 800,000, and even more preferably 100,000 to 500,000.

[0032] When the useful minute substance is an extracellular vesicle, the γ-globulin permeability of the hollow fiber membrane module of the present disclosure is preferably 5 to 90%, more preferably 10 to 80%, from the viewpoint of purification and concentration efficiency. In the present disclosure, the method for measuring the permeability of impurities in a solution containing the useful minute substance, including the aforementioned γ-globulin, is not particularly limited, and conventionally known methods can be employed. Specifically, a method for measuring the permeability of a standard substance having a molecular structure similar to that of the impurity and a molecular weight comparable to that of the impurity can be employed. When measuring the permeability, a hollow fiber membrane module may be used, or a single hollow fiber membrane may be directly used in the hollow fiber membrane module. Furthermore, the permeability of an internal pressure hollow fiber membrane module may be substituted for the permeability measured by applying pressure from the outside of a single hollow fiber membrane to permeate the impurity to the inside.

[0033] The number of hollow fiber membranes used in the hollow fiber membrane module of the present disclosure is not particularly limited. The number of membranes incorporated into the module can be designed appropriately to obtain the required effective membrane area depending on the intended use of the present disclosure and the weight of the useful minute substance to be purified and concentrated.

[0034] The hollow fiber membranes are preferably bundled together with an adhesive or the like and housed in the container. In the present disclosure, when a liquid containing a useful micro-substance is supplied from the first liquid inlet / outlet and / or the second liquid inlet / outlet of the hollow fiber membrane module, the liquid flows into one side of the hollow fiber membrane bundle in the container (inside or outside the hollow fiber membrane bundle) and is purified and concentrated by the hollow fiber membrane bundle. The permeated liquid that permeates the hollow fiber membrane bundle flows out the other side of the hollow fiber membrane bundle (inside or outside the hollow fiber membrane bundle) and is discharged from the third liquid inlet / outlet. At this time, the concentrated liquid that remains inside or outside the hollow fiber membrane bundle without permeating can be discharged (recovered) from the hollow fiber membrane module through the first liquid inlet / outlet and / or the second liquid inlet / outlet.

[0035] An internal pressure type hollow fiber membrane module is one in which a liquid containing useful micro-substances is supplied to the inside of a hollow fiber membrane bundle. Hereinafter, an example of an internal pressure type hollow fiber membrane module will be described as one embodiment of the hollow fiber membrane module of the present disclosure.

[0036] FIG. 2 is a schematic cross-sectional view showing an example of an internal pressure type hollow fiber membrane module 33. The hollow fiber membrane module 33 contains a hollow fiber membrane bundle 35, which is a bundle of multiple hollow fiber membranes, housed within a cylindrical container 34. The multiple hollow fiber membranes are bound together with a biocompatible sealing adhesive (e.g., a polyurethane-based sealing adhesive) to form the hollow fiber membrane bundle 35, and are bonded and fixed to both longitudinal ends of the cylindrical container 34 with adhesive layers 36a, 36b made of the sealing adhesive. From the perspective of transparency, the cylindrical container 34 may be made of a material such as polycarbonate, poly(meth)acrylate, or polysulfone. Note that both end faces 34a, 34b of the cylindrical container 34 are open to the multiple hollow fiber membranes. An inlet / outlet 33a for a first liquid and an inlet / outlet 33b for a second liquid are provided at both longitudinal ends of the hollow fiber membrane module 33. In addition, third liquid inlets and outlets 33c and 33d are provided on the longitudinal side of the hollow fiber membrane module 33. The liquid supplied from the first liquid inlet and outlet 33a and / or the second liquid inlet and outlet 33b is sent to the inside of the hollow fiber membrane bundle 35 and purified and concentrated. The permeated liquid that has permeated to the outside of the hollow fiber membrane bundle 35 is discharged from the third liquid inlets and outlets 33c and 33d provided on the side of the cylindrical container 34. It is preferable that the third liquid inlets and outlets be provided in at least two locations, one at the upper side and one at the lower side of the cylindrical container 34. In FIG. 2, the third liquid inlets and outlets 33c and 33d are provided on only one side of the hollow fiber membrane module 33, but this is not a limitation. The third liquid inlets and outlets 33c and 33d may be provided separately on one side and the other side.

[0037] (Filling rate of hollow fiber membrane module) The filling rate of the hollow fiber membrane module of the present disclosure is preferably 5 to 55%, more preferably 20 to 55%, and even more preferably 25 to 55%. The filling rate refers to the percentage of the total cross-sectional area of ​​the outer diameter portion of the hollow fiber membrane bundle relative to the cross-sectional area of ​​the hollow fiber membrane module, generally a cylindrical container, in the short direction. If the filling rate is 5 to 45%, useful minute substances deposited on the membrane surfaces of the hollow fiber membranes can be more efficiently recovered while optimizing the size of the hollow fiber membrane module.

[0038] The apparatus 1 in Fig. 3 is equipped with a third tank 80 connected to the first flow path 10 and configured to recover the liquid in the first flow path 10. The third tank 80 is connected to a flow path 81B, which is connected to a flow path 81A via an on-off valve 76. The flow path 81A is further connected to the first flow path 10. An on-off valve 75 is also provided in the first flow path 10. While the liquid is being purified and concentrated, the on-off valve 75 is open and the on-off valve 76 is closed. When the purification and concentration process is completed, the on-off valve 75 is closed and the on-off valve 76 is opened. As a result, the concentrated liquid in the first flow path 10 is entirely recovered in the third tank 80.

[0039] The recovery of the concentrated liquid into the third tank 80 can be controlled by the second control unit. In the apparatus 1 of FIG. 3 , the second control unit can first open the on-off valve 76 and send clean gas from the air vent using the first pump 31 and the second pump 32. Furthermore, while sending the gas, the second control unit can close the on-off valve 75 at any timing to recover the concentrated liquid in the first flow path 10 into the third tank 80. Furthermore, the second control unit can operate the first pump 31 and the second pump 32 so that the concentrated liquid in the first flow path 10 circulates through the circulation path 30a. That is, the first pump 31 and the second pump 32 can be operated so that their liquid sending directions are the same clockwise. By closing the on-off 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. Furthermore, the second control unit can start the recovery process after determining that the concentration process in the first flow path 10 has been completed using the liquid level monitoring device 11 described below. Alternatively, the operation of recovering the concentrated liquid in the first flow path 10 can be manually performed by a person.

[0040] <Second Flow Path> The second flow path 20 supplies the liquid containing the useful micro substance to the first flow path 10. Several representative schematic configurations of the second flow path 20 are shown in Figures 4 to 8. The purification and concentration apparatus 1 of the present disclosure may include safety devices such as valves, automatic valves, check valves, orifices, flow meters, pressure gauges, level gauges, and rupture discs, which are omitted from the schematic configuration diagrams. The sensor units of the flow meters and pressure gauges are preferably configured to come into contact with the liquid containing the useful micro substance via a tube (i.e., not to come into direct contact with the liquid), from the viewpoint of easy attachment and detachment. Furthermore, as the flow meter, for example, a clamp-on integrating flow meter or the like can be preferably used.

[0041] In the apparatus 1 of Fig. 4, a raw material reservoir 50 that stores a raw material liquid containing minute useful substances is connected to the second flow path 20. In the apparatus 1 of Fig. 4, the second flow path 20 is divided into flow paths 20A and 20B with an on-off valve 70 in between, and flow path 20A is connected to the raw material reservoir 50, and flow path 20B is connected to the first flow path 10. In the apparatus 1 of Fig. 4, the raw material liquid in the raw material reservoir 50 can be supplied to the first flow path 10 by opening the on-off valve 70. Note that the direction of gravity is not reflected in the drawings of the present disclosure.

[0042] (Raw material storage section) The raw material storage section 50 has a container for storing the raw material liquid. The container may be fixed to the apparatus 1 or may be installed so as to be easily removable. If the container is installed so as to be easily removable, the raw material storage section 50 may include various equipment such as a fixing member for fixing the container.

[0043] The shape and volume of the container for storing the raw material liquid are not particularly limited and can be determined depending on the type of useful minute substance, the amount of the raw material liquid, etc. In one embodiment, when purifying and concentrating a raw material liquid containing a useful minute substance selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, nucleic acids, etc., the volume of the container may be 50 to 5,000 mL, or 200 to 2,000 mL. The container may be configured as an infusion pack so that it can be easily removed for each batch.

[0044] In the apparatus 1 of Fig. 5, a carrier column 21 is interposed in the second flow path to remove impurities from the raw material liquid supplied from the raw material reservoir 50. In the apparatus 1 of Fig. 5, the second flow path 20 is divided into a flow path 20A connected to the raw material reservoir 50, a flow path 20C that supplies the raw material liquid to the carrier column 21, and a flow path 20B that supplies the liquid treated in the carrier column 21 (hereinafter referred to as the "roughly purified liquid") to the first flow path 10, and an on-off valve 70 is disposed between the flow paths 20A and 20C. When the on-off valve 70 is opened, the raw material liquid in the raw material reservoir 50 is supplied to the carrier column 21. In addition, a three-way valve 71 is interposed in the flow path 20B, and one side of the three-way valve 71 is connected to a flow path 20E that flows the liquid toward the waste liquid tank 23.

[0045] (Carrier Column) The carrier column 21 is filled with a material capable of removing impurities other than useful micro-substances from the raw liquid. The material is preferably an adsorbent capable of adsorbing and removing impurities from the raw liquid. The adsorbent is preferably a porous granular material capable of adsorbing and retaining the impurities. Porous granular materials refer to granular substances with numerous pores on their surface and interior, and polymeric porous granular materials or inorganic porous granular materials can be used. When the useful micro-substances contained in the raw liquid to be treated by the apparatus 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, the porous granular materials preferably have positively charged pores inside. These porous granular materials can be used singly or in combination with multiple materials having different adsorption capabilities. Examples of multiple porous granular materials with different adsorption capabilities include those with different pore sizes, specific surface areas, average particle sizes, types of substrates constituting the porous granular materials, and types or densities of functional groups present on the inner surfaces of the pores of the porous granular materials. Examples of such porous granular materials include those based on polysaccharides such as cellulose, agarose, starch, amylose, dextran, pullulan, and glucomannan, synthetic polymers such as polyacrylic acid or derivatives thereof, polyvinyl alcohol, nylon, polysulfone, polyacrylnitrile, polyethylene, polypropylene, and polystyrene, glass, porous glass, silica gel, and hydroxyapatite. These may be used alone or in combination of two or more.

[0046] The porous granular material can be selected from those having an average pore diameter in the range of 0.1 to 1,000 nm depending on the type of impurities contained in the raw material liquid. The average pore diameter can be measured by mercury intrusion porosimetry or inverse size exclusion chromatography. The porous granular material may also be selected from those having an average particle diameter in the range of 1 to 1,000 μm depending on the type of impurities contained in the raw material liquid. The average particle diameter can be measured by laser diffraction scattering.

[0047] In one embodiment, when the useful micro-substances contained in the raw material solution to be treated by the apparatus 1 are biological substances such as extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids, the carrier column 21 may include an antibody column. An "antibody column" refers to a column packed with carriers onto which antibodies capable of binding to antigens (impurities) are immobilized. When the raw material solution contains the aforementioned biological substances, an antibody column packed with carriers onto which antibodies capable of binding (adsorbing) to impurities other than the biological substances can be used. Note that raw material solutions containing the aforementioned biological molecules as useful micro-substances often contain multiple types of impurities, so multiple antibody columns corresponding to the types of impurities may be used.

[0048] Alternatively, a column module formed by combining multiple carrier columns may be used instead of the carrier column 21. When a column module is used, the types of adsorbents contained in the carrier columns may be the same or different. The inner diameters and lengths of the carrier columns may also be the same or different.

[0049] In the apparatus 1 of FIG. 6 , a filter 22 is interposed in the second flow path to remove impurities from the raw material liquid supplied from the raw material reservoir 50. In the apparatus 1 of FIG. 6 , the raw material liquid is treated with the filter 22 before being supplied to the carrier column 21. In the apparatus 1 of FIG. 6 , the second flow path 20 is divided into a flow path 20A connecting the raw material reservoir 50 and the filter 22, a flow path for supplying the liquid treated by the filter 22 (hereinafter referred to as the "pre-treated liquid") to the carrier column 21, and a flow path 20D connected to an on-off valve 70, a flow path 20C connecting the on-off valve 70 and the carrier column 21, and a flow path 20B for supplying the roughly purified liquid to the first flow path 10. When the on-off valve 70 is opened, the pre-treated liquid is supplied to the carrier column 21 via the flow path 20C. With this configuration, the pre-treated liquid pre-treated by the filter 22 can be supplied to the carrier column 21. Although the device 1 in FIG. 6 is configured to have the filtering member 22 and the carrier column 21, it may also be configured to have only the filtering member 22.

[0050] (Filtering Member) The filtering member 22 is a member that filters the raw material liquid. When the useful minute substances contained in the raw material liquid include very small biomolecules such as extracellular vesicles, the filtering member 22 is preferably a filter equipped with a microfiltration membrane (microfiltration membrane module). From the viewpoint of efficiently filtering out impurities such as cells in the raw material liquid and biomolecules larger than the target extracellular vesicles, the average pore size of the microfiltration membrane (microfiltration membrane module) is preferably 0.10 to 0.50 μm, and more preferably 0.15 to 0.30 μm. Furthermore, preferred materials for the microfiltration membrane include polyethersulfone and polyvinylidene fluoride. Furthermore, commercially available microfiltration membranes made of these materials can be used.

[0051] The apparatus 1 of FIG. 7 includes a first tank 60 that stores the raw material liquid (roughly purified liquid) after impurities have been removed and the liquid in the first flow path 10. The first tank 60 is connected to a flow path 61A, and the flow path 61A is connected to a flow path 61B via an on-off valve 72. In the apparatus 1 of FIG. 7, the roughly purified liquid processed in the carrier column 21 is sent to flow path 61B via flow path 20B, then flows in an inflow direction 62a and is supplied to the first tank 60. The roughly purified liquid stored in the first tank 60 is supplied to flow path 61A, then flows in an inflow direction 62b and is sent to 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 drawing. In the apparatus 1 of FIG. 7, the flow path that flows in the inflow direction 62b of flow paths 61A and 61B is referred to as the "third flow path 62b."

[0052] (First Tank) In the apparatus 1 of the present disclosure, the first tank 60 is a tank that stores the roughly purified liquid (or pre-treated liquid) and the liquid in the first flow path 10. In one embodiment, the first tank 60 may be configured as an infusion pack so that it can be easily removed for each batch. Hereinafter, the liquid stored in the first tank 60 will be referred to as the "first stored liquid." The first tank 60 is preferably transparent so that the liquid level of the first stored liquid can be observed. Furthermore, from the viewpoint of preventing the stored liquid in the tank from adhering to and remaining on the inner wall surface of the tank, it is more preferable that the first tank 60 be made of a water-repellent material. Specifically, the first tank 60 is preferably made of a material including polyacrylonitrile; poly(meth)acrylate such as poly(meth)acrylic acid ester; polycarbonate; fluororesin, etc.

[0053] The flow paths for processing a raw material liquid in the apparatus 1 of FIG. 7 will be specifically described. By opening the on-off valve 70, the raw material liquid in the raw material reservoir 50 is supplied to the carrier column 21 via the flow paths 20A and 20C. After impurities are removed in the carrier column 21, by opening the three-way valve 71 to the flow path 20B side, a crudely 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 crudely purified liquid supplied to the flow path 61B flows in the inflow direction 62b and is sent to the first flow path 10. On the other hand, if a purification and concentration process is being performed in the first flow path 10, the crudely purified liquid flows in the inflow direction 62a and is sent toward the flow path 61A. By opening the on-off valve 72, the crudely purified liquid is stored in the first tank 60. By opening the on-off valve 72, the crudely purified liquid in the first tank 60 can be supplied to the first flow path 10 via the third flow path 62b.

[0054] The apparatus 1 of FIG. 8 includes the configuration of FIG. 7 and further includes a second tank 90 for storing diluent. The second tank 90 has a fourth flow path 71A for supplying the diluent in the tank to the hollow fiber membrane module 33 and a fifth flow path 71B for supplying the diluent to flow path 61B, each connected to a three-way valve 74. The apparatus 1 of FIG. 8 also includes a liquid level monitoring device 11 installed in flow path 61B, which forms the third flow path 62b. An integrating flow meter 63 may also be installed in flow path 61A connected to the first tank 60. In one embodiment, from the viewpoint of achieving a more compact and simple design of the apparatus 1, it is particularly preferable that the liquid volume of the first tank 60 be controlled by a flow rate measured by a clamp-on flow meter. That is, it is particularly preferable that the integrating flow meter 63 be a clamp-on integrating flow meter.

[0055] In the apparatus 1 of FIG. 8 , the fourth flow path 71A is connected to a flow path 40 that is connected to the third liquid inlets and outlets 33c and 33d of the hollow fiber membrane module 33. In the apparatus 1 of FIG. 8 , the flow path 40 is equipped with an on-off valve 73 and a flow meter 41. When supplying the diluent to the fourth flow path 71A, the three-way valve 74 is opened toward the fourth flow path 71A, and the on-off valve 73 is closed. As a result, the diluent is supplied to the third liquid inlets and outlets 33c and 33d of the hollow fiber membrane module 33 via the flow path 40. The amount of diluent supplied to the hollow fiber membrane module 33 is managed by the flow meter 41. On the other hand, when supplying the diluent to the fifth flow path 71B, the three-way valve 74 is opened toward the fifth flow path 71B, and the on-off valve 72 is opened. As a result, the diluent flows in 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 by the fifth flow path 71B. It is preferable that the flow meter 41 be a clamp-on type flow meter from the viewpoint of making the design of the device 1 more compact and simple. The fifth flow path 71B may be connected to the top of the first tank 60. The liquid can be delivered by means of a tube pump, high-pressure gas, potential energy (hydraulic head difference), or the like.

[0056] (Diluted Solution) The diluted solution stored in the second tank 90 can be, for example, pure water; filtered water such as tap water, groundwater, or river water; various aqueous solutions, etc., depending on the purpose of the present disclosure and the type of useful micro-substance. When the useful micro-substance is exosomes, it is preferable to use physiological saline or a medical or biochemical buffer solution. Examples of the buffer solution that can be used include phosphate buffer solution (PBS), Tris-HCl buffer solution, sodium citrate buffer solution, citrate phosphate buffer solution, acetate buffer solution, and borate buffer solution. Note that, depending on the purpose of the present disclosure, it is preferable that the diluted solution does not contain impurities. However, if it is difficult to obtain a diluted solution that does not contain impurities, a diluted solution with a low impurity concentration (e.g., an impurity concentration of less than 500 ppm) can be used.

[0057] As described above, the apparatus 1 in Fig. 8 includes the configuration in Fig. 7 and the second tank 90. ​​The first control unit can control the timing of supplying the diluted solution in the second tank 90 to the hollow fiber membrane module 33 based on the output from the liquid level position monitoring device 11.

[0058] (Liquid Level Position Monitoring Device) The device 1 of FIG. 8 includes a liquid level position monitoring device 11. In the device 1 of FIG. 8, the liquid level position monitoring device 11 is installed in the third flow path 62b. In one embodiment, the liquid level position monitoring device 11 is preferably installed in the flow path 61B that forms the third flow path 62b, immediately adjacent to the connection with the first flow path 10. When the liquid level position monitoring device 11 is installed, the portion of the flow path in which the liquid level position monitoring device 11 is installed, which is located at the bottom of the device, can be included in the capacity of the first flow path 10. That is, in the device 1 of FIG. 8, the capacity of the first flow path 10 can include the portion that forms the circulation path 30 and the flow path below the liquid level position monitoring device 11. In the device 1 of FIG. 8, the flow path 61B is made of a transparent material. The liquid level position monitoring device 11 includes a light sensor that irradiates light toward the flow path 61B and detects the light transmittance. The optical sensor includes a light-emitting element and a light-receiving element. The light-emitting element emits light into the flow path 61A, and the light-receiving element detects the intensity of the transmitted light (transmittance). The detected transmittance is then sent to the detection unit. When the detection unit detects a change in transmittance, the first control unit can control the three-way valve 74 to supply the diluted solution in the second tank 90 to the fourth flow path 71A or the fifth flow path 71B. The liquid level position monitoring device 11 may be a fiber sensor.

[0059] (First control unit) 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 by the first control unit is shown in the flowchart of Figure 10. Figure 10 is a flowchart when a liquid purification and concentration process is performed using the apparatus 1 of Figure 9.

[0060] The relationship between the liquid level position monitoring device 11 and the first control unit will be described in more detail. In the device 1 of FIG. 8 , the first stored liquid in the first tank 60 flows in the inflow direction 62b due to its own weight, is supplied to the flow path 61B, and then is sent to the first flow path 10. The first stored liquid is purified and concentrated in the first flow path 10 while circulating through the circulation path 30. As described above, the purification and concentration process in the first flow path 10 can be performed by operating one or more pumps interposed in the first flow path 10 to circulate the liquid. This allows the first stored liquid to be continuously supplied from the first tank 60 toward the first flow path 10.

[0061] When the amount of the first stored liquid in the first tank 60 decreases, the gas in the first tank 60 is discharged toward the third flow path 62b. At this time, the amount of liquid in the first flow path 10 decreases, and when air bubbles reach the liquid level monitor 11 disposed in flow path 61B, the light transmittance in the flow path changes. When the liquid level monitor 11 detects this change in transmittance, the first control unit determines that the amount of liquid in the first flow path 10 has decreased and opens the three-way valve 74 toward the fourth flow path 71A. Furthermore, the first control unit can operate one or more pumps in the first flow path 10 (preferably two or more pumps in the first flow path 10) to create a negative pressure in the hollow fiber membrane module 33. In the case of the device 1 shown in FIG. 8 , the first control unit can operate the first pump 31 so that the liquid flows counterclockwise in the flow path, and the second pump 32 so that the liquid flows clockwise in the flow path. Furthermore, it is preferable to operate the first pump 31 and the second pump 32 so that their pump outputs are the same. This operation creates a negative pressure in the hollow fiber membrane module 33. When the pressure inside the hollow fiber membrane module 33 becomes negative, the diluent in the second tank 90 is supplied through the fourth flow path 71A from the third liquid inlets 33c and 33d of the hollow fiber membrane module 33 into the first flow path 10. At this time, the on-off valve 73 of the flow path 40 is closed. The amount of diluent supplied to the hollow fiber membrane module 33 can be managed by the flow meter 41.

[0062] The diluted solution supplied into the module from the third liquid inlets 33c and 33d flows from the outside to the inside of the hollow fiber membrane bundle 35. This allows the diluted solution to flow into the first flow path 10 from the first liquid inlet 33a and the second liquid inlet 33b, while recovering minute useful substances deposited on the membrane surfaces of the hollow fiber membranes, particularly the membrane surfaces inside the hollow fiber membranes. The diluted solution supplied into the first flow path 10 continues to flow in the inflow direction 62a to the first tank 60 while diluting the liquid. This process of supplying diluted solution from the permeate side of the hollow fiber membrane module 33 is referred to as a "backwashing process." The backwashing process peels off minute useful substances and impurities deposited on the hollow fiber membrane surfaces, facilitating the recovery of the liquid permeation rate to almost its original value. Furthermore, diluting the liquid with the diluted solution uniformly distributes the impurities, improving removal efficiency. As a result, the efficiency of liquid purification and concentration can be further improved.

[0063] The first control unit can also supply the diluent in the second tank 90 to the first tank 60 via the fifth flow path 71B. For example, when diluting the liquid in the first flow path 10 without performing a backwashing process, the first control unit can open the three-way valve 74 to the fifth flow path 71B and supply the diluent toward the flow path 61B. This process is referred to as a "dilution process." When performing the dilution process, the first control unit can open the three-way valve 74 to the fifth flow path 71B when it determines, based on information from the liquid level monitoring device 11, that the amount of liquid in the first flow path 10 has decreased. Furthermore, the first control unit can stop one or more pumps in the first flow path 10. This allows the diluent supplied to the flow path 61B to be supplied into the first flow path 10 and dilute the liquid in the flow path. The diluent supplied into the first flow path 10 flows in the inflow direction 62a and is stored in the first tank 60 while diluting the liquid in the first flow path 10. The amount of liquid stored in the first tank 60 by the dilution process can also be managed by an integrating flow meter 63. As described above, from the viewpoint of making the device 1 more compact and simple in design, it is particularly preferable that the integrating flow meter 63 be a clamp-on type integrating flow meter.

[0064] In the device 1 of FIG. 8 , all of the flow paths from the first flow path to the fifth flow path can be configured to be detachable. 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 all be configured with resin tubes, and the resin tubes can be connected to each element. By configuring all of the flow paths from the first flow path to the fifth flow path to be detachable from other elements in this way, all of the flow paths from the first flow path to the fifth flow path can be removed from other elements and discarded after the purification and concentration process of the liquid containing the useful micro substance. This configuration allows the first flow path to the fifth flow path to be discarded and replaced with a new flow path for each batch, making it less likely that foreign matter will be mixed in. This also allows the device 1 to have a simpler configuration. Furthermore, it is preferable that the sensor parts of the flow meter and pressure gauge be configured to come into contact with the liquid containing the useful micro substance via the tubes (i.e., not directly contact the liquid) in order to simplify the attachment and detachment process. As the flow meter, for example, a clamp-on type integrating flow meter or the like can be preferably used.

[0065] In the purification and concentration apparatus according to the first embodiment of the present disclosure, one or more elements including the first flow path, the second flow path, and the hollow fiber membrane module may be configured to be disposed within a housing. From the viewpoint of creating an apparatus that is less susceptible to contamination by foreign matter, it is also possible to configure the apparatus so that all elements are disposed within the housing. A management unit for managing the cleanliness level within the housing may also be provided. This configuration allows the interior of the purification and concentration apparatus of the present disclosure to be kept clean, making it easier to prevent contamination of the liquid containing minute useful substances. The management unit may include a fan filter device that supplies filtered air into the housing, and a UV lamp. Combining these elements can further increase the cleanliness level within the housing.

[0066] (Fan filter device) The fan filter device has a configuration that allows filtered air to be supplied into the housing. The fan filter device can be a filter unit with a fan that has a built-in pre-filter. As the pre-filter, a HEPA filter, ULPA filter, chemical filter, fiber activated carbon filter, etc. can be used depending on the cleanliness level inside the housing.

[0067] (UV Lamp) The UV lamp is not particularly limited as long as it can sterilize the inside of the housing by irradiating UV (ultraviolet) rays. The number of UV lamps arranged in the housing is also not particularly limited, and two or more UV lamps may be arranged depending on the cleanliness level inside the housing.

[0068] (Cleanliness) When the purification and concentration apparatus of the present disclosure is configured such that each element is disposed within a housing, it is preferable to increase the cleanliness within the housing using the aforementioned management unit. In one embodiment, the cleanliness within the housing may be managed to be Class 8 or less, or Class 5 or less, according to the cleanliness classes defined in ISO 14644-1:2015. Furthermore, because the purification and concentration apparatus 1 of the present disclosure can be made compact, it can be easily carried into a facility where cleanliness is controlled, such as a clean booth or clean room, and operation can be more easily performed.

[0069] (Micro-useful substance) The micro-useful substance is preferably selected from extracellular vesicles, antibodies, viruses, proteins, enzymes, and nucleic acids.

[0070] <Cartridge> In the purification and concentration apparatus 1, each element of the apparatus can be configured as a removable cartridge. With this configuration, each element can be easily replaced simply by replacing the cartridge. A specific example will be described below with reference to Figures 11 to 13. In Figures 11 to 13, the purification and concentration apparatus 1 includes a frame 301 within a housing 300.

[0071] 11 , the hollow fiber membrane module 33 and the first flow path 10 are housed in a case 101 of a membrane treatment cartridge 100 that is detachable from a frame 301. That is, in detail, the membrane treatment cartridge 100 houses the hollow fiber membrane module 33 and the first flow path 10 in the case 101, and the case 101 is detachable from the frame 301 of the purification and concentration apparatus 1 via an attachment member (not shown). With this configuration, the hollow fiber membrane module 33 and the first flow path 10 can be easily replaced by simply replacing the membrane treatment cartridge 100.

[0072] In this purification and concentration apparatus 1, the first flow path 10 is at least partially composed of a resin tube. The first pump 31 and the second pump 32 are tube pumps that can lock a portion of the first flow path 10, and the tube pumps are attached to a frame 301. The tube pump causes liquid to flow in a tube that constitutes part of the first flow path 10 and is a device equipped with a roller that presses the tube to cause the liquid inside it to flow in one direction. This tube pump is provided with a guide frame surrounding the roller, and the tube that constitutes part of the first flow path 10 is hooked onto the roller along the guide frame when in use. The third liquid inlet / outlet ports 33c and 33d of the hollow fiber membrane module 33 are connected to a flow path 40 leading to a waste liquid tank via a connecting member as necessary when the membrane treatment cartridge 100 is attached to the frame 301.

[0073] The purification and concentration apparatus 1 preferably has a cartridge installation section on the front side of the apparatus where the membrane treatment cartridge 100 can be placed. By having the cartridge installation section on the front side of the apparatus, the cartridge can be replaced more easily.

[0074] As a modification of the above configuration, the first pump 31 and the second pump 32 may be housed within 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 housed within a detachable case 101 of the membrane treatment cartridge 100. According to this configuration, the hollow fiber membrane module 33, the first flow path 10, the first pump 31, and the second pump 32 can be easily replaced simply by replacing the membrane treatment cartridge 100. In this case, the used first pump 31 and second pump 32 can be reused after being subjected to treatment such as cleaning.

[0075] 11 , the second flow path 20 and the carrier column 21, together with the open valve 70 and the three-way valve 71 provided in the second flow path 20, are housed in a pretreatment cartridge 200 that is detachable from a frame 301. That is, in detail, the pretreatment cartridge 200 houses the second flow path 20 (open valve 70 and three-way valve 71) and the carrier column 21 in a case 201, and the case 201 is detachable from the frame 301 of the purification and concentration apparatus 1 via a mounting member (not shown). With this configuration, the second flow path 20 and the carrier column 21 can be easily replaced simply by replacing the pretreatment cartridge 200. When the pretreatment cartridge 200 is attached to the frame 301, the second flow path 20 is connected, if necessary, via a connecting member, to a raw material reservoir 50 that stores the raw material liquid.

[0076] The pretreatment cartridge 200 and the membrane treatment cartridge 100 can be connected by connecting the first flow path 10 of the membrane treatment cartridge 100 to the second flow path 20 of the pretreatment cartridge 200 via a connecting member as necessary. The connecting member may be provided in the membrane treatment cartridge 100 and / or the pretreatment cartridge 200, or may be provided in the frame 301.

[0077] The purification and concentration apparatus 1 preferably has a cartridge installation section on the front side of the apparatus where the pretreatment cartridge 200 can be placed. By having the cartridge installation section on the front side of the apparatus, the cartridge can be replaced more easily.

[0078] 12 , the hollow fiber membrane module 33, the first flow path 10, the second flow path 20, and the carrier column 21, together with the open valve 70 and the three-way valve 71 provided in the second flow path 20, are housed in a case 401 of the pretreatment and membrane treatment cartridge 400A, which is detachable from the frame 301. That is, in detail, the pretreatment and membrane treatment cartridge 400A houses 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 case 401, and the case 401 is detachable from the frame 301 of the purification and concentration apparatus 1 via an attachment member (not shown). With this configuration, the step of connecting the membrane treatment cartridge 100 and the pretreatment cartridge 200 can be omitted when attaching the cartridge to the frame 301. Furthermore, by simply replacing one cartridge, it is possible to simultaneously and easily replace the hollow fiber membrane module 33, the first flow path 10, the second flow path 20, and the carrier column 21. The contents described in the sections on the membrane treatment cartridge 100 and the pretreatment cartridge 200 also apply here.

[0079] 13 , the hollow fiber membrane module 33, the carrier column 21, the filtration member 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 air vent line 65, the on-off valves 72, 75, 76, 77A, 77B, 78, and the three-way valves 71, 74 are housed in a case 401 of the pretreatment and membrane treatment cartridge 400B, which is detachable from the frame 301. With this configuration, by simply replacing one cartridge, all of the elements that are normally replaced for each batch in the purification and concentration apparatus 1 can be easily replaced at once.

[0080] 13, the first flow path 10 is at least partially formed of a resin tube, as described above, and the first pump 31 and the second pump 32 are tube pumps. As described above, a tube pump includes rollers and a guide frame, and is used by aligning the tube, which is part of the first flow path 10, along the guide frame and hooking it onto the rollers.

[0081] In the purification and concentration apparatus 1 of Fig. 13, the third flow paths 61A and 61B and the fourth flow path 71A are also at least partially constituted by resin tubes. The liquid level monitoring device 11, the flow meter 41, and the integrating flow meter 63 are attached to the frame 301 and have tube installation sections where tubes can be installed, and the tubes that constitute part of the third flow paths 61A and 61B or the fourth flow path 71A are installed in the tube installation sections for use. It is also preferable that at least a portion of each of the other flow paths is also constituted by resin tubes.

[0082] When the pretreatment and membrane treatment cartridge 400 is installed in the frame 301, the third flow path 61A, the fourth flow path 71A, and the flow paths 40 and 81B are connected to the first tank 60, the second tank 90, the third tank 80, or the waste liquid tank 24, respectively, via connecting members as necessary. The second flow paths 20, 20E, and the flow paths 53A and 54A are connected to the raw material reservoir 50, the cleaning liquid reservoir tank 51, the equilibration liquid reservoir tank 52, or the waste liquid tank 23, respectively, via connecting members as necessary. The connecting members may be provided in the pretreatment and membrane treatment cartridge 400 or in the frame 301.

[0083] As a variation of the above configuration, one or more other elements selected from the first pump 31, the second pump 32, the liquid level monitoring device 11, and the integrating flow meter 63 may be housed in the membrane treatment and pretreatment cartridge, and the cartridge consisting of the hollow fiber membrane module 33, each flow path, and the other elements may be detachably attached to the frame 301.

[0084] The purification and concentration apparatus 1 preferably has a cartridge installation section on the front side of the apparatus where the pretreatment and membrane treatment cartridges 400A and 400B can be placed. By having the cartridge installation section on the front side of the apparatus, the cartridges can be replaced more easily.

[0085] Although one embodiment of the device 1 of the present disclosure has been described, the device 1 of the present disclosure is not limited to the combination of the above-described components. One of the features of the device 1 of the present disclosure is that it can be designed compactly and simply. Therefore, from the perspective of obtaining a device 1 with a more compact and simple design, it is possible to omit some or all of the above-described preferred components. In other words, from the perspective of making it easier to obtain a device 1 with a more compact and simple design, it is preferable to omit the above-described management unit (including the fan filter device and UV lamp).

[0086] [Method for producing a liquid in which useful micro substances are purified and concentrated] A method for producing a liquid in which useful micro substances are purified and concentrated using the apparatus 1 of the present disclosure will be described below with reference to Figure 9. Figure 9 is a configuration diagram showing an example of the overall configuration of the apparatus 1 of the present disclosure. The production method of the present disclosure is a method for producing a liquid in which useful micro substances are purified and concentrated using the apparatus 1, and includes supplying a raw material liquid containing the useful micro substances to the apparatus 1, and purifying and concentrating the liquid using the hollow fiber membrane module, and is characterized in that the purification and concentration of the liquid are performed while the liquid is circulated reversibly through the circulation path including the hollow fiber membrane module.

[0087] The manufacturing method of the present disclosure includes supplying a raw material liquid to the apparatus 1. The raw material liquid may be a culture solution or a dispersion liquid containing a useful micro substance. The concentration of the useful micro substance in the raw material liquid is not particularly limited.

[0088] In the apparatus 1 shown in Fig. 9, the raw material liquid is stored in the raw material reservoir 50. Then, the raw material liquid is supplied to the filtration member 22 via the flow path 20A. After the raw material liquid is pretreated in the filtration member 22, it is supplied to the flow path 20D. When the on-off valve 70 is opened, the pretreatment liquid is supplied to the carrier column 21 via the flow path 20C.

[0089] 9, a tank 51 storing a cleaning solution (hereinafter referred to as the "cleaning solution storage tank 51") and a tank 52 storing an equilibration solution (hereinafter referred to as the "equilibration solution storage tank 52") are connected to the flow path 20C. These are used to wash and equilibrate the carrier column 21. That is, the manufacturing method of the present disclosure may include washing and equilibrating the carrier column 21.

[0090] When washing and equilibrating the carrier column 21, first, the on-off valve 77A (such as an electromagnetic valve) is opened to supply the washing solution in the washing solution storage tank 51 to the flow path 20C via the flow paths 53A and 53B. After washing the carrier column 21, the washing solution supplied to the flow path 20C is discarded into the waste tank 23 via the flow path 20E. After the washing process, the on-off valve 77B (such as an electromagnetic valve) is opened to supply the equilibration solution in the equilibration solution storage tank 52 to the flow path 20C via the flow paths 54A and 54B. After equilibrating the carrier column 21, the equilibration solution supplied to the flow path 20C is discarded into the waste tank 23 via the flow path 20E.

[0091] (Washing Solution and Equilibration Solution) Examples of the washing solution used to wash the carrier column 21 include aqueous sodium chloride solutions, hydrochloric acid, citric acid, aqueous sodium hypochlorite solutions, and aqueous sodium hydroxide solutions. From the viewpoint of safety, it is preferable to use aqueous sodium chloride solutions. When the washing solution flows into the carrier column 21, impurities adhering to the adsorbent in the carrier column 21 are removed. After removal with the washing solution, the carrier column 21 is equilibrated. Examples of the equilibration solution that can be used include physiological saline, phosphate buffer solution (PBS), Tris-HCl buffer solution, sodium citrate buffer solution, citrate phosphate buffer solution, acetate buffer solution, and borate buffer solution. The materials of the washing solution storage tank 51 and the equilibration solution storage tank 52 that store the washing solution and the equilibration solution are not particularly limited, and may have the same structure as the first tank 60 and the second tank 90 described above, for example. From the viewpoint of making it easy to visually check the amount of solution stored in the tanks, it is preferable that the washing solution storage tank 51 and the equilibration solution storage tank 52 be transparent.

[0092] The washing and equilibration of the carrier column 21 can be set arbitrarily depending on the amount of the raw material solution treated in the carrier column 21. In one embodiment, the washing and equilibration of the carrier column 21 may be performed when the amount of raw material solution treated by the carrier column 21 relative to the total mass of the raw material solution is 5 to 100 times the volume of the carrier column. For example, when treating 1,000 mL of raw material solution, it is preferable to perform the washing and equilibration treatment of the carrier column 21 every time 20 to 150 mL, preferably every 50 to 100 mL of raw material solution is treated in the carrier column 21.

[0093] (Impurity Removal Treatment) The pretreatment liquid supplied to the carrier column 21 has impurities adsorbed and removed by the adsorbent in the carrier column 21 to form a roughly purified liquid, which is then sent to the flow path 61B via the flow path 20B. In one embodiment, the impurity removal treatment by the carrier column 21 may be to reduce the concentration of impurities in the raw material liquid or the pretreatment liquid to 1,000 ppm or less, preferably to 500 ppm or less.

[0094] (Purification and concentration process) The partially purified liquid supplied to flow path 61B flows in inflow direction 62b and can be stored in first tank 60. The first stored liquid can be supplied to first flow path 10 via third flow path 62b depending on the progress of the purification and concentration process of the liquid in first flow path 10. The liquid supplied to first flow path 10 (first stored liquid) is purified and concentrated while circulating through circulation paths 30a and 30b.

[0095] By operating one or more pumps interposed in the first flow path 10, the liquid in the first flow path 10 can be circulated through the circulation path 30. In the device 1 of FIG. 9, when the first pump 31 and the second pump 32 are both tube pumps, to circulate the liquid through the circulation path 30a, the liquid sending directions of the two pumps are set 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 pump. In one embodiment, the main pump is preferably a pump located upstream of the liquid when the hollow fiber membrane module 33 is taken as the starting point. In the device 1 of FIG. 9, when circulating the liquid through the circulation path 30a, the output of the first pump 31 is preferably set higher than the output of the second pump 32. Furthermore, when circulating the liquid through the circulation path 30b, the output of the second pump 32 is preferably set higher than the output of the first pump 31. Furthermore, the difference in pump output in this case is preferably 2 to 15%, more preferably 4 to 20%. When the first pump 31 and the second pump 32 are tube pumps, the outputs of these pumps can be set by changing the discharge flow rate (mL / min) or the output (number of revolutions).

[0096] In the manufacturing method of the present disclosure, it is preferable to circulate the liquid in the first flow path 10 alternately through the circulation paths 30a and 30b. The timing of switching the circulation direction of the liquid may be controlled according to the purification and concentration time of the liquid, or may be controlled according to the flow rate of the liquid circulated in the circulation path 30. Specifically, it can be set appropriately depending on the fouling tendency of the liquid containing the useful micro substance to the hollow fiber membrane. In one embodiment, when the circulation direction is switched based on the purification and concentration time, it is preferably every 1 second to 10 minutes, and more preferably every 3 seconds to 5 minutes. Furthermore, when the circulation direction is switched according to the flow rate of the liquid (the accumulated flow rate since the circulation direction of the liquid is switched), it is preferably every 5 mL to 300 mL, and more preferably every 10 mL to 200 mL.

[0097] The liquid in the first flow path 10 can be purified and concentrated while being alternately circulated through the circulation path 30. The liquid that enters the hollow fiber membrane module 33 is purified and concentrated by alternating tangential flow filtration. The permeated liquid can be discharged from the third liquid inlets and outlets 33c and 33d of the hollow fiber membrane module 33 through the flow path 40 to the waste liquid tank 24.

[0098] In addition, the viscosity of the liquid containing the minute useful substance during operation is －１ When the liquid viscosity is 10 to 30 mPa·s, a hollow fiber membrane having a large diameter and an average inner diameter of 1.0 mm to 1.4 mm can be used.

[0099] In one embodiment, the liquid in the first flow path 10 may be circulated so 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, and more preferably 0.03 to 0.12 MPa. The pressure between the hollow fiber membranes is the differential pressure between the pressure applied to the inner surface and the pressure applied to the outer surface of the hollow fiber membrane.

[0100] (Backwashing Process) The manufacturing method of the present disclosure may include backwashing the hollow fiber membranes to recover minute useful substances deposited on the membrane surface of the hollow fiber membranes while performing alternating tangential flow filtration of the liquid in the hollow fiber membrane module 33. The backwashing process can be performed by supplying the diluted liquid in the second tank 90 to the third liquid inlet / outlet side of the hollow fiber membrane module 33 when the liquid volume in the first flow path 10 falls below a set value. For example, when using the apparatus 1 shown in FIG. 9 , the liquid volume in the first flow path 10 may be set to the liquid volume when the liquid level monitoring device 11 detects a change in transmittance. In this case, the first control unit can open the three-way valve 74 to the fourth flow path 71A side, thereby performing the backwashing process. Furthermore, the liquid delivery direction of the first pump 31 can be set counterclockwise and the liquid delivery direction of the second pump 32 can be set clockwise to create a negative pressure inside the hollow fiber membrane module 33. Furthermore, it is preferable to operate the first pump 31 and the second pump 32 with the same output (the difference in output between the first pump 31 and the second pump 32 is in the range of 0 to 4%). The outputs of the first pump 31 and the second pump 32 at this time can be set arbitrarily. From the viewpoint of energy conservation, the outputs of the first pump 31 and the second pump 32 may be 30% or less, or may be 20% or less. In one embodiment, the backwashing process can be performed appropriately even when the amount of liquid in the first flow path 10 falls below a set value. Examples of such timing include when there is concern about a decrease in the permeation rate due to pressure fluctuations or flow rate fluctuations, when a tendency for the permeation rate to decrease can be determined, or when safety operations are performed to prevent blockage of the hollow fiber membrane.

[0101] When performing backwashing, it is preferable to supply a diluent that is 2 to 200 times the liquid volume set by the liquid level monitoring device 11 from the hollow fiber membrane module 33 into the first flow path 10. At this time, the excess diluent flows in the inflow direction 62a while diluting the liquid in the first flow path 10 and is stored in the first tank 60. It is also preferable to manage the amount of diluent with a flow meter 41. In one embodiment, it is more preferable that the amount of diluent is 2 to 20 times the liquid volume. By performing such backwashing, it is possible to recover minute useful substances deposited on the membrane surface of the hollow fiber membrane while also diluting the concentrated liquid, which is preferable. Furthermore, in the initial stage of the purification and concentration treatment (e.g., the stage of purification and concentration treatment of a crudely purified solution), the amount of the dilution liquid may be kept at 2 to 10 times the aforementioned liquid volume, and in the intermediate or later stages of the purification and concentration treatment (e.g., the stage of repurifying and concentrating a solution that has been diluted one or more times), the amount of the dilution liquid may be increased to 2 to 15 times the amount of the dilution liquid supplied in the initial stage, thereby improving the efficiency of purification and concentration.

[0102] Before backwashing, a flushing treatment can be carried out in advance with a cleaning liquid (diluted liquid) to prevent the permeated liquid remaining in the hollow fiber membrane module from mixing with the backwashing liquid and flowing into the concentrated liquid side, and the permeated liquid remaining in the hollow fiber membrane module can be replaced with the cleaning liquid (diluted liquid).

[0103] In this case, the amount of the washing liquid (diluted liquid) used is preferably 1 to 8 times, more preferably 2 to 6 times, the volume of the permeated liquid remaining in the hollow fiber membrane module.

[0104] When concentrating and purifying certain extracellular vesicles from certain culture supernatants, performing a flushing operation before backwashing can sometimes reduce the amount of remaining impurity proteins to less than half of that when no flushing operation is performed.

[0105] When the hollow fiber membrane module 33 is an internal pressure type, the permeate remaining in the hollow fiber membrane module can be replaced with a cleaning liquid (diluted liquid) using either the third liquid inlet / outlet 33c or 33d in FIG. 2 as an inlet and the other as an outlet. Because the hollow fiber membranes have a resistance to liquid flow, supplying the diluted liquid through either the third liquid inlet / outlet 33c or 33d allows the permeate remaining in the hollow fiber membrane module 33 to be replaced with the diluted liquid with minimal leakage into the membrane. For example, stopping the flow of the first pump 31 and the second pump 32 stops the flow of the first liquid through the inlet / outlet 33a and the second liquid through the inlet / outlet 33b. In this state, supplying the diluted liquid through either the third liquid inlet / outlet 33c or 33d using a pump or the like (not shown) allows the diluted liquid to be discharged from the other third liquid inlet / outlet 33c or 33d. This allows the permeate remaining in the hollow fiber membrane module 33 to be replaced with the diluted liquid. Furthermore, with the first pump 31 and the second pump 32 stopped from passing liquid, a valve (not shown) is closed to stop the flow of the liquid through the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b. In this state, by supplying a diluent through one of the third liquid inlets / outlets 33c and 33d using a pump (not shown) or the like, the diluent is discharged from the other third liquid inlet / outlet 33c or 33d. This allows the permeated liquid remaining in the hollow fiber membrane module 33 to be replaced with the diluent.

[0106] The timing of the flushing process is controlled by the first control unit. That is, based on the output of the liquid level position monitoring device, the first control unit can supply the diluent in the second tank 90 from one of the at least two third liquid inlets 33c and 33d of the hollow fiber membrane module 33 while stopping the flow of liquid through the first pump 31 and the second pump 32 and closing valves (not shown) to stop the flow of the liquid through the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b. At this time, the on-off valve 73 provided in the flow path 40 connected to the waste liquid tank is open. Then, the valves are opened to allow flow through the first liquid inlet / outlet 33a and the second liquid inlet / outlet 33b, and the diluent in the second tank 90 can be supplied from one of the at least two third liquid inlets / outlets 33c and 33d of the hollow fiber membrane module 33 using the first pump 31 and the second pump 32 or another pump (not shown), etc., to perform backwashing. At this time, the on-off valve 73 provided in the flow path 40 connected to the waste liquid tank is closed.

[0107] (Dilution Treatment) In the manufacturing method of the present disclosure, the purified and concentrated concentrate can be further purified and concentrated while being diluted. In one embodiment, the dilution ratio of the concentrate is preferably 2 to 200 times, more preferably 2 to 20 times. The dilution treatment can be performed when the amount of liquid (concentrated liquid) in the first flow path 10 falls below a set value. When using the apparatus 1 of FIG. 9, the liquid volume can be set to the amount when the liquid level position monitoring device 11 detects a change in transmittance, as in the backwashing treatment described above. When performing the dilution treatment, the first control unit can also open the three-way valve 74 to the fifth flow path 71B side. Furthermore, the first control unit preferably stops the first pump 31 and the second pump 32 when performing the dilution treatment. In one embodiment, the purification and concentration treatment can be performed in parallel with the dilution treatment. When performing the purification and concentration treatment in parallel with the dilution treatment, the fifth flow path 71B is preferably connected to the top of the first tank 60. Furthermore, the diluted liquid can be delivered to the first tank 60 using a tube pump, high-pressure gas, potential energy (hydraulic head difference), or other means. Furthermore, when the first pump 31 and the second pump 32 are stopped during the dilution process, the dilution liquid supplied to the fifth flow path 71B is supplied from the flow path 61B into the first flow path 10, and then flows in the inflow direction 62a and is supplied to the first tank 60.

[0108] In the manufacturing method of the present disclosure, it is preferable to remove impurities from the raw material liquid in the carrier column 21 and purify and concentrate the liquid in the hollow fiber membrane module 33 in parallel. In this case, a liquid in which useful micro substances have been purified and concentrated can be produced more efficiently. In the apparatus 1 of Figure 9, the roughly purified liquid from which impurities have been removed in the carrier column 21 flows in the inflow direction 62a and is stored in the first tank 60. As the purification and concentration process progresses, the first stored liquid in the first tank 60 can be supplied to the first flow path 10 via the third flow path 62b.

[0109] (Recovery Process) The manufacturing method of the present disclosure may include recovering the entire amount of concentrated liquid in the first flow path 10. The entire amount of concentrated liquid in the first flow path 10 is recovered into the third tank 80 via the flow paths 81A and 81B by opening the on-off valve 76 and closing the on-off valve 75 at any time while feeding clean gas from the air vent using the first pump 31 and the second pump 32. In the apparatus 1 of FIG. 9 , the second control unit opens the on-off valve 76 and also opens the air vent valve 64 and the on-off valve 78. The air vent valve 64 is connected to the flow path 61B via the air vent line 65. The air vent line 65 is also provided with the on-off valve 78. The second control unit can then recover the concentrated liquid by closing the on-off valve 75 at any time. Furthermore, the first pump 31 and the second pump 32 can be operated so that the concentrated liquid in the first flow path 10 circulates through the circulation path 30a. That is, the first pump 31 and the second pump 32 can be operated to send liquid in the same clockwise direction. The outputs of the first pump 31 and the second pump 32 at this time can be set as desired. In one embodiment, from the viewpoint of energy conservation, the outputs of the first pump 31 and the second pump 32 are preferably 50% or less, and more preferably 30% or less. Furthermore, it is preferable that the difference in output between the first pump 31 and the second pump 32 is approximately the same (for example, in the range of 0 to 4%). The operation of recovering the concentrated liquid in the first flow path 10 can also be performed manually by a person.

[0110] (Washing-out Operation of Useful Micro Substances Deposited on the Hollow Fiber Membrane Surface Immediately Before the Recovery Process) The manufacturing method of the present disclosure may include a washing-out operation of useful micro substances deposited on the hollow fiber membrane surface immediately before the recovery process. For example, when concentrating and purifying certain extracellular vesicles from certain culture supernatants, the recovery rate may be low because the extracellular vesicles that are relatively firmly deposited on the hollow fiber membrane surface are not dispersed in the concentrate. In particular, when the amount of concentrate in the first flow path 10 is small, the efficiency of purification and concentration decreases if the extracellular vesicles that are relatively firmly deposited on the hollow fiber membrane surface cannot be recovered. Therefore, an operation step of redispersing the extracellular vesicles deposited on the hollow fiber membrane surface into the concentrate using a small amount of backwashing liquid (dilution liquid) can be performed immediately before the recovery process. In this case, it is preferable to perform the above-mentioned flushing operation sufficiently and use a small amount of backwashing liquid (dilution liquid). For example, the volume of backwashing liquid (dilution liquid) used is preferably half to twice the volume of the first flow path. The liquid passing rate at this time can be set to be the same as the liquid passing rate in the purification and concentration step.

[0111] Example 1: Here, we investigated a method for further improving the recovery rate of useful micromaterials by coating the surface of hollow fiber membranes with biomolecules. 790 mL of mesenchymal stem cell culture supernatant with a protein concentration of 0.49 mg / mL and an exosome concentration of 166.4 pg / mL was used as the raw material solution. The exosome concentration was quantitatively analyzed using a CD9 / CD63 ELISA (EXH0102EL, Cosmo Bio) and corresponds to the amount of CD9 / CD63 fusion standard protein used to create the calibration curve. 79 mL of carrier (a porous granular material with positively charged pores and capable of adsorbing and retaining negatively charged waste materials excluding extracellular vesicles) equilibrated with 50 mM Tris-HCl (pH 7.2) solution was added to 790 mL of culture supernatant, and the mixture was mixed by inversion at room temperature for 10 minutes using a rotator. Thereafter, the carrier was removed from the carrier-cell supernatant mixture using a 0.22 μm filtration unit (S2GPU11RE, Merck Millipore) to obtain a carrier-treated culture supernatant.

[0112] Next, the carrier-treated culture supernatant was purified and concentrated by filtration using an alternating tangential flow system at a filtration pressure of 30 to 40 kPa. The filtration membrane used was a hollow fiber membrane module made of cellulose acetate (molecular weight cutoff: 300,000, membrane area: 0.062 m). ２ ) was used. The filtration membrane used had its surface coated with biomolecules. The coating treatment was performed by passing a 1.3 mg / mL solution of skim milk dissolved in PBS through the filtration membrane at a flow rate of 200 mL / min for 10 minutes. This resulted in a coated membrane in which certain biomolecules in the skim milk were adsorbed and bound to the membrane surface as a thin layer. The filtration membrane was then washed with 250 mL of PBS to remove excess skim milk that had simply accumulated on the thin layer.

[0113] Here, the binding state between the filtration membrane and certain biomolecules in skim milk is strong enough that they do not come off under washing and operating conditions, and furthermore, there is almost no decrease in filtration performance even after long-term operation.

[0114] Alternating tangential flow filtration was repeated until the volume of the concentrate reached 10 mL, after which the concentrate was diluted by supplying 90 mL of PBS to the hollow fiber membrane module. This concentration, filtration, and dilution process was repeated two more times, and then 11.9 mL of purified concentrate (extracellular vesicle purified product) was obtained by alternating tangential flow filtration. The extracellular vesicles in the final exosome concentrate were quantitatively analyzed using the CD9 / CD63 ELISA method (EXH0102EL, Cosmo Bio), and the CD9 / CD63 fusion standard protein used to create the calibration curve was found to be 3,746.7 pg / mL, with a recovery rate of 33.9%.

[0115] Comparative Example 1 When the same procedure was carried out using a hollow fiber membrane that had not been coated with skim milk, the recovery rate was 7.4%.

[0116] The results of Example 1 and Comparative Example 1 showed that the recovery rate of minute useful substances can be improved by using a hollow fiber membrane coated with a biomolecule (i.e., a hollow fiber membrane having a type of biomolecule different from the minute useful substance attached to at least a portion of the membrane).

[0117] Example 2 Here, a method for further improving the purity of useful minute substances in the obtained purified concentrate was investigated.

[0118] As a raw material solution, 40 mL of culture supernatant of amniotic membrane-derived mesenchymal stem cells with a protein concentration of 1.49 mg / mL and an exosome concentration of 196 pg / mL was used. The exosomes in the culture supernatant were separated into 33 hollow fiber membrane modules made of cellulose acetate (molecular weight cutoff: 300,000, membrane area: 0.01 m). ２ Using a purification and concentration device equipped with a permeate volume (i.e., 10 mL of hollow fiber extracapillary space (ECS)), separation, purification, and concentration were performed using an alternating tangential flow method with a filtration pressure of 30 to 40 kPa. After concentrating the culture supernatant to approximately 10 mL, 40 mL of diluent (PBS) was injected through the third liquid inlet / outlet 33c of the hollow fiber membrane module and discharged through the third liquid inlet / outlet 33d. This flushed out impurities in the permeate remaining in the ECS with PBS. Then, 90 mL of the same diluent (PBS) was forced from the permeate side to the concentration side of the hollow fiber membrane module 33 to perform backpressure washing (backwashing). The separation and purification operation was performed again under the same conditions, and when the culture supernatant reached approximately 10 mL, the same flushing and backwashing operation was performed two more times. After three backwashings, the culture supernatant was concentrated to 5.5 mL. The amount of remaining protein in the purified concentrate was measured by BCA assay, and the protein concentration was found to be 0.16 mg / mL. The results are shown in Figure 14. The absorbance of the purified concentrate was also measured using an Infinite M Nano+ absorbance microplate reader manufactured by TECAN. The results are shown in Figure 15.

[0119] Comparative Example 2: In the same separation and purification procedure as in Example 2, the culture supernatant was concentrated to 5.2 mL without flushing the ECS with PBS. The remaining protein amount in the resulting purified concentrate was measured by BCA assay, and the protein concentration was found to be 0.36 mg / mL. The results are shown in Figure 14. The absorbance of the resulting purified concentrate was also measured in the same manner as in Example 2. The results are shown in Figure 15.

[0120] In Figures 14 and 15, "Before" refers to the sample before the purification and concentration process, "with ECF" refers to the purified concentrate obtained by the method of Example 2, and "without ECS" refers to the purified concentrate obtained by the method of Comparative Example 2. As is clear from Figure 14, the method of Example 2 further reduces the concentration of protein impurities, thereby further improving the purity of exosomes. In Figure 15, the peak at a wavelength of approximately 557 nm is a peak derived from phenol red, which is contained in the culture medium as a pH indicator and corresponds to an impurity in the purified concentrate. As shown in Figure 15, the method of Example 2 further reduces the concentration of phenol red as an impurity, thereby further improving the purity of exosomes.

[0121] These results indicate that flushing the ECS with a diluent such as PBS or saline to remove impurities remaining in the ECS before the backwashing process of the hollow fiber membrane module can further reduce the concentration of impurities in the resulting purified concentrate, thereby further improving the purity of the useful micro-substances. A non-limiting reason for this is thought to be that flushing prevents impurities in the ECS from being reconstituted in the purified concentrate during the subsequent backwashing process with the diluent.

[0122] [Example 3] As a raw material solution, 200 mL of culture supernatant of amniotic membrane-derived mesenchymal stem cells with a protein concentration of 1.25 mg / mL and an exosome concentration of 138 pg / mL was used. The exosomes in the culture supernatant were separated into 200 mL of a cellulose acetate hollow fiber membrane module (molecular weight cutoff: 300,000, membrane area: 0.01 m). ２Separation, purification, and concentration were performed using an alternating tangential flow method using a purification and concentration device equipped with an ECS (10 mL). After concentrating the culture supernatant to approximately 17 mL, 40 mL of diluent (PBS) was injected through the third liquid inlet / outlet 33c of the hollow fiber membrane module and discharged through the third liquid inlet / outlet 33d. This flushed impurities in the permeate remaining in the ECS with PBS. Then, 160 mL of the same diluent (PBS) was pressure-fed from the permeation side to the concentration side of the hollow fiber membrane module 33 to perform backpressure washing (backwashing). The separation and purification operation was performed again under the same conditions, and when the culture supernatant reached approximately 17 mL, the same flushing and backwashing operation was performed two more times. The culture supernatant after backwashing was concentrated to 50 mL. Backwashing was performed again to wash out the micro-useful substances adhering to the hollow fiber membrane surface into the purified concentrate, and 50 mL of purified concentrate was recovered. The extracellular vesicles in the purified concentrate were quantitatively analyzed using the CD9 / CD63 ELISA method (EXH0102EL, Cosmo Bio). The concentration of the CD9 / CD63 fusion standard protein used to create the calibration curve was 486 pg / mL, resulting in a recovery rate of 88%. These results demonstrate that the recovery rate of useful micro-substances can be increased to 80% or higher by further supplying a diluent to the hollow fiber membrane module immediately before collecting the purified concentrate and washing out the useful micro-substances.

[0123] DESCRIPTION OF SYMBOLS 1 Purification and concentration apparatus, 10 First flow path, 11 Liquid level position monitoring device, 20 (20A to 20E) Second flow path, 21 Carrier column, 22 Filtration 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-on type flow meter), 50 Raw material storage section, 51 Cleaning liquid storage tank, 52 Equilibration liquid storage tank, 60 First tank, 61A, 61B Flow path, 62a, 62b Inflow direction, 63 Integrating flow meter (clamp-on type integrating flow meter), 64 Air vent valve, 65 Air vent line, 80 Third tank, 90 Second tank, 100 Membrane treatment cartridge, 200 Pretreatment cartridge, 300 Housing, 301 Frame, 400A, 400B Pretreatment and membrane treatment cartridges, 101, 201, 401 cases

Claims

1. a hollow fiber membrane module in which a plurality of hollow fiber membranes are accommodated within a container, the container having a first liquid inlet / outlet and a second liquid inlet / outlet for supplying a liquid containing a micro useful substance to the hollow fiber membranes, and a third liquid inlet / outlet for discharging the liquid that has permeated the hollow fiber membranes; a first flow path that is connected to an inlet / outlet for the first liquid and an inlet / outlet for 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 disposed in the first flow path for reversibly circulating the liquid.

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

3. The purification and concentration apparatus according to claim 1 or 2, wherein the first flow path has a capacity of 5 to 100 mL.

4. The purification and concentration apparatus according to claim 1 or 2, wherein the pump comprises at least a first pump arranged on an inlet / outlet side of the first liquid in the first flow path and a second pump arranged on an inlet / outlet side of the second liquid.

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

6. The purification and concentration apparatus according to claim 1 , wherein the first flow path is configured to be detachable.

7. 3. The purification and concentration apparatus according to claim 1, wherein the second flow path is connected to a raw material reservoir that stores a raw material liquid of the liquid containing the useful micro substance.

8. 8. The purification and concentration apparatus according to claim 7, further comprising a carrier column disposed in the second flow path for removing impurities from the liquid supplied from the raw material reservoir.

9. The purification and concentration apparatus according to claim 7 , further comprising a filtering member disposed in the second flow path for removing impurities from the liquid supplied from the raw material reservoir.

10. a first tank configured to store the liquid after impurities in the liquid have been removed and the liquid in the first flow path; The purification and concentration apparatus according to claim 8 , wherein 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 toward the first flow path.

11. The purification and concentration apparatus according to claim 10 , further comprising a second tank configured to store a dilute solution and to which a fourth flow path is connected for supplying the dilute solution to the hollow fiber membrane module.

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

13. The purification and concentration apparatus according to claim 12 , wherein all of the first flow path to the fifth flow path are configured to be detachable.

14. a liquid level monitoring device including an optical sensor that irradiates light toward the third flow path and detects the transmittance of the light; The purification and concentration apparatus according to claim 11 , further comprising: a first control unit that controls a timing for supplying the dilution liquid in the second tank to the hollow fiber membrane module based on an output of the liquid level monitoring device.

15. The purification and concentration apparatus according to claim 14 , wherein the first control unit controls a timing at which the dilution liquid in the second tank is supplied from the inlet / outlet for the third liquid of the hollow fiber membrane module.

16. The purification and concentration apparatus according to claim 1 or 2, further comprising a third tank connected to the first flow path and configured to recover the liquid in the first flow path.

17. 3. The purification and concentration apparatus according to claim 1, wherein one or more elements of the first flow path, the second flow path, and the hollow fiber membrane module are disposed within a housing.

18. a management unit for managing a cleanliness level inside the housing, The purification and concentration apparatus according to claim 17 , wherein the management unit includes a fan filter device that supplies filtered air.

19. The purification and concentration apparatus according to claim 18 , wherein the management unit comprises a UV lamp.

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

21. 3. The purification and concentration apparatus according to claim 1, wherein at least the hollow fiber membrane module and the first flow path are housed in a detachable cartridge case.

22. 9. The purification and concentration apparatus according to claim 8, wherein at least the second flow path and the carrier column are housed in a removable cartridge case.

23. The purification and concentration apparatus according to claim 22 , wherein the hollow fiber membrane module and the first flow path are housed in the case.

24. The hollow fiber membrane module has at least two inlets and outlets for the third liquid, 16. The purification and concentration apparatus according to claim 15, wherein the first control unit supplies the diluting liquid from one of the at least two inlets and outlets for a third liquid while stopping the flow of the liquid through the inlet and outlet for the first liquid and the inlet and outlet for the second liquid of the hollow fiber membrane module, and then supplies the diluting liquid from one of the at least two inlets and outlets for a third liquid while opening the flow of the liquid through the inlet and outlet for the first liquid and the inlet and outlet for the second liquid of the hollow fiber membrane module.

25. A method for producing a purified and concentrated solution containing minute useful substances using the purification and concentration apparatus according to claim 1 or 2, comprising the steps of: Supplying a raw material liquid containing minute useful substances to the purification and concentration device; purifying and concentrating the liquid in the hollow fiber membrane module; The method for producing a purified and concentrated liquid containing useful micro substances comprises purifying and concentrating the liquid while reversibly circulating the liquid through the circulation path including the hollow fiber membrane module.