Method for producing concentrated liquid containing minute useful substance

Abstract

Provided is a method for producing a concentrated liquid of a minute useful substance.　The method for producing a concentrated liquid containing a minute useful substance involves: adding an oxyethylene adduct of a sorbitan unsaturated fatty acid ester to a primary liquid raw material containing a minute useful substance to obtain a secondary liquid raw material; and applying the secondary liquid raw material to a hollow fiber membrane to concentrate the secondary liquid raw material.

Description

Method for producing a concentrated solution containing minute useful substances

[0001] The present disclosure relates to a method for producing a concentrate containing small useful substances.

[0002] As a method for concentrating useful minute substances such as extracellular vesicles, 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 useful minute substance may be selected from the group consisting of proteins, viruses, exosomes, and nucleic acids.

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

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

[0006] Generally, the concentration of useful micro substances in liquid raw materials is not high in most cases, so there is a demand for a liquid containing useful micro substances at a high concentration (a concentrated liquid of useful micro substances).

[0007] The present disclosure has been made in view of the above circumstances, and aims to provide a method for effectively producing a concentrated solution of minute useful substances.

[0008] In order to solve the above problems, the present disclosure can employ the following means: [1] A method for producing a concentrated liquid containing useful micro substances, the method comprising: adding an oxyethylene adduct of a sorbitan unsaturated fatty acid ester to a primary liquid feedstock containing useful micro substances to obtain a secondary liquid feedstock; applying the secondary liquid feedstock to a hollow fiber membrane; and concentrating the secondary liquid feedstock. [2] A method for concentrating a liquid feedstock containing useful micro substances, the method comprising: adding an oxyethylene adduct of a sorbitan unsaturated fatty acid ester to a primary liquid feedstock containing useful micro substances to obtain a secondary liquid feedstock; and applying the secondary liquid feedstock to a hollow fiber membrane.

[0009] According to the present disclosure, a method for effectively producing a concentrated solution containing minute useful substances can be provided.

[0010] FIG. 1 is a schematic diagram illustrating an example of an apparatus used in the manufacturing method of the present disclosure.

[0011] An example of the present disclosure will be described below using the drawings. However, each configuration and combination thereof in each embodiment is merely an example, 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. Each aspect disclosed in this specification can be combined with any other feature disclosed in this specification. Furthermore, when multiple upper and lower limits are described for a specific parameter, any of these upper and lower limits can be combined to form a suitable numerical range. Furthermore, the lower and / or upper limits of a numerical range described in this disclosure are numerical values ​​within that numerical range and may be replaced with numerical values ​​shown in the examples. In this specification, the expression "X to Y" indicating a numerical range means "greater than or equal to X and less than or equal to Y."

[0012] [Method for Producing a Concentrated Liquid Containing a Micro-Useful Substance] A method for producing a concentrated liquid containing a micro-useful substance according to one embodiment of the present disclosure includes adding an oxyethylene adduct of a sorbitan unsaturated fatty acid ester to a primary liquid feedstock containing the micro-useful substance to obtain a secondary liquid feedstock, and applying the secondary liquid feedstock to a hollow fiber membrane to concentrate the secondary liquid feedstock. By concentrating the secondary liquid feedstock, in which an oxyethylene adduct of a sorbitan unsaturated fatty acid ester is added to the primary liquid feedstock containing the micro-useful substance, using a hollow fiber membrane, the recovery efficiency (concentration) of the micro-useful substance is likely to be improved. This allows for the effective production of a concentrated liquid containing the micro-useful substance. While the mechanism behind this is unclear at this stage, one possible non-limiting mechanism is that the micro-useful substance contributes to structural stabilization in the liquid. A concentrated liquid containing a micro-useful substance refers to a liquid in which the concentration of the micro-useful substance is higher than that of the liquid feedstock. The concentration of the useful micro substance in the concentrated solution depends on the concentration of the useful micro substance in the liquid raw material, but is preferably 0.1 to 500 ng / ml, more preferably 0.5 to 200 ng / ml, and even more preferably 1 to 100 ng / ml. Each term is explained in detail below. Details of each step will be described later.

[0013] (Micro-useful substance) Micro-useful substance refers to a micro-substance including nucleic acids (microRNA, messenger RNA, DNA, etc.), proteins, lipids, and / or various metabolic products. The diameter of the micro-useful substance is preferably 10 to 1000 nm, more preferably 15 to 500 nm, and even more preferably 20 to 200 nm. In one embodiment, the micro-useful substance is preferably a particle covered with a lipid bilayer, and examples of particles covered with a lipid bilayer include extracellular vesicles. Extracellular vesicles are a collective term for particles without a nucleus that are covered with a lipid bilayer and are released outside of cells, and include nucleic acids, proteins, lipids, metabolic products, etc., and include exosomes, microvesicles, and apoptotic vesicles.

[0014] (Primary Liquid Source) The primary liquid source is not particularly limited as long as it contains a micro-substance, and examples thereof include a cell culture medium or dispersion. The concentration of the micro-substance in the primary liquid source is not particularly limited. The culture medium is preferably a cell culture supernatant, and more preferably a mesenchymal stem cell culture supernatant. Mesenchymal stem cells containing extracellular vesicles can be derived from various sources, such as bone marrow, blood, fat, umbilical cord, umbilical cord blood, periosteum, perichondrium, and other somatic tissues. Culturing methods are described, for example, in JP 2011-67175 A and JP 2003-52360 A.

[0015] (Secondary Liquid Raw Material) The secondary liquid raw material contains a useful micro substance and an oxyethylene adduct of a sorbitan unsaturated fatty acid ester. The concentration of the useful micro substance in the secondary liquid raw material is not particularly limited. In one embodiment, the secondary liquid raw material may contain pure water; filtered water such as tap water, groundwater, or river water; various aqueous solutions, etc. for dilution. 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 Tris-HCl buffer (TBS), phosphate buffer solution (PBS), sodium citrate buffer solution, citrate-phosphate buffer solution, acetate buffer solution, borate buffer solution, etc.

[0016] (Oxyethylene Adducts of Sorbitan Unsaturated Fatty Acid Esters) Examples of oxyethylene adducts of sorbitan unsaturated fatty acid esters include oxyethylene adducts of esters of sorbitan and monounsaturated or polyunsaturated fatty acids. Examples of monounsaturated or polyunsaturated fatty acids include unsaturated fatty acids having 10 to 30 carbon atoms (preferably 12 to 28, more preferably 13 to 27, even more preferably 15 to 25, and particularly preferably 18 to 22). Specific examples of oxyethylene adducts of sorbitan unsaturated fatty acid esters include polyoxyethylene sorbitan oleate, polyoxyethylene sorbitan linolenate, polyoxyethylene sorbitan arachidonate, polyoxyethylene sorbitan eicosapentaenoate, and polyoxyethylene sorbitan docosahexaenoate. Of these, oxyethylene adducts of esters of monounsaturated fatty acids and sorbitan are preferred, and polyoxyethylene sorbitan oleate is more preferred. The concentration of the oxyethylene adduct of sorbitan unsaturated fatty acid ester in the secondary liquid raw material is preferably 1 to 1000 ppm, more preferably 10 to 500 ppm, and even more preferably 50 to 250 ppm.

[0017] (Hollow fiber membrane) The material of the hollow fiber membrane can be appropriately designed depending on the purpose of use of the present disclosure and the properties of the liquid containing the useful micro-substance. Specifically, organic polymer materials such as polyvinyl fluoride (PVDF), polyethersulfone (PES), polysulfone (PSf), polyolefins (PE, PP, PTFE, etc.), polyamide, polyacrylonitrile, cellulose acetate (CA), and their modified forms can be applied. Among 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). By using a hollow fiber membrane made of CA (hereinafter sometimes referred to as "CA membrane"), it is possible to suppress the decrease in filtration rate during concentration treatment and further improve the recovery effect of the useful micro-substance 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.

[0018] The average inner diameter of the hollow fiber membrane can be designed as needed depending on the intended use of the present disclosure, the properties of the liquid containing the useful micro-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 cut surface, and the average value is defined as the "average inner diameter."

[0019] In one embodiment, the hollow fiber membrane 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."

[0020] In the intended use of this embodiment, 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.

[0021] The molecular weight cutoff of the hollow fiber membrane can be designed as appropriate depending on the intended use, the properties of the liquid raw material 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. It is preferable to use a hollow fiber membrane with a molecular weight cutoff slightly lower than that of the minute useful substance (for example, a hollow fiber membrane having a molecular weight cutoff approximately 10 to 50% lower than the molecular weight of the minute useful substance). 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. The molecular weight cutoff can be evaluated by the gamma globulin permeability (%) ((gamma globulin concentration in permeate / gamma globulin concentration in solution (100 mg / L) × 100) when a 100 mg / L solution of gamma globulin (bovine serum gamma globulin manufactured by SIGMA, molecular weight 150,000) in a phosphate buffer solution is cross-flow permeated through the hollow fiber membrane 30 at a filtration pressure of 0.1 MPa (membrane surface velocity: 0.2 m / s). The hollow fiber membrane 30 preferably has a gamma globulin permeability of 5% to 95%, more preferably 10 to 80%, and even more preferably 10 to 70%. Examples of the hollow fiber membrane 30 that can be used include FUS5082 (polyethersulfone membrane; molecular weight cutoff 500,000, gamma globulin permeability 70%) manufactured by Daisen Membrane Systems Co., Ltd. and FUC1582 (cellulose acetate membrane; molecular weight cutoff 150,000, gamma globulin permeability 10%) manufactured by Daisen Membrane Systems Co., Ltd.

[0022] (Hollow Fiber Membrane Module) In one embodiment, the hollow fiber membrane may be a hollow fiber membrane module in which a plurality of hollow fiber membranes are housed in a case housing having a plurality of liquid inlets and outlets.

[0023] The number of hollow fiber membranes used in the hollow fiber membrane module is not particularly limited. The number of membranes to be incorporated into the module can be designed appropriately depending on the intended use of the present disclosure, the weight of the useful minute substance to be concentrated, etc., so as to obtain the required effective membrane area.

[0024] 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 concentrated in 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.

[0025] 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 concentration efficiency.

[0026] The packing 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 packing 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 packing rate is 5 to 55%, useful minute substances deposited on the membrane surfaces of the hollow fiber membranes can be more efficiently recovered while optimizing the size of the hollow fiber membrane module.

[0027] Each step in the manufacturing method of this embodiment will be briefly described below: (Step of Preparing Primary Liquid Source) First, a primary liquid source containing minute useful substances is prepared.

[0028] (Secondary Liquid Feedstock Preparation Step) In the secondary liquid feedstock preparation step, an oxyethylene adduct of a sorbitan unsaturated fatty acid ester is added to a primary liquid feedstock containing useful micro-substances. The timing of adding the oxyethylene adduct of a sorbitan unsaturated fatty acid ester is not particularly limited. The secondary liquid feedstock may be obtained by adding the oxyethylene adduct of a sorbitan unsaturated fatty acid ester to the primary liquid feedstock before the liquid feedstock is fed into the hollow fiber membrane, or by adding the oxyethylene adduct of a sorbitan unsaturated fatty acid ester so that the primary liquid feedstock and the oxyethylene adduct of a sorbitan unsaturated fatty acid ester are mixed within the hollow fiber membrane. In the latter case, for example, the oxyethylene adduct of a sorbitan unsaturated fatty acid ester may be added through an inlet separate from the inlet for the primary liquid feedstock to the hollow fiber membrane, and the primary liquid feedstock and the oxyethylene adduct of a sorbitan unsaturated fatty acid ester are mixed within the hollow fiber membrane to obtain the secondary liquid feedstock. Furthermore, in the latter case, the timing of starting to feed the primary liquid feedstock to the hollow fiber membrane and the timing of adding the oxyethylene adduct of a sorbitan unsaturated fatty acid ester may be the same or different. The addition method is not limited, and may be, for example, addition to the primary liquid raw material while stirring at room temperature (e.g., 25° C.) The amount of the oxyethylene adduct of sorbitan unsaturated fatty acid ester added to the primary liquid raw material can be appropriately adjusted so that the concentration of the oxyethylene adduct of sorbitan unsaturated fatty acid ester in the secondary liquid raw material is 1 to 1,000 ppm, 10 to 500 ppm, or 50 to 250 ppm.

[0029] (Secondary Liquid Feedstock Concentration Process) In the secondary liquid feedstock concentration process, the secondary liquid feedstock is applied to a hollow fiber membrane to concentrate the secondary liquid feedstock. Applying the secondary liquid feedstock to a hollow fiber membrane means passing the secondary liquid feedstock through at least a portion of the inside of the hollow fiber membrane, and in one embodiment, means sending the liquid from one end to the other end. Concentrating means increasing the concentration of a specific substance in the target liquid. In the secondary liquid feedstock concentration process, it means increasing the concentration of a useful micro substance in the secondary liquid feedstock. Here, when the secondary liquid feedstock is applied to a hollow fiber membrane, the liquid that permeates the hollow fiber membrane is referred to as the "permeate," and the liquid that does not permeate the hollow fiber membrane and remains in the hollow fiber membrane is referred to as the "concentrate."

[0030] The step of concentrating the secondary liquid raw material according to one embodiment of the present disclosure will be described below with reference to FIG. 1. The concentrating apparatus shown in FIG. 1 includes a first tank 10, a second tank 20, and a hollow fiber membrane 30. In this example, a concentrated liquid recovery tank 35 for recovering a concentrated liquid (final concentrated liquid) is connected to the hollow fiber membrane 30 via a three-way stopcock 40. (First filtration step) First, a previously prepared secondary liquid raw material is placed in the first tank 10. A pump (not shown) or the like is operated (for example, a liquid transfer pump (not shown) is used) or a gas (nitrogen (N ２ ) gas, etc.) into the first tank 10), the secondary liquid raw material stored in the first tank 10 is forced into the inside of the hollow fiber membrane 30. At this time, the useful micro-substances are filtered without passing through the hollow fiber membrane 30, and the secondary liquid raw material is concentrated. In this manner, the first filtration step can be performed. In this embodiment, the process of filtering the liquid in the first tank 10 through the hollow fiber membrane 30 and sending it to the second tank 20 is all referred to as the first filtration step.

[0031] The gas used for pressurization may be selected from the group consisting of inert gases such as nitrogen gas, argon, and helium, carbon dioxide, and clean air filtered through a HEPA filter, etc. The permeated liquid that has permeated the hollow fiber membrane 30 is discharged to the outside of the system, and the concentrated liquid (first concentrated liquid) is sent to the second tank 20. When the first concentrated liquid is contained in the second tank 20, a space where no first concentrated liquid exists remains above the second tank 20.

[0032] 1 , the hollow fiber membrane 30 is arranged so as to connect the liquid inlet / outlet of the first tank 10 with the liquid inlet / outlet at the connection part of the second tank 20. The hollow fiber membrane 30 can be connected to the liquid inlet / outlet of the first tank 10, for example, by fitting the open end of the hollow fiber membrane 30 into a thin tube such as a syringe needle fixed to the liquid inlet / outlet side of the first tank 10. The hollow fiber membrane 30 can also be connected to the liquid inlet / outlet of the second tank 20 in a similar manner.

[0033] Although FIG. 1 shows one hollow fiber membrane 30, multiple hollow fiber membranes may be used, for example, as a hollow fiber membrane bundle consisting of 2 to 150 membranes. Alternatively, a hollow fiber membrane module may be used in which multiple hollow fiber membranes 30 (hollow fiber membrane bundle) are housed in a case housing having multiple liquid inlets and outlets. When used as a hollow fiber membrane bundle, one or both ends can be joined together with an adhesive. When using the hollow fiber membrane module, the multiple liquid inlets and outlets of the hollow fiber membrane module can be connected to the liquid inlets and outlets of the first tank 10 and the second tank 20, and the liquid permeation outlet of the hollow fiber membrane module can be connected to a permeate tank.

[0034] In the first filtration step, filtration is preferably performed at a membrane surface velocity in the range of 0.3 m / sec to 4.0 m / sec, and more preferably at a membrane surface velocity in the range of 0.5 m / sec to 3.5 m / sec. If the membrane surface velocity is below 0.3 m / sec, the purification efficiency decreases. Conversely, if it exceeds 4 m / sec, the pressure level required to increase the membrane surface velocity becomes too high, and the shear force applied to the useful micro-substance during filtration becomes too high, which may result in deterioration of the useful micro-substance. To maintain the membrane surface velocity within the above range, the inlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet 10a side of the first tank 10) is preferably adjusted to 0.01 MPa to 0.2 MPa, more preferably adjusted to 0.015 MPa to 0.15 MPa, and even more preferably adjusted to 0.02 MPa to 0.12 MPa.

[0035] (Second Filtration Step) In the second filtration step, a pump (not shown) or the like is operated to supply gas (nitrogen gas in this example) from a gas supply source (not shown) into the second tank 20, pressurizing the liquid (first concentrated liquid) in the second tank 20 and passing it through the inside of the hollow fiber membrane 30 for filtration. In this embodiment, the entire process of filtering the liquid in the second tank 20 through the hollow fiber membrane 30 and sending it to the first tank 10 is referred to as the second filtration step. The filtered permeate is stored in the permeate tank, and the concentrated liquid (second concentrated liquid) containing the useful micro-substances is sent to the first tank 10.

[0036] Although the first tank 10 and the second tank 20 are cylindrical in shape in FIG. 1 , this is not a limitation, and the shape and volume can be determined depending on the installation location and the processing volume. The first tank 10 and the second tank 20 are preferably transparent so that the liquid level inside can be visually observed. The materials of the first tank 10 and the second tank 20 are not limited, and may be, for example, glass, metal or alloy, or resin (e.g., acrylic resins such as polyacrylonitrile and polyacrylic ester; polycarbonate; fluororesin, etc.). The first tank 10 and the second tank 20 preferably have the same shape and volume. The first tank 10 and the second tank 20 can be arranged at the same height with a gap between them.

[0037] The membrane surface velocity in the second filtration step is preferably in the same range as the membrane surface velocity in the first filtration step. To maintain the membrane surface velocity within this range, the inlet pressure of the hollow fiber membrane 30 (the liquid inlet / outlet side of the second tank 20) ​​in the second filtration step is preferably adjusted to 0.01 MPa to 0.2 MPa, more preferably 0.015 MPa to 0.15 MPa, and even more preferably 0.02 MPa to 0.12 MPa. To maintain the membrane surface velocity within this range, the outlet pressure of the hollow fiber membrane 30 (the liquid inlet / outlet side of the first tank 10) in the second filtration step is preferably adjusted to 0.03 MPa or less, more preferably 0.01 MPa or less, and even more preferably 0 MPa.

[0038] The liquid containing the useful micro-substances can then be concentrated by repeating the first and second filtration steps multiple times. When the first and second filtration steps are repeated multiple times, the dilution factor of the target solution in the first tank 10 of the first filtration step with the buffer solution (i.e., the dilution factor of the target solution in the first filtration step) can be increased, for example, by 2 to 15 volumetric times, and preferably by 2 to 10 volumetric times. Alternating tangential flow filtration, which alternately performs the first and second filtration steps in this manner, can produce a concentrated liquid with an increased concentration of useful micro-substances.

[0039] The concentration step is preferably carried out so that the concentration of the useful microsubstance in the resulting concentrate is 5 times or more, more preferably 7 times or more, and even more preferably 10 times or more, compared to the concentration of the secondary liquid raw material. The recovery rate of the useful microsubstance is preferably 50% or more, more preferably 60% or more, and even more preferably 70% or more. The amount of the useful microsubstance can be measured using an ELISA kit (e.g., CD9-Capture Human Exosome ELISA Kit (Fujifilm Wako Pure Chemical Industries, Ltd.)). Alternatively, it can be measured using the ExoScreen method described in Cytometry Research 26(1):1-6, 2016, "New Developments in Liquid Biopsy Using Exosomes," by Yusuke Yoshioka and Takahiro Ochiya.

[0040] [Method for concentrating a liquid raw material containing useful micro substances] A method for concentrating a liquid raw material containing useful micro substances according to one embodiment of the present disclosure includes adding an oxyethylene adduct of sorbitan unsaturated fatty acid ester to a primary liquid raw material containing useful micro substances to obtain a secondary liquid raw material, and applying the secondary liquid raw material to a hollow fiber membrane. The process for obtaining the secondary liquid raw material and the process for applying the secondary liquid raw material to a hollow fiber membrane according to this embodiment are the same as those in the embodiment of the method for producing a concentrated liquid containing useful micro substances, and therefore, the description thereof will be omitted.

[0041] The present disclosure will be explained in more detail below by showing examples, but interpretation of the present disclosure is not limited to these examples.

[0042] The various raw materials used in the examples and the methods for producing them are as follows. (Primary liquid raw materials) Tris-buffered saline (TBS, manufactured by Nippon Gene Co., Ltd.) Exosome solution "COLO201 cell-derived exosomes" (exosome concentration: 10 μg / mL) (manufactured by Fujifilm Wako Pure Chemical Corporation) (Secondary liquid raw materials) Polyoxyethylene sorbitan oleate "Polyoxyethylene (20) sorbitan monooleate" (manufactured by Fujifilm Wako Pure Chemical Corporation) Bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene sorbitan laurate "Polyoxyethylene (20) sorbitan monolaurate" (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene sorbitan stearate "Polyoxyethylene (20) sorbitan monostearate" (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene sorbitan palmitate "Polyoxyethylene (20) sorbitan monopalmitate" (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0043] [Example 1] As a primary liquid raw material, 2 μl of exosome solution (exosomes: 20 ng) was added to 150 ml of TBS to prepare a secondary liquid raw material. Next, polyoxyethylene sorbitan oleate was added to the primary liquid raw material to a concentration of 100 ppm to obtain a secondary liquid raw material. The secondary liquid raw material was concentrated at room temperature (approximately 20°C) using the apparatus 1 shown in Figure 1. - First tank 10 and second tank 20 Material: Glass Length: 25 cm Inner diameter: 3 cm Capacity: 180 cm ３ ・Concentrated liquid collection tank 35 Capacity: 50 cm ３ ・Hollow fiber membrane 30 Inner diameter: 0.8 mm Outer diameter: 1.3 mm Length: 60 cm Membrane area: 15 cm ２ Molecular weight cutoff: 150,000 Material: Cellulose acetate (CA) manufactured by Daisen Membrane Systems Co., Ltd.

[0044] (First filtration step) (1) 150 ml of the secondary liquid feedstock was filled into the first tank 10. (2) Nitrogen gas was supplied to the upper space of the first tank at a pressure of 0.025 MPa, and tangential flow filtration was performed while the secondary liquid feedstock was passed inside the hollow fiber membrane 30. At this time, the membrane surface velocity flowing inside the hollow fiber membrane 30, i.e., the membrane surface velocity, was 3.3 m / s. The membrane surface velocity was calculated from the rate of increase in the amount of concentrated liquid in the second tank. The permeated liquid permeated the hollow fiber membrane and was discharged outside the system, and the concentrated liquid (first concentrated liquid) was transferred to the second tank.

[0045] (Second Filtration Step) (3) When the secondary liquid raw material in the first tank 10 passed through the inside of the hollow fiber membrane 30 and was filtered, and most of it transferred to the second tank 20, nitrogen gas was supplied to the second tank 20, and at the same time, the pressure in the first tank was released. (4) Through this operation, filtration was performed while the first concentrated solution was transferred from the second tank 20 to the first tank 10, the permeated solution permeated the hollow fiber membrane and was discharged outside the system, and the concentrated solution (second concentrated solution) was transferred into the first tank 10. Alternating tangential flow filtration was performed, in which the same first filtration step and second filtration step were repeated multiple times. Alternating tangential flow filtration was performed until the liquid volume was approximately 10 ml, and the concentrated solution with an increased exosome concentration (final concentrated solution) was discharged into the concentrated solution recovery tank 35 and recovered.

[0046] The amount of exosomes in the final concentrate was measured using a CD9-Capture human exosome ELISA kit (Fujifilm Wako Pure Chemical Industries, Ltd.), and the exosome recovery rate was 73%. The exosome concentration in the secondary liquid raw material was 133 pg / ml, and the exosome concentration in the final concentrate was 1293 pg / ml, indicating that the exosome concentration in the final concentrate (concentration factor) was 9.7 times higher than that in the secondary liquid raw material.

[0047] Comparative Example 1 Alternating tangential flow filtration was performed in the same manner as in Example 1, except that polyoxyethylene sorbitan oleate was not added, in which the first filtration step and the second filtration step were repeated multiple times to obtain approximately 10 mL of a final concentrate, and the recovery rate and concentration ratio were determined.

[0048] Comparative Example 2-5 Alternating tangential flow filtration was performed in the same manner as in Example 1, except that the additives shown in Table 1 were added instead of polyoxyethylene sorbitan oleate, in which the first filtration step and the second filtration step were repeated multiple times to obtain approximately 10 mL of a final concentrate, and the recovery rate and concentration ratio were determined.

[0049] The results are shown in Table 1.

[0050] As shown above, the addition of polyoxyethylene sorbitan oleate not only achieved a higher recovery rate and concentration factor than when no additive was added or when bovine serum albumin was added, but also achieved a higher recovery rate and concentration factor than when polyoxyethylene sorbitan laurate, polyoxyethylene sorbitan stearate, or polyoxyethylene sorbitan palmitate, which have similar structures, were added.

[0051] The manufacturing method of the present disclosure can be used to manufacture a concentrated solution containing minute useful substances.

[0052] 1 Concentrator 10 First tank 20 Second tank 30 Hollow fiber membrane 35 Concentrate recovery tank 40 Three-way stopcock

Claims

1. A method for producing a concentrated liquid containing useful micro substances, comprising: adding an oxyethylene adduct of a sorbitan unsaturated fatty acid ester to a primary liquid raw material containing useful micro substances to obtain a secondary liquid raw material; and applying the secondary liquid raw material to a hollow fiber membrane to concentrate the secondary liquid raw material.

2. The method for producing a concentrated liquid containing minute useful substances according to claim 1, wherein concentrating the secondary liquid raw material comprises filtration using a hollow fiber membrane, and the filtration is tangential flow filtration in one direction or alternating directions.

3. A method for producing a concentrate containing micro-useful substances as described in claim 2, wherein the tangential flow filtration comprises: a first filtration step in which the secondary liquid raw material is pressurized into a first opening on one end side of the hollow fiber membrane, filtered, and separated into a permeate and a first concentrate; and a second filtration step in which the first concentrate is pressurized into a second opening on the other end side of the hollow fiber membrane, filtered, and separated into a permeate and a second concentrate, and the first filtration step and the second filtration step are alternately performed multiple times.

4. A method for producing a concentrated solution containing minute useful substances as described in claim 3, wherein the first filtration step and the second filtration step are carried out by introducing one or more gases selected from nitrogen gas, inert gas, carbon dioxide, and air filtered through a HEPA filter.

5. The method for producing a concentrated solution containing minute useful substances according to claim 3, wherein the hollow fiber membrane has an inner diameter of 0.2 mm to 1.4 mm and a molecular weight cutoff of 5,000 to 3,000,000.

6. A method for producing a concentrate containing minute useful substances according to any one of claims 1 to 5, wherein the oxyethylene adduct of sorbitan unsaturated fatty acid ester is polyoxyethylene sorbitan oleate.

7. A method for producing a concentrated solution containing minute useful substances according to any one of claims 1 to 5, wherein the minute useful substances comprise particles covered with a lipid bilayer membrane.

8. The method for producing a concentrated solution containing minute useful substances according to claim 7, wherein the particles covered with a lipid bilayer membrane are extracellular vesicles.

9. A method for producing a concentrated solution containing minute useful substances described in any one of claims 1 to 5, wherein the hollow fiber membrane is a hollow fiber membrane module in which multiple hollow fiber membranes are housed in a case housing having multiple liquid inlets and outlets.

10. A method for producing a concentrated solution containing minute useful substances according to any one of claims 1 to 5, wherein the primary liquid raw material comprises a cell culture supernatant.

11. A method for producing a concentrated solution containing minute useful substances according to any one of claims 1 to 5, wherein the primary liquid raw material comprises a culture supernatant of mesenchymal stem cells.

12. A method for concentrating a liquid raw material containing a useful micro substance, comprising: preparing a secondary liquid raw material by adding an oxyethylene adduct of a sorbitan unsaturated fatty acid ester to a primary liquid raw material containing the useful micro substance; and applying the secondary liquid raw material to a hollow fiber membrane.

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