Method and apparatus for separating and purifying useful minute substances

Abstract

To provide a separating / purifying method of micro useful substance.SOLUTION: A separating / purifying method of micro useful substance in which liquid containing micro useful substance is filtered by a hollow fiber membrane, where the hollow fiber membrane has an inner diameter of 0.2 to 1.4 mm and a molecular weight cut-off of 100,000 to 1,000,000. The filtration method has: a first filtration process in which liquid containing micro useful substance is press-fitted and filtered from a first opening part on one end side of the hollow fiber membrane, and separated into permeated liquid and first concentrated liquid; and a second filtration process in which the first concentrated liquid is press-fitted and filtered from a second opening part on the other end side of the hollow fiber membrane, and separated into permeated liquid and second concentrated liquid, where concentrated liquid with increased concentration of the micro useful substance is obtained by filtration of alternately executing the first filtration process and the second filtration process for plural times at the membrane surface velocity of 0.3 to 2 m / sec.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a separation and purification method for separating and purifying minute useful substances selected from exosomes, antibodies, viruses, proteins, nucleic acids, etc., and a separation and purification apparatus for carrying out the separation and purification method.

Background Art

[0002] As a method for separating and purifying useful substances from a culture solution, a method using a separation membrane is known. Patent Document 1 describes an invention of a method for concentrating a single-cell algal culture solution by cross-flow filtration using a hollow fiber type ultrafiltration membrane (UF membrane) module with a molecular weight cut-off of 10,000 to 1,000,000, in which periodic dead-end washing is carried out. When the culture solution is concentrated by cross-flow filtration, a concentrated solution exists outside the UF membrane, and a permeate enters the inside of the UF membrane. When dead-end washing is carried out, the washing water enters the inside of the UF membrane and then exits to the outside of the UF membrane, thereby washing the UF membrane.

[0003] Patent Document 2 includes a bleeding step of discharging a culture solution from a cell culture tank and adding the same amount of fresh medium as the discharged culture solution to the culture tank, and a filtration step of filtering the culture solution extracted from the culture tank using a porous membrane substantially having no dense layer, wherein the filtration in the filtration step is tangential flow filtration and the permeate velocity in the filtration step is 1.0 LMH or less. An invention of a method for recovering useful substances is described. It is described that the useful substances are selected from the group consisting of proteins, viruses, exosomes, and nucleic acids. It is described that the tangential flow filtration can also be alternating tangential flow filtration.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present disclosure aims to provide a separation and purification method for separating and purifying minute useful substances selected from exosomes, antibodies, viruses, proteins, nucleic acids, etc., and a separation and purification apparatus for carrying out the separation and purification method.

Means for Solving the Problems

[0006] The present disclosure is a method for separating and purifying minute useful substances by filtering a liquid containing minute useful substances through a hollow fiber membrane, wherein the hollow fiber membrane has an inner diameter of 0.2 mm to 1.4 mm and a fractional molecular weight of 100,000 to 1,000,000, and the filtration method is a first filtration step of press-filtrating the liquid containing the minute useful substances from a first opening on one end side of the hollow fiber membrane to separate it into a permeate and a first concentrate, a second filtration step of press-filtrating the first concentrate from a second opening on the other end side of the hollow fiber membrane to separate it into a permeate and a second concentrate, and a method of obtaining a concentrate with an increased concentration of the minute useful substances by alternately performing the first filtration step and the second filtration step a plurality of times by crossflow filtration, wherein the membrane surface velocity in the first filtration step and the second filtration step is 0.3 m / sec to 2 m / sec, and a separation and purification apparatus for carrying out the separation and purification method are provided.

Advantages of the Invention

[0007] According to the separation and purification method and separation and purification apparatus for minute useful substances of the present disclosure, minute useful substances such as exosomes, antibodies, viruses, proteins, nucleic acids, etc. can be concentrated to a high concentration.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0009] An embodiment of a method for separating and purifying minute useful substances will be described by the manufacturing flow using the separation and purification apparatus 1 shown in FIG. 1. In the first step, a liquid containing minute useful substances is put into the first tank 10. When the liquid containing minute useful substances is a culture solution containing minute useful substances, it is preferable to put the supernatant into the first tank 10. Examples of the minute useful substances include those selected from exosomes, antibodies, viruses, proteins, nucleic acids, and the like.

[0010] In FIG. 1, the first tank 10 is in a cylinder shape, but it is not limited thereto, and the shape and volume can be determined according to the situation of the installation location and the throughput. The first tank 10 preferably has transparency that allows visual observation of the liquid level inside, and further preferably consists of a material having water repellency in order to prevent the liquid from adhering to and remaining on the inner wall surface of the first tank 10. Part or all of the first tank 10 preferably consists of an acrylic resin such as polyacrylonitrile or polyacrylate ester, polycarbonate, or fluororesin. A take-out line for taking out the final concentrated liquid from inside the first tank 10 can be provided in the first tank 10.

[0011] As shown in Fig. 2, the connection portion 11 between the hollow fiber membrane 30 of the portion including the liquid inlet / outlet 10a of the first tank 10 has a conical inclined surface 11a and a cylindrical vertical surface 11b whose diameter becomes smaller from the first tank 10 side to the hollow fiber membrane 30 side. In Fig. 2, the connection portion 11 has a conical inclined surface 11a and a cylindrical vertical surface 11b. However, as long as it has the conical inclined surface 11a, the cylindrical vertical surface 11b may not be provided. Having the connection portion 11 shown in Fig. 2 is preferable because it can prevent the liquid containing the minute useful substance or its concentrated solution from staying on the bottom side of the first tank 10 and can increase the recovery rate of the minute useful substance.

[0012] Before the first step, a pretreatment step using a microfiltration membrane (microfiltration membrane module) can be carried out as necessary. The microfiltration membrane (microfiltration membrane module) preferably has a pore size of 0.1 μm to 0.5 μm. In the pretreatment step, the filtrate (pretreatment liquid) obtained by filtering the liquid containing the minute useful substance with a microfiltration membrane (microfiltration membrane module) is sent to the first tank 10.

[0013] In the second step, with the opening / closing valve (such as an electromagnetic valve) 46 open, the buffer liquid in the buffer liquid tank 40 is supplied into the first tank 10 from the buffer liquid supply line 45 to dilute the liquid containing the minute useful substance (or the pretreatment liquid). When the diluted liquid of the liquid containing the minute useful substance (or the pretreatment liquid) is in the first tank 10, there remains a space in the upper part of the first tank 10 where the diluted liquid does not exist. The buffer liquid in the buffer liquid tank 40 is preferably a medical or biochemical buffer liquid, and phosphate buffer (PBS), Tris-HCl buffer, sodium citrate buffer, citrate-phosphate buffer, acetate buffer, borate buffer, etc. can be used. When replenishing the buffer liquid to the buffer liquid tank 40, it is replenished from the buffer liquid replenishment line 41. Incidentally, the order of the first step and the second step may be reversed. After supplying the buffer solution in the buffer solution tank 40 into the first tank 10 from the buffer solution supply line 45, the liquid containing the minute useful substance can be put into the first tank 10. Alternatively, the first step and the second step may be combined into one step. After adding and mixing the liquid containing the minute useful substance and the buffer solution in a separately provided mixing tank to prepare a diluted solution of the liquid containing the minute useful substance, the diluted solution can be put into the first tank 10.

[0014] In the third step, a pump (not shown) or the like is operated to supply gas from a gas supply source (not shown) into the first tank 10, and by pressurizing the liquid containing the minute useful substance in the first tank 10, a first filtration step is carried out in which the liquid containing the minute useful substance is press-fitted inside the hollow fiber membrane 30 for filtration. In the present disclosure, all steps of filtering the liquid in the first tank 10 with the hollow fiber membrane 30 and sending it to the second tank 20 are referred to as the first filtration step. The gas for pressurization is sent through the gas supply line 52 equipped with the pressure gauge 51 and the first tank gas supply line 53 by switching the three-way valve 61. At this time, the on-off valve 46 and the on-off valve 62 of the first gas vent line 55 are closed, and the on-off valve 63 of the second gas vent line 56 is opened. As the gas, a gas selected from inert gases such as nitrogen gas, argon, and helium, carbon dioxide, clean air filtered by a HEPA filter, etc. can be used. The filtered permeate is stored in the permeate tank 35, and the concentrated solution (first concentrated solution) containing the minute useful substance is sent to the second tank 20. When the first concentrated solution is in the second tank 20, a space where the first concentrated solution does not exist remains at the upper part of the second tank 20.

[0015] The inner diameter of the hollow fiber membrane 30 is preferably 0.2 mm to 1.4 mm, more preferably 0.2 mm to 1.0 mm, and even more preferably 0.4 mm to 1.0 mm. The hollow fiber membrane 30 is preferably an ultrafiltration membrane with a fractional molecular weight of 100,000 to 1,000,000, more preferably an ultrafiltration membrane with a fractional molecular weight of 200,000 to 800,000, and even more preferably an ultrafiltration membrane with a fractional molecular weight of 300,000 to 600,000. The fractional molecular weight is evaluated by the transmittance % of γ-globulin ((γ-globulin concentration in the permeate / γ-globulin concentration in the solution (100 mg / L) × 100)) when a solution of 100 mg / L of γ-globulin (bovine serum γ-globulin manufactured by SIGMA, molecular weight 150,000) in a phosphate buffer 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). For the hollow fiber membrane 30, the transmittance of γ-globulin is preferably 5% to 95%, more preferably 10% to 80%, and even more preferably 30% to 70%. The hollow fiber membrane 30 may be a hydrophobic membrane such as a polyethersulfone membrane or a hydrophilic cellulose-based membrane, but a hydrophilic cellulose-based membrane is preferred. Examples of the hydrophilic cellulose-based membrane include cellulose acetate membrane, regenerated cellulose membrane, cellulose propionate membrane, cellulose butyrate membrane, cellulose benzoate membrane, and the like. As the hollow fiber membrane 30, FUS5082 (polyethersulfone membrane; fractional molecular weight 500,000, γ-globulin transmittance 70%) of Daisen Membrane Systems Co., Ltd., FUC1582 (cellulose acetate membrane; fractional molecular weight 150,000, γ-globulin transmittance 10%) of Daisen Membrane Systems Co., Ltd., etc. can be used.

[0016] The hollow fiber membrane 30 is arranged by connecting the liquid inlet / outlet 10a of the first tank 10 and the liquid inlet / outlet 20a of the second tank 20. The connection between the hollow fiber membrane 30 and the liquid inlet / outlet 10a of the first tank 10 is connected, for example, by fitting the open end of the hollow fiber membrane 30 into a thin tube such as an injection needle fixed to the liquid inlet / outlet 10a side of the first tank 10. The connection between the hollow fiber membrane 30 and the liquid inlet / outlet 20a of the second tank 20 can be carried out in the same manner. The permeate tank 35 is for storing the permeate obtained by filtration in the hollow fiber membrane 30. In FIG. 1, the permeate tank 35 is shown small, but it can also be a large tank into which most of the hollow fiber membrane 30 can fit.

[0017] In FIG. 1, one hollow fiber membrane 30 is shown, but a plurality of hollow fiber membranes may be used, and for example, it can be used as a hollow fiber membrane bundle composed of 2 to 150 hollow fiber membranes. Alternatively, a hollow fiber membrane module in which a plurality of hollow fiber membranes (hollow fiber membrane bundle) 30 are housed in a case housing having a plurality of liquid inlets and outlets may be used. When used as the hollow fiber membrane bundle, one end or both ends can be integrated with an adhesive. When using the hollow fiber membrane module, connect a plurality of liquid inlets and outlets of the hollow fiber membrane module, the liquid inlet and outlet 10a of the first tank 10, and the liquid inlet and outlet 20a of the second tank 20, and further connect the remaining liquid inlets and outlets (liquid permeation outlets) of the hollow fiber membrane module to the permeate tank 35.

[0018] For the filtration in the third step, it is preferably filtered in the range where the membrane surface velocity is 0.3 m / sec to 2 m / sec, and more preferably filtered in the range where the membrane surface velocity is 0.5 m / sec to 1.5 m / sec. If the membrane surface velocity is less than 0.3 m / sec, the purification efficiency will decrease. Conversely, if it exceeds 2 m / sec, the pressure level for increasing the membrane surface velocity will become too high, and the shear force applied to the minute useful substances during filtration will also become too high, resulting in a risk of altering the minute useful substances. The method for maintaining the membrane surface velocity within the above range is preferably to adjust the pressure of the inlet pressure of the hollow fiber membrane 30 (on the liquid inlet and outlet 10a side of the first tank 10) to 0.01 MPa to 0.2 MPa, more preferably to 0.02 MPa to 0.15 MPa, and even more preferably to 0.03 MPa to 0.12 MPa. The method for maintaining the membrane surface velocity within the above range is preferably to adjust the pressure of the outlet pressure of the hollow fiber membrane 30 (on the liquid inlet and outlet 20a side of the second tank 20) to 0.03 MPa or less, more preferably to 0.01 MPa or less, and even more preferably to 0 MPa.

[0019] In the fourth step, pumps (not shown) are operated to supply gas from a gas supply source (not shown) into the second tank 20, and the liquid (first concentrated liquid) containing the minute useful substances in the second tank 20 is pressurized, and a second filtration step is carried out in which the liquid containing the minute useful substances is passed through the inside of the hollow fiber membrane 30 for filtration. In the present disclosure, all steps of filtering the liquid in the second tank 20 with the hollow fiber membrane 30 and sending it to the first tank 10 are referred to as the second filtration step. The filtered permeate is stored in the permeate tank 35, and the concentrated liquid (second concentrated liquid) containing the minute useful substances is sent to the first tank 10.

[0020] The second tank 20 is cylindrical in FIG. 1, but is not limited thereto, and the shape and volume can be determined according to the situation of the installation location and the processing amount. The second tank 20 preferably has transparency that allows the internal liquid level to be visually observed, and further preferably has water repellency to prevent the liquid from adhering to and remaining on the inner wall surface of the second tank 20. Part or all of the second tank 20 is preferably made of an acrylic resin such as polyacrylonitrile or polyacrylate ester, polycarbonate, or fluororesin. The first tank 10 and the second tank 20 preferably have the same shape and the same volume. The first tank 10 and the second tank 20 are arranged at the same height with a space therebetween.

[0021] The gas for pressurization is sent through the gas supply line 52 and the second tank gas supply line 54 by switching the three-way valve 61. At this time, the on-off valves 63 and 46 of the second gas vent line 56 are closed, and the on-off valve 62 of the first gas vent line 55 is opened. As the gas, a gas selected from inert gases such as nitrogen gas, argon, and helium, clean air filtered by carbon dioxide, a HEPA filter, etc. can be used.

[0022] The membrane surface velocity in the fourth step is preferably in the same range as the membrane surface velocity in the third step. The inlet pressure of the hollow fiber membrane 30 in the fourth step (on the liquid inlet / outlet 20a side of the second tank 20) is preferably adjusted to 0.01 MPa to 0.2 MPa, more preferably adjusted to 0.02 MPa to 0.15 MPa, and even more preferably adjusted to 0.03 MPa to 0.12 MPa. The pressure of the outlet pressure of the hollow fiber membrane 30 in the fourth step (on the liquid inlet / outlet 10a side of the first tank 10) is preferably adjusted to 0.03 MPa or less, more preferably adjusted to 0.01 MPa or less, and even more preferably adjusted to 0 MPa. The third step and the fourth step can be continuously carried out by switching the three-way valve 61 while continuously supplying gas from the gas supply source through the gas supply line 52.

[0023] Thereafter, the micro useful substances in the liquid containing micro useful substances are separated and purified by repeating the first filtration step (the third step) and the second filtration step (the fourth step) a plurality of times. When repeating the first filtration step and the second filtration step a plurality of times, as the number of repetitions increases, it is preferable to increase the dilution multiple of the buffer solution to be filtered in the first tank 10 in the first filtration step. For example, it can be increased in the range of 2 to 15 volume times, preferably in the range of 2 to 10 volume times. In this way, a concentrated solution with an increased concentration of micro useful substances can be obtained by alternating tangential flow filtration in which the first filtration step and the second filtration step are alternately carried out.

[0024] Each aspect disclosed in this specification can be combined with any other features disclosed in this specification. Each configuration and their combinations in each embodiment are examples, and within the scope not departing from the gist of the disclosure of the present invention, additions, omissions, substitutions and other changes of the configuration can be made as appropriate. This disclosure is not limited by the embodiments, but only limited by the claims.

Example

[0025] Example 1 (Separation and purification apparatus 1 shown in FIG. 1) ·First tank 10 and second tank 20 Material: Polyacrylonitrile Size: Length 25 cm, inner diameter 0.25 cm, capacity 120 cm 3 ·Buffer tank 40 Capacity: 1.6 L ·Hollow fiber membrane 30 Inner diameter 0.8 mm, outer diameter 1.3 mm, length 50 cm, membrane area 12.6 cm 2 , Hollow fiber membrane made of polyethersulfone (PES) with a fractional molecular weight of 5 million (product name FUS5081, manufactured by Daisen Membrane Systems Co., Ltd.)

[0026] <Preparation of exosome medium> Ultra ExoM (registered trademark) Culture Medium for Extracellular Vesicles (EVs) (manufactured by Santejya Co., Ltd.) Basal medium and supplement were mixed at a mixing ratio of 9:1 (volume ratio) to prepare 50 ml of exosome medium. When the proteins contained in this medium were analyzed by SDS-PAGE (CBB staining) method, it was found that the protein mainly had a molecular weight of about 70,000. Also, as a result of quantitative analysis by the Bradford method, the total amount of protein in the medium was 107 mg. In addition, when the insulin contained in this medium was quantified by the (ELISA) method, it was 3531 μg.

[0027] <Preparation of liquid sample containing exosomes> To 50 ml of the exosome medium obtained above, Lonza exosome HansaBioMed (Human plasma of health donors 100 μg [number of particles >1×10 10 )]) was added to prepare 50 ml of a liquid sample containing micro-useful substances (exosomes).

[0028] <Implementation of a method for separating and purifying exosomes from a liquid sample containing minute useful substances (exosomes)> Using the separation and purification apparatus shown in Fig. 1, exosomes were separated and purified from a liquid sample containing minute useful substances (exosomes). The separation and purification were carried out at room temperature (about 20°C). (1) The entire amount of the liquid sample was filtered through an MF membrane with a pore size of 0.22 μm (PES made by Millipore, Millex - GP material) of the type set in a syringe cylinder to obtain a filtrate (pretreatment liquid). (2) In a mixing container (not shown in Fig. 1), 25 ml of the filtrate and 25 ml of a phosphate - buffered saline (PBS) were mixed to prepare 50 ml of a liquid sample diluent. (3) 50 ml of the liquid sample diluent in the mixing container was placed into the first tank 10. (4) Nitrogen gas was supplied to the upper space of the first tank 10 at a pressure of 0.1 MPa, and tangential flow filtration was carried out while passing the above - mentioned liquid sample diluent through the inside of the hollow fiber membrane 30. At this time, the second tank 20 was open to the atmosphere with the opening - closing valve 56 open, and the pressure was zero. Also, the membrane surface linear velocity flowing through the inside of the hollow fiber membrane 30 was 1.0 m / s. The membrane surface linear velocity was calculated from the increasing rate of the concentrated liquid amount in the second tank 20. The permeate was stored in the permeate tank 35, and the concentrated liquid (first concentrated liquid) was transferred into the second tank 20 (first filtration step). (5) When most of the liquid sample diluent in the first tank 10 passed through the inside of the hollow fiber membrane 30 and was filtered and transferred to the second tank 20, by switching the three - way valve 61, nitrogen gas was supplied to the second tank 20, and at the same time, the pressure of the first tank 10 was released by opening the opening - closing valve 62. (6) By this operation, filtration was carried out while the first concentrated liquid was transferred from the second tank 20 to the first tank 10. The permeate was stored in the permeate tank 35, and the concentrated liquid (second concentrated liquid) was transferred into the first tank 10 (second filtration step). When most of the first concentrated solution in the second tank 20 had transferred to the first tank 10, by switching the three-way valve 61, the second concentrated solution in the first tank 10 was filtered again by the hollow fiber membrane 30, and the concentrated solution was transferred into the second tank 20 (first filtration step). Crossflow filtration was performed by repeating the same first filtration step and second filtration step multiple times.

[0029] While repeating the first filtration step and second filtration step (crossflow filtration) of (4) to (6) above, when the liquid volume of the concentrated solution in the first tank 10 became approximately half (about 25 ml), 50 ml of phosphate buffer (calcium- and magnesium-free phosphate buffered saline) (10× PBS Buffer manufactured by Nippon Gene Co., Ltd.) in the buffer tank 40 was added. In addition, nitrogen gas corresponding to the reduced amount of the buffer solution was enclosed in the gas phase part (the space part where no buffer solution was present) of the buffer tank 40.

[0030] The separation and purification steps (crossflow filtration) of (4) to (6) above were repeated a total of 9 times, and finally, a total of 450 ml of phosphate buffer was added to the initial 50 ml of the liquid sample. After adding the phosphate buffer for the 9th time (concentrated solution volume 75 ml), without dilution, filtration was performed by crossflow filtration until it reached 6 ml, and a concentrated solution with an increased exosome concentration was obtained.

[0031] When the surface of the hollow fiber membrane after separation and purification was observed with an electron microscope (10,000 times magnification), exosomes with a size of approximately 100 nm were observed. By filtering using a hollow fiber membrane with a fractional molecular weight of 500,000 (equivalent membrane pore size 20 nm), it was determined that the membrane permeation of exosomes was blocked and the exosomes were sufficiently recovered by the membrane. On the other hand, the total protein amount in the final 6 ml of the sample concentrated solution was 5.7 mg, and the insulin amount was 20 μg. Compared with the 50 ml of the liquid sample containing exosomes (total protein amount 107 mg, insulin amount 3531 μg), the total protein amount could be reduced to 5.3% and the insulin amount could be reduced to 0.6%.

[0032] In the above cross - tangential flow filtration process, the filtrate volume was sampled over time, and the filtration rate was calculated from the mass change thereof. For 1 hour, with a membrane area of 1 m 2 , the conversion filtration rate per 0.1 MPa of pressure, although it decreased significantly initially, from around 25 minutes after the start of filtration, it was approximately 200 - 230 (average 210) L / m 2 h and became almost constant, and the separation and purification were completed approximately 88 minutes after the start of filtration.

[0033] Comparative Example 1 The length of the hollow fiber membrane in Example 1 was changed from 50 cm to 10 cm, and exosomes were separated and purified in the same manner as in Example 1 except that it was not diluted with phosphate buffer. For 1 hour, with a membrane area of 1 m 2 , the conversion filtration rate per 0.1 MPa of pressure, the initial measured value was 275 L / m 2 h or more, but it gradually decreased, and when it exceeded 200 minutes after the start of filtration, it was less than 25 L / m 2 h, and the separation and purification operation was aborted.

[0034] Example 2 Instead of the hollow fiber membrane in Example 1, a hollow fiber membrane made of cellulose acetate (CA) with an inner diameter of 0.8 mm, an outer diameter of 1.3 mm, and a molecular weight cut - off of 150,000 ((product name FUC1582, manufactured by Daisen Membrane Systems Co., Ltd.)) was used, and exosomes were separated and purified using the same apparatus as in Example 1. At that time, 27 ml of phosphate buffer was added to 3 ml of the liquid sample containing exosomes to make 30 ml of the liquid sample diluent. For 1 hour, with a membrane area of 1 m 2 , the conversion filtration rate per 0.1 MPa of pressure, the initial measured value was 500 L / m 2 h and did not decrease significantly, and was approximately 420 L / m 2It was stable at h, and the filtration rate was about twice that of Example 1, and exosomes could be efficiently recovered. On the other hand, the protein content in the permeate was lower than that in Example 1, but the total protein amount in the final 0.1 ml of sample concentrate was 0.25 mg, and the total protein amount could be reduced to 6.6% with respect to 3 ml of the liquid sample containing exosomes (total protein amount was 3.8 mg).

[0035] Comparative Example 2 Exosomes were separated and purified in the same manner as in Example 2, except that no buffer was added in the operation of Example 2. For 1 hour, with a membrane area of 1 m 2 , the conversion filtration rate per 0.1 MPa of pressure decreased significantly from the initial value of 550 L / m 2 h, and decreased significantly to about 200 L / m 2 h at 6 minutes after the start of filtration.

Industrial Applicability

[0036] The separation and purification method of the present disclosure can be used when separating and purifying substances selected from exosomes, antibodies, viruses, proteins, nucleic acids, etc. from a culture solution.

Explanation of Symbols

[0037] 1 Separation and purification device 10 First tank 20 Second tank 30 Hollow fiber membrane 35 Permeate tank 40 Buffer tank

Claims

1. A method for separating and purifying a micro-useful substance by filtering a liquid containing the micro-useful substance through a hollow fiber membrane, wherein the hollow fiber membrane has an inner diameter of 0.2 mm to 1.4 mm and a fractional molecular weight of 100,000 to 1,000,000, and the filtration method is, by pressurizing the inlet pressure on one end side of the hollow fiber membrane to 0.01 MPa to 0.2 MPa, the liquid containing the micro-useful substance is press-fitted from the first opening on one end side of the hollow fiber membrane for filtration and separated into a permeate and a first concentrate; a first filtration step, by pressurizing the inlet pressure on the other end side of the hollow fiber membrane to 0.01 MPa to 0.2 MPa, the first concentrate is press-fitted from the second opening on the other end side of the hollow fiber membrane for filtration and separated into a permeate and a second concentrate; a second filtration step, a method for obtaining a concentrate with an increased concentration of the micro-useful substance by alternately performing the first filtration step and the second filtration step a plurality of times by crossflow filtration, wherein the membrane surface velocity in the first filtration step and the second filtration step is 0.3 m / sec to 2 m / sec. A method for separating and purifying a micro-useful substance.

2. The filtration method is a step of filtering the liquid containing the micro-useful substance through a precision filtration membrane with a pore size of 0.1 μm to 0.5 μm, and then using the filtrate of the precision filtration membrane to alternately perform the first filtration step and the second filtration step a plurality of times. The method for separating and purifying a micro-useful substance according to Claim 1.

3. When performing the first filtration step, after adding a buffer solution to dilute the liquid containing the micro-useful substance or the second concentrate obtained in the second filtration step, the first filtration step is performed. The method for separating and purifying a micro-useful substance according to Claim 1 or 2.

4. The first filtration step and the second filtration step are performed by press-fitting by introducing a gas selected from nitrogen gas, inert gas, carbon dioxide, and air filtered by a HEPA filter. The method for separating and purifying a micro-useful substance according to any one of Claims 1 to 3

5. The micro-useful substance is selected from exosomes, antibodies, viruses, proteins, and nucleic acids. The method for separating and purifying a micro-useful substance according to any one of Claims 1 to 4.

6. The hollow fiber membrane is a hollow fiber membrane module in which a plurality of hollow fiber membranes are accommodated in a case housing having a plurality of liquid inlets and outlets. The method for separating and purifying a micro-useful substance according to any one of Claims 1 to 5.

7. The method for separating and purifying a micro useful substance according to any one of claims 1 to 6, wherein when the first filtration step and the second filtration step are alternately performed, the dilution multiple of the filtration target in the first filtration step is increased as the number of times of execution increases.

8. The method for separating and purifying a micro useful substance according to any one of claims 1 to 6, wherein when the first filtration step and the second filtration step are alternately performed, the dilution multiple of the filtration target in the first filtration step is increased in the range of 2 to 15 volume times as the number of times of execution increases.

9. A separation and purification apparatus for carrying out the method for separating and purifying a micro useful substance according to claim 1, comprising: a first tank into which a liquid containing the micro useful substance enters; a second tank arranged at an interval from the first tank; a hollow fiber membrane arranged to connect a liquid inlet / outlet of the first tank and a liquid inlet / outlet of the second tank; a buffer tank connected to be liquid-feeding-capable to the first tank by a liquid-feeding line; a permeate tank for storing the permeate filtered by the hollow fiber membrane; A separation and purification apparatus for separating and purifying a micro useful substance, having a pressurizing device capable of pressurizing one of the liquid inside the first tank and the liquid inside the second tank.

10. A separation and purification apparatus for carrying out the method for separating and purifying a micro useful substance according to claim 2, comprising: a precision filtration membrane having a pore diameter of 0.1 μm to 0.5 μm; a first tank into which the liquid containing the micro useful substance enters, connected to an outlet of the filtrate of the precision filtration membrane by a liquid-feeding line; a second tank arranged at an interval from the first tank; a hollow fiber membrane arranged to connect a liquid inlet / outlet of the first tank and a liquid inlet / outlet of the second tank; a buffer tank connected to be liquid-feeding-capable to the first tank by a liquid-feeding line; a permeate tank for storing the permeate filtered by the hollow fiber membrane; A separation and purification apparatus for carrying out the method for separating and purifying a micro useful substance, having a pressurizing device capable of pressurizing one of the liquid inside the first tank and the liquid inside the second tank.

11. The separation and purification apparatus for carrying out the method for separating and purifying a micro useful substance according to claim 9 or 10, wherein the first tank and the second tank have transparency such that the liquid level inside can be visually observed.

12. The connection portion between the hollow fiber membrane of the portion including the liquid inlet / outlet of the first tank has a conical inclined surface whose diameter decreases from the first tank side to the hollow fiber membrane side. The separation and purification apparatus for carrying out the method for separating and purifying a minute useful substance according to claim 9 or 10, wherein the connection portion between the hollow fiber membrane of the portion including the liquid inlet / outlet of the second tank has a conical inclined surface whose diameter decreases from the second tank side to the hollow fiber membrane side.

13. The hollow fiber membrane is a hollow fiber membrane module in which a bundle of 5 to 50 hollow fiber membranes, at least one end portion of which is sealed with an adhesive, is accommodated in a case housing. The case housing has three liquid inlet / outlets, one of the three liquid inlet / outlets is a permeate port, and the permeate port is connected to a permeate tank. The separation and purification apparatus for carrying out the method for separating and purifying a minute useful substance according to any one of claims 9 to 12, wherein the two liquid inlet / outlets excluding the permeate port are respectively connected to the liquid inlet / outlet of the first tank and the liquid inlet / outlet of the second tank.

Images