Filter unit, purification apparatus, and method for producing purified liquid

Abstract

The present invention provides a filter unit, a purification apparatus, a purified liquid, and a method for producing the purified liquid. 【Solution means】 The filter unit of the present invention has a separation functional layer a portion having an average pore diameter D a a separation functional layer b portion having an average pore diameter D b and a separation functional layer c portion having an average pore diameter D c and an inlet for introducing a liquid to be treated into the separation functional layer a portion, and the average pore diameters D a , D b and D c satisfy the following conditions A and B, and are characterized in that each separation functional layer is arranged in the order of the separation functional layer a portion, the separation functional layer b portion, and the separation functional layer c portion from the inlet. Condition A: 80 μm ≥ D a > D b > D c ≥ 1 μm Condition B: 6.5 ≥ D a / D b ≥ 1.5

Description

[Technical Field]

[0001] The present invention relates to a filter unit, a purification apparatus, and a method for producing a purified liquid. [Background technology]

[0002] With the recent advancements in cell culture technology, there is a growing need for the efficient purification of solutions containing biologically derived substances produced by cells and other organisms. In particular, the need for the culture and purification of viral vectors has increased with the development of gene therapy and vaccine development. Among the methods commonly used in gene therapy, those employing viral vectors are especially promising, and efficient purification of solutions containing viral vectors is desirable when manufacturing them. Similarly, efficient purification of solutions containing viral vectors is also desirable when manufacturing vaccines.

[0003] For example, Patent Document 1 discloses a method for purifying a viral vector by sequentially using filters with pores of a size that allows the viral vector to pass through but not cells and other cell culture components (impurities). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2017-529070 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, while Patent Document 1 discloses a method for purifying cell fragments using multiple depth filters and screen filters with different pore sizes, the combination of pore sizes of each filter was not considered, and clogging occurred prematurely, sometimes leading to a rapid increase in pressure. Furthermore, in such cases, a large filter had to be used to process large volumes, which presented a problem.

[0006] The present invention provides a filter unit, a purification apparatus, a purified liquid, and a method for producing a purified liquid, in which a filter suitable for efficient purification of a solution containing a bio-derived substance, specifically a biopharmaceutical (antibody drug, gene therapy drug, nucleic acid drug, virus vector, etc.), a food ingredient or a beverage ingredient, particularly a virus vector, is arranged.

Means for Solving the Problems

[0007] The present invention for solving the above problems has the following configurations (1) to (10). (1) A separation functional layer a portion having an average pore diameter D a , a separation functional layer b portion having an average pore diameter D b , and a separation functional layer c portion having an average pore diameter D c , and an introduction port for introducing a liquid to be treated into the separation functional layer a portion, and the average pore diameters D a , D b , and D c satisfy the following conditions A and B, and are arranged in this order from the introduction port: the separation functional layer a portion, the separation functional layer b portion, and the separation functional layer c portion. A filter unit. Condition A: 80 μm ≥ D a > D b > D c ≥ 1 μm Condition B: 6.5 ≥ D a / D b ≥ 1 (2) The separation functional layer a portion has a thickness T a , the separation functional layer b portion has a thickness T b , and the upper limits of the thickness T a and the thickness T b are each 5 mm or less and satisfy the following conditions C and D. The filter unit according to (1). Condition C: Thickness T a ×1000 / average pore diameter D a ≥ 5 Condition D: Thickness T b ×1000 / average pore diameter D b ≥ 15 (3) The separation functional layer c is made of a nonwoven fabric with an average fiber diameter of 0.3 to 1.5 μm, as described in (1) or (2) of the filter unit. (4) The separation functional layer a, the separation functional layer b, or the separation functional layer c supports a polymer having a unit selected from the group consisting of a monocarboxylate vinyl ester unit, a vinylpyrrolidone unit, a vinylcaprolactam unit, a vinylacetamide unit, and an acrylamide unit, the filter unit according to (1) or (2). (5) A filter unit according to (1) or (2) for the purification of biologically derived substances. (6) A filter unit according to (1) or (2), having a housing having the above-mentioned inlet and outlet, wherein the housing incorporates a bonded filter in which the above-mentioned separation function layer a, the above-mentioned separation function layer b, and the above-mentioned separation function layer c are joined together. (7) A purification apparatus for purifying biological substances, comprising the filter unit described in (6), a pump, and a pressure gauge, wherein the pressure gauge is connected to the connection between the pump and the inlet of the filter unit. (8) The purification apparatus described in (7) used for the purification of biopharmaceuticals, food components, or beverage components. (9) Bio-derived substances purified by the purification apparatus described in (7) (10) Average pore diameter D a Step X to obtain a processed liquid X by passing the liquid to be processed through the separation functional layer a having a linear velocity in the range of 0.01 to 1.5 m / hour, and the average pore diameter D b Step Y involves passing the above-mentioned processing liquid X through the separation functional layer b having the above-mentioned processing liquid X at a linear velocity in the range of 0.001 to 1.5 m / hour to obtain the processing liquid Y, and the average pore diameter D c The process includes a step Z to obtain a purified liquid by passing the above processing liquid Y through the separation functional layer c having the above average pore size D at a linear velocity in the range of 0.0005 to 1.5 m / hour, wherein the average pore size D a , D b and D c This is a method for producing a purified liquid containing biologically derived substances that satisfies the following conditions A and B. Condition A: 80 μm ≥ D a >D b >Dc ≥1μm Condition B: 6.5≧D a / D b ≥1.5 [Effects of the Invention]

[0008] According to the present invention, when purifying biological substances, specifically biopharmaceuticals (antibody drugs, gene therapy drugs, nucleic acid drugs, viral vectors, etc.), food components, or beverage components from solutions containing cell fragments such as cell culture media and cell lysates, clogging of the filter unit or pressure increase can be suppressed, enabling efficient purification. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram showing an embodiment of the filter unit of the present invention. [Figure 2] This is a schematic diagram showing another embodiment of the filter unit of the present invention. [Figure 3] This is a schematic diagram showing another embodiment of the filter unit of the present invention. [Figure 4] This is a schematic diagram showing the purification apparatus of the present invention. [Figure 5] This is a schematic diagram showing another embodiment of the filter unit of the present invention. [Modes for carrying out the invention]

[0010] In this specification, "~" represents a range that includes its lower and upper limits.

[0011] (Filter unit) The filter unit of the present invention has an average pore size D a A separation functional layer a, having an average pore size D b Separation functional layer b having an average pore diameter D c It comprises a separation function layer c having a pore size D, and an inlet for introducing the liquid to be processed into the separation function layer a, wherein the average pore size D a , Db and D c The following conditions A and B are met, and the separation function layer a, separation function layer b, and separation function layer c are arranged in that order from the inlet. Condition A: 80 μm ≥ D a >D b >D c ≥1μm Condition B: 6.5≧D a / D b ≥1.5

[0012] A "filter unit" refers to a separation device that combines multiple separation functional layers so that fluid flows in one direction, and can be used to remove impurities contained in fluids such as liquids or gases. The separation functional layers used in a filter unit may be a combination of multiple separation functional layers with the same material and average pore size, or a combination of multiple separation functional layers with different materials and average pore sizes.

[0013] Furthermore, the filter unit includes units in which multiple separation functional layers are housed in the same housing, as well as units in which multiple housings containing individual or multiple separation functional layers are connected. Any commonly used connection method may be used for connecting each housing, and the connection conditions are arbitrary. Due to its simple structure and ease of maintenance, the filter unit is suitably used as an inline filter in purification equipment. It can also be used in both batch production (single use) and continuous production, and is not limited by the production method.

[0014] The filter unit of the present invention may have an additional separation functional layer, an adsorbent, or other component or support material used for purification on either the inlet or outlet side of the filter unit. Furthermore, by purifying the purified liquid produced by the filter unit of the present invention with a filter having an average pore size of 0.02 to 0.30 μm, a higher purity of bio-derived material can be obtained.

[0015] The "separation function layer" refers to the porous structure portion of a filter that exhibits separation performance. The entire filter may be the separation function layer, or a portion of the filter may be the separation function layer with the remaining portion being the support layer. Furthermore, if the pore size is distributed asymmetrically, or if the filter is integrated with a filter having multiple pore sizes, a single filter may have multiple separation function layers, or the layer with the smallest pore size may be designated as the separation function layer.

[0016] Average pore diameter D a The separation functional layer a, which has the following properties, removes cells from the cell culture medium or undisrupted cells and large cell fragments from the cell lysate. Also, the average pore size D b The separation functional layer b, which has the following properties, removes relatively small cell fragments that have passed through the separation functional layer a. Furthermore, the average pore size D c The separation functional layer c, which has the following properties, removes impurities such as small cell fragments that have passed through the separation functional layer b. To satisfy the purpose of these removals, the average pore size D a , D b and D c It is set to satisfy the following conditions A and B. Condition A: 80 μm ≥ D a >D b >D c ≥1μm Condition B: 6.5≧D a / D b ≥1.5

[0017] Furthermore, the average pore size D b and D c It is more preferable that the following conditions A and B are met. Condition AB:8.5≧D b / D c ≥2.0

[0018] Furthermore, in order to satisfy the purpose of removal, the separation functional layers a, b, and c are arranged in the order of separation functional layer a, separation functional layer b, and separation functional layer c, starting from the inlet where the liquid to be treated is introduced.

[0019] The separation functional layer a has a thickness T. a It has a separation function layer b with a thickness Tb It has a thickness T a and thickness T b The upper limits for each are 5 mm or less, and the following conditions C and D are met. Condition C: Thickness T a ×1000 / average pore diameter D a ≥5 Condition D: Thickness T b ×1000 / average pore diameter D b ≥15

[0020] Furthermore, the separation functional layer c has a thickness T c It has a thickness T c The upper limit is 5 mm or less, and it is even more preferable if the following conditions CD are met. Condition CD: Thickness T c ×1000 / average pore diameter D c ≥30

[0021] It is preferable that the separation functional layer a, separation functional layer b, and separation functional layer c are each composed of fibrous filters. Furthermore, the average porosity of separation functional layer a is 60% to 95%, and is preferably 70% to 90% from the viewpoint of balancing separation performance and processing speed. Furthermore, the average porosity of separation functional layer b and separation functional layer c is 70% to 95%, and is preferably 75% to 90% from the viewpoint of balancing separation performance and processing speed. Moreover, from the viewpoint of such pressure, it is even more preferable that the average porosity increases in the order of separation functional layer a, separation functional layer b, and separation functional layer c.

[0022] These separation function layers a and b may be either a depth filter that removes the target object internally or a screen filter that captures the target object on its surface. Of these, a depth filter is preferred because it is less prone to clogging.

[0023] The average porosity can be measured by the method described later in "Measurement of Porosity of Nonwoven Fabrics".

[0024] Specifically, the separation functional layer a removes cells from the cell culture medium or undisrupted cells and large cell fragments from the cell lysate, with an average pore size of D a Thickness T is 18-80 μm. a The average pore size is 0.2 mm or larger. This average pore size prevents rapid blockage of surface openings even when aggregates formed from multiple cells or cell fragments are present. Representative cultured cells that produce biopharmaceuticals as biologically derived substances include CHO cells and HEK293 cells, which are 10-30 μm in size. However, depending on the cell culture and lysis conditions, cells and cell fragments may aggregate, forming aggregates larger than a single cell.

[0025] From the perspective of achieving both separation performance and processing speed, the average pore size D a A thickness of 25-350 μm is more preferable. a A thickness of 0.3 to 1.5 mm is more preferable, and 0.5 to 1.0 mm is even more preferable.

[0026] The average pore size can be measured by the method described later in "Measurement of Pore Size of Nonwoven Fabrics".

[0027] The average pore diameter and thickness of the separation functional layer a can be controlled, for example, by the temperature of the air blown at the nozzle outlet during nonwoven fabric manufacturing or by laminating the nonwoven fabric.

[0028] The separation functional layer (a) of the filter unit of the present invention has an average fiber diameter of 3.0 to 20.0 μm and a basis weight of 20 g / m², from the viewpoint of reducing pressure loss and efficiently capturing cells, cell fragments, and aggregates thereof of a certain size or larger. 2 The nonwoven fabric described above is preferable. From the viewpoint of balancing separation performance and processing speed, an average fiber diameter of 4.0 to 15.0 μm is more preferable. The basis weight is 40 to 100 g / m². 2 More preferably, 55-65 g / m 2 That is even more preferable.

[0029] The average fiber diameter can be measured by the method described later in "Measurement of Fiber Diameter of Nonwoven Fabrics".

[0030] Examples of materials constituting the separation functional layer a include polyester or polyolefin. In particular, the main component of the separation functional layer a is preferably polyolefin, and it is more preferable that the main component of the material constituting the separation functional layer a is polypropylene, which is inexpensive, has excellent durability, and possesses hydrophobic adsorption properties.

[0031] "Main component" refers to the component that makes up 50% or more of the total mass of the material that constitutes the filter.

[0032] The separation functional layer b removes relatively small cell fragments that have passed through the separation functional layer a, and the average pore size D b Thickness T is 6-17 μm. b The average pore size D is 0.2 mm or larger. From the perspective of balancing separation performance and processing speed, b A thickness of 9-13 μm is more preferable. b A thickness of 0.3 to 1.5 mm is more preferable, and 0.5 to 1.0 mm is even more preferable.

[0033] The average pore diameter and thickness of the separation functional layer b can be controlled, for example, by the temperature of the air blown at the nozzle outlet during nonwoven fabric manufacturing or by laminating the nonwoven fabric.

[0034] The separation functional layer b of the filter unit of the present invention has an average fiber diameter of 1.6 to 3.0 μm and a basis weight of 24 g / m², from the viewpoint of reducing pressure loss and efficiently removing cell fragments larger than a certain size. 2 The nonwoven fabric described above is more preferable. From the viewpoint of balancing separation performance and processing speed, an average fiber diameter of 2.0 to 2.5 μm is more preferable. In addition, the basis weight should be 40 to 80 g / m². 2 More preferably, 55-65 g / m 2 That is even more preferable.

[0035] Examples of materials constituting the separation functional layer b include polyester or polyolefin. In particular, the main component of the separation functional layer b is preferably polyolefin, and more preferably polypropylene, which is inexpensive, has excellent durability, and possesses hydrophobic adsorption properties, is the main component of the material constituting the separation functional layer b.

[0036] The separation functional layer c removes impurities such as small cell fragments that have passed through the separation functional layer b, and the average pore size D c Thickness T is 1-5 μm. c The average pore size D is 0.2 mm or larger. From the perspective of balancing separation performance and processing speed, b The thickness T is more preferably 1 to 4 μm. c A thickness of 0.2 to 1.5 mm is more preferable, and 0.4 to 1.0 mm is even more preferable.

[0037] The separation functional layer c of the filter unit of the present invention has an average fiber diameter of 0.3 to 1.5 μm and a basis weight of 20 g / m², from the viewpoint of reducing pressure loss and efficiently removing cell fragments larger than a certain size. 2 The nonwoven fabric described above is more preferable. From the viewpoint of balancing separation performance and processing speed, an average fiber diameter of 0.5 to 0.9 μm is more preferable. In addition, the basis weight should be 30 to 80 g / m². 2 More preferably, 55-65 g / m 2 That is even more preferable.

[0038] The material constituting the separation function layer c is preferably a thermoplastic resin. Examples of thermoplastic resins include polyester, polyolefin, polyamide, or polyphenylene sulfide. Among these, the main component of the separation function layer c is preferably polyolefin, and more preferably polypropylene, which is easier to make into an average fiber diameter.

[0039] While known types of polypropylene can be used, when manufactured by the melt-blown method described later, it is preferable that the melt flow rate (hereinafter referred to as "MFR") is in the range of 10 g / 10 min to 2000 g / 10 min. The MFR, which indicates the fluidity of the molten resin, can be measured by the standard test method of JIS K7210-1:2014. For polypropylene, the measurement is performed under the measurement conditions of 2.16 kg and 230°C (conditions specified for polypropylene in JIS K6921-2:2018).

[0040] The separation functional layer c is manufactured by, for example, a melt spinning method, a dry spinning method, a wet spinning method, or a direct spinning method (such as a melt-blown method, a spunbond method, or an electrostatic spinning method), and is not particularly limited, but is preferably a melt-blown nonwoven fabric. In the melt-blown method, when the molten resin is extruded in a fibrous form from the spinning nozzle, compressed gas such as air is applied to both sides of the extruded fibrous molten resin, and the average fiber diameter can be made finer by accompanying the gas. The melt-blown method is preferred because it is easy to obtain a nonwoven fabric consisting of ultrafine fibers with an average fiber diameter of 0.8 μm or less.

[0041] The filter unit of the present invention is not particularly limited, but from the viewpoint of suppressing clogging by using multiple separation functional layers, it is preferable that the nonwoven fabrics used as separation functional layer a, separation functional layer b, and separation functional layer c have a uniform structure rather than an asymmetric or other non-uniform structure.

[0042] The nonwoven fabric used as the separation functional layer a, separation functional layer b, and separation functional layer c of the filter unit of the present invention preferably has a polymer containing a monocarboxylate vinyl ester unit, a vinylpyrrolidone unit, a vinylcaprolactam unit, a vinylacetamide unit, or an acrylamide unit supported on it, and more preferably on the surface or back surface of the nonwoven fabric. Furthermore, it is not necessary for the polymer to be supported on all of the separation functional layer a, separation functional layer b, and separation functional layer c, but it may be supported on any one of the separation functional layer a, separation functional layer b, or separation functional layer c.

[0043] The polymer containing monocarboxylate vinyl ester units, vinylpyrrolidone units, vinylcaprolactam units, vinylacetamide units, or acrylamide units is preferably supported on at least the surface that first comes into contact with the liquid being treated, and more preferably on both the front and back surfaces, the polymer containing monocarboxylate vinyl ester units, vinylpyrrolidone units, vinylcaprolactam units, vinylacetamide units, or acrylamide units is supported on both surfaces.

[0044] Furthermore, it is preferable that a polymer containing monocarboxylate vinyl ester units, vinylpyrrolidone units, vinylcaprolactam units, vinylacetamide units, or acrylamide units is supported inside the nonwoven fabric. By supporting a polymer containing monocarboxylate vinyl ester units, vinylpyrrolidone units, vinylcaprolactam units, vinylacetamide units, or acrylamide units on all surfaces of the nonwoven fabric—the surface, back surface, and interior—the adhesion of proteins and other substances can be effectively suppressed.

[0045] A "monocarboxylic acid" refers to a compound consisting of one carboxyl group and a hydrocarbon group bonded to the carbon atom of that carboxyl group, i.e., a compound represented as "R-COOH" (where R is the hydrocarbon group). The hydrocarbon group R may be either an aliphatic hydrocarbon group or an aromatic hydrocarbon group, but from the viewpoint of ease of synthesis, an aliphatic hydrocarbon group, particularly a saturated aliphatic hydrocarbon group, is preferred. Furthermore, from the viewpoint of manufacturing cost, a linear or branched structure is preferred for saturated aliphatic hydrocarbon groups, with a linear structure being more preferred. Examples of monocarboxylic acids where R is an aromatic hydrocarbon group include benzoic acid or its derivatives. Examples of monocarboxylic acids where R is a saturated aliphatic hydrocarbon group include acetic acid, propanoic acid, or butyric acid.

[0046] The saturated aliphatic hydrocarbon group may be a linear structure such as an ethyl group, n-propyl group, n-butyl group, n-pentyl group, or n-hexyl group, as well as a branched structure such as an isopropyl group or tert-butyl group, or a cyclic structure such as a cyclopropyl group or cyclobutyl group. Furthermore, it may contain ether bonds or ester bonds within the aliphatic chain. The hydrocarbon group R may have hydrogen atoms substituted with any substituent, but if the terminal hydrogen atom is substituted with an anionic functional group such as a sulfonic acid group, it may destabilize the protein structure and induce adhesion to the hollow fiber membrane surface; therefore, it is preferable that the terminal hydrogen atom is not substituted with an anionic functional group.

[0047] A low number of carbon atoms in the hydrocarbon group R is preferable because it reduces the hydrophobicity of the monocarboxylic acid and minimizes hydrophobic interactions with proteins, thus preventing adhesion. Therefore, when the hydrocarbon group R is an aliphatic hydrocarbon group or an aromatic hydrocarbon group, the number of carbon atoms is preferably 1 to 20, more preferably 1 to 9, and even more preferably 2 to 5. When the hydrocarbon group R is a saturated aliphatic hydrocarbon group, the compound with 1 carbon atom is acetic acid, and the compound with 2 carbon atoms is propanoic acid.

[0048] "Unit" refers to a repeating unit in a homopolymer or copolymer obtained by polymerizing monomers, and "carboxylate vinyl ester unit" refers to a repeating unit obtained by polymerizing carboxylate vinyl ester monomers, i.e., a repeating unit represented as "-CH(OCO-R)-CH2-". R is the same as described above for monocarboxylic acids, and preferred examples are also the same as above.

[0049] Specific examples of the vinyl ester unit of a monocarboxylic acid in which the hydrocarbon group R is a saturated aliphatic group include a vinyl propionate unit, a vinyl pivalate unit, a vinyl decanoate unit, a vinyl methoxyacetate unit, and the like. Since it is preferable that the hydrophobicity is not too strong, a vinyl acetate unit (R: CH3), a vinyl propionate unit (R: CH2CH3), a vinyl butyrate unit (R: CH2CH2CH3), a vinyl pentanoate unit (R: CH2CH2CH2CH3), a vinyl pivalate unit (R: C(CH3)3), or a vinyl hexanoate unit (R: CH2CH2CH2CH2CH3) is preferable. Specific examples of the vinyl ester unit of a monocarboxylic acid in which the hydrocarbon group R is an aromatic group include a vinyl benzoate unit or a substituted product thereof.

[0050] That the polymer containing the vinyl ester unit of a monocarboxylic acid is supported on the front surface, back surface, and inside of the nonwoven fabric can be confirmed by combining composition analysis by time-of-flight secondary ion mass spectrometry (hereinafter referred to as "TOF-SIMS") and measurement by X-ray photoelectron spectroscopy (hereinafter referred to as "XPS"). Specifically, first, since a peak derived from a carboxylic acid ion of the vinyl ester unit of a monocarboxylic acid can be detected by composition analysis by TOF-SIMS, the structure of the monocarboxylic acid can be specified by analyzing its mass (m / z).

[0051] In the composition analysis by TOF-SIMS, for example, when Bi3 is used as the primary ion species and secondary negative ions are detected, the peak at m / z = 59.02 corresponds to C2H3O2, that is, acetic acid (number of aliphatic chain carbons: 1). Further, the peak at m / z = 73.04 corresponds to C3H5O2, that is, propionic acid (number of aliphatic chain carbons: 2). ++ - -

[0052] Furthermore, when XPS measurement is performed, a peak of carbon derived from an ester group (COO) is CH x ​​​Since it appears at +4.0 to 4.2 eV from the main peak of C-C (around 285 eV), it can be seen that the above carboxylic acid forms an ester bond. As the measurement angle of XPS, the value measured at 90° is used. When measured at a measurement angle of 90°, a region with a depth from the surface of about 10 nm is detected.

[0053] From the above two measurement results, it can be confirmed that polymers containing vinyl monocarboxylate units are supported on the front surface, back surface, and inside of the non-woven fabric used as the separation functional layer a part, the separation functional layer b part, and the separation functional layer c part.

[0054] From the viewpoints of sufficiently suppressing protein adhesion and avoiding a decrease in the introduction efficiency into the hollow fiber membrane, the number average molecular weight of the polymer containing vinyl monocarboxylate units is preferably from 1,000 to 1,000,000, more preferably from 5,000 to 500,000, and even more preferably from 10,000 to 100,000. The number average molecular weight of the homopolymer or copolymer can be measured by gel permeation chromatography.

[0055] From the viewpoint of achieving both separation performance and water permeability, the present invention's filter unit preferably uses the treatment amount per product of the contact area and the separation functional layer thickness of 0.8 L / cm for the separation functional layer a part. 3 Hereinafter, 0.8 L / cm for the separation functional layer b part. 3 Hereinafter, 1.0 L / cm for the separation functional layer c part. 3 It is preferably used under the condition of being as follows.

[0056] Also, from the viewpoint of achieving both separation performance and water permeability, the present invention's filter unit preferably uses the condition that the ratio of the contact area (m 2 ) to the separation functional layer thickness (m) is 7 or less for the separation functional layer a part, 7 or less for the separation functional layer b part, and 10 or less for the separation functional layer c part.

[0057] From the viewpoint of miniaturizing the filter unit, the present invention's filter unit has the contact area of the separation functional layer b part (hereinafter referred to as "S A ") with respect to the contact area of the separation functional layer a part (hereinafter referred to as "SB The ratio S of " A / S B The values ​​are 1 to 10, and S A Contact area of ​​the separation functional layer c (hereinafter referred to as "S") C The ratio S of " A / S C It is preferable that the value is between 1 and 20.

[0058] Generally, clogging is most likely to occur when filtering solutions containing the most impurities in cell fragments, resulting in the largest membrane area required for filtration and making it difficult to miniaturize the filter unit. On the other hand, as described above, the filter unit of the present invention reduces the required membrane area of ​​each filter by appropriately adjusting the average pore diameter and thickness of separation functional layer a, separation functional layer b, and separation functional layer c, thereby enabling miniaturization of the filter unit.

[0059] The filter unit of the present invention will be described in detail below, but the present invention is not limited to these embodiments.

[0060] The shape of the housing is not particularly limited, but cylindrical, disc-shaped (piece-shaped), cartridge-shaped, or capsule-shaped are preferred, with cylindrical housing being preferred. The material of the housing is also not particularly limited, but materials with sterilization resistance are preferred, specifically polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, or polystyrene. As the adhesive for fixing the filter to the housing, general adhesive materials such as polyurethane resin, epoxy resin, and silicone resin can be preferably used.

[0061] Figure 1 shows one embodiment of the filter unit of the present invention. In Figure 1, a housing 100 having an inlet and an outlet is shown, and a filter (101) having a separation function layer a is built into the housing (200) having an inlet for introducing the liquid to be treated. Also shown is a housing 100 having an inlet and an outlet, and a housing 201 having a separation function layer b, and a filter (102) having a separation function layer b is built into the housing (201) having an inlet and an outlet. Furthermore, a housing 100 having an inlet and an outlet is shown, and a housing 102 having a separation function layer c, and a filter (103) having a separation function layer c is built into the housing (202) having an inlet and an outlet.

[0062] The filter unit of the present invention shown in Figure 1 is an example of a filter unit 300 in which the housings of the separation function layer a portion 200, the housings of the separation function layer b portion 201, and the housings of the separation function layer c portion 202 are connected by a connecting portion 104. In this way, the filter unit 300 of the present invention can be in a form in which each housing containing each filter is connected.

[0063] In this connected configuration, the separation function layers a, b, and c are arranged in the order of separation function layer a, separation function layer b, and separation function layer c, starting from the inlet for introducing the liquid to be treated.

[0064] Figure 2 shows a second embodiment of the filter unit of the present invention. In Figure 2, a housing 100 having an inlet and an outlet is shown, with a filter (101) having a separation function layer a on the inlet side and a filter (103) having a separation function layer c on the outlet side, and the housing 203 containing the separation function layers a, b, and c is shown, with the filter (101) having a separation function layer a, the filter (102) having a separation function layer b, and the separation function layer c (103) joined together in that order.

[0065] By forming a bonded filter in which the separation function layer a, separation function layer b, and separation function layer c are joined together, it becomes possible to further miniaturize the entire filter unit. Therefore, it is preferable that the housing of the filter unit incorporates the bonded filter formed by joining the separation function layer a, separation function layer b, and separation function layer c. Furthermore, the bonded filter may undergo advanced processing such as pleating or calendering.

[0066] "Joining" refers to a state in which multiple filters are integrated into one, and includes states in which multiple filters are stacked or bonded together with adhesive or the like. Furthermore, for example, a state in which a spacer filter or the like is provided between separation functional layer a and separation functional layer b and they are integrated together is also included in the state in which separation functional layer a and separation functional layer b are joined.

[0067] Figure 3 shows a third embodiment of the filter unit of the present invention. In Figure 3, a housing 100 having an inlet and an outlet is shown, in which a filter (101) having a separation function layer a is placed on the inlet side and a filter (103) having a separation function layer c is placed on the outlet side, and a housing (204) containing separation function layers a, b and c is shown, with the filters (101), b and c being stacked in the order of a, b and c, respectively.

[0068] Since the separation functional layer a, separation functional layer b, and separation functional layer c are each composed of fibrous filters, the fibers intertwine and can be stacked without separation without the use of spacers or adhesives. Therefore, it is preferable that the entire filter unit be made smaller and that the probability of generating impurities such as elutes and leaches during the purification process be reduced, and that a laminated filter in which the separation functional layer a, separation functional layer b, and separation functional layer c are stacked without separation be housed in the same housing. Furthermore, the laminated filter may be subjected to advanced processing such as pleating or calendering.

[0069] Figure 4 shows one embodiment of the purification apparatus of the present invention. Figure 4 is an example of a purification apparatus 301 for purifying biological substances, in which a pump 105 and a housing (204) containing separation function layers a, b, and c are connected by a connection part 104, and each connection part 104 is equipped with a pressure gauge 106.

[0070] Figure 5 shows a fourth embodiment of the filter unit of the present invention. Figure 5 shows a cylindrical housing 110 having an inlet and an outlet, in which a cylindrical inner core 111 is arranged inside, and a filter (101) having a separation function layer a, a filter (102) having a separation function layer b, and a filter (103) having a separation function layer c are stacked without being joined or separated and are wound around the cylindrical inner core 111 to house a separation function layer a, b, and c housing 205. In this cylindrical housing 205 with a separation function layer a, b, and c, the filter (101) having a separation function layer a is arranged on the inlet side, and the filter (103) having a separation function layer c is arranged on the outlet side.

[0071] In the filter unit 300 of the second or third embodiment of the present invention, from the viewpoint of increasing the effective area of ​​the separation functional layer and the size of the housing, the filling rate of the separation functional layer a, separation functional layer b, and separation functional layer c, calculated from the following formula (1), is preferably 30% or more and 95% or less, and more preferably 60% or more and 95% or less. Note that when calculating the filling rate of a filter unit that does not have internal components other than the filter, such as an inner core, the following formula (1) shall be used. Filling rate (%) = Film area × Film thickness × 100 / Volume inside the enclosure ... Equation (1)

[0072] In the filter unit 300 of the fourth embodiment of the present invention, from the viewpoint of increasing the effective area of ​​the separation functional layer and the size of the housing, the filling rates of the separation functional layer a, separation functional layer b, and separation functional layer c, calculated from the following formula (2), are preferably 30% or more and 95% or less, and more preferably 60% or more and 95% or less. The following formula (2) shall be used to calculate the filling rate of the filter unit having an inner core. Filling rate (%) = Film area × Film thickness × 100 / (Volume inside the housing - Volume of the inner core) ... Equation (2)

[0073] In the filter unit 300 of the fourth embodiment of the present invention, it is preferable to satisfy the following condition E from the viewpoint of increasing the effective area of ​​the separation functional layer and pressure loss inside the inner core. Condition E: 50 ≥ SA / IA > 1 SA: Surface area of ​​the separation functional layer a on the inlet side of the cylindrical housing IA: Inner surface area of ​​the cylindrical core at the outlet side of the cylindrical housing

[0074] Furthermore, it is even preferable if the following condition EE is met. Condition EE: 30 ≥ SA / IA ≥ 10

[0075] In the filter unit 300 of the fourth embodiment of the present invention, the filter (101) having separation functional layer a, the filter (102) having separation functional layer b, and the filter (103) having separation functional layer c, all located within a cylindrical housing 205 containing separation functional layers a, b, and c, may be laminated without joining or separation and then wound around a cylindrical inner core 111 in a pleated manner to form pleats, thereby forming a pleated nonwoven fabric. When forming pleats, from the viewpoint of increasing the effective area of ​​the separation functional layer and suppressing clogging, it is preferable that the height of the pleats be 12 mm or more and 30 mm or less, and the number of pleats be 30 or more and 70 or less. Furthermore, it is more preferable that the height of the pleats be 12 mm or more and 20 mm or less, and the number of pleats be 50 or more and 70 or less.

[0076] In the filter unit 300 of the fourth embodiment of the present invention, the cylindrical housing 205 containing the separation function layers a, b, and c preferably satisfies the following condition F from the viewpoint of processing speed and clogging suppression. Condition F: 4000 ≥ ALV / ILV ≥ 30 ALV: Linear velocity in the separation function layer a on the inlet side of the cylindrical housing. ILV: Linear velocity in the inner core of the cylindrical housing on the outlet side.

[0077] Furthermore, it is more preferable that the cylindrical housing 205 containing the separation function layers a, b, and c satisfy the following condition FF. Condition FF: 1000 ≥ ALV / ILV ≥ 100

[0078] In the above embodiment, the combination of housings containing the separation function layer a, separation function layer b, and separation function layer c is merely an example and is not particularly limited.

[0079] The filter unit and purification apparatus of the present invention can be suitably used for the purification of biologically derived substances. Furthermore, the purification apparatus of the present invention can be used for the purification of biologically derived substances, specifically biopharmaceuticals (antibody drugs, gene therapy drugs, nucleic acid drugs, viral vectors, etc.), food components, or beverage components, and is particularly useful for the purification of biopharmaceuticals, including gene therapy drugs or viral vectors.

[0080] In particular, it can be suitably used for crude purification applications, such as recovering viral vectors from cell lysates containing viral vectors. Furthermore, it can be suitably used for the purification of biological substances in cell culture media that have not undergone lysation.

[0081] The bio-derived substances purified by the purification apparatus of the present invention have their initial turbidity of the treated liquid reduced to less than half, thereby improving the efficiency of the subsequent purification or concentration process.

[0082] When used to recover viral vectors from cell lysates containing viral vectors, from an economic standpoint, a viral vector recovery rate of 80% or higher is preferable, and 90% or higher is more preferable. Furthermore, from the viewpoint of facilitating further purification, the turbidity of the purified solution is preferably 40 NTU or less, more preferably 30 NTU or less, and even more preferably 20 NTU or less. 20 NTU is merely an example and is not limited to this value, as it depends on the turbidity of the solution being treated.

[0083] (Method for producing purified liquid) The present invention provides a method for producing a purified solution containing a biologically derived substance, with an average pore size of D aStep X to obtain a processed liquid X by passing the liquid to be processed through the separation functional layer a having a linear velocity in the range of 0.01 to 1.5 m / hour, and the average pore diameter D b Step Y involves passing a processing liquid X through a separation functional layer b having a linear velocity in the range of 0.001 to 1.5 m / hour to obtain a processing liquid Y, and the average pore diameter D c The process includes a step Z to obtain a purified liquid by passing a processing liquid Y through a separation functional layer c having a linear velocity in the range of 0.0005 to 1.5 m / hour, wherein the average pore size D a and D b It satisfies the following conditions A and B. Condition A: 80 μm ≥ D a >D b >>D c ≥1μm Condition B: 6.5≧D a / D b ≥1.5

[0084] In the method for producing the purified liquid of the present invention, more specifically, the average pore size D a Thickness T is 18-80 μm. a Step X involves passing a cell lysate through a separation functional layer a, which has a diameter of 0.2 mm or more, to obtain a processed solution X, and the average pore size D b Thickness T is 6-17 μm. b Step Y involves passing the processing liquid X through the separation functional layer b, which has a diameter of 0.2 mm or more, to obtain the processing liquid Y, and the average pore size D c Thickness T is 1-5 μm. c The method for producing a purified liquid containing biologically derived substances comprises a step Z of passing a processing liquid Y through a separation functional layer c having a thickness of 0.2 mm or more to obtain a purified liquid.

[0085] In the above process, each separation functional layer may be housed in a separate housing, or two or more may be housed in the same housing. The shape of the housing is not particularly limited, but cylindrical, disc-shaped (piece-shaped), cartridge-shaped, or capsule-shaped housings are preferred, with cylindrical housings being preferred. Furthermore, in order to efficiently carry out the air venting process, it is more preferable that the housing has an air vent or air trap structure.

[0086] The "air venting process" is a process performed to remove air (bubbles) that has accumulated inside the housing. This process reduces the contact area between the liquid being processed and the filter, preventing a decrease in processing efficiency and a sudden increase in pressure. While the air venting process is not mandatory, it is preferable to perform it from the viewpoint of processing efficiency. The air venting process is particularly important in housings that contain hollow fiber membranes. When a hollow fiber membrane is contained in the housing, it is preferable to position the housing so that the inlet side is at the top in the direction of gravity and the outlet side is at the bottom in the direction of gravity, and then supply liquid from the inlet side to remove the air that has accumulated in the cylindrical housing. The liquid supplied in the air venting process is not particularly limited, but pure water or the same solvent as the liquid being processed is preferred.

[0087] In order to prevent air generated during liquid flow from accumulating inside the housing and to move it to the upper part of the housing, in step X, if the separation function layer a is built into the housing, it is preferable to pass the processing liquid X through the housing with the housing positioned such that the inlet side is on the upper side in the direction of gravity and the outlet side is on the lower side in the direction of gravity. In step Y, if the separation function layer b is built into the housing, it is preferable to pass the processing liquid X through the housing with the housing positioned such that the inlet side is on the upper side in the direction of gravity and the outlet side is on the lower side in the direction of gravity. In step Z, if the separation function layer c is built into the housing, it is preferable to pass the processing liquid Y through the housing with the housing positioned such that the inlet side is on the upper side in the direction of gravity and the outlet side is on the lower side in the direction of gravity.

[0088] Here, "the housing is positioned such that the upper side is in the direction of gravity and the outlet side is in the direction of gravity" means that when the inlet and outlet are positioned at the top and bottom of the central part of the housing, the arrangement of the inlet and outlet is within the range of 0 to 89 degrees, where 0 degrees is parallel to the direction of gravity and 90 degrees is perpendicular. Preferably, in all housings containing the separation function layer a, separation function layer b, and separation function layer c, the arrangement of the inlet and outlet of the housing is parallel to the direction of gravity.

[0089] As for the liquid flow method, the dead-end filtration method is preferred from the viewpoint of shortening the processing time. When purifying a large amount of liquid to be processed over a long period of time, the tangential flow filtration (TFF) method is preferred from the viewpoint of extending the lifespan of the membrane.

[0090] Below, we will specifically describe an example of a method for producing purified adeno-associated virus from a high-cell-density suspension.

[0091] A high-cell-density suspension containing a high concentration of adeno-associated virus can be obtained, for example, by infecting a high-cell-density suspension obtained by the method disclosed in International Publication 2010 / 060719 with adeno-associated virus using a known method.

[0092] Generally, when purifying adeno-associated virus particles from a high-cell-density suspension, there is a step of lysing most of the cells in the cell suspension to obtain a cell lysate. The lysation step can be, for example, freeze-thaw, solid shear, hypertonic and / or hypotonic dissolution, liquid shear, sonication, high-pressure extrusion, dissolution with a surfactant, or a combination thereof, and the means are not limited. Among these, the method using a dissolving surfactant is preferred because it is simple and easy to measure. Examples of surfactants include Tween 20 and Triton and / or polysorbate 80. In this application, in order to reduce the effects of changes over time after preparation, the amount of cell lysate used for testing was individually frozen after preparation and thawed before passing through the solution to ensure uniform conditions during passage.

[0093] The average pore size D of the cell lysate (treated solution) obtained in the dissolution process a Step X is performed to obtain a processed liquid X by passing the liquid to be processed through the separation functional layer a, which has the following properties, at a linear velocity in the range of 0.01 to 1.5 m / hour. Next, the average pore size D b Step Y is performed to obtain a processed solution Y by passing the processing solution X through the separation functional layer b, which has the above average pore size D, at a linear velocity in the range of 0.001 to 1.5 m / hour. Finally, step Z is performed to obtain a purified solution by passing the processing solution Y through the separation functional layer c, which has the above average pore size D a , D b and D c By going through steps X, Y, and Z, which satisfy the following conditions A and B, adeno-associated viruses can be purified with high efficiency. Condition A: 80 μm ≥ D a >D b >D c ≥1μm Condition B: 6.5≧D a / D b ≥1.5

[0094] More preferably, the cell lysate (treated solution) obtained in the dissolution step is given an average pore size D a Thickness T is 18-80 μm. a Step X involves passing the liquid through a separation functional layer a, which has a diameter of 0.2 mm or more, to obtain a processed liquid X, and the average pore size D b Thickness T is 6-17 μm. b Step Y involves passing the processing liquid X through the separation functional layer b, which has a diameter of 0.2 mm or more, to obtain the processing liquid Y, and the average pore size D c By passing a processing solution Y through a separation functional layer c, which is 1-5 μm thick and 0.2 mm or thicker, to obtain a purified solution (step Z), adeno-associated viruses can be purified with high efficiency.

[0095] The purified product obtained by the method for producing the purified solution of the present invention can be used as a biological substance, specifically a biopharmaceutical (antibody drug, gene therapy drug, nucleic acid drug, viral vector, etc.), a food component, or a beverage component, particularly as a vector or gene therapy drug. [Examples]

[0096] The filter unit and manufacturing method of the present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0097] <Measuring the thickness of nonwoven fabric> For test specimens of nonwoven fabric cut to 250 mm x 250 mm, the thickness (mm) of the center of each side was measured using a dial thickness gauge, and the average value was calculated from the obtained values. The thickness values ​​of the test specimens were rounded to the third decimal place.

[0098] <Measuring the basis weight of nonwoven fabrics> Three test pieces were taken from nonwoven fabric cut to 250 mm x 250 mm. The mass of each test piece was measured using an electronic balance, and the average value was calculated. This average value was then multiplied by 16 to calculate the result. The mass of each test piece was rounded to the first decimal place.

[0099] <Measuring the fiber diameter of nonwoven fabrics> The surface of the nonwoven fabric was observed at a magnification of 3000x using a scanning electron microscope (SEM, model S-5500, manufactured by Hitachi High-Technologies Corporation), and photographs were taken. Using the electron microscope's length-measuring function, the fiber diameter (μm) of 20 fibers that were in focus and not fused was measured, and the average value was calculated to determine the average fiber diameter. The individual fiber diameter values ​​were rounded to two decimal places.

[0100] <Measurement of average pore size of nonwoven fabric> The pore size distribution was calculated based on ASTM-316-86 using an automatic pore size distribution analyzer (model: CFP-1200AEXCS, manufactured by Porous Materials, Inc.). Specifically, nonwoven fabric cut to a size of 25 mm square or larger was used as a test specimen, and when lamination was performed, the nonwoven fabric sheets were cut so that they were all facing the same direction. A dry test specimen was placed, and the air pressure applied to one side was gradually increased to measure the dry flow curve, which shows the relationship between pressure and flow rate when air permeates through the dry test specimen. At this time, the pressure at which air began to permeate through the dry test specimen was defined as P1. Next, a half-dry flow curve was created based on the above dry flow curve, with the permeation flow rate set to 1 / 2. Then, after immersing the above test specimen in a reagent (GALWICK, surface tension 15.9 dyn / cm = 15.9 mN / m), the same measurement was performed to obtain a wet flow curve.

[0101] The average pore size was calculated using the following equation (3) from the pressure P2 at the intersection of the half-dry flow curve and the wet flow curve, and the differential pressure Pc between P1 and P2. Average pore diameter (μm)=Cr / Pc...Equation (3) C: Pressure constant (2860) r: Surface tension of the liquid (15.9 mN / m) Pc: Differential pressure (P2 - P1, Pa) Note that the average pore size value was rounded to two decimal places.

[0102] <Measurement of average porosity of nonwoven fabric> Dry test pieces were held in a holder and observed using a high-resolution 3DX-ray microscope (model: nano3DX, Rigaku Holdings Co., Ltd.) under the following conditions: X-ray source: Cu, tube voltage: 40kV, tube current: 30mAm, resolution: 0.63μm / voxel. Images were acquired, and ambient occlusion was calculated using image processing software (Avizo (ThermoFisherScientific)). The contour was extracted along the morphology of the sample surface, and the average porosity inside the sample was calculated.

[0103] <Preparation of cell disruption solution> The suspended HEK293 cells had a cell density of 1.0 × 10⁶ 6 Cells were cultured for 3 days in a bioreactor (CR0003L200, Merck) to a cell / mL concentration. A plasmid encoding the adeno-associated virus gene was transfected using a gene transfer reagent, and the culture was continued to produce the virus. After 72 hours, Triton-X100 and endonuclease were added, and the mixture was stirred for 1 hour to obtain a cell lysate containing adeno-associated virus.

[0104] <Evaluation of filterability of cell disruption solution> The flow rate is 0.24 mL / min / cm² relative to the contact area of ​​the separation functional layer a. 2 A pump configured to the specified settings was connected to the inlet of the filter unit, and a pressure measuring unit was connected between the pump and the filter unit. 200 mL of cell lysate was then filtered, and the pressure rise was measured. If the pressure rise exceeded 100 kPa, filtration was stopped, and it was determined that clogging had occurred. Furthermore, the processing volume (L / m³) was measured relative to the contact area of ​​the separation functional layer a. 2 The following tests were conducted on the purified solution obtained before clogging occurred: "turbidity measurement" and "viral vector recovery rate measurement," which will be described later.

[0105] <Turbidity Measurement> Each processed solution was measured three times using a digital turbidimeter (TBD700, manufactured by AS ONE Corporation), and the average value was calculated from the obtained values. The individual turbidity values ​​were rounded to two decimal places.

[0106] <Measurement of viral vector recovery rate> Using an adeno-associated virus vector titer determination kit (AAVpo Titration Kit (for Real Time PCR), manufactured by Takara Bio Inc.), the cell lysates and purified solutions were measured, and the viral vector recovery rate was calculated from the obtained values ​​using the following formula (4).

[0107] Viral vector recovery rate (%) = Viral titer of purified solution (VG / mL) / Viral titer of cell lysate (VG / mL) × 100 ... Equation (4) Note that the individual viral vector recovery rates were rounded to the first decimal place.

[0108] The nonwoven fabric used in this invention can be manufactured by, for example, melt spinning, dry spinning, wet spinning, or direct spinning (meltblown, spunbond, electrostatic spinning, etc.). The type of fibers constituting the nonwoven fabric and the manufacturing method are not particularly limited, and known fibers and manufacturing methods can be used. For example, a meltblown nonwoven fabric can be manufactured by melt-extruding a thermoplastic resin, spinning it from a meltblown spinneret, blow-spinning it as a fiber stream with a high-temperature, high-speed gas, collecting the fibers as a web with a collection device, heat-treating the resulting web, and thermally fusing the fibers together. The high-temperature, high-speed gas used in meltblown spinning is usually an inert gas such as air or nitrogen gas. The gas temperature is generally in the range of 200 to 500°C and the pressure is generally in the range of 0.1 to 6.5 kgf / cm2. Furthermore, the separation functional layer c is preferably manufactured by the meltblown method from the viewpoint that an ultrafine fiber nonwoven fabric layer can be easily and densely formed.

[0109] <Preparation of meltblown nonwoven fabric> Using polypropylene (Achieve™6936G2, manufactured by Exxon Mobil) as the raw material, a melt-blown nonwoven fabric manufacturing apparatus was used. The die temperature was set to 200°C, and the discharge rate per hole of the 0.15 mm diameter spinning nozzle was 0.0075 g / min. Heated and compressed air (temperature: 175°C, discharge rate per unit area: 57 Nm³) was discharged from both sides of the spinning nozzle of the above manufacturing apparatus. 3 / sec / m 2 The ) is sprayed onto the device, and the yarn is spun into a collection device 100 mm away from the spinning nozzle, with a basis weight of approximately 15 g / m². 2 A melt-blown nonwoven fabric C1 (average fiber diameter 0.72 μm) was obtained.

[0110] [Example 1] Nonwoven fabric A1 (average fiber diameter 4.3 μm, basis weight 20 g / m²) is used as the separation functional layer a. 2Three layers of nonwoven fabric B1 ("Toraymicron" (registered trademark), EM02010, manufactured by Toray Industries) are laminated without gaps as separation functional layer b, and four layers of nonwoven fabric C1 are laminated without gaps as separation functional layer c. Separation functional layer a is placed on the inlet side and separation functional layer c is placed on the outlet side, and with separation functional layer a, separation functional layer b, and separation functional layer c laminated without gaps in that order, the diameter is 47 mm (effective film area 1380 mm²). 2 A filter unit 1 was fabricated, which was built into the housing of the ). The average porosity of the nonwoven fabrics was 77.7% for nonwoven fabric A1, 81.1% for nonwoven fabric B1, and 86.6% for nonwoven fabric C1.

[0111] For the production of the purified liquid using filter unit 1, an air removal process was performed before the liquid was passed through, and the liquid to be treated was passed through from the inlet side to produce the purified liquid. The linear velocities for each separation functional layer during production were 0.14 m / Hour for separation functional layer a, 0.14 m / Hour for separation functional layer b, and 0.14 m / Hour for separation functional layer c.

[0112] [Example 2] A filter unit 2 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without gaps, except that six nonwoven fabric A1 sheets were stacked without gaps as the separation function layer a. The production of the purified liquid using filter unit 2 was also carried out in the same manner as in Example 1.

[0113] [Example 3] Nonwoven fabric A2 (average fiber diameter 12.1 μm, basis weight 9 g / m²) is used as the separation functional layer a. 2 A filter unit 3 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in the same order without gaps, except that 10 layers of the ) were stacked without gaps. The production of the purified liquid using the filter unit 3 was also carried out in the same manner as in Example 1.

[0114] [Example 4] A filter unit 4 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without separation, except that two nonwoven fabric A1 layers were stacked without separation to form separation function layer a. The production of the purified liquid using filter unit 4 was also carried out in the same manner as in Example 1.

[0115] [Example 5] The separation functional layer b consists of nonwoven fabric B2 (average fiber diameter 2.0 μm, basis weight 30 g / m²). 2 A filter unit 5 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in the same order without any gaps between them, except that two layers of the ) were stacked without any gaps between them. The production of the purified liquid using the filter unit 5 was also carried out in the same manner as in Example 1.

[0116] [Example 6] A filter unit 6 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without gaps, except that two nonwoven fabric B1 layers were stacked without gaps to form separation function layer b. The production of the purified liquid using the filter unit 6 was also carried out in the same manner as in Example 1.

[0117] [Example 7] A filter unit 7 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without gaps, except that 10 sheets of nonwoven fabric C1 were stacked without gaps as separation function layer c. The production of the purified liquid using the filter unit 7 was also carried out in the same manner as in Example 1.

[0118] [Example 8] A filter unit 8 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without separation, except that two nonwoven fabric C1 layers were stacked without separation to form separation function layer c. The production of the purified liquid using the filter unit 8 was also carried out in the same manner as in Example 1.

[0119] [Example 9] Each roll of raw material (50 cm wide x 50 m long) was loaded into a reciprocating folding machine (model 3471, manufactured by Hoptech Co., Ltd.) so that it was laminated in the following order: one porous polypropylene support material, three nonwoven fabrics A1 laminated without spacing to form separation functional layer a, three nonwoven fabrics B1 laminated without spacing to form separation functional layer b, four nonwoven fabrics C1 laminated without spacing to form separation functional layer c, and one polypropylene support material. This formed pleats with a height of 12 mm, and pleated nonwoven fabric was produced.

[0120] The pleated nonwoven fabric was formed into a cylindrical shape with 60 pleats, with the separation functional layer a on the outside and the separation functional layer c on the inside, and its ends were joined with molten polypropylene to form a cylindrical filter.

[0121] A cylindrical filter is built into a cylindrical housing approximately 80mm in diameter and 120mm in length (the inside of the housing is cylindrical with a diameter of 60mm and a length of 75mm, and the inside of the inner core is cylindrical with a diameter of 30mm and a length of 75mm) of filter unit 9 (effective film area 108,000 mm²). 2 ) was created.

[0122] For the production of the purified solution using filter unit 9, an air removal process was performed before passing the liquid through, and the liquid to be treated was passed through from the inlet side to produce the purified solution. The linear velocities for each separation functional layer during production were 0.14 m / Hour at separation functional layer a, 0.14 m / Hour at separation functional layer b, and 0.14 m / Hour at separation functional layer c. In Example 9, the filterability of the cell lysate was evaluated using 15,000 mL of cell lysate.

[0123] [Example 10] Three layers of nonwoven fabric A1 are laminated without gaps to form separation functional layer a, three layers of nonwoven fabric B1 are laminated without gaps to form separation functional layer b, and four layers of nonwoven fabric B1 are laminated without gaps to form separation functional layer c. Separation functional layer a is placed on the inlet side and separation functional layer c is placed on the outlet side, and with separation functional layer a, separation functional layer b, and separation functional layer c laminated without gaps in that order, the diameter is 47 mm (effective film area 1380 mm²). 2The nonwoven fabric was housed in a casing, and an aqueous solution of vinylpyrrolidone / vinyl propanoate random copolymer (40% mole fraction of vinyl propanoate units, number average molecular weight 16,500) dissolved in ethanol at a concentration of 100 ppm and 200 ppm was passed through the casing's inlet to coat the entire nonwoven fabric. Subsequently, 25 kGy of gamma rays was irradiated to produce a filter unit 10 containing nonwoven fabrics A3, B3, and C2 coated with vinylpyrrolidone / vinyl propanoate random copolymer. The production of the purified solution using the filter unit 10 was carried out in the same manner as in Example 1.

[0124] [Comparative Example 1] A filter unit 11 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in that order without any gaps between them, except that three sheets of nonwoven fabric A2 were used as the separation functional layer a. The production of the purified liquid using the filter unit 11 was also carried out in the same manner as in Example 1.

[0125] [Comparative Example 2] A filter unit 12 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in that order without any gaps between them, except that one nonwoven fabric B1 was used as the separation functional layer a. The production of the purified liquid using the filter unit 12 was also carried out in the same manner as in Example 1.

[0126] [Comparative Example 3] A filter unit 13 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without separation, except that six sheets of nonwoven fabric A1 were stacked without separation as the separation function layer b. The production of the purified liquid using the filter unit 13 was also carried out in the same manner as in Example 1.

[0127] [Comparative Example 4] A filter unit 14 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in that order without any gaps between them, except that one nonwoven fabric C1 was used as the separation functional layer b. The production of the purified liquid using the filter unit 14 was also carried out in the same manner as in Example 1.

[0128] [Comparative Example 5] A filter unit 15 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in that order without any gaps between them, except that one nonwoven fabric B1 was used as the separation functional layer b. The production of the purified liquid using the filter unit 15 was also carried out in the same manner as in Example 1.

[0129] [Comparative Example 6] A filter unit 16 was prepared in the same manner as in Example 1, with separation function layer a, separation function layer b, and separation function layer c stacked in the same order without separation, except that two nonwoven fabric B2 layers were stacked without separation to form separation function layer c. The production of the purified liquid using the filter unit 16 was also carried out in the same manner as in Example 1.

[0130] [Comparative Example 7] A filter unit 17 was fabricated in the same manner as in Example 1, by stacking the separation function layers a, b, and c in the order of separation function layer c without any gaps, except that one PES membrane filter (ULTA Disc HC, 47 mm, 0.6 μm / 0.2 μm, manufactured by Cytiva) was used as the separation function layer c, with the 0.6 μm side facing the separation function layer b and the 0.2 μm side facing the outlet. The production of the purified solution using the filter unit 17 was also carried out in the same manner as in Example 1.

[0131] [Comparative Example 7] A filter unit 18 was prepared in the same manner as in Example 1, with separation functional layer a, separation functional layer b, and separation functional layer c stacked in that order without any gaps between them, except that one nonwoven fabric C1 was used as the separation functional layer c. The production of the purified liquid using the filter unit 18 was also carried out in the same manner as in Example 1.

[0132] The results of evaluating the filterability of the cell lysate, measuring turbidity, and measuring the viral vector recovery rate using the obtained filter units 1 to 18 are shown in Tables 1, 2, 3, and 4.

[0133] [Table 1]

[0134] [Table 2]

[0135] [Table 3]

[0136] [Table 4]

[0137] As shown in the results of Example 1, by using the filter unit of the present invention, it was possible to obtain a purified solution with high efficiency and high precision from a cell lysate containing a viral vector while suppressing the pressure rise.

[0138] In Example 10, by coating all three separation functional layers—a, b, and c—with a vinylpyrrolidone / vinyl propanoate random copolymer, it was possible to further suppress the pressure increase and recover the viral vector with even greater efficiency when obtaining a purified solution from a cell lysate containing a viral vector, compared to Example 1.

[0139] In Comparative Example 1, clogging occurred. This is presumed to be because the nonwoven fabric A2 used in the separation functional layer a had a large average pore diameter and was thin, causing clogging of the separation functional layer b by substances that passed through without being removed in the separation functional layer a. Furthermore, a decrease in the viral vector recovery rate was observed due to adhesion to the clogging substances.

[0140] In Comparative Example 2, clogging occurred. This is presumed to be because the average pore size of the nonwoven fabric B1 used in the separation functional layer a was small, causing clogging in the separation functional layer a. Furthermore, a decrease in the viral vector recovery rate was observed due to adhesion to the clogging material.

[0141] In Comparative Example 3, clogging occurred. This is presumed to be because the average pore size of nonwoven fabric A1 used in the separation functional layer b was large, causing clogging in the separation functional layer c. Furthermore, a decrease in the viral vector recovery rate was observed due to adhesion to the clogging material.

[0142] In Comparative Example 4, clogging occurred. This is presumed to be because the average pore size of the nonwoven fabric C1 used in the separation functional layer b was small, causing clogging in the separation functional layer b. Furthermore, a decrease in the viral vector recovery rate was observed due to adhesion to the clogging material.

[0143] In Comparative Example 5, clogging occurred. This is presumed to be because the nonwoven fabric B1 used in the separation functional layer b was too thin, causing clogging in that layer. Furthermore, a decrease in the viral vector recovery rate was observed due to adhesion to the clogging material.

[0144] In Comparative Example 6, the entire cell lysate could be processed, but because the average pore size of the nonwoven fabric B2 used in the separation function c was large, the removal performance of the separation function c was insufficient, and the reduction of turbidity in the purified solution was insufficient.

[0145] In Comparative Example 7, clogging occurred. This is presumed to be because the average pore size of the PES membrane filter used in the separation functional layer c was small, causing clogging in the separation functional layer c. Furthermore, a decrease in the viral vector recovery rate was observed due to adhesion to the clogging material.

[0146] In Comparative Example 8, the entire cell lysate could be processed, but because the nonwoven fabric C1 used in the separation function c was thin, the removal performance of the separation function c was insufficient, and the reduction of turbidity in the purified solution was inadequate. [Explanation of symbols]

[0147] 100...Housing, 101...Separation function layer a, 102...Separation function layer b, 103...Separation function layer c, 104...Connection part, 105...Pump, 106...Pressure gauge, 110...Cylindrical housing, 111...Cylindrical inner core, 200...Housing with separation function layer a, 201...Housing with separation function layer b, 202...Housing with separation function layer c, 203...Housing with separation function layers a, b, and c, 204...Housing with separation function layers a, b, and c, 205...Cylindrical housing with separation function layers a, b, and c, 300...Filter unit, 301...Purification device

Claims

1. Average pore diameter D ａ A separation functional layer a, having an average pore diameter D ｂ Separation functional layer b having an average pore diameter D ｃ It comprises a separation function layer c having a separation function layer a, and an inlet for introducing the liquid to be processed into the separation function layer a, The average pore diameter D ａ , D ｂ and D ｃ The following conditions A and B are met, A filter unit in which the separation function layer a, the separation function layer b, and the separation function layer c are arranged in that order from the inlet. Condition A: 80 μm ≥ D ａ > D ｂ > D ｃ ≥ 1 μm Condition B: 6.5≧D ａ / D ｂ ≥ 1.5

2. The separation functional layer a portion has a thickness T ａ It has, The separation functional layer b has a thickness T ｂ It has, The thickness T ａ and the thickness T ｂ The filter unit according to claim 1, wherein the upper limits of each are 5 mm or less, and the following conditions C and D are satisfied. Condition C: thick みT ａ ×1000 / Average fine pore diameter D ａ ≧5 Condition D: thick みT ｂ ×1000 / Average fine pore diameter D ｂ ≥15

3. The filter unit according to claim 1 or 2, wherein the separation functional layer c is made of a nonwoven fabric with an average fiber diameter of 0.3 to 1.5 μm.

4. The filter unit according to claim 1 or 2, wherein the separation functional layer a, the separation functional layer b, or the separation functional layer c supports a polymer having a unit selected from the group consisting of a monocarboxylate vinyl ester unit, a vinylpyrrolidone unit, a vinylcaprolactam unit, a vinylacetamide unit, and an acrylamide unit.

5. A filter unit according to claim 1 or 2, for the purification of biologically derived substances.

6. The housing has the aforementioned inlet and outlet, The filter unit according to claim 1 or 2, wherein the housing incorporates a bonded filter formed by joining the separation function layer a, the separation function layer b, and the separation function layer c.

7. The filter unit according to claim 6, the pump, and the pressure gauge are provided, A purification apparatus for purifying biologically derived substances, wherein the pressure gauge is connected to the connection between the pump and the inlet of the filter unit.

8. The purification apparatus according to claim 7, used for the purification of biopharmaceuticals, food components, or beverage components.

9. A biological substance purified by the purification apparatus described in claim 7.

10. Average pore diameter D ａ Step X to obtain a processed liquid X by passing the liquid to be processed through a separation functional layer a having a linear velocity in the range of 0.01 to 1.5 m / hour, Average pore diameter D ｂ Step Y involves passing the processing liquid X through the separation functional layer b having the following properties at a linear velocity in the range of 0.001 to 1.5 m / hour to obtain a processing liquid Y; Average pore diameter D ｃ The process includes a step Z to obtain a purified liquid by passing the processing liquid Y through a separation functional layer c having a linear velocity in the range of 0.0005 to 1.5 m / hour, The average pore diameter D ａ , D ｂ and D ｃ A method for producing a purified liquid containing biologically derived substances that satisfies the following conditions A and B. Condition A: 80 μm ≥ D ａ > D ｂ > D ｃ ≥ 1 μm Condition B: 6.5≧D ａ / D ｂ ≥ 1.5

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