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 an average pore diameter D a and a thickness T a for a separation function layer a part, and an average pore diameter D b and a thickness T b for a separation function layer b part, and an inlet for introducing a liquid to be treated into the separation function layer a part. The average pore diameters D a and D b satisfy the following conditions A and B, and the thicknesses T a and T b have an upper limit of 10 mm or less each and satisfy the following conditions C and D, and are arranged in this order: the separation function layer a part, the separation function layer b part, from the inlet. Condition A: 30 μm ≥ D a > D b ≥ 1 μm Condition B: 6 ≥ D a / D b ≥ 2.5 Condition C: Thickness T a × 1000 / average pore diameter D a ≥ 15 Condition D: Thickness T b × 1000 / average pore diameter D b ≥ 60

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 Initiative] [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 biological substance, specifically a biopharmaceutical (antibody drug, gene therapy drug, nucleic acid drug, virus vector, etc.), a food component or a beverage component, particularly a virus vector, is disposed.

Means for Solving the Problems

[0007] The present invention for solving the above problems has the following configurations (1) to (9). (1) A separation functional layer a portion having an average pore diameter D a and a thickness T a and a separation functional layer b portion having an average pore diameter D b and a thickness T b 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 and D b satisfy the following conditions A and B, and the thicknesses T a and T b have an upper limit of 10 mm or less, respectively, and satisfy the following conditions C and D, and a filter unit in which the introduction port, the separation functional layer a portion, and the separation functional layer b portion are arranged in this order. Condition A: 30 μm ≥ D a > D b ≥ 1 μm Condition B: 6 ≥ D a / D b ≥ 2.5 Condition C: Thickness T a × 1000 / average pore diameter D a ≥ 15 Condition D: Thickness T b × 1000 / average pore diameter D b ≥ 60 (2) The filter unit according to (l), wherein the separation functional layer b portion is made of a nonwoven fabric having an average fiber diameter of 0.3 to 1.5 μm. (3) The filter unit according to (1) or (2), wherein the separation functional layer a or the separation functional layer b 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. (4) A filter unit according to (1) or (2) for the purification of biological substances. (5) The 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 functional layer a and the above-mentioned separation functional layer b are joined, and the filling rate of the bonded filter in the housing is 30% or more and 95% or less. (6) A purification apparatus for purifying biological substances, comprising the filter unit described in (5), 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. (7) The purification apparatus described in (6), used for the purification of biopharmaceuticals, food components, or beverage components. (8) A biological substance purified by the purification apparatus described in (7). (9) Average pore diameter D a and thickness T 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 and thickness T b The process includes step Y, which involves passing the above-mentioned processing liquid X through the separation functional layer b having the above-mentioned average pore diameter D at a linear velocity in the range of 0.001 to 1.5 m / hour to obtain a processing liquid Y, wherein the average pore diameter D a and D b The following conditions A and B are met, and the above thickness T a and T b A method for producing a purified liquid containing biologically derived substances, wherein the upper limits of each are 10 mm or less, and the following conditions C and D are met. Condition A: 30 μm ≥ D a >D b ≥1μm Condition B: 6≧D a / Db ≥2.5 Condition C: Thickness T a ×1000 / average pore diameter D a ≥15 Condition D: Thickness T b ×1000 / average pore diameter D b ≥60 [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 a solution containing cell fragments such as 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 and thickness T a A separation functional layer a and an average pore diameter D b and thickness T bIt comprises a separation functional layer b having a pore size D, and an inlet for introducing the liquid to be processed into the separation functional layer a, wherein the average pore size D a and D b The following conditions A and B are met, and the above thickness T a and T b The upper limits of each are 10 mm or less, and the following conditions C and D are met, and each separation functional layer is arranged in the order of part a and part b from the above-mentioned inlet. Condition A: 30 μm ≥ D a >D b ≥1μm Condition B: 6≧D a / D b ≥2.5 Condition C: Thickness T a ×1000 / average pore diameter D a ≥15 Condition D: Thickness T b ×1000 / average pore diameter D b ≥60

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

[0013] Furthermore, the filter unit includes units in which multiple filters are housed in the same enclosure, as well as units in which multiple enclosures containing individual filters or multiple filters are connected together. Any commonly used connection method may be used for connecting each enclosure, and the connection conditions are arbitrary. Due to its simple structure and ease of maintenance, the filter unit is suitably used as an in-line 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 additional separation functional layers, adsorbents, or other members or support materials used for purification at the inlet and outlet 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 above characteristics, removes undisrupted cells and 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. To satisfy the purpose of these removals, the average pore size D a and D b It satisfies the following conditions A and B, and has a thickness T a and T b The upper limits for each are 10 mm or less, and are set to satisfy the following conditions C and D. Condition A: 30 μm ≥ D a >D b ≥1μm Condition B: 6≧D a / D b ≥2.5 Condition C: Thickness T a ×1000 / average pore diameter D a ≥15 Condition D: Thickness T b ×1000 / average pore diameter D b ≥60

[0017] Furthermore, in order to satisfy the purpose of removal, the separation functional layers a and b are arranged in the order of separation functional layer a followed by separation functional layer b, starting from the inlet through which the liquid to be treated is introduced.

[0018] It is preferable that the separation functional layer a and the separation functional layer b are each composed of fibrous filters. Furthermore, the average porosity of the separation functional layer a and the separation functional layer b 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 of the separation functional layer a is smaller than the average porosity of the separation functional layer b.

[0019] These separation functional 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.

[0020] The average porosity can be measured by the method described later in "Measurement of Porosity of Nonwoven Fabrics".

[0021] Specifically, the separation functional layer a removes undisrupted cells and cell fragments from the cell lysate, with an average pore size of D a Thickness T is 7-30 μm. a The average pore size D is 0.2 mm or larger. From the perspective of balancing separation performance and processing speed, a A thickness of 9-15 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.

[0022] The average pore diameter can be measured by the method described later in "Measurement of Pore Diameter of Nonwoven Fabrics".

[0023] 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.

[0024] The separation functional layer (a) 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 capturing cell fragments 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 2.0 to 2.5 μm is more preferable. The basis weight is 40 to 80 g / m². 2 More preferably, 55-65 g / m 2 That is even more preferable.

[0025] The average fiber diameter can be measured by the method described later in "Measurement of Fiber Diameter of Nonwoven Fabrics".

[0026] 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.

[0027] "Main component" refers to the component that makes up 50% or more of the total mass of the material that constitutes the filter.

[0028] 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 1-6 μm. b The average pore size D is 0.3 mm or larger. From the perspective of balancing separation performance and processing speed, b A thickness of 1 to 4 μm is more preferable. b A thickness of 0.3 to 1.5 mm is more preferable, and 0.4 to 1.0 mm is even more preferable.

[0029] 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.

[0030] The separation functional layer b 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 40 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 0.8 μm is more preferable. In addition, the basis weight is 45 to 80 g / m². 2 More preferably, 55-65 g / m 2 That is even more preferable.

[0031] The average fiber diameter of the separation functional layer b can be controlled by, for example, the amount of resin discharged per spinning nozzle or the amount of air blown per unit area at the nozzle outlet, and the basis weight can be controlled by the amount of resin discharged per spinning nozzle, etc.

[0032] The material constituting the separation functional layer b is preferably a thermoplastic resin. Examples of thermoplastic resins include polyester, polyolefin, polyamide, or polyphenylene sulfide. Among these, the main component of the separation functional layer b is preferably polyolefin, and more preferably polypropylene, which is easier to make into an average fiber diameter.

[0033] 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).

[0034] The separation functional layer b 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.

[0035] The filter unit of the present invention is not particularly limited, but from the viewpoint of suppressing clogging by using a multi-stage filter, it is preferable that the nonwoven fabric used as the separation functional layer a and separation functional layer b have a uniform structure rather than an asymmetric or other non-uniform structure.

[0036] The nonwoven fabric used as the separation functional layer a and separation functional layer b 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 back or front surface of the nonwoven fabric. Furthermore, it is not necessary for the polymer to be supported on all of the separation functional layer a and separation functional layer b, but it may be supported on either the separation functional layer a or the separation functional layer b.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] "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.

[0043] Specific examples of monocarboxylate vinyl ester units in which the hydrocarbon group R is saturated aliphatic include vinyl propanoate units, vinyl pivalate units, vinyl decanoate units, or vinyl methoxyacetate units. Since it is preferable that the hydrophobicity is not too strong, vinyl acetate units (R:CH3), vinyl propanoate units (R:CH2CH3), vinyl butyrate units (R:CH2CH2CH3), vinyl pentanoate units (R:CH2CH2CH2CH3), vinyl pivalate units (R:C(CH3)3), or vinyl hexanoate units (R:CH2CH2CH2CH2CH3) are preferred. Specific examples of monocarboxylate vinyl ester units in which the hydrocarbon group R is aromatic include vinyl benzoate units or their substituted products.

[0044] The presence of polymers containing monocarboxylate vinyl ester units on the surface, back surface, and interior of nonwoven fabrics can be confirmed by combining compositional analysis using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and measurement using X-ray photoelectron spectroscopy (XPS). Specifically, first, compositional analysis using TOF-SIMS allows for the detection of peaks originating from carboxylate ions of the monocarboxylate vinyl ester units. By analyzing the mass (m / z) of these peaks, the structure of the monocarboxylic acid can be identified.

[0045] Compositional analysis by TOF-SIMS revealed, for example, Bi3 as a primary ion species. ++ When detecting secondary negative ions using this method, the peak at m / z = 59.02 is C2H3O2. - This corresponds to acetic acid (aliphatic chain carbon number: 1). Also, the peak at m / z = 73.04 is C3H5O2. - In other words, it corresponds to propanoic acid (aliphatic chain carbon number: 2).

[0046] Further XPS analysis revealed a peak originating from the ester group (COO) of CH x Since it appears at +4.0 to 4.2 eV from the main peak of CC (around 285 eV), it can be seen that the carboxylic acid is forming an ester bond. The XPS measurement angle used is 90°. When measured at a 90° angle, a region up to a depth of approximately 10 nm from the surface is detected.

[0047] From the two measurement results above, it can be confirmed that a polymer containing monocarboxylate vinyl ester units is supported on the surface, back surface, and interior of the nonwoven fabric used as separation functional layer a and separation functional layer b.

[0048] The number-average molecular weight of the polymer containing the monocarboxylate vinyl ester unit is preferably 1,000 to 1,000,000, more preferably 5,000 to 500,000, and even more preferably 10,000 to 100,000, from the viewpoint of sufficiently suppressing protein adhesion and avoiding a decrease in the efficiency of introduction into the hollow fiber membrane. The number-average molecular weight of the homopolymer or copolymer can be measured by gel permeation chromatography.

[0049] The filter unit of the present invention achieves both separation performance and water permeability, with a processing capacity per unit area of ​​contact area and thickness of separation functional layer portion a of 0.8 L / cm². 3 Below, 0.5L / cm in separation functional layer b part 3 It is preferable to use it under the following conditions.

[0050] Furthermore, the filter unit of the present invention has a contact area (m²) that balances separation performance and water permeability. 2 It is preferable to use the product under conditions where the ratio of the material to the thickness of the separation functional layer (m) is 7 or less in the separation functional layer a section and 5 or less in the separation functional layer b section.

[0051] The filter unit of the present invention has a contact area (hereinafter referred to as "S") of the separation functional layer a, from the viewpoint of miniaturizing the filter unit. A The contact area of ​​the separation functional layer b with respect to the (hereinafter referred to as "S B The ratio S of " A / S B It is preferable that the value is between 1 and 10.

[0052] 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 the separation functional layer a and separation functional layer b, thereby enabling miniaturization of the filter unit.

[0053] The filter unit of the present invention will be described in detail below, but the present invention is not limited to these embodiments.

[0054] 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.

[0055] 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 is built into the housing (201) having a filter (102) having a separation function layer b.

[0056] The filter unit of the present invention shown in Figure 1 is an example of a filter unit 300 in which the housings 200 containing the separation function layer a and the housing 201 containing the separation function layer b are connected by a connecting part 103. In this way, the filter unit of the present invention can be configured in a form in which each housing containing each filter is connected.

[0057] In this connected configuration, the separation function layers a and b are arranged in the order of separation function layer a and separation function layer b, starting from the inlet for introducing the liquid to be treated.

[0058] Figure 2 shows a second embodiment of the filter unit 300 of the present invention. Figure 2 shows a housing 100 having an inlet and an outlet, in which a filter (101) having a separation function layer a is placed on the inlet side and a filter (102) having a separation function layer b is placed on the outlet side and joined together, forming a housing 202 with a separation function layer a and b built-in.

[0059] By forming a bonded filter in which the separation function layer a and the separation function layer b 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 in which the separation function layer a and the separation function layer b are joined together. The bonded filter may undergo advanced processing such as pleating or calendering.

[0060] "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.

[0061] A third embodiment of the filter unit 300 of the present invention is shown in Figure 3. Figure 3 shows a housing 100 having an inlet and an outlet, in which a filter (101) having a separation function layer a is placed on the inlet side and a filter (102) having a separation function layer b is placed on the outlet side, with the two filters stacked without any gap between them, forming a housing (203) housing with separation function layers a and b.

[0062] Since both the separation functional layer a and the separation functional layer b are fibrous filters, the fibers can intertwine and be laminated without separation without the use of spacers or adhesives. This allows for further miniaturization of the entire filter unit and reduces the probability of impurities such as elutes and leaches being generated during the purification process. Therefore, it is preferable that a laminated filter, in which the separation functional layer a and the separation functional layer b are laminated without separation, be housed in the same housing. Furthermore, the laminated filter may undergo advanced processing such as pleating or calendering.

[0063] Figure 4 shows one embodiment of the purification apparatus 301 of the present invention. Figure 4 is an example of a purification apparatus 301 for purifying biological substances, in which a pump 104 and a housing 203 containing separation function layers a and b are connected by a connection part 103, and a pressure gauge 105 is provided at the connection part 103.

[0064] A fourth embodiment of the filter unit 300 of the present invention is shown in Figure 5. In Figure 5, a cylindrical inner core 111 is placed inside a cylindrical housing 110 having an inlet and an outlet, and a filter (101) having a separation function layer a and a filter (102) having a separation function layer b are joined together and wound around the cylindrical inner core 111 to form a cylindrical housing 204 with built-in separation function layers a and b. In the cylindrical housing 204 with built-in separation function layers a and b, the separation function layer a is located on the inlet side and the separation function layer b is located on the outlet side.

[0065] 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 and the separation functional layer b, 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)

[0066] 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 housing size, the filling rate of the separation functional layer a and separation functional layer b, calculated from the following formula (2), is preferably 30% or more and 95% or less, and more preferably 60% or more and 95% or less.

[0067] Furthermore, the filling rate of a filter unit having an inner core shall be calculated using the following formula (2). Filling rate (%) = Film area × Film thickness × 100 / (Volume inside the housing - Volume of the inner core) ... Equation (2)

[0068] In the filter unit 300 of the fourth embodiment of the present invention, the cylindrical housing 204 containing the separation functional layer a and b preferably satisfies the following condition E from the viewpoint of increasing the effective area of ​​the separation functional layer and reducing 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

[0069] Furthermore, the cylindrical housing 204 containing the separation function layers a and b is even more preferable if it satisfies the following condition EE. Condition EE: 30 ≥ SA / IA ≥ 10

[0070] In the filter unit 300 of the fourth embodiment of the present invention, the filter (101) having separation functional layer a and the filter (102) having separation functional layer b within the cylindrical housing 204 containing the separation functional layer a and b may be joined together and then wound around the 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, the height of the pleats is preferably 12 mm or more and 30 mm or less, and the number of pleats is preferably 30 or more and 70 or less. Furthermore, the height of the pleats is more preferably 12 mm or more and 20 mm or less, and the number of pleats is more preferably 50 or more and 70 or less.

[0071] In the filter unit 300 of the fourth embodiment of the present invention, the cylindrical housing 204 containing the separation function layers a and b 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.

[0072] Furthermore, it is more preferable that the cylindrical housing 204 containing the separation function layers a and b satisfy the following condition FF. Condition FF: 1000 ≥ ALV / ILV ≥ 100

[0073] In the above embodiment, the combination of the housing containing the separation function layer a and the separation function layer b is just one example and is not particularly limited.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] When used for the purpose of recovering a viral vector from a cell lysate containing the viral vector, from the viewpoint of facilitating further purification, the turbidity of the solution after purification is preferably 40 NTU or less, more preferably 30 NTU or less, and even more preferably 25 NTU or less. 25 NTU is merely an example and is not limited to this value because it depends on the turbidity of the liquid to be treated.

[0078] (Method for producing a purified solution) The method for producing a purified solution containing a biologically-derived substance of the present invention involves passing a liquid to be treated through a separation functional layer a portion having an average pore diameter D a and a thickness T a at a linear velocity in the range of 0.01 to 1.5 m / Hour to obtain a treatment liquid X in step X, and passing the treatment liquid X through a separation functional layer b portion having an average pore diameter D b and a thickness T b at a linear velocity in the range of 0.001 to 1.5 m / Hour to obtain a treatment liquid Y in step Y. The average pore diameters D a and D b satisfy the following conditions A and B, and the upper limits of the thicknesses T a and T b are each 10 mm or less and satisfy the following conditions C and D. Condition A: 30 μm ≥ D a > D b ≥ 1 μm Condition B: 6 ≥ D a / D b ≥ 2.5 Condition C: Thickness T a × 1000 / average pore diameter D a ≥ 15 Condition D: Thickness T b × 1000 / average pore diameter D b ≥ 60

[0079] In the method for producing a purified solution of the present invention, more specifically, in step X, a cell lysate is passed through a separation functional layer a portion having a pore diameter D a of 7 to 30 μm and a thickness T a of 0.2 mm or more to obtain a treatment liquid X, and in a separation functional layer having a pore diameter D b of 1 to 6 μm and a thickness T bThe method for producing a purified liquid containing a bio-product comprises a step Y of passing a processing liquid X through a separation functional layer b having a thickness of 0.3 mm or more to obtain a processing liquid Y.

[0080] In the above process, each separation functional layer may be housed in a separate housing, or two 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.

[0081] 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.

[0082] 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 above, 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 above, 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.

[0083] 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. In all housings containing the separation function layer a and separation function layer b, it is preferable that the arrangement of the inlet and outlet of the housing is parallel to the direction of gravity.

[0084] 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.

[0085] Below, we will specifically describe an example of a method for producing purified adeno-associated virus from a high-cell-density suspension.

[0086] 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.

[0087] 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 lysing 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 Triton and Triton and / or polysorbate 80.

[0088] The average pore size D of the cell lysate (treated solution) obtained in the dissolution process a and thickness T aStep 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 and thickness T b Step Y is performed to obtain a processing solution Y by passing the processing solution X through the separation functional layer b having the above average pore size D at a linear velocity in the range of 0.001 to 1.5 m / hour. a and D b The following conditions A and B are met, and the above thickness T a and T b The upper limits for each are 10 mm or less, and the following conditions C and D are met. By going through steps X and Y, adeno-associated virus can be purified with high efficiency. Condition A: 30 μm ≥ D a >D b ≥1μm Condition B: 6≧D a / D b ≥2.5 Condition C: Thickness T a ×1000 / average pore diameter D a ≥15 Condition D: Thickness T b ×1000 / average pore diameter D b ≥60

[0089] More preferably, the cell lysate (treated solution) obtained in the dissolution step is given an average pore size D a Thickness T is 7-30 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 1-6 μm. b By passing the processing solution X through the separation functional layer b, which has a thickness of 0.3 mm or more, to obtain processing solution Y, adeno-associated viruses can be purified with high efficiency through step Y.

[0090] 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]

[0091] 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.

[0092] <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.

[0093] <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.

[0094] <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.

[0095] <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 nonwoven fabric 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.

[0096] 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.

[0097] <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.

[0098] <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.

[0099] <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 turbidity was calculated, and the purified liquid obtained before clogging occurred was also subjected to the "turbidity measurement" described later.

[0100] <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.

[0101] <Production 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 2The ) 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 meltblown nonwoven fabric B1 was obtained.

[0102] [Example 1] Three layers of nonwoven fabric ("Toremicron" (registered trademark), EM02010, manufactured by Toray Industries, Inc., hereinafter referred to as "nonwoven fabric A1") are laminated as the separation functional layer a, with a diameter of 47 mm (effective film area of ​​1380 mm²). 2 It is housed in a casing of the following size, with four layers of nonwoven fabric B1 laminated as the separation function layer b, and has a diameter of 47 mm (effective film area 1380 mm²). 2 A filter unit 1 was fabricated by connecting the housings of the nonwoven fabrics in the order of separation function layer a and separation function layer b. The average porosity of the nonwoven fabrics was 81.1% for nonwoven fabric A1 and 86.6% for nonwoven fabric B1.

[0103] For the production of the purified liquid using filter unit 1, the liquid to be treated was passed through the housing containing separation function layer a, followed by the housing containing separation function layer b, in that order to produce the purified liquid. Before passing the liquid through the housings containing separation function layer a and separation function layer b, an air removal process was performed. The linear velocity for each separation function layer during production was 0.14 m / hour for separation function layer a and 0.14 m / hour for separation function layer b.

[0104] [Example 2] A filter unit 2 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation functional layer a and separation functional layer b, except that two nonwoven fabrics A1 were laminated as the separation functional layer a.

[0105] For the production of the purified liquid using filter unit 2, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0106] [Example 3] A filter unit 3 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation functional layer a and separation functional layer b, except that three layers of nonwoven fabric B1 were laminated as the separation functional layer b.

[0107] For the production of the purified liquid using filter unit 3, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / hour for separation functional layer a and 0.14 m / hour for separation functional layer b.

[0108] [Example 4] 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, separation functional layer a made by laminating three nonwoven fabrics A1, separation functional layer b made by laminating four nonwoven fabrics B1, and one polypropylene support material. After separation functional layer a and separation functional layer b, with a peak height of 12 mm, were joined together, pleats were formed to produce pleated nonwoven fabric.

[0109] The pleated nonwoven fabric was formed into a cylindrical shape with 60 pleats, nonwoven fabric A1 on the outside and nonwoven fabric B1 on the inside, and the ends were joined with molten polypropylene to create a cylindrical filter.

[0110] A cylindrical filter is built into a cylindrical housing approximately 80 mm in diameter and 120 mm in length (the inside of the housing is cylindrical with a diameter of 60 mm and a length of 75 mm, and the inside of the inner core is cylindrical with a diameter of 30 mm and a length of 75 mm), and the filter unit 4 consists of a housing containing separation function layer a and separation function layer b (effective film area 108,000 mm²). 2 ) was created.

[0111] For the production of the purified solution using filter unit 4, the liquid to be treated was passed through separation functional layer a and then separation functional layer b in that order to produce the purified solution. Before passing the liquid through the housings containing separation functional layer a and separation functional layer b, an air removal process was performed. The linear velocity for each separation functional layer during production was 0.14 m / Hour for separation functional layer a and 0.14 m / Hour for separation functional layer b. In Example 3, the filterability of the cell lysate was evaluated using 15,000 mL of cell lysate.

[0112] [Example 5] Three layers of nonwoven fabric A1 are laminated together, with a diameter of 47 mm (effective film area 1380 mm²). 2 The device 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 50 ppm and 200 ppm was passed through the casing's inlet and outlet to coat the entire nonwoven fabric. Subsequently, 25 kGy of gamma rays was irradiated, and the nonwoven fabric A2 coated with vinylpyrrolidone / vinyl propanoate random copolymer was designated as the separation functional layer a.

[0113] Four layers of nonwoven fabric B1 are laminated as the separation functional layer b, with a diameter of 47 mm (effective film area 1380 mm²). 2 The device 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 50 ppm and 200 ppm was passed through the casing's inlet and outlet to coat the entire nonwoven fabric. Subsequently, 25 kGy of gamma rays was irradiated, and the nonwoven fabric B2 coated with vinylpyrrolidone / vinyl propanoate random copolymer was designated as the separation functional layer b.

[0114] Apart from the above, a filter unit 5 was fabricated by connecting the housings in the order of separation function layer a and separation function layer b, similar to Example 1.

[0115] For the production of the purified liquid using filter unit 5, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0116] [Comparative Example 1] A filter unit 6 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation functional layer a and separation functional layer b, except that one glass fiber filter (ULTA Disc GF, 47 mm, 5.0 μm, manufactured by Cytiva) was used as the separation functional layer a.

[0117] For the production of the purified liquid using filter unit 6, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0118] [Comparative Example 2] A filter unit 7 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation functional layer a and separation functional layer b, except that one sheet of "Akstar" (registered trademark, G2200-1S BK0, manufactured by Toray Industries, Inc., hereinafter referred to as "nonwoven fabric A2") was used as the separation functional layer a.

[0119] For the production of the purified liquid using filter unit 7, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0120] [Comparative Example 3] One PES membrane filter (ULTA Disc HC, 47 mm, 0.6 μm / 0.2 μm, manufactured by Cytiva) is used as the separation functional layer b, with the 0.6 μm side as the separation functional layer a and the 0.2 μm side as the outlet side, with a diameter of 47 mm (effective membrane area 1380 mm²). 2A filter unit 8 was manufactured in the same manner as in Example 1, with the separation function layer a and separation function layer b connected in that order within the housing, except that it was built into the housing.

[0121] For the production of the purified liquid using filter unit 8, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0122] [Comparative Example 4] A filter unit 9 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation functional layer a and separation functional layer b, except that three layers of nonwoven fabric A1 were laminated as separation functional layer b.

[0123] For the production of the purified liquid using filter unit 9, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0124] [Comparative Example 5] A filter unit 10 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation function layer a and separation function layer b, except that one sheet of nonwoven fabric A1 was used as the separation function layer a.

[0125] For the production of the purified liquid using the filter unit 10, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / hour for separation functional layer a and 0.14 m / hour for separation functional layer b.

[0126] [Comparative Example 6] A filter unit 11 was fabricated in the same manner as in Example 1, by connecting the housings in the order of separation function layer a and separation function layer b, except that two nonwoven fabric B1 layers were laminated as separation function layer b.

[0127] For the production of the purified liquid using the filter unit 11, the liquid to be treated was passed through in the same order as in Example 1 to produce the purified liquid. The linear velocity for each separation functional layer during production was 0.14 m / Hour at separation functional layer a and 0.14 m / Hour at separation functional layer b.

[0128] Tables 1 and 2 show the results of evaluating the filterability and measuring the turbidity of the cell lysate using the obtained filter units 1 to 13.

[0129] [Table 1]

[0130] [Table 2]

[0131] 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 from a cell lysate containing a viral vector while suppressing the pressure increase.

[0132] In Example 5, by coating the separation functional layer a and separation functional layer b with a vinylpyrrolidone / vinyl propanoate random copolymer, the pressure increase when obtaining a purified solution from a cell lysate containing a viral vector was further suppressed compared to Example 1.

[0133] In Comparative Example 1, clogging occurred in the separation functional layer a. This is presumed to be because the average pore size of the glass fiber filter used in the separation functional layer a was small.

[0134] In Comparative Example 2, clogging occurred in the separation functional layer b. This is presumed to be because the average pore size of the nonwoven fabric A3 used in the separation functional layer a was large, causing clogging in the separation functional layer b due to substances that passed through without being removed in the separation functional layer a.

[0135] In Comparative Example 3, clogging occurred in the separation functional layer b. This is presumed to be because the average pore size of the PES membrane filter used in the separation functional layer b was small.

[0136] In Comparative Example 4, the entire cell lysate could be processed, but the removal performance was insufficient due to the large average pore size of the nonwoven fabric A1 used in the separation functional layer b, resulting in insufficient reduction of the turbidity of the purified solution.

[0137] In Comparative Example 5, clogging occurred in the separation functional layer b. This is presumed to be because the nonwoven fabric A1 used in the separation functional layer a was thin, causing clogging in the separation functional layer b due to substances that passed through without being removed in the separation functional layer a.

[0138] In Comparative Example 6, although the entire cell lysate could be processed, the removal performance was insufficient due to the thinness of the nonwoven fabric B1 used in the separation functional layer b, resulting in insufficient reduction of the turbidity of the purified solution. [Explanation of Symbols]

[0139] 100...Housing, 101...Separation function layer a, 102...Separation function layer b, 103...Connection part, 104...Pump, 105...Pressure gauge, 110...Cylindrical housing, 111...Cylindrical inner core, 200...Housing with separation function layer a built-in, 201...Housing with separation function layer b built-in, 202...Housing with separation function layers a and b built-in, 203...Housing with separation function layers a and b built-in, 204...Cylindrical housing with separation function layers a and b built-in, 300...Filter unit, 301...Purification device

Claims

1. Average pore diameter D ａ and thickness T ａ A separation functional layer a and an average pore diameter D ｂ and thickness T ｂ It comprises a separation function layer b 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 and D ｂ The following conditions A and B are met, The aforementioned thickness T ａ and T ｂ The upper limits are 10 mm or less each, and the following conditions C and D are met, A filter unit in which the separation function layer a and the separation function layer b are arranged in order from the inlet. Condition A: 30 μm ≥ D ａ > D ｂ ≥ 1 μm Condition B: 6≧D ａ / D ｂ ≥ 2.5 Condition C: thick みT ａ ×1000 / Average fine pore diameter D ａ ≥15 Condition D: thick みT ｂ ×1000 / Average fine pore diameter D ｂ ≥60

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

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

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

5. The housing has the aforementioned inlet and outlet, The housing incorporates a bonding filter in which the separation function layer a and the separation function layer b are joined, The filter unit according to claim 1 or 2, wherein the filling rate of the bonding filter in the housing is 30% or more and 95% or less.

6. The filter unit according to claim 5, 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.

7. The purification apparatus according to claim 6, used for the purification of biopharmaceuticals, food components, or beverage components.

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

9. Average pore diameter D ａ and thickness T 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 and thickness T ｂ The process includes a step Y to obtain a processing liquid Y by passing the processing liquid X through a separation functional layer b having a linear velocity in the range of 0.001 to 1.5 m / hour, The average pore diameter D ａ and D ｂ The following conditions A and B are met, The aforementioned thickness T ａ and T ｂ A method for producing a purified liquid containing biologically derived substances, wherein the upper limits of each are 10 mm or less, and the following conditions C and D are met. Condition A: 30 μm ≥ D ａ >D ｂ ≥ 1 μm Condition B: 6≧D ａ / D ｂ ≥ 2.5 Condition C: thick みT ａ ×1000 / Average fine pore diameter D ａ ≥15 Condition D: thick みT ｂ ×1000 / Average fine pore diameter D ｂ ≥60

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