Porous membrane, method for producing same , and method for removing virus

Abstract

Provided is a porous membrane for removing a virus, which comprises a pre-filter layer and a dense layer, wherein the pre-filter layer has an average degree of anisotropy of 2.0 to 3.0 and the dense layer has an average degree of anisotropy less than 2.0.

Description

Porous membrane, method for producing the same, and method for removing viruses

[0001] The present invention relates to a porous membrane, a method for producing the same, and a method for removing viruses.

[0002] In recent years, the use of plasma-derived products and biopharmaceuticals has become widespread as pharmaceuticals due to their low incidence of side effects and high therapeutic efficacy. However, because plasma-derived products are derived from human blood and biopharmaceuticals are derived from animal cells, there is a risk of contamination of these drugs with pathogenic substances such as viruses.

[0003] To prevent viral contamination of pharmaceuticals, virus removal or inactivation is always performed. Methods for virus removal or inactivation include heat treatment, optical treatment, and chemical treatment. Due to issues such as protein denaturation, the efficiency of virus inactivation, and chemical contamination, membrane filtration methods that are effective against all viruses, regardless of their thermal and chemical properties, are attracting attention.

[0004] Viruses that should be removed or inactivated include poliovirus, which has a diameter of 25-30 nm, and parvovirus, the smallest virus, which has a diameter of 18-24 nm. Relatively large viruses include HIV, which has a diameter of 80-100 nm. In recent years, there has been a growing need to remove small viruses, especially parvovirus.

[0005] The primary performance requirement for a virus removal membrane is safety. Safety includes ensuring that pathogenic substances such as viruses do not contaminate plasma-derived products and biopharmaceuticals, and ensuring that foreign substances such as elutes from the virus removal membrane do not contaminate them. To ensure safety by preventing contamination by pathogenic substances such as viruses, it is important that the virus removal membrane sufficiently removes viruses. Non-patent document 1 states that the target clearance (LRV) for mouse microvirus and porcine parvovirus should be 4, but in recent years, from the perspective of further safety regarding viruses, an LRV of 5 or higher is desired (Non-patent document 2). Furthermore, to ensure safety by preventing contamination by foreign substances such as elutes, it is important that no elutes are released from the virus removal membrane.

[0006] The second performance requirement for virus removal membranes is productivity. Productivity refers to the efficient recovery of proteins such as albumin (5 nm size) and antibodies (10 nm size). Ultrafiltration membranes and hemodialysis membranes with pore sizes of a few nanometers, as well as reverse osmosis membranes with even smaller pore sizes, are unsuitable as virus removal membranes because proteins clog the pores during filtration. In particular, when the goal is to remove small viruses such as parvovirus, it has been difficult to achieve both safety and productivity because the size of the virus and the size of the protein are similar.

[0007] Patent Document 1 discloses a porous membrane containing a hydrophobic polymer and a water-insoluble hydrophilic polymer, which has a dense layer in the downstream part of the membrane, a gradient asymmetric structure in which the average pore diameter increases from the downstream part to the upstream part of the membrane, and a gradient index of the average pore diameter from the dense layer to the coarse layer is 0.5 or more and 12.0 or less.

[0008] International Publication No. 2016 / 031834

[0009] PDA Journal of GMP and Validation in Japan, Vol. 7, No. 1, p. 44 (2005) Kayukawa.T et al., Particle-based analysis elucidates the real retention capacities of virus filters and enables optimal virus clearance study design with evaluation systems of diverse virological characteristics Biotechnol. Prog. 2022, 38, (2), e3237.

[0010] However, in recent years, there has been a growing demand not only for safety and productivity, but also for improved resistance to impurities such as protein aggregates in protein solutions. The present invention aims to provide a porous membrane with excellent protein filtration rate, recovery rate, and virus removal performance.

[0011] A porous membrane having a pre-filter layer having a predetermined anisotropy and a dense layer having a predetermined anisotropy can solve the above problem.

[0012] The present invention includes the following embodiments: [1] A porous membrane for removing viruses, comprising a prefilter layer and a dense layer, wherein the prefilter layer has an average anisotropy of 2.0 or more and 3.0 or less, and the dense layer has an average anisotropy of less than 2.0. [2] The porous membrane according to [1], wherein the prefilter layer is located on the upstream side of the filtration surface than the dense layer. [3] The porous membrane according to [1] or [2], wherein the prefilter layer has a structural size of 40 nm or more and 65 nm or less. [4] The porous membrane according to any one of [1] to [3], wherein the dense layer has a structural size of 8 nm or more and 25 nm or less. [5] The porous membrane according to any one of [1] to [4], wherein the porous membrane comprises a hydrophobic polymer and a water-insoluble hydrophilic polymer. [6] The porous membrane according to [5], wherein the hydrophobic polymer comprises at least one selected from the group consisting of polyolefins, polyamides, polyimides, polyesters, polyketones, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone polymers. [7] The porous membrane according to [5] or [6], wherein the water-insoluble hydrophilic polymer comprises at least one selected from the group consisting of vinyl polymers, polysaccharides, polyethylene glycol and its derivatives, block copolymers of ethylene glycol and hydrophobic monomers, copolymers of ethylene glycol and propylene glycol or ethylbenzyl glycol, hydrophilized polyethylene terephthalate, hydrophilized polyethersulfone, and hydrophilized methacrylate resins. [8] The porous membrane according to any one of [1] to [7], wherein the porous membrane comprises a hydrophobic polymer into which hydrophilic groups have been introduced. [9] The porous membrane according to any one of [1] to [8], wherein the porous membrane is a hollow fiber membrane.

[10] A porous membrane according to any one of [1] to [9], wherein the prefilter layer has a thickness of 5 μm or more and 30 μm or less.

[11] A porous membrane according to any one of [1] to

[10] , wherein the dense layer has a thickness of 2 μm or more and 30 μm or less.

[12] A method for removing a virus, characterized by filtering a protein solution containing a virus with a porous membrane according to any one of [1] to

[11] .

[13] A method for removing a virus, comprising filtering a protein solution containing a virus through the hollow fiber membrane described in [9], wherein the protein solution flows from the inner surface to the outer surface of the hollow fiber membrane.

[14] A method for producing a porous membrane according to any one of [1] to

[11] , comprising: an extrusion step of extruding a film-forming stock solution containing a polymer and a first non-solvent and an internal coagulation liquid containing a second non-solvent in a vertical direction from a spindle, wherein the distance between two points, the Hansen solubility parameter of the polymer and the Hansen solubility parameter of the first non-solvent, is 10.0 or more, the amount of the first non-solvent is 30% by mass or more and 60% by mass or less based on the mass of the film-forming stock solution, and the amount of the second non-solvent is 15% by mass or more and 40% by mass or less based on the mass of the internal coagulation liquid; an air-running step of running the extruded film-forming stock solution in an air-running section, wherein the anisotropic draw ratio is 1.1 or more and 2.0 or less; and a coagulation step of introducing the air-running film-forming stock solution into a coagulation liquid.

[15] The manufacturing method according to

[14] , wherein the film-forming stock solution has a viscosity of 80 Pa·s or more and 110 Pa·s or less at 25°C.

[16] The manufacturing method according to

[14] or

[15] , wherein the coagulation solution contains a third non-solvent, and the amount of the third non-solvent is 70% by mass or more based on the mass of the coagulation solution.

[17] A porous membrane for removing viruses, comprising a pre-filter layer and a dense layer, wherein the average anisotropy of the pre-filter layer is greater than the average anisotropy of the dense layer.

[18] The porous membrane according to

[17] , wherein the ratio of the average anisotropy of the pre-filter layer to the average anisotropy of the dense layer (average anisotropy of the pre-filter layer / average anisotropy of the dense layer) is 1.2 or more and 3.0 or less.

[19] The porous membrane according to

[17] or

[18] , wherein the ratio of the average anisotropy of the prefilter layer to the average anisotropy of the dense layer (average anisotropy of the prefilter layer / average anisotropy of the dense layer) is 1.3 or more and 2.5 or less.

[0013] The present invention can provide a porous membrane with excellent protein filtration and recovery rates, as well as virus removal performance.

[0014] This shows an image of a hollow fiber membrane and a test specimen made from the membrane. This shows the relationship between each position (distance from the outer surface) in the film thickness direction of the porous membrane of Example 1 and the anisotropy corresponding to each position. This shows the relationship between each position (distance from the outer surface) in the film thickness direction of the porous membrane of Example 1 and the structural size corresponding to each position. This shows a method for measuring the change in the outer diameter of the film-forming solution in the empty section when the film-forming solution is extruded from the spindle and introduced into the external coagulation solution. This shows the relationship between each region of the empty section and the outer diameter of the film-forming solution corresponding to each region.

[0015] The embodiments of the present invention will be described in detail below, but the present invention is not limited to these, and various modifications are possible without departing from its essence.

[0016] <Porous Membrane> The present invention relates to a porous membrane for removing viruses, comprising a pre-filter layer and a dense layer, wherein the pre-filter layer has an average anisotropy of 2.0 or more and 3.0 or less, and the dense layer has an average anisotropy of less than 2.0. The present invention also relates to a porous membrane for removing viruses, comprising a pre-filter layer and a dense layer, wherein the average anisotropy of the pre-filter layer is greater than the average anisotropy of the dense layer.

[0017] To purify protein preparations in a short time using porous membranes, high protein permeability and high virus removal capabilities are required simultaneously. However, protein preparations easily form aggregates with even slight stimulation from acid or heat, and these aggregates clog the porous structure inside the membrane, significantly reducing the filtration performance inherent to the porous membrane. In the porous membrane according to this embodiment, a pre-filter layer with a predetermined anisotropy captures aggregates, and then a dense layer with a predetermined anisotropy captures viruses, thereby achieving excellent protein filtration and recovery rates, as well as excellent virus removal performance. Furthermore, the anisotropy of the porous membrane according to this embodiment is measured by small-angle X-ray scattering using a microbeam, as described later. Transmission small-angle X-ray scattering allows for obtaining average structural information in the thickness direction of the test piece (theoretically on the order of millimeters) by transmitting X-rays in the thickness direction of the porous membrane test piece. Compared to the SEM method, which is a two-dimensional image analysis, it provides information as a true three-dimensional structure, including the Y-axis direction (longitudinal direction of the hollow fiber) as shown in Figure 1. Furthermore, by using microbeam X-rays, it is possible to analyze the local structure of the hollow fiber in the film thickness direction, allowing for precise analysis of the structure of specific membrane components that are important for the performance of the membrane, such as the dense layer and the pre-filter layer. By controlling the average anisotropy of the dense layer and pre-filter layer within a predetermined range, it is possible to provide porous membranes with excellent protein filtration rate and recovery rate, as well as virus removal performance, for various types of protein preparations that may induce diverse aggregation phenomena. In addition, by controlling the average anisotropy of the dense layer and pre-filter layer within a predetermined range, it is possible to provide porous membranes with high safety, productivity, and excellent resistance to contaminants such as protein aggregates.

[0018] In this invention, a layer with a structural size of 40 nm to 65 nm obtained by small-angle X-ray scattering is used as the pre-filter layer.

[0019] The porous membrane according to this embodiment has a pre-filter layer. The pre-filter layer is intended to remove protein aggregates contained in the protein solution.

[0020] In this invention, a dense layer is defined as a layer with a structural size of 8 nm or more and 25 nm or less obtained by small-angle X-ray scattering.

[0021] The porous membrane according to this embodiment has a dense layer. The dense layer is intended to remove viruses contained in the protein solution.

[0022] In this specification, “structural size” refers to the correlation length Ξ obtained from fitting the sector-average SAXS profile in the circumferential direction using equation (1) below. Equation (1) is a model sometimes used for structures with smooth interfaces and randomly separated into two phases (see, for example, R.-J. Roe, “Method of X-ray and Neutron Scattering in Polymer Science”, Oxford University Press, New York (2000)). In equation (1), I ０ θ is a constant, t corresponds to the interface thickness, and q is the absolute value of the scattering vector (scattering vector q = 4πsinθ / λ, where λ is the X-ray wavelength and 2θ is the scattering angle).

[0023] The fitting was performed using WaveMetrics' Igor Pro8, with a fitting range of 0.02 < q (nm). -1 ) < 0.4 was set as the initial value for fitting. ０ A value of q = 0.02 nm⁻¹ was used for Ξ, 15 nm for Ξ, and 1 nm for t.

[0024] In this specification, "anisotropy" refers to the anisotropy of the porous structure of a porous membrane. The orientation of the pores allows for shorter, more efficient removal of material, while also enabling a larger pore cross-sectional area, thus maintaining high permeability of protein preparations. The formula for calculating anisotropy will be described later.

[0025] The average anisotropy of the prefilter layer can be determined by arithmetically averaging the anisotropy of the region of the prefilter layer based on a graph (e.g., FIG. 2) showing the relationship between each position (distance from the outer surface) in the film thickness direction of the porous membrane and the anisotropy corresponding to each position, obtained by measuring the anisotropy. The specific determination method is as described in the examples below.

[0026] The average anisotropy of the prefilter layer is 2.0 or more and 3.0 or less, preferably 2.1 or more and 2.9 or less, and more preferably 2.2 or more and 2.8 or less.

[0027] If the average anisotropy of the prefilter layer is 2.0 or more, it means that the minor axis of the pores is small in the structure of the prefilter layer, and aggregates can be efficiently captured. When the average anisotropy of the prefilter layer is 3.0 or less, in the structure of the prefilter layer in the region irradiated with X-rays specified by the small-angle X-ray scattering method, the orientation in the fiber axis direction is moderately suppressed, so that the high strength of the porous membrane can be maintained.

[0028] The average anisotropy of the dense layer can be determined by arithmetically averaging the anisotropy of the dense layer based on a graph (e.g., FIG. 2) showing the relationship between each position (distance from the outer surface) in the film thickness direction of the porous membrane and the anisotropy corresponding to each position, obtained by measuring the anisotropy. The specific determination method is as described in the examples below.

[0029] The average anisotropy of the dense layer is less than 2.0, preferably less than 1.8, and more preferably less than 1.6.

[0030] When the average anisotropy of the dense layer is less than 2.0, the pore shape is close to circular in the structure of the dense layer in the region irradiated with X-rays specified by the small-angle X-ray scattering method, so that the virus can be captured while allowing the protein to pass through.

[0031] [Arrangement of Each Layer] The porous membrane according to the present embodiment has a prefilter layer and a dense layer. These layers are preferably positioned perpendicular or substantially perpendicular to the membrane thickness direction of the porous membrane. Thereby, when the protein solution flows from the filtration upstream surface side to the filtration downstream surface side of the porous membrane, it will pass through the prefilter layer and the dense layer.

[0032] When the protein solution passes through the porous membrane, when the protein solution enters from the first surface of the porous membrane, passes through the interior, and exits from the second surface, the first surface is the filtration upstream surface and the second surface is the filtration downstream surface. On the other hand, when the direction of flowing the protein solution is reversed, the protein solution enters from the second surface and exits from the first surface, so the second surface is the filtration upstream surface and the first surface is the filtration downstream surface. Thus, depending on the direction of flowing the protein solution, the first surface of the porous membrane can be either the filtration upstream surface or the filtration downstream surface. The same applies to the second surface of the porous membrane.

[0033] The prefilter layer is preferably positioned on the filtration upstream surface side rather than the dense layer. That is, it is preferable that the protein solution passes through the dense layer after passing through the prefilter layer. By adopting such an arrangement, it is possible to efficiently remove protein aggregates and viruses.

[0034] The specific position of the prefilter layer is not particularly limited, but in the membrane thickness direction, it is preferably at a position of preferably 30% or more and 95% or less, more preferably 35% or more and 85% or less, still more preferably 40% or more and 75% or less from the filtration downstream surface. For example, when the membrane thickness of the porous membrane is 100 μm, the position of 40% or more and 75% or less from the filtration downstream surface means a position of 40 μm or more and 75 μm or less from the filtration downstream surface toward the filtration upstream surface. When the porous membrane is a hollow fiber membrane, the filtration downstream surface is preferably the outer surface. Note that the prefilter layer being at a predetermined position means that at least a part of the prefilter layer is at that position, and it is preferable that all of the prefilter layer is at that position.

[0035] The specific location of the dense layer is not particularly limited, but it is preferably located at a position 0% to 40% from the downstream surface of the filter in the film thickness direction, more preferably at a position 0% to 35%, and even more preferably at a position 0% to 30%. For example, when the film thickness of the porous membrane is 100 μm, the position 0% to 30% from the downstream surface of the filter means a position 0 μm to 30 μm from the downstream surface of the filter toward the upstream surface of the filter. When the porous membrane is a hollow fiber membrane, the downstream surface of the filter is preferably the outer surface. Note that "the dense layer is in a predetermined position" means that at least a part of the dense layer is in that position, and it is preferable that the entire dense layer is in that position.

[0036] [Materials] In a first embodiment, the porous membrane according to this embodiment preferably contains a hydrophobic polymer and a water-insoluble hydrophilic polymer.

[0037] (Hydrophobic Polymers) In this specification, "hydrophobic polymer" means a polymer in which the contact angle when PBS (9.6 g of Dulbecco PBS (-) powder "Nissui," commercially available from Nissui Pharmaceutical Co., Ltd., is dissolved in water to make a total volume of 1 L) is brought into contact with the polymer film is 80 degrees or more. The contact angle is the angle formed by the surface of a water droplet when it is dropped onto the film surface, and is defined in JIS R3257. The measurement is performed at a temperature of 25°C.

[0038] Examples of hydrophobic polymers include polyolefins, polyamides, polyimides, polyesters, polyketones, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone polymers.

[0039] The hydrophobic polymer is preferably a polysulfone polymer, more preferably an aromatic polysulfone polymer, and even more preferably an aromatic polysulfone polymer having a structural unit represented by the following general formula (1) and / or general formula (2): -O-Ar-C(CH ３ ) ２ -Ar-O-Ar-SO ２ -Ar- (1) -O-Ar-SO ２-Ar- (2) [In the formula, Ar is independently a divalent aromatic group, preferably a phenylene group, and more preferably a p-phenylene group.] Preferred examples of aromatic polysulfone polymers include polysulfones and polyethersulfones.

[0040] The constituent units represented by the above general formulas (1) and (2) may be substituted with substituents. Examples of substituents include alkyl groups and halogens.

[0041] The hydrophobic polymer may be a single type or a combination of two or more types.

[0042] (Hydrophilic polymers) In this specification, "hydrophilic polymers" means those in which the contact angle is measured by the method described in the (hydrophobic polymers) section above and the contact angle is less than 80 degrees.

[0043] The contact angle of the hydrophilic polymer is preferably 60 degrees or less, and more preferably 40 degrees or less. Including a hydrophilic polymer with a contact angle of 60 degrees or less makes the porous membrane more easily wettable with water. Including a hydrophilic polymer with a contact angle of 40 degrees or less makes the porous membrane significantly more easily wettable with water.

[0044] (Water-insoluble hydrophilic polymer) In this specification, "water-insoluble" is measured by the weight change before and after immersion as follows: A dried polymer plate (or film) with an effective surface area of ​​3.3 cm². ２ The material is then weighed. After that, it is immersed in 100 mL of pure water at 100°C for 1 hour and then vacuum dried. The weight of the polymer plate is measured, and if the weight change before and after immersion is 10% or less, it is classified as "insoluble in water".

[0045] In this specification, "water-insoluble hydrophilic polymer" refers to a polymer that satisfies the above-mentioned weight change before and after immersion and contact angle.

[0046] Water-insoluble hydrophilic polymers are preferably electrically neutral from the viewpoint of preventing protein adsorption. "Electrically neutral" means that the molecule has no charge, or that the amount of cations and anions within the molecule is equal.

[0047] Examples of water-insoluble hydrophilic polymers include vinyl polymers, polysaccharides, polyethylene glycol and its derivatives, block copolymers of ethylene glycol and hydrophobic monomers, copolymers of ethylene glycol and propylene glycol or ethylbenzyl glycol, hydrophilized polyethylene terephthalate, hydrophilized polyethersulfone, and hydrophilized methacrylate resins.

[0048] The water-insoluble hydrophilic polymer is preferably a vinyl polymer.

[0049] Examples of vinyl polymers include hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, acrylamide, dimethylacrylamide, glucoxyoxyethyl methacrylate, 3-sulfopropyl methacryloxyethyl dimethylammonium betaine, 2-methacryloyloxyethyl phosphorylcholine, and homopolymers of 1-carboxydimethylmethacryloyloxyethyl methaneammonium.

[0050] Furthermore, examples of vinyl polymers include random copolymers, graft copolymers, and block copolymers of hydrophobic monomers such as styrene, ethylene, propylene, propyl methacrylate, butyl methacrylate, ethylhexyl methacrylate, octadecyl methacrylate, benzyl methacrylate, and methoxyethyl methacrylate, and hydrophilic monomers such as hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, acrylamide, dimethylacrylamide, glucoxyoxyethyl methacrylate, 3-sulfopropyl methacryloxyethyldimethylammonium betaine, 2-methacryloyloxyethyl phosphorylcholine, and 1-carboxydimethylmethacryloyloxyethylmethaneammonium.

[0051] Furthermore, examples of vinyl polymers include copolymers of cationic monomers such as dimethylaminoethyl methacrylate and diethylaminoethyl methacrylate, anionic monomers such as acrylic acid, methacrylic acid, vinyl sulfonic acid, sulfopropyl methacrylate, and phosphooxyethyl methacrylate, and the hydrophobic monomers mentioned above. It is preferable that the cationic monomers and anionic monomers are included in a ratio that is electrically neutral.

[0052] The water-insoluble hydrophilic polymer is preferably a random copolymer of a hydrophilic monomer such as a homopolymer of hydroxyethyl methacrylate, a homopolymer of hydroxypropyl methacrylate, or 3-sulfopropyl methacryloxyethyldimethylammonium betaine, 2-methacryloyloxyethyl phosphorylcholine, or 1-carboxydimethylmethacryloyloxyethylmethaneammonium, and a hydrophobic monomer such as butyl methacrylate or ethylhexyl methacrylate.

[0053] The water-insoluble hydrophilic polymer may be a single type or a combination of two or more types.

[0054] The total amount of hydrophobic polymer and water-insoluble hydrophilic polymer is preferably 80% by mass or more, more preferably 90% by mass or more, even more preferably 95% by mass or more, and particularly preferably 100% by mass, based on the mass of the material constituting the porous membrane.

[0055] (Hydrophilic Group-Introduced Polymer) In a second embodiment, the porous membrane according to this embodiment preferably contains a hydrophobic polymer into which hydrophilic groups have been introduced (hereinafter also referred to as "hydrophilic group-introduced polymer"). A hydrophilic group-introduced polymer is a hydrophobic polymer into which hydrophilic groups have been introduced, thereby giving it hydrophilic properties.

[0056] The amount of hydrophilic group-introduced polymer is preferably 80% by mass or more, more preferably 90% by mass or more, even more preferably 95% by mass or more, and particularly preferably 100% by mass, based on the mass of the material constituting the porous membrane.

[0057] In this specification, "hydrophilic polymer" means a polymer whose contact angle is less than 80 degrees when measured using the same method as the definition of "hydrophobic polymer" described above.

[0058] Examples of hydrophilic group-introduced polymers include those obtained by copolymerizing a water-soluble hydrophilic polymer, which is produced by copolymerizing a monomer having an azide group in its side chain with a hydrophilic monomer such as 2-methacryloyloxyethyl phosphorylcholine, onto a hydrophobic polymer substrate film, and then heat-treating it to covalently bond the water-soluble hydrophilic polymer to the substrate film, thereby making the water-soluble hydrophilic polymer insoluble, and those obtained by graft polymerization of a hydrophilic monomer such as 2-hydroxyalkyl acrylate onto a hydrophobic polymer substrate film.

[0059] The hydrophilic group-introduced polymer may be a single type or a combination of two or more types.

[0060] Methods for hydrophilizing a substrate film include, for example, coating the substrate film with a water-insoluble hydrophilic polymer, grafting, and crosslinking.

[0061] The water-insoluble hydrophilic polymer may be mixed with a hydrophobic polymer (blended film formation). The hydrophilic polymer used in the blended film formation is not particularly limited, as long as it is compatible with the hydrophobic polymer in a good solvent.

[0062] [Shape] The shape of the porous membrane according to this embodiment is not particularly limited as long as it can filter a protein solution, but is preferably a hollow fiber membrane or a flat membrane, and more preferably a hollow fiber membrane.

[0063] When the porous membrane is a hollow fiber membrane, it is preferable that the inner surface is the upstream surface of filtration and the outer surface is the downstream surface of filtration.

[0064] The thickness of the hollow fiber membrane is preferably 30 μm to 80 μm. The inner diameter of the hollow fiber membrane is preferably 200 μm to 400 μm.

[0065] The thickness of the pre-filter layer is preferably 5 μm to 30 μm, more preferably 10 μm to 30 μm, and even more preferably 18 μm to 30 μm.

[0066] When the pre-filter layer thickness is 5 μm or more, it is possible to efficiently remove impurities contained in the filtered solution, and protein flux attenuation tends to be suppressed. When the pre-filter layer thickness is 30 μm or less, it tends to be possible to achieve a balance between permeability and removal performance.

[0067] If the anisotropy and structural size deviations in adjacent X-ray irradiated regions in the thickness direction of the pre-filter layer are small (for example, when scanning X-rays in 2 μm steps, the anisotropy and structural size deviations between the region at a distance of 40 μm from the outer surface and the region at 42 μm), the pre-filter layer can be evaluated as having homogeneity in the overall structure of the film in each X-ray irradiated region. A structure that has homogeneity in the overall structure of the film in each X-ray irradiated region improves the efficiency of removing impurities contained in the filtered solution.

[0068] The coefficient of variation of the prefilter layer is obtained by dividing the standard deviation of the prefilter layer by its mean value. When the anisotropy deviation in the thickness direction of the prefilter layer is small, the prefilter layer has homogeneity throughout its thickness direction, and the variation in pore size in the film thickness direction is small. Therefore, it can provide stable virus removal performance in solution treatment containing various types of protein preparations that may induce a wide variety of aggregation phenomena. The coefficient of variation of the anisotropy of the prefilter layer is preferably 0 to 0.30, more preferably 0 to 0.25, and most preferably 0 to 0.20 throughout its thickness direction.

[0069] In this specification, "thickness of the pre-filter layer" means the thickness of the region with a structural size of 40 nm or more and 65 nm or less in the film thickness direction. If there are multiple pre-filter layers, the thickness of the pre-filter layer means the sum of the thicknesses of the multiple pre-filter layers.

[0070] The thickness of the pre-filter layer can be determined by calculating the length of the region corresponding to the pre-filter layer based on a graph (for example, Figure 3) showing the relationship between each position in the film thickness direction of the porous membrane, obtained by measuring the structural size, and the structural size corresponding to each position. The specific determination method is described in the examples below.

[0071] The thickness of the dense layer is preferably 2 μm to 30 μm, more preferably 5 μm to 25 μm, and even more preferably 8 μm to 20 μm.

[0072] If the density layer thickness is 2 μm or more, viruses can be sufficiently removed, resulting in a high virus removal rate. If the density layer thickness is 30 μm or less, the decrease in flow velocity can be suppressed, resulting in a high rate.

[0073] The coefficient of variation of the dense layer is obtained by dividing the standard deviation of the dense layer by its mean value. When the anisotropy deviation is small in the thickness direction of the dense layer, the dense layer has homogeneity throughout its thickness direction, and the variation in pore size in each region in the film thickness direction is small. Therefore, it can provide stable virus removal performance in solution treatment containing various types of protein preparations that may induce a wide variety of aggregation phenomena. The coefficient of variation of the anisotropy of the dense layer is preferably 0 to 0.30, more preferably 0 to 0.25, and most preferably 0 to 0.20 throughout its thickness direction.

[0074] In this specification, "thickness of the dense layer" means the thickness of the region with a structural size of 8 nm or more and 25 nm or less in the film thickness direction. If there are multiple dense layers, the thickness of the dense layer means the sum of the thicknesses of the multiple dense layers.

[0075] [Applications] The porous membrane according to this embodiment is preferably used to remove viruses from protein solutions that may contain viruses. The protein is preferably an antibody.

[0076] <Method for Manufacturing a Porous Membrane> One embodiment of the present invention relates to a method for manufacturing a porous membrane, comprising a discharge step, a free-running step, and a solidification step. The manufacturing method according to this embodiment may further include a water washing step, a high-pressure hot water treatment step, a hydrophilization treatment step, a drying step, and the like. A preferred manufacturing method for the case where the porous membrane is a hollow fiber membrane will be described below.

[0077] [Dispensing Process] The dispensing process is a process of dispensing a film-forming stock solution containing a polymer and a non-solvent (also called the "first non-solvent") vertically from the spindle. Specifically, it is preferable to simultaneously dispensing the film-forming stock solution from the annular portion of the double-tube nozzle (spindle) and the internal coagulation solution from the center.

[0078] The spinneret temperature is preferably between 20°C and 50°C.

[0079] (Film-forming stock solution) The polymers contained in the film-forming stock solution include those mentioned above as materials for porous membranes.

[0080] The amount of polymer is preferably 10% to 40% by mass, more preferably 15% to 35% by mass, and even more preferably 20% to 30% by mass, based on the mass of the film-forming stock solution.

[0081] When the amount of polymer is within the aforementioned range, there is a tendency to improve film strength and permeability.

[0082] The non-solvent contained in the film-forming stock solution can be any solvent that does not dissolve the polymer, and can be selected according to the type of polymer. Examples of non-solvents include glycerin, water, alcohols, and diol compounds. The non-solvent is preferably a diol compound. Examples of diol compounds include diethylene glycol (DEG), triethylene glycol (TriEG), tetraethylene glycol (TetraEG), and polyethylene glycol (PEG). These non-solvents are more preferable when the polymer is a polysulfone polymer.

[0083] The amount of non-solvent is preferably 30% to 60% by mass, more preferably 35% to 55% by mass, and even more preferably 40% to 50% by mass, based on the mass of the film-forming stock solution.

[0084] When the amount of non-solvent is within the aforementioned range, it tends to be possible to form a film structure with a desirable anisotropy.

[0085] The film-forming stock solution further contains a solvent. The solvent can be any solvent that dissolves polymers, and can be selected according to the type of polymer. Examples of solvents include N-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide, and ε-caprolactam. The solvent is preferably NMP, DMF, or DMAc, and more preferably NMP. These solvents are even more preferred when the polymer is a polysulfone polymer.

[0086] The amount of solvent is preferably 15% to 45% by mass, more preferably 20% to 40% by mass, and even more preferably 25% to 35% by mass, based on the mass of the film-forming stock solution.

[0087] When the amount of solvent is within the aforementioned range, a favorable film structure tends to be formed.

[0088] The viscosity of the film-forming stock solution at 25°C is preferably 80 Pa·s to 110 Pa·s, more preferably 85 Pa·s to 105 Pa·s, and even more preferably 90 Pa·s to 100 Pa·s.

[0089] When the viscosity of the film-forming stock solution is within the aforementioned range, the anisotropic draw ratio tends to be controlled to an appropriate range.

[0090] The viscosity of the film-forming stock solution can be measured using a rotational viscometer. The specific measurement method is described in the examples below.

[0091] The distance between two points (inter-plot distance) of the Hansen solubility parameter (HSP value) between the polymer and the non-solvent contained in the film-forming stock solution is preferably 10.0 or higher, more preferably 10.5 or higher, and even more preferably 11.0 or higher.

[0092] The difference in HSP values ​​being within the aforementioned range tends to cause phase separation on the inner surface side of the free-running section.

[0093] The HSP value can be calculated using the Hansen SP & QSPR model, an add-on for the commercially available software Winmostar 9.4.11. The specific measurement method is described in the examples below.

[0094] The preparation of the film-forming stock solution is preferably carried out under an inert gas atmosphere. Examples of inert gases include nitrogen and argon.

[0095] The film-forming solution is preferably degassed from the viewpoint of preventing yarn breakage during spinning and suppressing the formation of macrovoids after film formation. Degassing can be performed, for example, by reducing the pressure in a tank containing the completely dissolved film-forming solution to 2 kPa and letting it stand for at least one hour, repeating this process seven or more times. To improve degassing efficiency, the film-forming solution may be stirred during degassing.

[0096] (Internal Coagulation Solution) The internal coagulation solution preferably contains a solvent and a non-solvent (also referred to as a "second non-solvent"). Examples of solvents and non-solvents included in the internal coagulation solution include those mentioned above as solvents and non-solvents included in the film-forming stock solution. It is preferable that the solvents and non-solvents included in the internal coagulation solution are of the same type as those included in the film-forming stock solution.

[0097] An increase in the amount of solvent in the internal solidification solution slows down the solidification process and promotes the formation of the membrane structure slowly. An increase in the amount of non-solvent in the internal solidification solution slows down the diffusion of the solution due to its thickening effect, thereby slowing down the solidification process and promoting the formation of the membrane structure slowly. The amount of non-solvent is preferably 15% by mass or more and 40% by mass or less, based on the mass of the internal solidification solution. An increase in the amount of water in the internal solidification solution has the effect of accelerating the solidification process.

[0098] In order to ensure proper coagulation and obtain a desirable membrane structure, the mass ratio of organic components (solvent and non-solvent) to water in the internal coagulation solution (organic component / water) is preferably 70 / 30 to 85 / 15.

[0099] [Idle run process] The idle run process is a process in which the discharged film-forming stock solution is allowed to run idle in the idle run section.

[0100] [Residence time in the idle section] In this specification, "residence time in the idle section" is the value obtained by dividing the distance from the discharge surface of the film-forming solution to the liquid surface of the external coagulation solution by the winding speed.

[0101] The dwell time in the idle section is preferably 2.0 seconds or more and 8.0 seconds or less, more preferably 2.0 seconds or more and 6.0 seconds or less, and even more preferably 2.3 seconds or more and 5.0 seconds or less.

[0102] When the residence time in the free-running portion is within the aforementioned range, a favorable membrane structure tends to be formed.

[0103] During the free-running process, the film-forming stock solution and the internal solidification solution come into contact, causing phase separation and forming a porous membrane structure. When the porous membrane structure (specifically, the pores) is formed, if the winding process is performed in a way that reduces the outer diameter of the porous membrane, the pores being formed deform, increasing the anisotropy. Therefore, when the pre-filter layer is formed, winding in a way that reduces the outer diameter of the porous membrane can increase the average anisotropy of the pre-filter layer. On the other hand, when the dense layer is formed, avoiding winding in a way that reduces the outer diameter of the porous membrane can lower the average anisotropy of the dense layer.

[0104] In this specification, the "anisotropic draw ratio" can be determined by measuring the change in outer diameter of the raw material extruded from the spindle until it is introduced into the coagulation bath, identifying the intersection point where the initial straight line and the final straight line intersect, and calculating the ratio of the outer diameter of the raw material at the distance from the extrusion point at that intersection (referred to as the "intermediate outer diameter") to the outer diameter of the raw material just before it hits the water. Specifically, the anisotropic draw ratio can be determined as described in the examples below.

[0105] The anisotropic draw ratio refers to the proportion of the material drawn during the formation of the pre-filter layer. By adjusting the anisotropic draw ratio, it is possible to perform winding that reduces the outer diameter of the porous membrane during the formation of the pre-filter layer, while avoiding winding during the formation of the dense layer.

[0106] The anisotropic draw ratio during the free-running process is preferably 1.1 or more and 2.0 or less, and more preferably 1.2 or more and 1.8 or less.

[0107] When the anisotropic draw ratio is within the aforementioned range, it tends to impart a desirable degree of anisotropy to the pre-filter layer.

[0108] The anisotropic draw ratio can also be adjusted, for example, by changing the winding speed of the porous membrane.

[0109] When the anisotropic draw ratio is within the aforementioned range, the degree of anisotropy in the dense layer tends to be suppressed.

[0110] The average anisotropy of the dense layer can be adjusted, for example, by a combination of the proportion of non-solvents in the stock solution and internal coagulation solution and the anisotropic draw ratio.

[0111] The draft ratio is preferably 1.2 to 5.0, more preferably 1.6 to 4.0, and even more preferably 2.0 to 3.0.

[0112] [Coagulation Process] The coagulation process involves introducing the film-forming stock solution, which has been run empty, into a coagulation bath containing the coagulation solution.

[0113] The coagulation solution preferably contains a solvent and a non-solvent (also referred to as a "third non-solvent"). Examples of solvents and non-solvents included in the coagulation solution include those mentioned above as solvents and non-solvents included in the film-forming stock solution. It is preferable that the solvents and non-solvents included in the coagulation solution are of the same type as those included in the internal coagulation solution.

[0114] The amount of non-solvent is preferably 70% by mass or more, more preferably 70% by mass or more and 90% by mass or less, and even more preferably 70% by mass or more and 80% by mass or less, based on the mass of the coagulation solution.

[0115] When the amount of non-solvent is within the aforementioned range, a favorable film structure tends to be formed.

[0116] The thickness of the dense layer can be adjusted, for example, by phase separation in the air-flow section or by the non-solvent concentration of the solidified liquid.

[0117] [Water Washing Process] The water washing process involves washing the porous membrane obtained through the solidification process with water. The water washing process removes any remaining solvent and non-solvent from the porous membrane.

[0118] The washing water is preferably warm water, and preferably warm water at 50°C or higher.

[0119] The porous membrane is preferably left in a water bath containing washing water for 80 seconds to 300 seconds.

[0120] [High-Pressure Hot Water Treatment Process] The high-pressure hot water treatment process is a process in which a porous membrane is treated with hot water under high pressure. In the high-pressure hot water treatment process, polymer fine particles remaining in the porous membrane are removed.

[0121] It is preferable that the porous membrane be subjected to high-pressure hot water treatment while being held firmly on a support to prevent sagging.

[0122] High-pressure hot water treatment is preferably carried out using a high-pressure steam sterilizer or the like at a temperature of 120°C or higher for 2 to 8 hours.

[0123] [Hydrophilic Treatment Process] The hydrophilic treatment process is a process of making the surface of the porous membrane hydrophilic. Hydrophilic treatment is preferably performed when the porous membrane is composed only of hydrophobic polymers, or when the hydrophilicity of the porous membrane is insufficient.

[0124] One method of hydrophilization is to coat, graft, or crosslink a porous membrane with a water-insoluble hydrophilic polymer.

[0125] [Drying Process] The drying process is a process of drying the porous membrane. The drying method is not particularly limited, but vacuum drying is preferred.

[0126] <Method for removing viruses> One embodiment of the present invention relates to a method for removing viruses, which includes filtering a protein solution containing a virus through the porous membrane described above to remove the virus.

[0127] If the porous membrane is a hollow fiber membrane, a method of flowing the protein solution from the inner surface to the outer surface of the hollow fiber membrane (internal pressure filtration method) may be adopted, or a method of flowing the protein solution from the outer surface to the inner surface of the hollow fiber membrane (external pressure filtration method) may be adopted, but the internal pressure filtration method is preferred.

[0128] The present invention will be described in more detail below using examples and comparative examples, but the technical scope of the present invention is not limited thereto.

[0129] The various values ​​in the examples may be preferred lower or upper limits in the embodiments of the present invention. Alternatively, two similar values ​​in the examples may be combined as appropriate to form a preferred numerical range.

[0130] <Measurement Method> [Microbeam Small-Angle X-ray Scattering (SAXS) Method] The local structural size and anisotropy distribution of hollow fibers in the film thickness direction were analyzed using the microbeam small-angle X-ray scattering method under the following equipment and measurement conditions. • Equipment: BL03XU 2nd Hatch, SPring-8 Synchrotron Radiation Facility • X-ray wavelength λ: 0.10 nm • Camera length: 7894 mm • Exposure time: 1 second • Beam stop size: 3 mm • X-ray beam full width at half maximum (FWHM): 8 μm (beam width in the Z direction) • Measurement temperature: Room temperature (20°C to 30°C) • Detector: PILATUS 1M

[0131] As shown in Figure 1, a hollow fiber was sliced ​​perpendicular to its length, and a section approximately 400 μm thick was prepared as a test specimen. The test specimen was set on the sample stage of a swivel stage. The angle of the test specimen was adjusted on the swivel stage to irradiate the cross-section of the test specimen perpendicularly with X-rays. Next, X-rays were scanned in the Z direction (direction perpendicular to the X-ray incidence direction) in 2 μm steps from the outer surface to the inner surface of the hollow fiber, and SAXS measurements were performed at each point. In this setting, because the beam shape is elliptical, the beam width is smaller in the film thickness direction (Z direction) than in the circumferential direction (X direction), so scanning was performed in the Z direction. To improve the accuracy of the position in the film thickness direction, a narrower beam width is preferable, and in this embodiment, the full width at half maximum (FWHM) of the X-ray beam was set to 8 μm (beam width in the Z direction). Also, in this embodiment, the position on the outer surface shown in Figures 2 and 3, that is, the position where the "distance from the outer surface" is 0 μm, was defined as the position where the X-ray transmittance began to decrease. Furthermore, the position of the inner surface was determined to be at a distance from the position of the outer surface equal to the average film thickness measured by microscopic observation. Details of the method for observing the average film thickness will be described later. If the specimen thickness is thin, for example 10 μm, the X-ray transmittance will be almost 100%, and the position of the outer surface cannot be determined. Also, if the specimen thickness is thick, for example 1 cm, the X-ray transmittance will be almost 0%, and the position of the outer surface cannot be determined. In order to determine the position of the outer surface from the X-ray transmittance in this measurement, the specimen thickness must be appropriate, and a thickness of about 100 μm to 1000 μm is preferable.

[0132] The two-dimensional SAXS pattern I(2θ, φ) obtained by the two-dimensional detector was used to calculate the fan-shaped average scattering intensity I(2θ) based on the following formula (2). The range of the fan-shaped average was set to φ ± 10 deg, and the azimuth angle φ was defined such that the 12 o'clock direction of the two-dimensional SAXS pattern was 0°, with the clockwise direction defined.

[0133] The obtained fan-shaped average scattering intensity I(2θ) was used in the following formula (3): {In the formula, I c (2θ): Scattering intensity corrected for the empty cell, I sample (2θ): Scattering intensity of the sample, I empty (2θ): Scattering intensity of the empty cell, t sample : Measurement time of the sample, t empty : Measurement time of the empty cell measurement, T: Transmittance, C: Instrument constant} to perform empty cell scattering correction and calculate the one-dimensional scattering intensity I c .

[0134] [Anisotropy of the prefilter layer and the dense layer] In the Porod region of the SAXS profile, scattering from the surface of the scatterer is obtained, and the scattering intensity I(q) in the Porod region is proportional to Sq -4 . Here, S is a constant. q is the absolute value of the scattering vector (scattering vector q = 4πsinθ / λ with X-ray wavelength λ and scattering angle 2θ). Also, the anisotropy of the structure was expressed by the following formula (4) as the ratio of S in the film thickness direction (Z direction) to that in the circumferential direction (X direction).

[0135] S_thickness direction and S_circumferential direction were obtained from fitting using the formula I(q)=Sq -4 for the fan-shaped average SAXS profile in the film thickness direction (Z direction) and the fan-shaped average SAXS profile in the circumferential direction (X direction). The fitting was performed using Igor Pro8 from WaveMetrics, and the fitting range was set to 0.3 < q (nm -1 ) < 0.5. The fan-shaped average SAXS profile in the film thickness direction (Z direction) here was obtained by performing a fan-shaped average in the direction of azimuth angle φ = 0°, and the fan-shaped average SAXS profile in the circumferential direction (X direction) was obtained by performing a fan-shaped average in the direction of azimuth angle φ = 90°.

[0136] [Thickness of pre-filter layer and dense layer] Based on the structural size in the film thickness direction (Z direction) obtained by the above [microbeam small-angle X-ray scattering (SAXS) method], in a graph plotting the distance from the outer surface on the horizontal axis and the structural size on the vertical axis, the thickness with a structural size of 40 nm to 65 nm was defined as the thickness of the pre-filter layer, and the thickness with a structural size of 8 nm to 25 nm was defined as the thickness of the dense layer.

[0137] [Average Anisotropy of Pre-filter Layer and Dense Layer] Based on the measurement results of the structural size and anisotropy distribution in the film thickness direction (Z direction) obtained by the above [Microbeam Small-Angle X-ray Scattering (SAXS) Method], in a graph plotting the distance from the outer surface on the horizontal axis and anisotropy on the vertical axis, the average value of the anisotropy of plots with a structural size of 40 nm to 65 nm was taken as the average anisotropy of the pre-filter layer, and the average value of the anisotropy of plots with a structural size of 8 nm to 25 nm was taken as the average anisotropy of the dense layer.

[0138] The ratio of average anisotropies is the value obtained by dividing the average anisotropy of the pre-filter layer by the average anisotropy of the dense layer.

[0139] The average anisotropy ratio is not particularly limited, but is preferably between 1.2 and 3.0, more preferably between 1.3 and 2.8, and even more preferably between 1.4 and 2.5. When the average anisotropy ratio is within these ranges, the amount of antibody solution that can be processed when filtering a solution containing aggregates tends to be higher.

[0140] The difference in average anisotropy is the value obtained by subtracting the average anisotropy of the dense layer from the average anisotropy of the pre-filter layer.

[0141] The difference in average anisotropy is not particularly limited, but is preferably between 0.3 and 5.0, more preferably between 0.5 and 3.0, and even more preferably between 0.9 and 2.0.

[0142] [Coefficient of Variation of Anisotropy] The mean and standard deviation of the anisotropy of the dense layer and the prefilter layer were determined. The coefficient of variation of the anisotropy was calculated by dividing the standard deviation by the mean.

[0143] [Hansen Solubility Parameters (HSP Values)] Calculation Method: The HSP of the modeled monomer structure was calculated using the Hansen SP & QSPR model, an add-on for the commercially available software Winmostar 9.4.11.

[0144] [Viscosity of film-forming stock solution] Viscosity was measured using a rotational viscometer (Viscotester iQ, manufactured by Thermo Fisher Japan) heated to 25°C, in accordance with the method specified in "JIS Z 8803 Viscosity of liquids - Measurement method".

[0145] [Anisotropic Draw Ratio] The film-forming solution was extruded vertically from the spindle, allowed to run freely in the free-running section, introduced into the coagulation bath for solidification, and then the film was continuously wound up at a predetermined speed. During this process, the change in the outer diameter of the film-forming solution in the free-running section was measured. Specifically, as shown in Figure 4, the vertical distance from the spindle to the coagulation bath was divided into n regions at 1 cm intervals, and the outer diameter of the film-forming solution in each region was measured using a sensor. Here, the region adjacent to the spindle was designated as the first region, and the regions were numbered sequentially in the vertical direction as the second region, the third region, and so on, until the region adjacent to the coagulation bath was designated as the nth region (n≧4). If n≦3, the width of the region was changed so that n=4 or greater. A graph (for example, Figure 5) was created showing the relationship between each region and the outer diameter of the film-forming solution corresponding to each region. A polynomial approximation of degree 2 was performed on these plots to obtain an approximation curve. Here, the plot corresponding to the first region was designated as the first plot, the plot corresponding to the second region as the second plot, the plot corresponding to the (n-1)th region as the (n-1)th plot, and the plot corresponding to the nth region as the nth plot. The first plot and the second plot were connected to obtain a straight line (referred to as the "initial straight line"), and the (n-1)th plot and the nth plot were connected to obtain a straight line (referred to as the "final straight line"). The intersection point where the initial straight line and the final straight line intersect was identified, the distance from the discharge part at that intersection point was determined, and the value of the approximate curve of the change in the outer diameter of the film-forming solution at the determined distance from the discharge part was identified as the "intermediate outer diameter". In addition, the outer diameter of the film-forming solution in the nth region was identified as the "final outer diameter". The anisotropic draw ratio was determined based on the following formula: Anisotropic draw ratio = Intermediate outer diameter / Final outer diameter

[0146] [Water permeability] The effective membrane area inside the coupler is 3.3 cm², calculated based on the inner surface area of ​​the hollow fiber. ２ A measurement module was fabricated by setting hollow fibers in a specific configuration, injecting urethane into the coupler, and bonding and solidifying the hollow fibers and urethane. The measurement module was used to measure the amount of pure water filtered at 25°C for 3 minutes using constant-pressure dead-end filtration at 1.0 bar, with n=10. The permeability per unit time and unit membrane area was calculated from the filtration time and effective membrane area based on the following formula: Permeability [L / m³] 2 / h,1bar,25℃] = Filtration amount [L] / Filtration time [h] / Membrane area [m 2 ]

[0147] [Protein Filtration] Heat-denatured aggregates were prepared by heating donated blood venoglobulin IH 5% intravenously (2.5 g / 50 mL) at 60°C for 3 hours. After pretreatment with a 0.22 μm sterilization filter, the aggregates were fractionated by size exclusion chromatography using AKTA avant25 (Cytiva). A Sephacryl300 HR 16 / 60 (Cytiva) column was used. 10 mM acetate buffer pH 4.5 and 300 mM ArgHCl were used as the mobile phase. The flow rate was 1.0 ml / min. Fractions with elution volumes of 41.2 ml to 42.2 ml were collected. From DLS measurement, the polydispersity index was 0.097 and the Z-mean particle size was 29.9 nm. The obtained aggregate fraction was added to a 50 mM acetate buffer pH 5.0, 200 mM NaCl, 10 mg / mL nivolumab solution to a concentration of 0.2 wt% to 0.8 wt%. The prepared solution was filtered through a dead end at a constant pressure of 196 kPa for 180 minutes. The cumulative nivolumab permeate over 180 minutes was calculated from the filtrate volume recovered over 180 minutes and the filter membrane area (converted to internal surface area).

[0148] [Protein Recovery Rate] In the protein filtration rate test, the absorbance of the prepared stock solution and the resulting filtrate pool was measured at a wavelength of 280 nm using a multi-plate reader BioTec Synergy H1 (Agilent Technologies). The protein recovery rate was calculated as follows: Protein Recovery Rate [%] = Filtrate Absorbance / Stock Solution Absorbance × 100.

[0149] [Viral Clearance (LRV)] Add 0.5% by volume of PPV solution to the solution prepared in the protein filtration rate test. Titer 6.5 to 7.0 Log 10 (TCID ５０ The solution sampled at the target of ) / mL was used as the filtrate. PPV was purified using the method described in Non-Patent Document 2. The prepared filtrate was filtered at a dead end under a constant pressure of 2.0 bar for 120 minutes. The titer (TCID) of the filtrate was measured. ５０ The value was measured by a viral assay. The viral clearance of PPV is LRV = Log １０ (TCID ５０ ) / mL (filtered solution) - Log １０ (TCID ５０ Calculated using the formula: ) / mL (filtrate).

[0150] [Average Film Thickness] Film thickness was determined by taking a microscope image of a cross-section of the film cut perpendicular to the length of the fiber. Perpendicular cross-sections were prepared by cutting a bundle of 20 hollow fibers with a razor blade. From the image of the perpendicular cross-section of one hollow fiber, the film thickness at 10 points was determined, and the average value was calculated as the "film thickness of one hollow fiber". The average film thickness was the average of the "film thickness of one hollow fiber" for each of the 20 hollow fibers.

[0151] <Manufacturing and Evaluation of Porous Membranes> [Example 1] 25 parts by mass of polyethersulfone (PES) (BASF ULTRASON® E6020P), 30 parts by mass of NMP (Kishida Chemical Co., Ltd.), and 45 parts by mass of TriEG (Kanto Chemical Co., Ltd.) were mixed at 20°C, and the solution was subjected to degassing under reduced pressure at 2 kPa seven times to obtain the film-forming stock solution. The viscosity of the film-forming stock solution at 25°C was 96 Pa·s. The spindle temperature was set to 25°C, and the film-forming stock solution was discharged from the annular part of a double-tube nozzle. From the center, a 25°C mixture consisting of 75 parts by mass of NMP and 25 parts by mass of water was discharged as the core liquid. The discharged film-forming stock solution and core liquid were introduced into a coagulation bath containing a 20°C coagulation solution consisting of 25 parts by mass of NMP and 75 parts by mass of water, via a 200 mm sealed empty section. The distance between the plotted HSP values ​​of PES and TriEG contained in the film-forming stock solution was 11.3. The anisotropic draw ratio in the free-running section was 1.1.

[0152] The membrane, extracted from the coagulation bath, was passed through a water washing tank set to 55°C, and then wound up in water using a skein. The spinning speed was 5 m / min, and the draft ratio was 2.3.

[0153] The wound-up film was cut at both ends of the skein, bundled together, and held securely on a support to prevent slack. After high-pressure hot water treatment at 128°C for 6 hours, it was vacuum-dried at 80°C for 6 hours to obtain a hollow fiber-like base film.

[0154] The obtained hollow fiber-like substrate film was immersed for 6 hours in a coating solution consisting of 1.5 parts by mass of polyhydroxyethyl methacrylate (hydroxyethyl methacrylate manufactured by Asahi Kasei Finechem Co., Ltd.) with a weight-average molecular weight of 140,000 and 98.5 parts by mass of methanol. After washing with a 10% by mass methanol aqueous solution, it was vacuum-dried at 50°C for 16 hours to obtain a hollow fiber-like porous film.

[0155] Figure 2 shows a graph illustrating the relationship between each position in the film thickness direction of the porous membrane and the anisotropy corresponding to each position. Figure 3 shows a graph illustrating the relationship between each position in the film thickness direction of the porous membrane and the structural size corresponding to each position.

[0156] [Examples 2-7, Comparative Examples 1-3] Hollow fiber porous membranes were obtained in the same manner as in Example 1, except that the conditions in Example 1 were changed as shown in Table 1.

[0157] Table 1 shows the manufacturing conditions and performance of the porous membranes obtained in the above-described examples and comparative examples.

Claims

1. A porous membrane for removing viruses, comprising a pre-filter layer and a dense layer, wherein the pre-filter layer has an average anisotropy of 2.0 or more and 3.0 or less, and the dense layer has an average anisotropy of less than 2.

0.

2. The porous membrane according to claim 1, wherein the pre-filter layer is located on the upstream side of the filtration surface than the dense layer.

3. The porous membrane according to claim 1, wherein the pre-filter layer has a structural size of 40 nm or more and 65 nm or less.

4. The porous membrane according to claim 1, wherein the dense layer has a structural size of 8 nm or more and 25 nm or less.

5. The porous membrane according to claim 1, wherein the porous membrane comprises a hydrophobic polymer and a water-insoluble hydrophilic polymer.

6. The porous membrane according to claim 5, wherein the hydrophobic polymer comprises at least one selected from the group consisting of polyolefins, polyamides, polyimides, polyesters, polyketones, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone polymers.

7. The porous membrane according to claim 5, wherein the water-insoluble hydrophilic polymer comprises at least one selected from the group consisting of vinyl polymers, polysaccharides, polyethylene glycol and its derivatives, block copolymers of ethylene glycol and hydrophobic monomers, copolymers of ethylene glycol and propylene glycol or ethylbenzyl glycol, hydrophilized polyethylene terephthalate, hydrophilized polyethersulfone, and hydrophilized methacrylate resins.

8. The porous membrane according to claim 1, wherein the porous membrane contains a hydrophobic polymer into which hydrophilic groups have been introduced.

9. The porous membrane according to claim 1, wherein the porous membrane is a hollow fiber membrane.

10. The porous membrane according to claim 3, wherein the pre-filter layer has a thickness of 5 μm or more and 30 μm or less.

11. The porous membrane according to claim 4, wherein the dense layer has a thickness of 2 μm or more and 30 μm or less.

12. A method for removing a virus, characterized by filtering a protein solution containing a virus through the porous membrane described in claim 1.

13. A method for removing a virus, comprising filtering a protein solution containing a virus through a hollow fiber membrane as described in claim 9, wherein the protein solution flows from the inner surface to the outer surface of the hollow fiber membrane.

14. A method for producing a porous membrane according to claim 1, comprising: an extrusion step of extruding a film-forming stock solution containing a polymer and a first non-solvent, and an internal coagulation solution containing a second non-solvent, in a vertical direction from a spindle, wherein the distance between two points, the Hansen solubility parameter of the polymer and the Hansen solubility parameter of the first non-solvent, is 10.0 or more, the amount of the first non-solvent is 30% by mass or more and 60% by mass or less based on the mass of the film-forming stock solution, and the amount of the second non-solvent is 15% by mass or more and 40% by mass or less based on the mass of the internal coagulation solution; an air-running step of running the extruded film-forming stock solution in an air-running section, wherein the anisotropic draw ratio is 1.1 or more and 2.0 or less; and a coagulation step of introducing the air-running film-forming stock solution into a coagulation solution.

15. The manufacturing method according to claim 14, wherein the film-forming stock solution has a viscosity of 80 Pa·s or more and 110 Pa·s or less at 25°C.

16. The manufacturing method according to claim 14, wherein the coagulation solution contains a third non-solvent, and the amount of the third non-solvent is 70% by mass or more, based on the mass of the coagulation solution.

17. A porous membrane for removing viruses, comprising a pre-filter layer and a dense layer, wherein the average anisotropy of the pre-filter layer is greater than the average anisotropy of the dense layer.

18. The porous membrane according to claim 17, wherein the ratio of the average anisotropy of the prefilter layer to the average anisotropy of the dense layer (average anisotropy of the prefilter layer / average anisotropy of the dense layer) is 1.2 or more and 3.0 or less.

19. The porous membrane according to claim 17, wherein the ratio of the average anisotropy of the prefilter layer to the average anisotropy of the dense layer ((average anisotropy of the prefilter layer / average anisotropy of the dense layer)) is 1.3 or more and 2.5 or less.

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