Virus removal membrane and methods of making and using same

Abstract

Virus removal membrane, and method for manufacturing and using the same. [Problem] To provide a virus removal membrane and the like. [Means for Solution] A virus removal membrane comprising a hydrophobic polymer and a hydrophilic polymer, the virus removal membrane having a virus removal rate of 4.0 or more at 2.0 bar and 0.1 g / m 2 When filtering a protein aggregate having an average particle size of 30 nm and a polydispersity index of 0.10, the ratio kf / km of the fouling resistance kf to the membrane resistance km is 0.04 or more and 1.0 or less, the capture width of the aforementioned aggregate observed by Proteostat staining is 4 μm or more and 10 μm or less, the log removal rate of porcine parvovirus of the virus removal membrane is 4.0 or more, and the thickness unevenness ratio of the virus removal membrane is 1.2 or less.

Description

Technical Field

[0001] This invention relates to virus removal membranes, their manufacturing methods, and their usage methods. Background Technology

[0002] In recent years, plasma fractionation preparations and biological drugs have become increasingly popular treatments due to their fewer side effects and higher therapeutic efficacy. However, plasma fractionation preparations are derived from human blood, and biological drugs are derived from animal cells, thus posing a risk of contamination by pathogenic substances such as viruses.

[0003] To prevent viral contamination of drugs, viral removal or inactivation is necessary. Methods for viral removal or inactivation include heat treatment, optical treatment, and chemical treatment. Due to issues such as protein denaturation, viral inactivation efficiency, and the risk of chemical contamination, membrane filtration methods, which are not limited by the thermal or chemical properties of viruses and are effective against all viruses, have attracted attention.

[0004] Viruses that should be removed or inactivated include poliovirus (25–30 nm in diameter), parvovirus (18–24 nm in diameter), and HIV (80–100 nm in diameter). In recent years, the need for removal of small viruses, particularly parvovirus, has increased.

[0005] The primary performance requirement for virus removal membranes is safety. Safety considerations include preventing the introduction of pathogenic substances such as viruses into plasma fractionation preparations and biopharmaceuticals, and preventing the introduction of foreign substances such as leachates from the virus removal membrane.

[0006] To ensure safety and prevent the introduction of pathogenic substances such as viruses, it is important to thoroughly remove viruses using a virus removal membrane. In Non-Patent Literature 1, the clearance rate (LRV) for mouse parvovirus and porcine parvovirus was set to 4.

[0007] In addition, for safety reasons, it is important that no foreign matter such as leachates is introduced, and that no leachates are released from the virus removal membrane.

[0008] The second performance requirement for virus removal membranes is productivity. Productivity refers to the efficient recovery of proteins such as albumin (5 nm) and globulin (10 nm). Ultrafiltration membranes and hemodialysis membranes with pore sizes of several nm, as well as reverse osmosis membranes with even smaller pore sizes, are unsuitable as virus removal membranes because proteins clog the pores during filtration. This is especially true when removing small viruses such as parvoviruses, whose size is close to that of proteins, making it difficult to balance safety and productivity.

[0009] Patent document 1 discloses a virus removal method using a membrane formed from regenerated cellulose.

[0010] Patent document 2 discloses a virus removal membrane formed by hydrophilizing the surface of a membrane made of polyvinylidene fluoride (PVDF) prepared by thermal phase separation through graft polymerization.

[0011] In addition, Patent Document 3 discloses a virus removal membrane having an initial logarithmic removal rate (LRV) of at least 4.0 for PhiX174 and a surface coated with hydroxyalkyl cellulose.

[0012] Patent document 4 discloses a virus removal membrane formed by a mixture of polysulfone polymers and polyvinylpyrrolidone (PVP).

[0013] Patent document 5 discloses a virus removal membrane formed by coating a polysaccharide or polysaccharide derivative onto a mixed membrane of a polysulfone polymer with a copolymer of vinylpyrrolidone and vinyl acetate.

[0014] Existing technical documents

[0015] Patent documents

[0016] Patent Document 1: Japanese Patent No. 4024041

[0017] Patent Document 2: International Publication No. 2004 / 035180

[0018] Patent Document 3: Japanese Patent No. 4504963

[0019] Patent Document 4: International Publication No. 2011 / 111679

[0020] Patent Document 5: Japanese Patent No. 5403444

[0021] Non-patent literature

[0022] Non-patent literature 1: PDA Journal of GMP and Validation in Japan, Vol.7, No.1, p.44 (2005)

[0023] Non-patent literature 2: Kayukawa.T et al., Particle-based analysis elucidates thereal retention capacities of virus filters and enables optimal virusclearance study design with evaluation systems of diverse virologicalcharacteristics Biotechnol.Prog. 2022, 38, (2), e3237. Summary of the Invention

[0024] The problem the invention aims to solve

[0025] Clogging of virus removal membranes due to protein aggregates means a sharp decrease in the flux of the filtration solution. Decreased flux significantly reduces the efficiency of protein recovery. Furthermore, protein processing is difficult to predict. However, research on the inhibition of flux reduction caused by aggregates is insufficient. Predicting protein processing refers to the time required to reach the target protein processing volume and the predicted amount of protein that can be processed per unit time.

[0026] The primary target proteins for the virus removal membrane are immunoglobulins, physiologically active substances found in plasma fractionation preparations and biopharmaceuticals, with a molecular size of approximately 10 nm. In plasma fractionation preparations and biopharmaceuticals, due to the selection of highly stable proteins, the protein aggregates that may be generated during the process are not very large, primarily below 30 nm. Furthermore, even if aggregates of various sizes are generated, aggregates significantly different in size from the target protein are easily removed by processes prior to the virus removal step due to significant differences in size, charge, and other physical properties. On the other hand, aggregates of similar size to the target protein, such as below 30 nm, are not significantly different in physical properties and are therefore difficult to remove by processes prior to the virus removal step. Therefore, the possibility of incorporation of aggregates below 30 nm is high in the virus removal step used in the final stage of drug purification. A virus removal membrane resistant to clogging caused by aggregates below 30 nm can achieve effective protein recovery and easily predict protein processing.

[0027] Furthermore, even when using the same manufacturing method to remove virus membranes, deviations in membrane thickness can occur. This deviation is represented by the thickness non-uniformity ratio calculated from the maximum membrane thickness / minimum membrane thickness. The closer the thickness non-uniformity ratio is to 1, the smaller the membrane thickness deviation; the larger the thickness non-uniformity ratio, the larger the membrane thickness deviation. If the thickness non-uniformity ratio is large, the solution does not flow uniformly during filtration, resulting in deviations in protein recovery efficiency. In constant pressure filtration, protein recovery efficiency corresponds to the time required to reach the target protein throughput. In other words, in constant pressure filtration, if the protein recovery efficiency deviates, the time required to reach the target protein throughput also deviates. Consequently, the required time for the manufacturing process varies, making it difficult to stably supply the formulation. Additionally, protein throughput becomes difficult to predict. Due to the large thickness non-uniformity ratio and the non-uniform flow of the solution during filtration, the solution concentrates in areas with thinner membranes, partially increasing the viral load in those areas. Therefore, viral leakage may occur from these areas. Quantitatively, a thickness non-uniformity ratio of 1.5 or higher becomes particularly problematic.

[0028] The problem to be solved by the present invention is to provide a virus removal membrane (porous membrane) that performs well in removing viruses contained in solution, suppresses the decrease in flux caused by protein aggregates contained in solution, has excellent protein permeability, suppresses deviations in protein recovery efficiency, and makes it easy to predict protein treatment, a method for manufacturing the virus removal membrane, and a virus removal method using the virus removal membrane.

[0029] Solution for solving the problem

[0030] In order to solve the above-mentioned problems, the inventors conducted in-depth research and discovered that the problems could be solved by using a virus removal membrane with a specific structure, thus completing the present invention. Furthermore, by eliminating the uneven absolute humidity of the vapor in contact with the film-forming solution in the air gap, the aforementioned virus removal membrane with the specific structure can be manufactured.

[0031] That is, the present invention is as described below. [1]

[0033] A virus removal membrane comprising hydrophobic and hydrophilic polymers.

[0034] The virus removal membrane was tested at 2.0 bar with a concentration of 0.1 g / m³. 2 When filtering protein aggregates with an average particle size of 30 nm and a polydispersity index of 0.10, the ratio of fouling resistance (kf) to membrane resistance (km), kf / km, is greater than 0.04 and less than 1.0.

[0035] The capture width of the aforementioned aggregates, observed by Proteostat staining, was greater than 4 μm and less than 10 μm.

[0036] The logarithmic removal rate of porcine parvovirus on the virus-removing membrane is above 4.0.

[0037] The thickness non-uniformity of the virus removal membrane is less than 1.2. [2]

[0039] According to the virus removal membrane described in [1], the aforementioned hydrophobic polymer is at least one selected from the group consisting of polyolefins, polyamides, polyimides, polyesters, polyketides, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile and polysulfone polymers. [3]

[0041] According to the virus removal membrane described in [1] or [2], the aforementioned hydrophilic polymer is selected from at least one of the following groups: vinyl polymers, polysaccharides, block copolymers of ethylene glycol and hydrophobic monomers, random copolymers or block copolymers of ethylene glycol and propylene glycol or phenylethylene glycol (1-Phenylethane-1,2-diol), polymers in which one or both ends of polyethylene glycol are replaced by hydrophobic groups and are not water-soluble, hydrophilized polyethylene terephthalate, hydrophilized polyethersulfone, and hydrophilic polymers obtained by introducing hydrophilic groups into the aforementioned hydrophobic polymers. [4]

[0043] The virus removal membrane according to any one of [1] to [3], wherein the shape of the aforementioned membrane is hollow fiber. [5]

[0045] The virus removal membrane according to any one of [1] to [3], wherein the shape of the aforementioned membrane is a flat membrane. [6]

[0047] A method for manufacturing a virus removal membrane, the virus removal membrane comprising a hydrophobic polymer and a hydrophilic polymer, the manufacturing method comprising the following steps:

[0048] The process of spraying a film-forming solution containing hydrophobic polymers into the air gap.

[0049] Steam is supplied to the aforementioned air gap in a flow direction opposite to that of the film-forming solution.

[0050] The absolute humidity of the aforementioned vapor is 3–34 g / m³. 3 ,

[0051] The aforementioned steam flow rate is 0.5–3 L / min.

[0052] The air gap distance of the aforementioned air gap section is 5 to 40 cm. [7]

[0054] According to the manufacturing method described in [6], the residence time of the aforementioned film-forming stock solution in the aforementioned air gap is 0.02 to 6.0 seconds. [8]

[0056] According to the manufacturing method described in [6] or [7], the aforementioned film-forming stock solution sprayed into the aforementioned air gap is introduced into the coagulation liquid. [9]

[0058] According to any one of [6] to [8], the manufacturing method wherein the aforementioned hydrophobic polymer is at least one selected from the group consisting of polyolefins, polyamides, polyimides, polyesters, polyketides, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile and polysulfone polymers.

[10]

[0060] According to any one of [6] to [9], the aforementioned hydrophilic polymer is selected from at least one of the following groups: vinyl polymers, polysaccharides, block copolymers of ethylene glycol and hydrophobic monomers, random copolymers or block copolymers of ethylene glycol and propylene glycol or phenylethylene glycol, polymers in which one or both ends of polyethylene glycol are replaced by hydrophobic groups and are not water-soluble, hydrophilic polyethylene terephthalate or polyethersulfone, and hydrophilic polymers obtained by introducing hydrophilic groups into the aforementioned hydrophobic polymers.

[11]

[0062] A method for removing virus particles from a protein solution containing virus particles using a virus removal membrane.

[0063] The virus removal membrane comprises hydrophobic and hydrophilic polymers.

[0064] The virus removal membrane was tested at 2.0 bar with a concentration of 0.1 g / m³. 2 When filtering protein aggregates with an average particle size of 30 nm and a polydispersity index of 0.10, the ratio of fouling resistance (kf) to membrane resistance (km), kf / km, is greater than 0.04 and less than 1.0.

[0065] The capture width of the aforementioned aggregates, observed by Proteostat staining, was greater than 4 μm and less than 10 μm.

[0066] The logarithmic removal rate of porcine parvovirus on the virus-removing membrane is above 4.0.

[0067] The thickness non-uniformity of the virus removal membrane is less than 1.2.

[0068] The effects of the invention

[0069] According to the present invention, a virus removal membrane is provided that exhibits sufficient performance in removing viruses and the like contained in solution, suppresses the decrease in flux caused by protein aggregates contained in solution, has excellent protein permeability, suppresses deviations in protein recovery efficiency, and facilitates predictability of protein treatment; a method for manufacturing the virus removal membrane is also provided; and a virus removal method using the virus removal membrane is provided. Detailed Implementation

[0070] The following describes a method for implementing the present invention (hereinafter referred to as "this embodiment"). The present invention is not limited to the following embodiment, and various modifications can be made within its scope.

[0071] The virus removal membrane of this embodiment contains hydrophobic polymers and water-insoluble hydrophilic polymers.

[0072] The shape of the virus removal membrane in this embodiment is not particularly limited; for example, it can be a hollow fiber membrane or a flat membrane. When the virus removal membrane is a hollow fiber membrane, it is preferable to use multiple hollow fiber membranes bundled together. When the virus removal membrane is a flat membrane, a single flat membrane can be used, or multiple flat membranes can be overlapped. When the virus removal membrane contains multiple flat membranes, as a whole (i.e., as a combination of multiple flat membranes), it is preferable to satisfy the various physical properties of the virus removal membrane described later.

[0073] In this embodiment, examples of hydrophobic polymers that serve as membrane substrates include polyolefins, polyamides, polyimides, polyesters, polyketides, polyvinylidene fluoride (PVDF), polymethyl methacrylate, polyacrylonitrile, and polysulfone polymers.

[0074] From the perspective of high film-forming properties and control over film structure, polysulfone-based polymers are preferred.

[0075] Hydrophobic polymers can be used alone or in combination of two or more.

[0076] Examples of polysulfone-based polymers include polysulfone (PSf) having repeating units as shown in Formula 1 below, and polyethersulfone (PES) having repeating units as shown in Formula 2 below, with polyethersulfone being preferred.

[0077] Formula 1:

[0078]

[0079] Formula 2:

[0080]

[0081] As a polysulfone polymer, it can contain functional groups, alkyl groups and other substituents in the structure of Formula 1 and Formula 2, and the hydrogen atoms of the hydrocarbon skeleton can be replaced by other atoms such as halogens and substituents.

[0082] Polysulfone polymers can be used alone or in combination of two or more.

[0083] The virus removal membrane of this embodiment contains a non-water-soluble hydrophilic polymer.

[0084] From the viewpoint of preventing a sharp decrease in filtration rate due to membrane clogging caused by protein adsorption, the virus removal membrane of this embodiment is hydrophilized by having a non-water-soluble hydrophilic polymer on the pore surface of a substrate membrane containing a hydrophobic polymer.

[0085] Methods for hydrophilizing the substrate film include coating, grafting, and crosslinking reactions after the substrate film is formed from hydrophobic polymers. Alternatively, the substrate film can be hydrophilized by coating, grafting, or crosslinking reactions after the film is formed from a mixture of hydrophobic and hydrophilic polymers.

[0086] In this embodiment, a polymer that forms a contact angle of less than 90 degrees when it comes into contact with PBS (9.6g of Dulbecco's PBS(-) powder "Nissui" commercially available from Nissui Pharmaceutical Co., Ltd. dissolved in water to form a total volume of 1L of PBS) on a polymer film is called a hydrophilic polymer.

[0087] In this embodiment, the contact angle of the hydrophilic polymer is preferably 60 degrees or less, and more preferably 40 degrees or less. When the membrane contains a hydrophilic polymer with a contact angle of 60 degrees or less, it is easily wetted by water. When the membrane contains a hydrophilic polymer with a contact angle of 40 degrees or less, the tendency to be easily wetted by water is further increased.

[0088] The contact angle refers to the angle formed on the surface of a water droplet when it falls onto the surface of a thin film, as defined in JIS R3257.

[0089] In this embodiment, "water-insoluble" refers to the material used to form an effective film area of ​​3.3 cm². 2 The filter, assembled in this manner, filters 100 mL of pure water at 25°C through a constant pressure dead-end filtration at 2.0 bar, with a dissolution rate of less than 0.1%.

[0090] The dissolution rate is calculated using the following method.

[0091] Filter 100 mL of pure water at 25°C to obtain a filtrate, which is then recovered and concentrated. Using the obtained concentrate, the carbon content is determined using a Total Organic Carbon (TOC-L) meter (manufactured by Shimadzu Corporation), and the dissolution rate originating from the membrane is calculated.

[0092] In this embodiment, non-water-soluble hydrophilic polymers refer to substances that satisfy the aforementioned contact angle and dissolution rate. Non-water-soluble hydrophilic polymers include not only hydrophilic polymers that are inherently non-water-soluble, but also hydrophilic polymers that are water-soluble or have undergone water-free dissolution during the manufacturing process. That is, even water-soluble hydrophilic polymers that satisfy the aforementioned contact angle and achieve the aforementioned dissolution rate in constant-pressure dead-end filtration after filter assembly through water-free dissolution during the manufacturing process are also included in the category of non-water-soluble hydrophilic polymers in this embodiment.

[0093] From the viewpoint of preventing the adsorption of proteins as solutes, non-water-soluble hydrophilic polymers are preferably electrically neutral.

[0094] In this embodiment, electroneutrality refers to the absence of charge within the molecule, or the equal amount of cations and anions within the molecule.

[0095] Examples of non-water-soluble hydrophilic polymers include vinyl polymers.

[0096] Examples of vinyl polymers include homopolymers of hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, and triethylene glycol methacrylate; homopolymers of hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, acrylamide, dimethacrylamide, glucosyl methacrylate, 3-sulfopropylmethacryloyloxyethyl dimethylammonium betaine (MPC), 2-methacryloyloxyethyl phosphorylcholine, and 1-carboxydimethylmethacryloyloxyethyl methaneammonium; and homopolymers of styrene, ethylene, and propylene. Hydrophobic monomers such as 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, dimethacrylamide, glucosyl methacrylate, 3-sulfopropylmethacryloyloxyethyl dimethylammonium betaine, 2-methacryloyloxyethyl phosphorylcholine, and 1-carboxydimethylmethacryloyloxyethyl methaneammonium, are used in random copolymers, graft copolymers, and block copolymers.

[0097] In addition, examples of vinyl polymers include cationic monomers such as dimethylaminoethyl methacrylate and diethylaminoethyl methacrylate, and anionic monomers such as acrylic acid, methacrylic acid, vinyl sulfonic acid, methyl methacrylate, phosphoryl oxyethyl methacrylate, and copolymers of the above-mentioned hydrophobic monomers. Polymers containing equal amounts of anionic and cationic monomers in an electrically neutral manner are also possible.

[0098] Examples of non-water-soluble hydrophilic polymers include cellulose as a polysaccharide and cellulose triacetate as a derivative thereof. Additionally, substances formed by cross-linking hydroxyalkyl cellulose (e.g., hydroxypropyl cellulose (HPC)) are also included as polysaccharides or their derivatives.

[0099] As a non-water-soluble hydrophilic polymer, it can be polyethylene glycol and its derivatives, or it can be a block copolymer of ethylene glycol and the above-mentioned hydrophobic monomers, a random copolymer or block copolymer of ethylene glycol and propylene glycol, phenylethylene glycol, etc. Alternatively, one or both ends of polyethylene glycol and the above-mentioned copolymers can be replaced with hydrophobic groups to achieve non-water solubility.

[0100] Compounds in which one or both ends of polyethylene glycol are replaced by hydrophobic groups include α,ω-dibenzyl polyethylene glycol, α,ω-di(dodecyl) polyethylene glycol, etc. In addition, copolymers of polyethylene glycol and hydrophobic monomers such as dichlorodiphenyl sulfone with halogen groups at both ends of the molecule can also be used.

[0101] Examples of non-water-soluble hydrophilic polymers include polyethylene terephthalate (PET) and polyethersulfone (PES), which are obtained through polycondensation, in which hydrogen atoms in the main chain are replaced by hydrophilic groups, and thus hydrophilized PET and PES. In hydrophilized PET and PES, hydrogen atoms in the main chain can be replaced by anionic or cationic groups, and the anionic and cationic groups can be in equal amounts.

[0102] It can also be a non-water-soluble hydrophilic polymer formed by ring-opening of the epoxy groups of bisphenol A type and phenolic varnish type epoxy resins, or a non-water-soluble hydrophilic polymer formed by introducing vinyl polymers, polyethylene glycol, etc. into the epoxy groups.

[0103] Alternatively, it can be a non-water-soluble hydrophilic polymer coupled with silane.

[0104] Non-water-soluble hydrophilic polymers can be used alone or in combination of two or more.

[0105] From the viewpoint of ease of manufacture, homopolymers of hydroxyethyl methacrylate, hydroxypropyl methacrylate, and dihydroxyethyl methacrylate are preferred as non-water-soluble hydrophilic polymers; random copolymers of hydrophilic monomers such as 3-sulfopropylmethacryloyloxyethyl dimethylammonium betaine (SB), 2-methacryloyloxyethyl phosphorylcholine, and 1-carboxydimethylmethacryloyloxyethyl methaneammonium, and hydrophobic monomers such as butyl methacrylate and ethylhexyl methacrylate. From the viewpoint of ease of solvent selection for coating solutions when coating non-water-soluble hydrophilic polymers, dispersibility and operability in coating solutions are more preferred as homopolymers of hydroxyethyl methacrylate and hydroxypropyl methacrylate; random copolymers of hydrophilic monomers such as 3-sulfopropylmethacryloyloxyethyl dimethylammonium betaine, 2-methacryloyloxyethyl phosphorylcholine, and hydrophobic monomers such as butyl methacrylate and ethylhexyl methacrylate.

[0106] As a method of creating a non-water-soluble hydrophilic polymer by non-water-soluble polymerization of a water-soluble hydrophilic polymer during membrane manufacturing, for example, a water-soluble hydrophilic polymer can be copolymerized with a monomer having an azide group on its side chain and a hydrophilic monomer such as 2-methacryloyloxyethylphosphorylcholine after coating a hydrophobic polymer substrate membrane with a water-soluble hydrophilic polymer, followed by heat treatment. This covalently bonds the water-soluble hydrophilic polymer to the substrate membrane, thereby non-water-solubleling the water-soluble hydrophilic polymer. Alternatively, a hydrophilic monomer such as 2-hydroxyalkyl acrylate can be grafted onto the hydrophobic polymer substrate membrane.

[0107] The virus removal membrane or the substrate membrane in this embodiment can be formed by mixing hydrophilic and hydrophobic polymers.

[0108] The hydrophilic polymer used in the film-forming process is not particularly limited if it is compatible with the hydrophobic polymer in a good solvent. Polyvinylpyrrolidone or copolymers containing vinylpyrrolidone are preferred as hydrophilic polymers.

[0109] Specifically, examples of polyvinylpyrrolidone include LUVITEC (trade name) K60, K80, K85, and K90, which are commercially available from BASF, with LUVITEC (trade name) K80, K85, and K90 being preferred.

[0110] From the viewpoint of compatibility with hydrophobic polymers and inhibition of protein interactions on the membrane surface, copolymers containing vinylpyrrolidone are preferred as copolymers.

[0111] From the perspective of protein adsorption on the membrane surface and interaction with polysulfone polymer membranes, the copolymerization ratio of vinylpyrrolidone and vinyl acetate is preferably 6:4 to 9:1.

[0112] As copolymers of vinylpyrrolidone and vinyl acetate, examples include LUVISKOL (trade name) VA64 and VA73, which are commercially available from BASF.

[0113] Hydrophilic polymers can be used alone or in combination of two or more.

[0114] In this embodiment, from the viewpoint of suppressing the leaching of foreign matter from the membrane during filtration, when a water-soluble hydrophilic polymer is used during membrane preparation, it is preferable to wash with hot water after membrane preparation. Washing removes the hydrophilic polymer that is not sufficiently entangled with the hydrophobic polymer from the membrane, thereby suppressing leaching during filtration.

[0115] As a washing process that utilizes hot water, it can be subjected to high-pressure hot water treatment and warm water treatment after coating.

[0116] The inner diameter and thickness of the virus removal membrane were determined by photographing a vertically cut section of the membrane using a stereomicroscope. The vertical cut section was created by cutting the virus removal membrane with a razor blade.

[0117] For membrane thickness, in order to avoid localized values, in the case of a flat membrane for virus removal, the value is set as the average of the "membrane thickness of 1 flat membrane" of 10 flat membranes. The membrane thickness of 1 flat membrane is set as the value obtained by measuring the membrane thickness at 10 points every 10 μm for a 100 μm flat membrane and averaging them.

[0118] In this embodiment, by using a virus removal membrane that can operate at high filtration pressure and whose time-dependent Flux reduction during filtration is suppressed, proteins can be recovered efficiently and the amount of protein processed can be easily predicted.

[0119] In addition, the virus removal membrane of this embodiment has a high permeability to pure water, which allows for more efficient protein recovery.

[0120] In this embodiment, by using a hydrophobic polymer with pressure resistance as a substrate, operation can be performed under high filtration pressure.

[0121] In addition, in this embodiment, when 1.5% by mass of immunoglobulin G is filtered at a constant pressure of 2.0 bar from the inner surface to the outer surface of the membrane, the cumulative permeation rate of immunoglobulin G is 4.0 kg / m³ after 180 minutes. 2The above allows for efficient protein recovery. In this embodiment, the cumulative immunoglobulin G permeation rate of 1.5% by mass immunoglobulin at constant pressure of 2.0 bar over 180 minutes was determined by the method described in the example as "(4) Filtration test of immunoglobulin (IgG)".

[0122] In addition, in this embodiment, when 1.5% by mass of immunoglobulin G is filtered at a constant pressure of 2.0 bar from the inner surface to the outer surface of the hollow fiber, the ratio of immunoglobulin G Flux F60 from 50 minutes after the start of filtration to 60 minutes after the start of filtration (F60 / F10) is 0.75 or more, resulting in a small decrease in Flux. This makes it easy to predict the time until the target protein processing amount is reached.

[0123] The permeability of pure water also serves as a benchmark for the filtration rate (flux) of protein solutions. Compared to pure water, the viscosity of a protein solution is higher, thus its permeability is lower; however, a higher permeability of pure water results in a higher filtration rate for the protein solution. Therefore, in this embodiment, by increasing the permeability of pure water, a virus removal membrane capable of achieving more efficient protein recovery can be formed.

[0124] In this embodiment, the permeability of the virus removal membrane to pure water is preferably 180–500 L / m³. 2 / h / bar.

[0125] The permeability of pure water is 180 L / m³. 2 A flow rate of 500 L / m³ or higher allows for efficient protein recovery. Additionally, the permeability of pure water is 500 L / m³. 2 Below / h / bar, it can provide sustained virus removal performance.

[0126] In this embodiment, the permeability of pure water is determined by the method described in the example as “(3) Permeability Measurement”.

[0127] The permeability of pure water can be adjusted, for example, by changing the absolute humidity of the steam supplied to the air gap, the airflow rate of the supplied steam, and the length of the air gap in the manufacturing process of the virus removal membrane. Specifically, increasing the absolute humidity of the steam, increasing the airflow rate of the supplied steam, or lengthening the air gap tends to increase the permeability.

[0128] In the purification processes of plasma fractionation preparations and biopharmaceuticals using membranes, filtration typically takes more than one hour, and sometimes more than three hours. For efficient protein recovery, it is important that the fluid filtration process does not decrease over a long period. However, when filtering proteins, the fluid filtration rate typically decreases over time, tending to decrease the amount of filtrate recovered. This is believed to be due to the clogging (blockage) of the pores over time during filtration. As the number of pores that become blocked over time increases, the number of pores in the membrane that can capture viruses decreases. Therefore, it is believed that even if the initial virus removal capacity is high, the clogging of the pores can lead to a gradual decrease in virus removal performance over time.

[0129] Virus removal membranes become clogged due to protein aggregates, leading to a sharp decrease in fluid. This fluid reduction significantly decreases protein recovery efficiency, but research on the inhibition of fluid reduction caused by aggregates is insufficient. Immunoglobulins, the main physiologically active substances targeted for filtration, have a molecular size of approximately 10 nm. Because formulations select highly stable proteins, the aggregates that may be generated during the process are not that large, with the main size being below 30 nm. Furthermore, even if aggregates of various sizes are generated, those significantly deviating from the target protein in size are easily removed by processes preceding the virus removal step due to their significantly different physical properties such as size and charge. On the other hand, aggregates smaller than 30 nm, which are close to the target protein in size, are not easily removed by processes preceding the virus removal step, as their physical properties are similar to those of the target protein. Therefore, there is a high possibility of aggregates smaller than 30 nm being introduced into the virus removal step at the final stage of drug purification. Virus removal membranes resistant to clogs caused by aggregates smaller than 30 nm can achieve efficient protein recovery and allow for easy prediction of protein processing.

[0130] Virus removal membrane at 2.0 bar pressure and 0.1 g / m 2The fouling resistance when filtering a solution containing 5 μg / ml of protein aggregates with a Z-average particle size of 30 nm and a polydispersity index of 0.10 as determined by dynamic light scattering is denoted as "kf" and the membrane resistance as "km". The value of kf / km in this invention is not particularly limited, but suitable examples are shown below. Specifically, as a lower limit, it is preferably 0.04 or more, more preferably 0.06 or more, 0.08 or more, or 0.10 or more. As an upper limit, it is preferably 1.0 or less, more preferably 0.95 or less, or 0.9 or less. As a range, it is preferably 0.04 to 1.0, more preferably 0.06 to 0.95, or 0.08 to 0.90. By having kf / km of 0.04 or more and 1 or less, the decrease in fluid over time can be suppressed, achieving highly efficient protein recovery and sustained virus removal performance, thereby forming a protein treatment membrane that performs adequately in removing viruses and the like contained in solution.

[0131] The fouling resistance kf and membrane resistance km are calculated as follows. Flux and resistance are usually expressed as constants using equation (1).

[0132] Flux=A / (km+kf)…(1)

[0133] If the flux of the buffer (which does not contain aggregates) is set to F0 before aggregate filtration, then kf = 0 because there is no blockage during buffer filtration.

[0134] Substituting this into (1) forms km = A / F0,

[0135] If we substitute it into (1), then we derive

[0136] kf = A / Flux - A / F0

[0137] form

[0138] kf / km=(F0-Flux) / Flux

[0139] kf / km represents the Flux during buffer filtering and the Flux at each time point during aggregate filtering. In this embodiment, kf / km during aggregate filtering is determined by the method described in the example as "(5) Aggregate Filtering Test" described later.

[0140] kf / km can be adjusted, for example, by changing the absolute humidity of the steam supplied to the air gap, the air volume of the supplied steam, and the length of the air gap in the manufacturing process of the virus removal membrane.

[0141] The clogging mechanism of aggregates in virus removal membranes is believed to be as follows: A solution containing aggregates passes through an aggregate trapping layer composed of multiple overlapping trapping surfaces perpendicular to the permeation direction. The pore sizes within this surface are distributed, with pores smaller than the aggregate size trapping the aggregates. Due to this mechanism, if the pore size distribution within the trapping surface is narrow, only that surface traps the aggregates, thus rapidly causing clogging. On the other hand, if the pore size distribution within the trapping surface is wide, multiple surfaces can trap the aggregates, and the pores larger than the aggregates on each surface will not trap the aggregates, thus maintaining a continuous flow path and reducing clogging. The pore size distribution within the trapping surface can be indirectly observed as the width of the trapped aggregates (the range of aggregates trapped in the virus removal membrane). A wider trapping width reduces clogging, while a narrower trapping width increases clogging. However, membranes with low virus removal efficiency cannot trap aggregates, thus narrowing the trapping width. The trapping width of aggregates in the virus removal membrane can be determined by specifically staining the aggregates using Proteostat.

[0142] The capture width of the aggregates in the virus removal membrane of the present invention is not particularly limited, and the following examples are suitable. Specifically, as a lower limit, from the viewpoint of preventing a decrease in protein recovery efficiency due to clogging, it is preferably 4 μm or more, and more preferably 5 μm or more. As an upper limit, from the viewpoint of preventing a decrease in protein recovery efficiency due to a decrease in pure water permeability, it is preferably 10 μm or less, and more preferably 9 μm or less. As a range, it is preferably 4 to 10 μm, and more preferably 5 to 9 μm. With a capture width of 4 μm or more and 10 μm or less for the aggregates, there is no clogging and the protein recovery efficiency does not decrease.

[0143] The capture width of the aggregate was determined by the method described in the examples as “(7) Determination of the capture width of the aggregate”.

[0144] The capture width of the aggregates can be adjusted, for example, by changing the absolute humidity of the steam supplied to the air gap, the airflow rate of the supplied steam, and the length of the air gap in the manufacturing process of the virus removal membrane. Specifically, increasing the absolute humidity of the steam, increasing the airflow rate of the supplied steam, or lengthening the air gap tends to widen the capture width of the aggregates.

[0145] Even when virus removal membranes are manufactured using the same method, membrane thickness may vary. This variation is expressed as the thickness non-uniformity ratio calculated using the maximum membrane thickness / minimum membrane thickness. The thickness non-uniformity ratio of the virus removal membrane in this invention is not particularly limited, but can be suitably exemplified as follows: As a lower limit, it is preferably 1.0 or higher. As an upper limit, it is preferably 1.2 or lower, more preferably 1.1 or lower. As a range, it is preferably 1.0 to 1.2, more preferably 1.0 to 1.1. The closer the thickness non-uniformity ratio is to 1, the smaller the membrane thickness deviation; the larger the thickness non-uniformity ratio, the larger the membrane thickness deviation. If the thickness non-uniformity ratio is large, the solution does not flow uniformly during filtration, resulting in a deviation in protein recovery efficiency. Protein recovery efficiency in constant pressure filtration corresponds to the time required to reach the target protein throughput. That is, if the protein recovery efficiency deviates in constant pressure filtration, the time required to reach the target protein throughput also deviates. This leads to variations in the required manufacturing time and makes it difficult to maintain a stable supply of the formulation. Furthermore, it becomes difficult to predict protein throughput. In this embodiment, the deviation of protein recovery efficiency is determined by the method described in the examples as "(6) Calculation of deviation of protein recovery efficiency" (described later). The deviation of protein recovery efficiency is preferably no more than 0.10. To ensure that the deviation of protein recovery efficiency is no more than 0.10, the thickness non-uniformity ratio is preferably set to 1.2 or less.

[0146] Furthermore, due to a large thickness non-uniformity and uneven solution flow during filtration, the solution concentrates in areas with thinner membranes, leading to a localized increase in the viral load at those areas. Therefore, viral leakage is possible from these areas. Thus, a membrane with a small thickness non-uniformity and uniform solution flow is preferred.

[0147] In this embodiment, the thickness non-uniformity ratio is determined by the method described in the embodiment as “(2) Calculation of thickness non-uniformity ratio” described later.

[0148] Uneven thickness can be adjusted, for example, by changing the absolute humidity of the steam supplied to the air gap, the air volume of the supplied steam, and the length of the air gap in the manufacturing process of the virus removal membrane.

[0149] The parvovirus clearance rate was determined through the following experiments.

[0150] (1) Preparation of the filtration solution

[0151] A solution was prepared using 5% intravenous immunoglobulin IH (2.5 g / 50 mL), commercially available from the Japan Blood Products Agency, with an immunoglobulin G concentration of 15 g / L, a sodium chloride concentration of 0.1 M, and a pH of 4.5. The solution obtained by adding 0.5% (v / v) of porcine parvovirus (PPV) solution to this solution was then used as the filtration solution.

[0152] (2) Sterilization of the membrane

[0153] For a membrane with an effective area of ​​3.3 cm², 2 The filters assembled in this manner are subjected to autoclaving at 122°C for 60 minutes.

[0154] (3) Filtration

[0155] The filtration solution adjusted in (1) was filtered for 120 minutes at a constant pressure of 2.0 bar using the dead-end method.

[0156] (4) Virus clearance rate

[0157] The titer (TCID) of the filtrate obtained by filtering the solution is determined using virus analysis. 50 The virus clearance rate of PPV is calculated using LRV = Log(TCID). 50 ) / mL (filtered solution))-Log(TCID) 50 The LRV is calculated as () / mL (filtrate). The preferred LRV is 4.0 or higher.

[0158] The parvovirus clearance rate was determined by the method described in the examples as “(8) Determination of porcine parvovirus clearance rate”.

[0159] The clearance rate of parvoviruses can be adjusted, for example, by changing the absolute humidity of the steam supplied to the air gap, the airflow rate of the supplied steam, and the length of the air gap in the manufacturing process of the virus removal membrane. Specifically, reducing the absolute humidity of the steam, reducing the airflow rate of the supplied steam, or shortening the air gap tends to increase the clearance rate.

[0160] In this embodiment, the shape of the virus removal membrane is not particularly limited. For example, if the virus removal membrane is formed as a porous hollow fiber membrane, it can be manufactured as follows. The following description will use a polysulfone-based polymer as the hydrophobic polymer as an example.

[0161] For example, polysulfone-based polymers, solvents, and non-solvents are mixed, dissolved, and degassed to form a film-forming stock solution. This stock solution, along with the core solution, is simultaneously ejected from the annular and central portions of a dual-tube nozzle (spinneret), passing through an air gap and being guided into a coagulation bath to form a membrane. The resulting membrane is then washed with water, wound up, the inner solution is extracted, heat-treated, and dried. Finally, it undergoes a hydrophilization treatment.

[0162] If the solvent used in the film-forming solution is a good solvent for polysulfone-based polymers such as N-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide, or ε-caprolactam, it can be widely used. Preferably, amide-based solvents such as NMP, DMF, and DMAc are preferred, and NMP is even more preferred.

[0163] Non-solvents are preferably added to the film-forming solution. Examples of non-solvents used in film-forming solutions include glycerol, water, and glycol compounds, with glycol compounds being preferred.

[0164] Diol compounds refer to compounds having hydroxyl groups at both ends of the molecule. As diol compounds, compounds having an ethylene glycol structure with repeating unit n of 1 or more as shown in Formula 3 below are preferred.

[0165] Examples of diol compounds include diethylene glycol (DEG), triethylene glycol (TriEG), tetraethylene glycol (TetraEG), and polyethylene glycol (PEG), with DEG, TriEG, TetraEG, and TriEG being preferred.

[0166] Formula 3:

[0167]

[0168] The detailed mechanism is unclear, but by adding non-solvents to the film-forming solution, the viscosity of the film-forming solution increases, which inhibits the diffusion rate of solvents and non-solvents in the coagulation solution. This controls the coagulation and facilitates the structural control preferred for virus removal membranes, making it suitable for the formation of the desired structure.

[0169] The solvent / non-solvent ratio in the film-forming solution is preferably 40 / 60 to 80 / 20 by mass.

[0170] From the perspective of membrane strength and permeability, the concentration of polysulfone polymer in the membrane preparation solution is preferably 15-35% by mass, more preferably 20-30% by mass.

[0171] The film-forming stock solution is obtained by dissolving a polysulfone-based polymer, a good solvent, and a non-solvent while stirring at a certain temperature. This temperature is above room temperature, preferably 30–80°C. Compounds containing nitrogen atoms of tertiary nitrogen content or lower (NMP, DMF, DMAc) are easily oxidized in air and further oxidized upon heating; therefore, 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; from a production cost perspective, nitrogen is preferred.

[0172] From the perspective of preventing yarn breakage during spinning and inhibiting the formation of large pores after film formation, it is preferable to degas the film-forming solution.

[0173] The degassing process can be performed as follows: Reduce the pressure to 2 kPa in a tank containing the completely dissolved film-forming stock solution and let it stand for at least 1 hour. Repeat this operation at least 7 times. To improve degassing efficiency, the solution can be stirred during degassing.

[0174] It is preferable to remove foreign matter from the film-forming solution until it exits the spinneret. Removing foreign matter prevents yarn breakage during spinning and allows for control of the membrane structure. To prevent foreign matter from entering through the liner of the film-forming solution tank, it is also preferable to install a filter before the film-forming solution exits the spinneret. Filters with different pore sizes can be installed in multiple stages without particular limitation; for example, it is suitable to sequentially install a 30μm pore size screen filter and a 10μm pore size screen filter from the side closest to the film-forming solution tank.

[0175] The composition of the core solution used in film formation is preferably the same as that of the good solvent used in the film formation solution and the coagulation solution.

[0176] For example, if NMP is used as the solvent for the film-forming solution and NMP / water is used as a good solvent / non-solvent for the coagulation solution, then the core solution is preferably composed of NMP and water.

[0177] Increasing the amount of solvent in the core solution slows down the coagulation process, resulting in a slower membrane structure formation. Conversely, increasing the amount of water accelerates the coagulation process. To achieve proper coagulation and control the membrane structure to obtain a preferred membrane structure for virus removal, the ratio of good solvent to water in the core solution is preferably 60 / 40 to 80 / 20 by mass.

[0178] To achieve the appropriate orifice size, the spinneret temperature is preferably 25–50°C.

[0179] After the membrane-forming solution is ejected from the spinneret, it is introduced into the coagulation bath through an air gap. The residence time in the air gap is preferably 0.02 to 6.0 seconds. By setting the residence time to 0.02 seconds or more, sufficient coagulation is achieved until the solution is introduced into the coagulation bath, resulting in an appropriate pore size. By setting the residence time to 6.0 seconds or less, over-coagulation can be prevented, and precise control of the membrane structure in the coagulation bath can be achieved.

[0180] Steam is supplied to the air gap in a flow direction opposite to that of the film-forming solution. This allows for a continuous supply of humidified steam to the solution. Moisture is supplied to the film-forming solution via diffusion through the steam in the air gap. Appropriate moisture supply to the air gap promotes phase separation on the outer surface of the ejected solution, allowing for proper and uniform solidification on the outer surface. As a result, stable spinning is achieved, and thickness unevenness is suppressed. Furthermore, there is a tendency that more moisture supply leads to greater phase separation and larger membrane pore size, while less moisture results in less phase separation and a smaller membrane pore size.

[0181] The absolute humidity of the supplied steam is preferably 3–34 g / m³. 3 More preferably 5-30 g / m 3 If the absolute humidity is below the aforementioned lower limit, the moisture supply is insufficient, failing to properly solidify the outer surface and increasing thickness unevenness. Furthermore, the aggregate capture width narrows, making it prone to clogging. On the other hand, if the absolute humidity is above the aforementioned upper limit, the moisture supply increases, thereby increasing the pore size of the virus capture layer and reducing virus removal efficiency.

[0182] The preferred steam supply airflow is 0.5–3 L / min. If the airflow is less than 0.5 L / min, the moisture supply will be insufficient, failing to properly solidify the outer surface and increasing thickness unevenness. Furthermore, the aggregate capture width will narrow, making clogging more likely. On the other hand, if the airflow is greater than 3 L / min, sloshing and increased thickness unevenness will occur.

[0183] The length of the air gap is preferably 5 to 40 cm. If the air gap is less than 5 cm, the moisture supply will be insufficient, the outer surface cannot be properly cured, and the thickness unevenness will increase. In addition, the aggregate capture width will become narrower, making it easier to cause blockage. On the other hand, if the air gap is greater than 40 cm, the moisture supply to the air gap will increase, thereby increasing the pore size of the virus capture layer and reducing the virus removal efficiency.

[0184] There are no particular restrictions on the spinning speed if the goal is to obtain a defect-free membrane. However, to ensure slow liquid exchange between the membrane and the coagulation bath and to control the membrane structure, it is preferable to spin as slowly as possible. Therefore, from the viewpoint of productivity and solvent exchange, a speed of 4 to 15 m / min is preferred.

[0185] The draw ratio refers to the ratio of the traction speed to the linear velocity of the film-forming solution ejected from the spinneret. A high draw ratio means a high stretch ratio after ejection from the spinneret.

[0186] In wet phase separation membrane fabrication, the membrane structure is largely determined as the membrane-forming solution passes through the air gap and exits the coagulation bath. The membrane interior consists of real regions formed by the entanglement of polymer chains and virtual regions forming pores devoid of polymers. The detailed mechanism is unclear, but if the membrane is overstretched before coagulation is complete—that is, if it is overstretched before the polymer chains become entangled—the entanglement is torn apart, the pores connect, resulting in excessively large pores; or the pores are fragmented, resulting in excessively small pores. Excessively large pores can lead to viral leakage, while excessively small pores can cause blockage.

[0187] From the perspective of structural control, the draw ratio is preferably as small as possible, preferably 1.1 to 6, more preferably 1.1 to 4.

[0188] Regarding the composition of the coagulation bath, since a good solvent has the effect of slowing down coagulation and water has the effect of accelerating coagulation, the ratio of good solvent to water by mass is preferably 50 / 50 to 5 / 95 in order to achieve coagulation at an appropriate rate, form an appropriate thickness of dense layer, and obtain a membrane with a preferred pore size. Furthermore, from the viewpoint of pore size control, the coagulation bath temperature is preferably 0 to 50°C.

[0189] The membrane lifted from the coagulation bath is washed with warm water.

[0190] In the washing process, it is preferable to effectively remove both good solvents and non-solvents. If drying is carried out while the membrane contains solvent, the solvent inside the membrane will be concentrated during drying, and the polysulfone polymer will dissolve or swell, which may alter the membrane structure.

[0191] To increase the diffusion rate of solvents and non-solvents that should be removed and improve the efficiency of water washing, the temperature of the warm water is preferably above 50°C.

[0192] To ensure thorough washing, the preferred residence time for the membrane in the water bath is 10 to 300 seconds.

[0193] The membrane, lifted from the water bath, is wound onto a spool using a winding machine. If the membrane is wound in the air at this time, it dries slowly, and although minimal, membrane shrinkage occurs. For the same membrane structure, to form a uniform membrane, it is preferable to wind the membrane in water.

[0194] The membrane, wound onto a spool, is cut at both ends to form a bundle, which is then held in a way that prevents it from loosening. The held membrane is then immersed in hot water for washing in a hot water treatment process.

[0195] A cloudy liquid remains in the hollow portion of the membrane while it is wound on a spool. This liquid contains nano- to micron-sized polysulfone polymer particles. If this cloudy liquid is not removed and the membrane is not dried, these fine particles may clog the membrane pores, potentially reducing membrane performance. Therefore, it is preferable to remove the liquid from the hollow portion.

[0196] In the hot water treatment process, since washing is also performed from the inside of the membrane, the good solvents and non-solvents that cannot be completely removed by the water washing process are effectively removed.

[0197] The preferred temperature of the hot water in the hot water treatment process is 50-100℃, and the preferred washing time is 30-120 minutes.

[0198] It is preferable to change the hot water several times during washing.

[0199] The wound membrane is preferably subjected to high-pressure hot water treatment. Specifically, it is preferable to load the membrane into a high-pressure steam sterilizer while it is completely immersed in water and treat it at above 120°C for 2 to 6 hours. The detailed mechanism is unclear, but high-pressure hot water treatment not only completely removes the solvent and non-solvent residues in the membrane, but also optimizes the entanglement and existence state of the polysulfone polymers in the dense layer region.

[0200] A substrate film made of polysulfone polymer is completed by drying the membrane after it has been treated with high-pressure hot water. The drying method can be air drying, vacuum drying, hot air drying, etc., and there are no particular restrictions, but it is preferred to dry the membrane while fixing both ends of the membrane in a way that prevents the membrane from shrinking during drying.

[0201] The virus removal membrane of this embodiment is formed by coating the substrate film.

[0202] For example, in the case of hydrophilization treatment by coating, the coating process includes an impregnation process of the substrate film with the coating liquid, a liquid removal process of the impregnated substrate film, and a drying process of the liquid-removed substrate film.

[0203] In the impregnation process, the substrate film is immersed in a hydrophilic polymer solution. There are no particular restrictions on whether the solvent of the coating solution is a good solvent for hydrophilic polymers or a poor solvent for polysulfone polymers; alcohol is preferred.

[0204] The concentration of the non-water-soluble hydrophilic polymer in the coating solution is preferably 1.0% by mass or more, from the viewpoint of fully covering the pore surface of the substrate membrane with the hydrophilic polymer and suppressing the time-related decrease in flux due to the adsorption of proteins during filtration. From the viewpoint of covering with an appropriate thickness, ensuring that the pore size is not too small and preventing the decrease in flux, it is preferably 10.0% by mass or less.

[0205] The preferred immersion time of the substrate film in the coating solution is 8 to 24 hours.

[0206] The substrate film, immersed in the coating solution for a specified time, is dehydrated by centrifugation during the dehydration process to remove excess coating solution adhering to the hollow portion and outer periphery of the film. From the viewpoint of preventing adhesion between the dried films due to residual hydrophilic polymers, it is preferable to set the centrifugal force to 10G or more and the centrifugation time to 30 minutes or more.

[0207] The virus-removing membrane of this embodiment can be obtained by drying the dehydrated membrane. The drying method is not particularly limited, but vacuum drying is preferred as it is most efficient.

[0208] From the perspective of ease of filter manufacturing, the inner diameter of the virus removal membrane is preferably 200-400 μm and the membrane thickness is preferably 30-80 μm.

[0209] [Example]

[0210] The present invention will be described in detail below through embodiments, but the present invention is not limited to the following embodiments. The test methods shown in the embodiments are as described below.

[0211] (1) Measurement of inner diameter, film thickness and calculation of inner surface area

[0212] The inner diameter and thickness of the virus removal membrane were determined by photographing a vertically cut section of the membrane using a stereomicroscope. The vertical cut section was created by cutting the bundled hollow fibers using a razor blade. Alternatively, the cut section of the flat membrane was created by vertically cutting a flat membrane that had been embedded in ice.

[0213] The inner diameter is set as the average of the inner diameters of the 20 hollow fibers. For the inner diameter of a single hollow fiber, the image of the vertical cross-section of the hollow fiber is divided 90 times in the circumferential direction of the hollow fiber, and the inner diameter of the 90 points is calculated and set as its average value.

[0214] The film thickness is set as the average of the film thickness of one hollow fiber in each of the 20 hollow fibers. For the film thickness of one hollow fiber, the image of the vertical cross-section of one hollow fiber is divided 90 times in the circumferential direction of the hollow fiber, and the film thickness of 90 points is calculated and set as its average value.

[0215] In addition, the inner surface area is calculated from the inner diameter and the effective length of the membrane.

[0216] (2) Calculation of thickness non-uniformity ratio

[0217] The thickness non-uniformity ratio of a single hollow fiber membrane is calculated by determining the maximum (Dmax) and minimum (Dmin) values ​​of the membrane thickness at 90 points using a single hollow fiber, and then dividing the maximum value by the minimum value. The thickness non-uniformity ratio is set as the value obtained by averaging the thickness non-uniformity ratio of each of the 20 hollow fibers.

[0218] Thickness non-uniformity ratio = Dmax / Dmin

[0219] (3) Measurement of water permeability

[0220] By using the method to form an effective membrane area of ​​3.3 cm² 2 The filtration capacity of the filter assembled in this manner, which is subjected to constant pressure dead-end filtration at 1.0 bar, to obtain pure water at 25°C is measured with n=10, and the water permeability is calculated from the filtration time.

[0221] (4) Immunoglobulin (IgG) filtration test

[0222] For a membrane with an effective area of ​​3.3 cm², 2 The filter, assembled in this manner, was autoclaved at 122°C for 60 minutes. The solution was prepared by diluting with water for injection using 5% intravenous immunoglobulin IH (2.5 g / 50 mL), commercially available from the Japan Blood Products Agency, to achieve an immunoglobulin G concentration of 15 g / L, a sodium chloride concentration of 0.1 M, and a pH of 4.5. The prepared solution was filtered at a constant pressure of 196 kPa for 180 minutes using a dead-end method. The cumulative immunoglobulin G permeation over 180 minutes was calculated from the filtrate recovery volume and the filter membrane area.

[0223] In addition, the ratio of immunoglobulin G Flux F60 to immunoglobulin G Flux F10 (F60 / F10) was measured from 50 minutes after the start of filtration until 60 minutes later.

[0224] (5) Aggregate filtration test

[0225] Heat-denatured aggregates were prepared by intravenously injecting 5% venous globulin IH (2.5 g / 50 mL) at 60 °C for 3 hours. Pretreatment was performed using a 0.22 μm sterile filter, and the aggregates were size-fractionated using size exclusion chromatography with an AKTA avant 25 (Cytiva) column. A Sephacryl 300HR16 / 60 (Cytiva) column was used. The mobile phase consisted of 10 mM acetate buffer (pH 4.5) and 300 mM ArgHCl. The flow rate was set to 1.0 mL / min. Fractions with elution volumes of 41.2 mL to 42.2 mL were collected. DLS analysis showed a polydispersity index of 0.097 and a Z-mean particle size of 29.9 nm. The obtained aggregate fractions were diluted to 5 μg / mL with 10 mM acetate buffer (pH 4.5) and 300 mM ArgHCl.

[0226] Using a filter prepared in the same manner as described in "(4) Filtration test of immunoglobulins (IgG)" above, 10 mM acetate buffer (pH 4.5) and 300 mM ArgHCl were filtered at a pressure of 2.0 bar for 15 minutes, and the average Flux at this point was set as the initial Flux (F0). Then, a 5 μg / ml aggregate solution was filtered at a pressure of 2.0 bar for 3 hours, and the kf / km was determined based on the above formula [kf / km = (F0 - Flux) / Flux].

[0227] (6) Deviation in protein recovery efficiency

[0228] Using multiple filters made using the same method as described in “(4) Filtration Test of Immunoglobulin (IgG)” above, the same filtration as described in “(4) Filtration Test of Immunoglobulin (IgG)” was performed. The standard deviation of the 1.5% immunoglobulin G treatment amount at 90 minutes after the start of filtration was divided by the average of the 1.5% immunoglobulin G treatment amount at 90 minutes after the start of filtration as the deviation of protein recovery efficiency.

[0229] (7) Determination of aggregate capture width

[0230] After filtration in the above "(5) Aggregate Filtration Test", the filter was disassembled, the membrane was removed, and it was immersed in 4% oligooxygen phosphate buffer at 4°C overnight to immobilize the captured aggregates. Then it was immersed in water. The water-replaced membrane was cryogenically embedded in the OCT complex, and 8 μm sections were prepared from the filtered membrane using a Leica CM1950 (Leica).

[0231] The sections were washed four times with water, followed by four washes with PBS-T. Proteostat (Enzo Lifesience) diluted 1000-fold with Dako Antibody Diluent was added, and the sections were incubated in the dark at room temperature for 40 minutes. The sections were then washed four times with PBS-T and sectioned. The stained sections were observed using a Leica DM18 (Leica) microscope. The excitation wavelength was set to 552 nm, and the observation wavelength to 578 nm. The exposure time was 10 ms. The capture width of the aggregates was determined from the obtained images.

[0232] (8) Determination of porcine parvovirus clearance rate

[0233] The solution prepared in “(4) Filtration test of immunoglobulin (IgG)” above was spiked with 0.5% by volume PPV solution to obtain the filtration solution. The PPV used was purified using the method described in Non-Patent Literature 2. The prepared filtration solution was filtered at a constant pressure of 2.0 bar for 120 minutes in a dead-end manner.

[0234] The titer (TCID) of the filtrate was determined using virus analysis. 50 The virus clearance rate of PPV is calculated using LRV = Log(TCID). 50 ) / mL (filtered solution) - Log(TCID) 50 Calculate by ) / mL (filtrate).

[0235] (Manufacturing example)

[0236] The copolymers of poly(hydroxyethyl methacrylate) (PHEMA), 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate (PMPC-PBMA), and 2-(N-3-sulfopropyl-N,N-dimethylammonium) ethyl methacrylate and n-butyl methacrylate used in the examples were synthesized by conventional free radical polymerization using α,α-azobisisobutyronitrile (manufactured by Kanto Chemical Co., Ltd.) as an initiator.

[0237] In addition, polyethylene glycol-b-polystyrene (PEG-PSt) was synthesized according to Biomaterials, Vol. 20, p. 963 (1999).

[0238] (Example 1)

[0239] A solution obtained by mixing 24 parts by weight of PES (BASF-manufactured ULTRASON (registered trademark) E6020P), 36 parts by weight of NMP (manufactured by KISHIDACHEMICAL Co., Ltd.), and 40 parts by weight of TriEG (manufactured by Kanto Chemical Co., Ltd.) at 35°C and then repeating the degassing process under reduced pressure at 2 kPa seven times was used as the film-forming stock solution. The film-forming stock solution was ejected from the annular portion of a dual-tube nozzle with the spinneret temperature set to 35°C. A mixture of 75 parts by weight of NMP and 25 parts by weight of water was ejected from the center portion as the core solution. The ejected film-forming stock solution and core solution were then introduced into an air gap section supplied with steam at a relative humidity of 30% in the opposite direction of the ejection direction, and then into a coagulation bath at 18°C ​​containing a coagulation solution containing 25 parts by weight of NMP and 75 parts by weight of water.

[0240] The membrane, extracted from the coagulation bath, is moved by a Nelson roller in a water washing tank set at 55°C, and then wound up in water using a spool. The spinning speed is set to 5 m / min and the draw ratio is set to 2.

[0241] The wound film is cut at both ends of the spool, formed into a bundle, and held in a way that prevents it from loosening. It is then immersed in hot water at 80°C and washed for 60 minutes. After washing, the film is subjected to high-pressure hot water treatment at 128°C for 3 hours, followed by vacuum drying, thereby obtaining a hollow fiber substrate film.

[0242] The obtained hollow fiber substrate membrane was immersed in a coating solution containing 2.5 parts by weight of poly(hydroxyethyl methacrylate) (manufactured by Kanto Chemical Co., Ltd.) and 97.5 parts by weight of methanol with a weight average molecular weight of 80 kDa for 24 hours, and then centrifuged at 12.5 G for 30 minutes to remove the liquid. After centrifugation and removal of liquid, it was vacuum dried for 18 hours to obtain a hollow fiber porous membrane.

[0243] The spinning conditions for the examples and comparative examples are shown in Tables 1 and 2. In addition, the measurement results of (1) to (8) of the obtained porous membranes are shown in Tables 3 and 4.

[0244] [Table 1]

[0245]

[0246] [Table 2]

[0247]

[0248] [Table 3]

[0249]

[0250] [Table 4]

[0251]

Claims

1. A virus removal membrane comprising a hydrophobic polymer and a hydrophilic polymer, The virus removal membrane was tested at 2.0 bar with a concentration of 0.1 g / m³. 2 The ratio of fouling resistance (kf) to membrane resistance (km) after filtration of protein aggregates with an average particle size of 30 nm and a polydispersity index of 0.10 for 3 hours was greater than 0.04 and less than 1.

0. The capture width of the aggregates, as observed by Proteostat staining, was greater than 4 μm and less than 10 μm. The aggregate capture width is the range within which aggregates are captured in the virus removal membrane. The logarithmic removal rate of porcine parvovirus on the virus-removing membrane is above 4.

0. The thickness non-uniformity of the virus removal membrane is less than 1.

2. The thickness non-uniformity ratio of the virus removal membrane is the maximum value of the membrane thickness divided by the minimum value of the membrane thickness.

2. The virus removal membrane according to claim 1, wherein, The virus removal membrane was tested at 2.0 bar with a concentration of 0.1 g / m³. 2 The ratio of fouling resistance (kf) to membrane resistance (km) when filtering protein aggregates with an average particle size of 30 nm and a polydispersity index of 0.10 was 0.08–0.

90.

3. The virus removal membrane according to claim 1, wherein, The capture width of the aggregates, as observed by Proteostat staining, was 5–9 μm.

4. The virus removal membrane according to claim 1, wherein, The thickness non-uniformity of the virus removal membrane is 1.0~1.

1.

5. The virus removal membrane according to claim 1, wherein, The inner diameter of the virus removal membrane is 200~400μm.

6. The virus removal membrane according to claim 1, wherein, The thickness of the virus removal membrane is 30~80μm.

7. The virus removal membrane according to claim 1, wherein, When 1.5% by mass immunoglobulin G is filtered at a constant pressure of 2.0 bar from the inner surface to the outer surface of the membrane, the cumulative permeation rate of immunoglobulin G is 4.0 kg / m³ after 180 minutes. 2 above.

8. The virus removal membrane according to claim 1, wherein, When 1.5% by mass of immunoglobulin G is filtered at a constant pressure of 2.0 bar from the inner surface to the outer surface of a hollow fiber, the ratio of immunoglobulin G Flux F60 to immunoglobulin GFlux F10, from 50 minutes after the start of filtration until 60 minutes later, is greater than or equal to 0.

75.

9. The virus removal membrane according to claim 1, wherein, The permeability of the virus removal membrane to pure water is 180~500 L / m³. 2 / h / bar.

10. The virus removal membrane according to claim 1, wherein, The hydrophobic polymer is selected from at least one group consisting of polyolefins, polyamides, polyimides, polyesters, polyketides, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone polymers.

11. The virus removal membrane according to claim 1, wherein, The virus removal membrane contains a non-water-soluble hydrophilic polymer.

12. The virus removal membrane according to claim 1, wherein, The hydrophilic polymer is selected from at least one of the following groups: vinyl polymers, polysaccharides, block copolymers of ethylene glycol and hydrophobic monomers, random copolymers or block copolymers of ethylene glycol and propylene glycol or phenylethylene glycol, polymers in which one or both ends of polyethylene glycol are replaced by hydrophobic groups and are not water-soluble, hydrophilized polyethylene terephthalate, hydrophilized polyethersulfone, and hydrophilic polymers obtained by introducing hydrophilic groups into the hydrophobic polymer.

13. The virus removal membrane according to claim 1, wherein, The membrane is in the shape of hollow fibers.

14. The virus removal membrane according to claim 1, wherein, The membrane is flat.

15. A method for manufacturing a virus removal membrane, the virus removal membrane comprising a hydrophobic polymer and a hydrophilic polymer, the manufacturing method comprising the following steps: The process of spraying a film-forming solution containing hydrophobic polymers into the air gap. Steam is supplied to the air gap in a flow direction opposite to that of the film-forming solution. The absolute humidity of the vapor is 3~34 g / m³. 3 , The steam flow rate is 0.5~3L / min. The air gap distance of the air gap section is 5~40cm.

16. The manufacturing method according to claim 15, wherein, The absolute humidity of the vapor is 5~30 g / m³. 3 .

17. The manufacturing method according to claim 15, wherein, The residence time of the film-forming stock solution in the air gap is 0.02 to 6.0 seconds.

18. The manufacturing method according to claim 15, wherein, The film-forming stock solution sprayed into the air gap is introduced into the coagulation solution.

19. The manufacturing method according to claim 15, wherein, The hydrophobic polymer is selected from at least one group consisting of polyolefins, polyamides, polyimides, polyesters, polyketides, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone polymers.

20. The manufacturing method according to claim 15, wherein, The hydrophilic polymer is selected from at least one of the following groups: vinyl polymers, polysaccharides, block copolymers of ethylene glycol and hydrophobic monomers, random copolymers or block copolymers of ethylene glycol and propylene glycol or phenylethylene glycol, polymers in which one or both ends of polyethylene glycol are replaced by hydrophobic groups and are not water-soluble, hydrophilized polyethylene terephthalate or polyethersulfone, and hydrophilic polymers obtained by introducing hydrophilic groups into the hydrophobic polymer.

21. A method for removing virus particles from a protein solution containing virus particles using a virus removal membrane. The virus removal membrane comprises hydrophobic and hydrophilic polymers. The virus removal membrane was tested at 2.0 bar with a concentration of 0.1 g / m³. 2 The ratio of fouling resistance (kf) to membrane resistance (km) after filtration of protein aggregates with an average particle size of 30 nm and a polydispersity index of 0.10 for 3 hours was greater than 0.04 and less than 1.

0. The capture width of the aggregates, as observed by Proteostat staining, was greater than 4 μm and less than 10 μm. The aggregate capture width is the range within which aggregates are captured in the virus removal membrane. The logarithmic removal rate of porcine parvovirus on the virus-removing membrane is above 4.

0. The thickness non-uniformity of the virus removal membrane is less than 1.

2. The thickness non-uniformity ratio of the virus removal membrane is the maximum value of the membrane thickness divided by the minimum value of the membrane thickness.

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