CN117942791A - Virus removal membrane and methods of making and using the same - Google Patents
Virus removal membrane and methods of making and using the same Download PDFInfo
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- CN117942791A CN117942791A CN202311406115.6A CN202311406115A CN117942791A CN 117942791 A CN117942791 A CN 117942791A CN 202311406115 A CN202311406115 A CN 202311406115A CN 117942791 A CN117942791 A CN 117942791A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Virus-removing membranes and methods of making and using the same. [ problem ] to provide a virus removal membrane and the like. [ solution ] A virus-removing membrane comprising a hydrophobic polymer and a hydrophilic polymer, wherein the membrane has a particle-to-membrane resistance km ratio kf/km of 0.04-1.0 inclusive, the particle-to-membrane resistance km being a ratio of kf/km of 0.1g/m 2 of protein aggregates having an average particle diameter of 30nm and a polydispersity index of 0.10 inclusive, the aggregate having a capture width of 4 [ mu ] m-10 [ mu ] m inclusive, the membrane has a porcine parvovirus log removal rate of 4.0-4.0 inclusive, and the membrane has a thickness unevenness ratio of 1.2 or less, as observed by Proteostat staining.
Description
Technical Field
The present invention relates to virus-removal membranes, methods of making and methods of using the same.
Background
In recent years, treatments using plasma fractionation preparations and biological drugs have been widely used as drugs because of few side effects and high therapeutic effects. However, since the plasma fraction preparation is derived from human blood and the biological drug is derived from animal cells, there is a risk that pathogenic substances such as viruses are mixed into the drug.
In order to prevent the contamination of the drug with the virus, it is necessary to remove or inactivate the virus. Examples of the method for removing or inactivating viruses include heat treatment, optical treatment, and chemical treatment. Due to the problems of protein denaturation, virus inactivation efficiency, chemical contamination, etc., membrane filtration methods that are effective for all viruses are attracting attention without being limited by the thermal and chemical properties of the viruses.
The viruses to be removed or inactivated include polioviruses having a diameter of 25 to 30nm and parvoviruses having a diameter of 18 to 24nm, which are the smallest viruses, and the relatively large viruses include HIV viruses having a diameter of 80 to 100 nm. In recent years, there has been an increasing demand for removal of small viruses such as parvovirus.
The first property required for virus removal membranes is safety. The safety includes safety against contamination of pathogenic substances such as viruses and foreign substances such as eluted substances derived from virus-removing membranes in plasma fractionation preparations and biopharmaceuticals.
It is important to sufficiently remove viruses by a virus-removing film as safety against contamination with pathogenic substances such as viruses. In non-patent document 1, the clearance (LRV) of the target mouse parvovirus and porcine parvovirus is set to 4.
In addition, it is important to prevent elution from the virus-removing membrane, as safety against contamination with foreign substances such as elution.
The second property required for virus removal membranes is productivity. The productivity means that proteins such as albumin having a size of 5nm and globulin having a size of 10nm are efficiently recovered. Ultrafiltration membranes and hemodialysis membranes having pore diameters of about several nm, and reverse osmosis membranes having further small pore diameters are unsuitable as virus removal membranes because proteins block pores during filtration. In particular, in the case of removing small viruses such as parvovirus, the virus size is close to the protein size, and therefore it is difficult to achieve both the safety and productivity.
Patent document 1 discloses a virus removal method using a membrane formed of regenerated cellulose.
Patent document 2 discloses a virus-removing film comprising a film made of polyvinylidene fluoride (PVDF) and formed by a thermal phase separation method, wherein the film is hydrophilized on the surface by a graft polymerization method.
Patent document 3 discloses a virus removal film having an initial log virus removal rate (LRV) of at least 4.0 with respect to PhiX174 and a surface coated with hydroxyalkyl cellulose.
Patent document 4 discloses a virus-removing membrane formed by membrane formation in a mixed state of a polysulfone-based polymer and polyvinylpyrrolidone (PVP).
Patent document 5 discloses a virus-removing film in which a polysaccharide or polysaccharide derivative is coated on a mixed film of a polysulfone polymer and a copolymer of vinylpyrrolidone and vinyl acetate.
Prior art literature
Patent literature
Patent document 1: japanese patent No. 4024041
Patent document 2: international publication No. 2004/035180
Patent document 3: japanese patent No. 4504963
Patent document 4: international publication No. 2011/111679
Patent document 5: japanese patent No. 5403444
Non-patent literature
Non-patent document 1: PDA Journal of GMP and Validation in Japan, vol.7, no.1, p.44 (2005)
Non-patent document 2: kayukawa 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.
Disclosure of Invention
Problems to be solved by the invention
The virus-removal membrane is clogged by the aggregates of proteins, meaning that the Flux (Flux) of the permeation rate of the filtered solution is drastically reduced. The reduction of Flux significantly reduces the efficiency of protein recovery, and it is difficult to predict protein treatment, but studies on inhibition of Flux reduction due to aggregates are insufficient, and prediction of protein treatment refers to prediction of the time until the target protein throughput is reached, the amount of protein that can be processed per unit time, and the like.
The target protein to be filtered, which is the main filtration target of the virus-removing membrane, is immunoglobulin, which is a physiologically active substance contained in a plasma fractionation preparation and a biological drug, and the immunoglobulin has a molecular size of about 10nm. In the plasma fractionation preparation and the biopharmaceutical, protein aggregates which may be generated in the process are not so large because of the selection of proteins with high stability, and the major dimension is 30nm or less. In addition, even if aggregates of various sizes are generated, aggregates of a size greatly different from that of the target protein are easily removed by the steps preceding the virus removal step due to the great difference in physical properties such as size and charge. On the other hand, aggregates having a size close to the target protein of 30nm or less, for example, are not significantly different from the target protein in physical properties, and therefore are not easily removed by the steps before the virus removal step. Therefore, in the virus removal step used in the final stage of the drug purification step, there is a high possibility that aggregates of 30nm or less are mixed. A virus-removing membrane resistant to clogging due to aggregates of 30nm or less can achieve efficient protein recovery and can easily predict protein processing.
In addition, even in the case of virus-removed films and film thicknesses produced by the same production method, there is a possibility that variations may occur. This deviation is expressed as a thickness unevenness ratio calculated by the maximum film thickness/minimum film thickness. The smaller the thickness variation is, the larger the thickness variation is. If the thickness unevenness is large, the solution does not flow uniformly during filtration, and the protein recovery efficiency varies. The protein recovery efficiency corresponds to the time to reach the target protein throughput in constant pressure filtration. That is, in the constant pressure filtration, if the protein recovery efficiency varies, the time to reach the target protein throughput varies. Thus, the time required for the production process varies, and it is difficult to stably supply the preparation. In addition, protein processing is difficult to predict. When the thickness unevenness is large and the solution does not flow uniformly during filtration, the solution concentrates on a portion having a small film thickness, and the load of viruses on the portion partially increases. Therefore, there is a possibility that virus leakage occurs from this site. Quantitatively, a thickness unevenness ratio of 1.5 or more is particularly problematic.
The present invention aims to provide a virus-removal membrane (porous membrane) which exhibits sufficient performance for removing viruses contained in a solution, suppresses a decrease in Flux (Flux) due to protein aggregates contained in the solution, has excellent protein permeability, suppresses variation in protein recovery efficiency, and makes it possible to easily predict protein treatment, a method for producing the virus-removal membrane, and a method for removing viruses using the virus-removal membrane.
Solution for solving the problem
The present inventors have intensively studied to solve the above problems, and as a result, have found that the above problems can be solved by a virus-removing film having a specific structure, and have completed the present invention. In addition, in the air gap (air gap) portion, the virus-removing film having the above-described specific structure can be produced by eliminating the unevenness of the absolute humidity of the vapor in contact with the film-forming stock solution.
Namely, the present invention is as follows.
[1]
A virus-removing film comprising a hydrophobic polymer and a hydrophilic polymer,
The ratio kf/km of the fouling resistance kf to the membrane resistance km of the virus-removing membrane obtained by filtering protein aggregates having an average particle diameter of 30nm and a polydispersity index of 0.10 at 2.0bar at 0.1g/m 2 is not less than 0.04 and not more than 1.0,
The capture width of the aggregate observed by Proteostat staining is 4 μm or more and 10 μm or less,
The logarithmic removal rate of the porcine parvovirus of the virus removal membrane is more than 4.0,
The thickness unevenness ratio of the virus-removing film is 1.2 or less.
[2]
The virus-removing membrane according to [1], wherein the hydrophobic polymer is at least one selected from the group consisting of polyolefin, polyamide, polyimide, polyester, polyketone, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile and polysulfone-based polymer.
[3]
The virus-removing film according to [1] or [2], wherein the hydrophilic polymer is at least one selected from the group consisting of a vinyl polymer, a polysaccharide, a block copolymer of ethylene glycol and a hydrophobic monomer, a random copolymer or a block copolymer of ethylene glycol and propylene glycol or phenyl ethylene glycol (1-PHENYLETHANE-1, 2-diol), a polymer in which one or both ends of polyethylene glycol are substituted with a hydrophobic group and not water-dissolved, a hydrophilized polyethylene terephthalate, a hydrophilized polyethersulfone, and a hydrophilic polymer obtained by introducing a hydrophilic group into the hydrophobic polymer.
[4]
The virus-removal membrane according to any one of [1] to [3], wherein the membrane has a hollow fiber shape.
[5]
The virus-removal membrane according to any one of [1] to [3], wherein the shape of the membrane is a flat membrane.
[6]
A method for producing a virus-removing film comprising a hydrophobic polymer and a hydrophilic polymer, the method comprising the steps of:
a step of ejecting a film-forming raw liquid containing a hydrophobic polymer to the air gap portion,
Vapor is supplied to the air gap portion in a flow direction opposite to the ejection direction of the film forming stock solution,
The absolute humidity of the vapor is 3-34 g/m 3,
The air volume of the vapor is 0.5-3L/min,
The air gap distance of the air gap part is 5-40 cm.
[7]
The production method according to [6], wherein the residence time of the film-forming raw liquid in the air gap portion is 0.02 to 6.0 seconds.
[8]
The production method according to [6] or [7], wherein the film-forming raw liquid discharged to the air gap is introduced into a coagulation liquid.
[9]
The production method according to any one of [6] to [8], wherein the hydrophobic polymer is at least one selected from the group consisting of polyolefin, polyamide, polyimide, polyester, polyketone, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile and polysulfone-based polymer.
[10]
The method according to any one of [6] to [9], wherein the hydrophilic polymer is at least one selected from the group consisting of a vinyl polymer, a polysaccharide, a block copolymer of ethylene glycol and a hydrophobic monomer, a random copolymer or block copolymer of ethylene glycol and propylene glycol or phenyl ethylene glycol, a polymer in which one or both ends of polyethylene glycol are substituted with a hydrophobic group and are not water-soluble, a hydrophilized polyethylene terephthalate or polyethersulfone, and a hydrophilic polymer obtained by introducing a hydrophilic group into the hydrophobic polymer.
[11]
A method for removing virus particles from a protein solution containing the virus particles using a virus-removing film,
The virus-removing film comprises a hydrophobic polymer and a hydrophilic polymer,
The ratio kf/km of the fouling resistance kf to the membrane resistance km of the virus-removing membrane obtained by filtering protein aggregates having an average particle diameter of 30nm and a polydispersity index of 0.10 at 2.0bar at 0.1g/m 2 is not less than 0.04 and not more than 1.0,
The capture width of the aggregate observed by Proteostat staining is 4 μm or more and 10 μm or less,
The logarithmic removal rate of the porcine parvovirus of the virus removal membrane is more than 4.0,
The thickness unevenness ratio of the virus-removing film is 1.2 or less.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, there are provided a virus-removal membrane which exhibits sufficient performance for removing viruses and the like contained in a solution, suppresses reduction of Flux due to protein aggregates contained in the solution, is excellent in protein permeability, suppresses variation in protein recovery efficiency, and is easy to predict protein treatment, a method for producing the virus-removal membrane, and a method for removing viruses using the virus-removal membrane.
Detailed Description
The mode for carrying out the present invention (hereinafter referred to as "the present embodiment") will be described below. The present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist thereof.
The virus-removing membrane of the present embodiment contains a hydrophobic polymer and a water-insoluble hydrophilic polymer.
The shape of the virus-removing membrane according to the present embodiment is not particularly limited, and may be, for example, a hollow fiber membrane or a flat membrane. When the virus-removing membrane is a hollow fiber membrane, a plurality of hollow fiber membranes are preferably bundled and used. When the virus-removing film is a flat film, 1 flat film may be used alone, or a plurality of flat films may be used in an overlapping manner. When the virus-removing film contains a plurality of flat films, the whole (i.e., a combination of a plurality of flat films) preferably satisfies various physical properties of the virus-removing film described later.
In this embodiment, examples of the hydrophobic polymer to be a membrane substrate include polyolefin, polyamide, polyimide, polyester, polyketone, polyvinylidene fluoride (PVDF), polymethyl methacrylate, polyacrylonitrile, and polysulfone-based polymer.
From the viewpoints of high membrane formability and membrane structure control, polysulfone-based polymers are preferable.
The hydrophobic polymer may be used alone or in combination of 2 or more kinds.
Examples of the polysulfone-based polymer include polysulfone (PSf) having a repeating unit represented by the following formula 1 and Polyethersulfone (PES) having a repeating unit represented by the following formula 2, and polyethersulfone is preferable.
Formula 1:
formula 2:
The polysulfone-based polymer may have a substituent such as a functional group and an alkyl group in the structures of formula 1 and formula 2, and a hydrogen atom of the hydrocarbon skeleton may be substituted with another atom such as halogen.
The polysulfone-based polymer may be used alone or in combination of 2 or more.
The virus-removing membrane of the present embodiment contains a water-insoluble hydrophilic polymer.
The virus-removing membrane of the present embodiment is hydrophilized by the presence of a water-insoluble hydrophilic polymer on the pore surface of a base membrane containing a hydrophobic polymer, from the viewpoint of preventing rapid decrease in filtration rate due to clogging of the membrane by adsorption of proteins.
Examples of the hydrophilization method of the base film include coating, grafting reaction, and crosslinking reaction after the base film is formed of a hydrophobic polymer. In addition, the substrate film may be hydrophilized by coating, grafting reaction, crosslinking reaction, or the like after the film is formed by mixing the hydrophobic polymer and the hydrophilic polymer.
In this embodiment, a polymer having a contact angle of 90 degrees or less when PBS (Dulbecco's PBS (-) powder "Nissui"9.6g, commercially available from Niwater pharmaceutical Co., ltd.) is contacted with a polymer film, and the total amount of the polymer film is dissolved in water to form 1L of PBS) is referred to as a hydrophilic polymer.
In this embodiment, the contact angle of the hydrophilic polymer is preferably 60 degrees or less, more preferably 40 degrees or less. When the hydrophilic polymer having a contact angle of 60 degrees or less is contained, the virus-removing film is easily wetted with water, and when the hydrophilic polymer having a contact angle of 40 degrees or less is contained, the tendency of being easily wetted with water is further remarkable.
The contact angle refers to an angle formed by the surface of a water droplet when the water droplet falls on the surface of a film, and is defined by JIS R3257.
In this embodiment, the term "insoluble" means that the dissolution rate is 0.1% or less when pure water at 25℃is filtered 100mL by constant pressure dead-end filtration at 2.0bar using a filter equipped so as to form an effective membrane area of 3.3cm 2.
The dissolution rate was calculated by the following method.
100ML of pure water at 25℃was filtered to obtain a filtrate, and the filtrate was recovered and concentrated. The obtained concentrate was used to measure the carbon content by total organic carbon meter TOC-L (manufactured by Shimadzu corporation) and calculate the dissolution rate of the originating membrane.
In the present embodiment, the water-insoluble hydrophilic polymer means a substance satisfying the contact angle and dissolution rate described above. The water-insoluble hydrophilic polymer includes a hydrophilic polymer which is water-insoluble in itself, and also includes a hydrophilic polymer which is water-insoluble in the production process. That is, even if the water-soluble hydrophilic polymer is a material that satisfies the contact angle, the water-insoluble hydrophilic polymer is included in the present embodiment if the dissolution rate is satisfied in constant pressure dead-end filtration after the filter is assembled by non-water dissolution in the manufacturing process.
The water-insoluble hydrophilic polymer is preferably electrically neutral from the viewpoint of preventing adsorption of a protein as a solute.
In this embodiment, the term "charge neutral" refers to a state in which no charge is present in a molecule, or an equivalent amount of cations and anions is present in a molecule.
Examples of the water-insoluble hydrophilic polymer include vinyl polymers.
Examples of the vinyl polymer include homopolymers of hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, and triethylene glycol methacrylate; homopolymers such as hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, acrylamide, dimethylacrylamide, gluconoethyl methacrylate, 3-sulfopropyl methacryloxyethyl dimethyl ammonium betaine (MPC), 2-methacryloxyethyl phosphorylcholine, 1-carboxydimethyl acryloxyethyl ammonium and the like; and random copolymers, graft copolymers, block copolymers, and the like of a hydrophobic monomer such as styrene, ethylene, propylene, propyl methacrylate, butyl methacrylate, ethylhexyl methacrylate, octadecyl methacrylate, benzyl methacrylate, methoxyethyl methacrylate, and a hydrophilic monomer such as hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, vinylpyrrolidone, acrylamide, dimethylacrylamide, gluconoethyl methacrylate, 3-sulfopropylmethacryloxyethyl dimethyl ammonium betaine, 2-methacryloxyethyl phosphorylcholine, 1-carboxydimethyl acryloxyethyl ammonium methyl methacrylate.
Examples of the vinyl polymer include copolymers of a cationic monomer such as dimethylaminoethyl methacrylate and diethylaminoethyl methacrylate, an anionic monomer such as acrylic acid, methacrylic acid, vinylsulfonic acid, sulfopropyl methacrylate and phosphonooxyethyl methacrylate, and the above-mentioned hydrophobic monomer, and polymers containing the anionic monomer and the cationic monomer in the same amount so as to form electric neutrality.
Examples of the water-insoluble hydrophilic polymer include cellulose as a polysaccharide, cellulose triacetate as a derivative thereof, and the like. Further, polysaccharides or derivatives thereof include those obtained by crosslinking hydroxyalkyl celluloses or the like (for example, hydroxypropyl cellulose (HPC)).
The water-insoluble hydrophilic polymer may be polyethylene glycol or a derivative thereof, or may be a block copolymer of ethylene glycol and the above-mentioned hydrophobic monomer, a random copolymer or a block copolymer of ethylene glycol, propylene glycol, phenyl ethylene glycol, or the like. Alternatively, the polyethylene glycol and one or both ends of the copolymer may be substituted with a hydrophobic group and non-water-dissolved.
Examples of the compound in which one or both ends of the polyethylene glycol are substituted with a hydrophobic group include α, ω -dibenzyl polyethylene glycol, α, ω -didodecyl polyethylene glycol, and the like, and copolymers of polyethylene glycol and a hydrophobic monomer such as dichlorodiphenyl sulfone having halogen groups at both ends in the molecule.
Examples of the water-insoluble hydrophilic polymer include polyethylene terephthalate, polyether sulfone, and the like, which are obtained by polycondensation, in which a hydrogen atom in a main chain of polyethylene terephthalate, polyether sulfone, and the like is substituted with a hydrophilic group, and which are hydrophilized. As the hydrophilized polyethylene terephthalate, polyethersulfone, or the like, the hydrogen atoms in the main chain may be substituted with anionic groups, cationic groups, or the like, which may be in equal amounts.
The water-insoluble hydrophilic polymer may be a bisphenol a type or novolak type epoxy resin in which the epoxy group is ring-opened, or a water-insoluble hydrophilic polymer in which a vinyl polymer, polyethylene glycol or the like is introduced into the epoxy group.
Alternatively, the polymer may be a water-insoluble hydrophilic polymer which is silane-coupled.
The water-insoluble hydrophilic polymer may be used alone or in combination of 2 or more.
The water-insoluble hydrophilic polymer is preferably a homopolymer of hydroxyethyl methacrylate, hydroxypropyl methacrylate, or dihydroxyethyl methacrylate, from the viewpoint of ease of production; the random copolymer of a hydrophilic monomer such as 3-sulfopropyl methacryloyloxyethyl dimethyl ammonium betaine (SB), 2-methacryloyloxyethyl phosphorylcholine, 1-carboxydimethyl acryloyloxyethyl ammonium methane, and a hydrophobic monomer such as butyl methacrylate or ethylhexyl methacrylate is more preferably a homopolymer of hydroxyethyl methacrylate or hydroxypropyl methacrylate from the viewpoint of ease of selection of a solvent of a coating liquid when a water-insoluble hydrophilic polymer is applied, dispersibility in the coating liquid, and handleability; random copolymers of hydrophilic monomers such as 3-sulfopropyl methacryloxyethyl dimethyl ammonium betaine and 2-methacryloxyethyl phosphorylcholine, and hydrophobic monomers such as butyl methacrylate and ethylhexyl methacrylate.
As the water-insoluble hydrophilic polymer obtained by non-water-solubilizing the water-soluble hydrophilic polymer in the film production process, for example, a water-soluble hydrophilic polymer obtained by copolymerizing a monomer having an azide group in a side chain and a hydrophilic monomer such as 2-methacryloyloxyethyl phosphorylcholine may be coated on a base film of a hydrophobic polymer, and then heat-treated, whereby the water-soluble hydrophilic polymer is covalently bonded to the base film, and thus the water-soluble hydrophilic polymer is non-water-solubilized. In addition, a hydrophilic monomer such as 2-hydroxyalkyl acrylate may be graft-polymerized to a base film of a hydrophobic polymer.
The virus-removing film according to the present embodiment or the substrate film according to the present embodiment may be formed by mixing a hydrophilic polymer and a hydrophobic polymer.
The hydrophilic polymer used for the mixed film is not particularly limited as long as it is compatible with the hydrophobic polymer in a good solvent, and polyvinylpyrrolidone or a copolymer containing vinylpyrrolidone is preferable as the hydrophilic polymer.
Specific examples of polyvinylpyrrolidone include LUVITEC (trade name) K60, K80, K85, and K90 commercially available from BASF corporation, and LUVITEC (trade name) K80, K85, and K90 are preferable.
The copolymer containing vinylpyrrolidone is preferably a copolymer of vinylpyrrolidone and vinyl acetate from the viewpoints of compatibility with a hydrophobic polymer and inhibition of interaction of a protein with a film surface.
The copolymerization ratio of vinylpyrrolidone and vinyl acetate is preferably 6:4 to 9:1 from the viewpoints of adsorption of protein on the membrane surface and interaction with the polysulfone-based polymer membrane.
Specific examples of the copolymer of vinylpyrrolidone and vinyl acetate include LUVISKOL (trade name) VA64 and VA73 commercially available from BASF corporation.
The hydrophilic polymer may be used alone or in combination of 2 or more.
In the present embodiment, in the case of using a water-soluble hydrophilic polymer in the mixing process, it is preferable to wash the membrane with hot water after the mixing process, from the viewpoint of suppressing elution of foreign matters from the membrane during filtration. The hydrophilic polymer which is insufficiently entangled with the hydrophobic polymer is removed from the membrane by washing, and elution during filtration is suppressed.
As washing with hot water, high-pressure hot water treatment and warm water treatment after coating may be performed.
The inner diameter and the film thickness of the virus-removing film were obtained by photographing the vertical cut surface of the virus-removing film with a solid microscope. The vertical cut surface is produced by cutting the virus-removing film with a razor blade.
In the case where the virus-removed film is a flat film, for example, the film thickness is a value obtained by averaging 10 Ping Mo pieces of "film thickness of 1 flat film" so as not to form a local value. The film thickness of 1 flat film was obtained by measuring 10 film thicknesses of 100 μm flat film per 10 μm and averaging them.
In this embodiment, the protein can be recovered with high efficiency by using the virus-removing membrane which can be operated under high filtration pressure and in which the decrease in Flux with time during filtration is suppressed, and the protein throughput can be easily predicted.
In addition, the virus-removing membrane of the present embodiment can recover proteins with higher efficiency by having high water permeability through pure water.
In this embodiment, the use of a pressure-resistant hydrophobic polymer as a base material enables the operation under high filtration pressure.
In the present embodiment, the total cumulative permeation amount of immunoglobulin G at the time of constant pressure filtration from the inner surface side to the outer surface side of the membrane at 2.0bar was 4.0kg/m 2 or more at 180 minutes, whereby efficient protein recovery was possible. In this embodiment, the cumulative immunoglobulin G permeation amount of 1.5 mass% of immunoglobulin at a constant pressure of 2.0bar for 180 minutes was measured by the method described in the following "(4) filtration test of immunoglobulin (IgG)".
In the present embodiment, when 1.5 mass% of immunoglobulin G is subjected to constant pressure filtration from the inner surface side to the outer surface side of the hollow fiber at 2.0bar, the ratio (F60/F10) of immunoglobulin G Flux F60 from 50 minutes after the start of filtration to 60 minutes after the elapse of 60 minutes to immunoglobulin G Flux F10 from the start of filtration is 0.75 or more, and Flux decreases little, so that the time until the target protein throughput is processed can be easily predicted.
The water permeability of pure water also serves as a reference for the filtration rate Flux of the protein solution. The protein solution has a higher viscosity than pure water and thus a lower water permeability than pure water, but the higher the water permeability of pure water, the higher the filtration rate of the protein solution. Therefore, in the present embodiment, by increasing the water permeability of pure water, a virus-removing film can be formed which can achieve more efficient protein recovery.
The water permeability of pure water in the virus-removing membrane of the present embodiment is preferably 180 to 500L/m 2/h/bar.
The water permeability of the pure water is more than 180L/m 2/h/bar, so that the efficient recovery of the protein can be realized. In addition, the water permeability of pure water is 500L/m 2/h/bar or less, whereby the sustained virus removal performance can be exhibited.
In this embodiment, the water permeability of pure water is measured by the method described in the examples as "(3) water permeability measurement" described later.
The water permeability of the pure water can be adjusted by changing, for example, the absolute humidity of the vapor supplied to the air gap, the air volume of the supplied vapor, and the length of the air gap in the process of producing the virus-removing film. Specifically, if the absolute humidity of the vapor is increased, the air volume of the vapor is increased, or the air gap is increased, the water permeability tends to increase.
In the purification step using a membrane for the plasma fractionation preparation and the biopharmaceutical, filtration is usually performed for 1 hour or more, and sometimes, 3 hours or more. In order to recover proteins efficiently, it is important that Flux not be reduced for a long period of time. However, in general, if proteins are filtered, flux tends to decrease with time, and the amount of filtrate recovered tends to decrease. This is thought to be due to clogging (blocking) of the pores during filtration and with time. As the number of holes that are blocked with time increases, the number of holes that can capture viruses contained in the membrane decreases. Therefore, it is considered that Flux decreases with time, and even if the initial virus removal capacity is high, there is a risk that the virus removal capacity may decrease with time due to clogging of the pores.
The virus-removal membrane is clogged by protein aggregates and the Flux is drastically reduced. Flux reduction significantly reduces the efficiency of protein recovery, but inhibition of Flux reduction due to aggregates was not well studied. The molecular size of the immunoglobulin as the main physiologically active substance to be filtered is about 10nm. Since a protein having high stability is selected, aggregates which may be generated in the process are not so large and have a major dimension of 30nm or less. In addition, even if aggregates of various sizes are generated, aggregates that significantly deviate from the size of the target protein are significantly different in physical properties such as size and charge, and therefore are easily removed by the steps before the virus removal step. On the other hand, aggregates having a size close to the target protein of 30nm or less, for example, are not easily removed by a step prior to the virus removal step in which the physical properties are close to the target protein. Therefore, there is a high possibility that aggregates of 30nm or less are mixed in the virus removal step which goes to the final stage of the drug purification step. A virus-removing membrane resistant to clogging due to aggregates of 30nm or less can achieve efficient protein recovery and can easily predict protein processing.
The fouling resistance when a solution containing 5. Mu.g/ml of protein aggregates having a Z average particle diameter of 30nm and a polydispersity index of 0.10, as measured by dynamic light scattering, was filtered at a pressure of 2.0bar at 0.1g/m 2 in a virus removal membrane was set to "kf" and the membrane resistance was set to "km". The value of kf/km in the present invention is not particularly limited, and the following can be suitably exemplified. That is, the lower limit is preferably 0.04 or more, more preferably 0.06 or more, 0.08 or more, or 0.10 or more. The upper limit is preferably 1.0 or less, more preferably 0.95 or less, and still more preferably 0.9 or less. The range is preferably 0.04 to 1.0, more preferably 0.06 to 0.95, and 0.08 to 0.90. When kf/km is 0.04 or more and 1 or less, deterioration of Flux time can be suppressed, and efficient recovery of protein and continuous exertion of virus removal performance can be achieved, whereby a protein-processing film exhibiting sufficient performance for removal of viruses and the like contained in a solution can be formed.
The fouling resistance kf and the membrane resistance km are calculated as follows. Generally, flux and drag are expressed by formula (1) with A as a constant.
Flux＝A/(km+kf)…(1)
If Flux in the case of buffer (buffer) filtration without aggregates is F0 before the aggregate filtration, kf=0 because there is no clogging in the buffer filtration,
Substituting it into (1) to form km=AF0,
If it is substituted into (1), then export
kf＝A/Flux-A/F0
Formation of
kf/km＝(F0-Flux)/Flux
The kf/km is represented by Flux at each moment of the buffer filtration and the aggregate filtration. In this embodiment, kf/km in the aggregate filtration was measured by the method described in the examples as "(5) aggregate filtration test" described later.
The kf/km can be adjusted by changing, for example, the absolute humidity of the vapor supplied to the air gap portion, the air volume of the supplied vapor, and the length of the air gap portion in the process of producing the virus-removing film.
The mechanism of aggregate blocking in the virus removal membrane is thought to be as follows. The solution containing the aggregates is passed through an aggregate trapping layer formed by overlapping a plurality of trapping surfaces perpendicular to the transmission direction. The size of the pores in the face is distributed and the aggregates are captured by pores smaller than the size of the aggregates. By this mechanism, if the distribution of the sizes of pores in the trapping surface is narrow, only the surface is used to trap aggregates, and thus clogging rapidly occurs. On the other hand, if the distribution of the sizes of the pores in the trapping surface is wide, the aggregates can be trapped by a plurality of surfaces, and the pores larger than the aggregates on each surface can keep the flow path without trapping the aggregates, so that clogging is less likely to occur. The distribution of the size of the pores in the trapping plane can be indirectly observed as the width of the trapping aggregates (the range of trapping aggregates in the virus-removing film). The wider the aggregate trapping width is, the less likely to cause clogging, and the narrower the trapping width is, the more likely to cause clogging. However, even when a membrane having low virus removal property is used, aggregates cannot be trapped, and thus the aggregate trapping width is narrowed. The capture width of aggregates in virus-removal membranes can be determined by specifically staining the aggregates with Proteostat.
The capture width of the aggregates of the virus-removal film in the present invention is not particularly limited, and the following may be suitably exemplified. That is, the lower limit is preferably 4 μm or more, more preferably 5 μm or more, from the viewpoint of preventing a decrease in protein recovery efficiency due to clogging. The upper limit is preferably 10 μm or less, more preferably 9 μm or less, from the viewpoint of preventing a decrease in protein recovery efficiency due to a decrease in pure water permeation amount. The range is preferably 4 to 10. Mu.m, more preferably 5 to 9. Mu.m. The capture width of the aggregates is set to 4 μm or more and 10 μm or less, and the protein recovery efficiency is not lowered without clogging.
The capture width of the aggregate was measured by the method described in the examples as "(7) measurement of capture width of aggregate" described later.
The capturing width of the aggregate can be adjusted by changing, for example, the absolute humidity of the vapor supplied to the air gap portion, the air volume of the supplied vapor, and the length of the air gap portion in the process of producing the virus-removing film. Specifically, if the absolute humidity of the vapor is increased, the air volume of the vapor is increased, or the air gap portion is extended, the capturing width of the aggregate tends to be widened.
Even in the case of virus-removing films and film thicknesses produced by the same production method, there is a possibility that the film thicknesses may vary. The deviation is expressed as a thickness unevenness ratio calculated by using the maximum film thickness and the minimum film thickness. The thickness unevenness ratio of the virus-removing film in the present invention is not particularly limited, and the following can be suitably exemplified. That is, the lower limit is preferably 1.0 or more. The upper limit is preferably 1.2 or less, and more preferably 1.1 or less. The range is preferably 1.0 to 1.2, more preferably 1.0 to 1.1. The smaller the thickness variation is, the larger the thickness variation is. If the thickness unevenness is large, the solution does not flow uniformly during filtration, and the protein recovery efficiency varies. The protein recovery efficiency corresponds to the time to reach the target protein throughput in constant pressure filtration. That is, when the protein recovery efficiency varies in the constant pressure filtration, the time to reach the target protein throughput varies. This makes it difficult to stably supply the preparation because of the time required for the manufacturing process. In addition, protein processing is difficult to predict. In this embodiment, the variation in protein recovery efficiency is measured by the method described in the examples as "(6) calculation of variation in protein recovery efficiency" described below. The variation in protein recovery efficiency is preferably not more than 0.10. In order to prevent the variation in protein recovery efficiency from exceeding 0.10, the thickness unevenness ratio is preferably 1.2 or less.
In addition, when the thickness unevenness is large and the solution does not flow uniformly during filtration, the solution concentrates on a portion having a small film thickness, and the amount of virus load on the portion locally increases. Therefore, there is a possibility that viruses leak from this site. Therefore, it is preferable that the thickness unevenness is small and the solution flows uniformly.
In this embodiment, the thickness unevenness ratio is measured by the method described in the example as "(2) calculation of thickness unevenness ratio" described later.
The thickness unevenness can be adjusted by changing, for example, the absolute humidity of the vapor supplied to the air gap portion, the air volume of the supplied vapor, and the length of the air gap portion in the process of producing the virus-removing film.
Parvovirus clearance was determined by the following experiment.
(1) Preparation of the filtered solution
Using 5% intravenous injection (2.5G/50 mL) of hemoglobin IH, commercially available from Japanese blood preparation institution, a solution was prepared in such a manner that the concentration of immunoglobulin G in the solution was 15G/L and the concentration of sodium chloride was 0.1M, pH to 4.5. A solution obtained by adding a labeled (spike) 0.5% by volume Porcine Parvovirus (PPV) solution to this solution was used as a filtered solution.
(2) Sterilization of membranes
The filter assembled in such a manner that the effective membrane area was formed to 3.3cm 2 was subjected to autoclaving at 122℃for 60 minutes.
(3) Filtration
The filtered solution adjusted in (1) was filtered in a dead-end manner for 120 minutes at a constant pressure of 2.0 bar.
(4) Viral clearance rate
The Titer (Titer) of the filtrate (TCID 50 value) obtained by filtering the filtered solution was measured by virus analysis. The viral clearance of PPV was calculated by lrv=log (TCID 50)/mL (filtered solution)) -Log (TCID 50)/mL (filtered solution). The LRV is preferably 4.0 or more.
Parvovirus clearance was measured by the method described in the examples as "(8) porcine parvovirus clearance measurement" described later.
The parvovirus removal rate can be adjusted by changing, for example, the absolute humidity of the vapor supplied to the air gap portion, the amount of the supplied vapor, and the length of the air gap portion in the process of producing the virus-removing film. Specifically, if the absolute humidity of the vapor is reduced, the air volume supplied with the vapor is reduced, or the air gap portion is shortened, the clearance tends to increase.
In the present embodiment, the shape of the virus-removing membrane is not particularly limited, and for example, when the virus-removing membrane is a porous hollow fiber membrane which is a hollow fiber-shaped porous membrane, the virus-removing membrane can be produced as follows. The case of using a polysulfone-based polymer as the hydrophobic polymer will be described below as an example.
For example, a polysulfone polymer, a solvent and a non-solvent are mixed, dissolved and defoamed to form a film-forming stock solution, which is discharged together with a core solution from the annular portion and the central portion of a double-tube nozzle (spinneret) and introduced into a coagulation bath through an air gap portion to form a film. The obtained film was washed with water, wound up, inner liquid was removed, heat-treated, and dried. Then hydrophilization treatment is performed.
The solvent used in the membrane stock solution is a good solvent for polysulfone-based polymers such as N-methyl-2-pyrrolidone (NMP), N-Dimethylformamide (DMF), N-dimethylacetamide (DMAc), dimethylsulfoxide, and ε -caprolactam, and is preferably an amide-based solvent such as NMP, DMF, DMAc, and more preferably NMP.
The film-forming stock solution is preferably added with a non-solvent. Examples of the non-solvent used in the film-forming stock solution include glycerin, water, and a glycol compound is preferable.
The diol compound is a compound having hydroxyl groups at both ends of the molecule, and preferably has an ethylene glycol structure having 1 or more repeating units n as shown in the following formula 3.
Examples of the diol compound include diethylene glycol (DEG), triethylene glycol (TriEG), tetraethylene glycol (TetraEG), and polyethylene glycol (PEG), and preferably DEG, triEG, tetraEG, and more preferably TriEG.
Formula 3:
The detailed mechanism is not clear, but by adding a non-solvent to the film-forming stock solution, the viscosity of the film-forming stock solution increases, and the diffusion rate of the solvent and non-solvent in the coagulating liquid is suppressed, so that the coagulation can be controlled, and the preferable structure control as a virus-removing film can be easily performed, and the film-forming stock solution is suitable for the formation of a desired structure.
The ratio of the solvent to the non-solvent in the film-forming stock solution is preferably 40/60 to 80/20 by mass.
The concentration of the polysulfone-based polymer in the membrane-forming stock solution is preferably 15 to 35% by mass, more preferably 20 to 30% by mass, from the viewpoints of membrane strength and permeability.
The membrane-forming stock solution is obtained by dissolving a polysulfone-based polymer, a good solvent, and a non-solvent while stirring them at a certain temperature. The temperature at this time is higher than normal temperature, preferably 30 to 80 ℃. The compound (NMP, DMF, DMAc) containing nitrogen of tertiary or less is oxidized in air and further easily oxidized by heating, so that the preparation of the film-forming stock solution is preferably carried out under an inert gas atmosphere. The inert gas may be nitrogen, argon, or the like, and is preferably nitrogen from the viewpoint of production cost.
The preparation of the film-forming dope is preferably deaerated from the viewpoint of preventing breakage during spinning and suppressing formation of macropores after film formation.
The defoaming step can be performed as follows. The pressure in the tank containing the completely dissolved film-forming stock solution was reduced to 2kPa and allowed to stand for 1 hour or more. This operation was repeated 7 or more times. In order to improve the defoaming efficiency, the solution may be stirred during defoaming.
The film-forming stock solution is preferably removed of foreign matter until it is discharged from the spinneret. By removing the foreign matter, breakage during spinning can be prevented, and structural control of the film can be performed. In order to prevent foreign matter from being mixed in from the pad of the film-forming raw liquid tank, it is also preferable to provide a filter before the film-forming raw liquid is discharged from the spinneret. The filters having different pore diameters may be provided in a multistage manner, and for example, a mesh filter having a pore diameter of 30 μm and a mesh filter having a pore diameter of 10 μm are preferably provided in this order from the side close to the raw film forming liquid tank.
The composition of the core liquid used in the film formation is preferably the same as that of the good solvent used in the film formation stock solution and the coagulation solution.
For example, when NMP is used as a solvent of the film-forming stock solution and NMP/water is used as a good solvent/non-solvent of the coagulation solution, the core solution is preferably composed of NMP and water.
If the amount of the solvent in the core liquid is increased, the progress of solidification is slowed, and the film structure is formed slowly, and if the amount of water is increased, the progress of solidification is accelerated. In order to properly solidify and control the membrane structure to obtain a preferable membrane structure of the virus-removing membrane, the ratio of good solvent/water in the core liquid is preferably 60/40 to 80/20 by mass.
The spinneret temperature is preferably 25 to 50 ℃ in order to form a suitable pore size.
The film-forming stock solution is discharged from the spinneret, and then introduced into the coagulation bath through the air gap. The residence time of the air gap portion is preferably 0.02 to 6.0 seconds. The residence time is set to 0.02 seconds or longer, so that solidification is sufficient until the coagulation bath is introduced, and an appropriate pore size can be formed. By setting the residence time to 6.0 seconds or less, excessive coagulation can be prevented, and precise control of the film structure in the coagulation bath can be achieved.
The vapor is supplied to the air gap portion in a flow direction opposite to the ejection direction of the film forming raw liquid. This makes it possible to continuously supply the raw liquid with the humidified vapor. Moisture is supplied from the vapor to the film forming stock solution by diffusion in the air gap portion. The proper water supply to the air gap portion promotes the phase separation of the air gap portion on the outer surface side of the discharged stock solution, and the outer surface side can be properly solidified in a uniform state. As a result, stable spinning can be realized, and the thickness unevenness can be suppressed. There is additionally the following tendency: the larger the amount of water supplied, the more phase separation is performed, the larger the pore diameter of the membrane is, and if the amount of water supplied is small, the phase separation is not performed and the pore diameter of the membrane is reduced.
The absolute humidity of the supplied vapor is preferably 3 to 34g/m 3, more preferably 5 to 30g/m 3. If the absolute humidity is less than the lower limit, the moisture supply amount is insufficient, the outer surface side cannot be properly cured, and the thickness unevenness increases. In addition, the aggregate trapping width is narrowed, and clogging is likely to occur. On the other hand, if the absolute humidity exceeds the upper limit, the amount of water supplied increases, and thus the pore size of the virus-trapping layer increases, and the virus-removing property decreases.
The air volume of the vapor supply is preferably 0.5 to 3L/min, and if the air volume is less than 0.5L/min, the moisture supply amount is insufficient, the outer surface side cannot be properly cured, and the thickness unevenness ratio increases. In addition, the aggregate trapping width is narrowed, and clogging is likely to occur. On the other hand, if the air volume is larger than 3L/min, the wire is rocked and the thickness unevenness ratio increases.
The length of the air gap portion is preferably 5 to 40cm, and if the air gap portion is smaller than 5cm, the moisture supply amount is insufficient, the outer surface side cannot be properly cured, and the thickness unevenness ratio increases. In addition, the aggregate trapping width is narrowed, and clogging is likely to occur. On the other hand, if the air gap is larger than 40cm, the amount of water supplied to the air gap increases, and thus the pore diameter of the virus-trapping layer increases, and the virus-removing property decreases.
The spinning speed is not particularly limited as long as it is a condition for obtaining a film without defects, but is preferably as slow as possible in order to control the film structure by slowing down the liquid exchange between the film in the coagulation bath and the coagulation bath. Therefore, from the viewpoints of productivity and solvent exchange, it is preferably 4 to 15 m/min.
The draft ratio is the ratio of the drawing speed to the linear speed of ejection of the dope from the spinneret. The draft ratio means a high draw ratio after ejection from the spinneret.
In general, when a film is produced by wet phase separation, most of the film structure is determined when the film-forming stock solution passes through the air gap and comes out of the coagulation bath. The membrane interior is composed of a real part formed by intertwining polymer chains and an imaginary part forming a pore part in which no polymer exists. The detailed mechanism is not clear, but if the film is excessively stretched before the completion of solidification, that is, if it is excessively stretched before the polymer chains intertwine, the intertwine of the polymer chains is torn, the pore portions are connected, thereby forming excessively large pores, or the pore portions are divided, thereby forming excessively small pores. Too large pores may cause virus leakage and too small pores may cause clogging.
From the viewpoint of structural control, the draft ratio is preferably as small as possible, and the draft ratio is preferably 1.1 to 6, more preferably 1.1 to 4.
Since the good solvent has an effect of slowing down coagulation and water has an effect of accelerating coagulation with respect to the composition of the coagulation bath, the ratio of the good solvent to water is preferably 50/50 to 5/95 by mass in order to obtain a film having a preferable pore diameter by performing coagulation at a proper speed and forming a proper thickness of a dense layer. The coagulation bath temperature is preferably 0to 50 ℃ from the viewpoint of pore diameter control.
The film lifted from the coagulation bath was washed with warm water.
In the water washing step, the good solvent and the non-solvent are preferably removed reliably. If the membrane is dried in a state containing a solvent, the solvent in the membrane is concentrated during the drying, and the polysulfone-based polymer is dissolved or swelled, whereby the membrane structure may be changed.
In order to increase the diffusion rate of the solvent and the non-solvent to be removed and to increase the washing efficiency, the temperature of the warm water is preferably 50℃or higher.
In order to sufficiently wash the membrane with water, the residence time in the water bath of the membrane is preferably 10 to 300 seconds.
The film lifted from the water bath is wound on a winding shaft by a winding machine. At this time, when the film is wound in air, the film is dried slowly, but the film may shrink although it is small. As the same film structure, in order to form a uniform film, the film is preferably wound in water.
The film wound around the winding shaft is cut at both ends to form a bundle, and is held by the support body so as not to be loosened. The gripped film is then immersed in hot water in a hot water treatment step, and washed.
The cloudy liquid remains in the hollow portion of the film wound around the winding shaft. The liquid has particles of polysulfone-based polymer having a size of nanometer to micrometer floating therein. If the clouding liquid is not removed and the membrane is dried, the fine particles may clog the pores of the membrane, and the membrane performance may be reduced.
In the hot water treatment step, the film is also washed from the inside, so that the poor solvent and the non-solvent which have not been removed in the water washing step are effectively removed.
The temperature of the hot water in the hot water treatment step is preferably 50 to 100 ℃, and the washing time is preferably 30 to 120 minutes.
The hot water is preferably replaced several times during the washing.
The wound film is preferably subjected to high-pressure hot water treatment. Specifically, it is preferable that the film is put into a high-pressure steam sterilizer in a state of being completely immersed in water, and is treated at 120℃or higher for 2 to 6 hours. The detailed mechanism is not clear, but by the high-pressure hot water treatment, not only the solvent and the non-solvent which are slightly remained in the membrane are completely removed, but also the intertwining and the existing state of the polysulfone-based polymer in the dense layer region are optimized.
The membrane treated with high-pressure hot water was dried to complete a base membrane made of a polysulfone-based polymer. The drying method is not particularly limited, and is air-drying, reduced pressure drying, hot air drying, or the like, but it is preferable to dry the film in a state where both ends of the film are fixed so that the film does not shrink during drying.
The substrate film is subjected to a coating step to form the virus-removing film of the present embodiment.
For example, in the case of hydrophilization treatment by coating, the coating step includes a step of impregnating the substrate film with a coating liquid, a step of removing the impregnated substrate film, and a step of drying the substrate film after removal of the liquid.
In the impregnation step, the base film is impregnated with a hydrophilic polymer solution. The solvent of the coating liquid is not particularly limited as long as it is a good solvent for the hydrophilic polymer or a poor solvent for the polysulfone-based polymer, and is preferably an alcohol.
The concentration of the water-insoluble hydrophilic polymer in the coating liquid is preferably 1.0 mass% or more in terms of sufficiently covering the pore surfaces of the substrate film with the hydrophilic polymer and suppressing the decrease in Flux with time due to adsorption of protein during filtration, and is preferably 10.0 mass% or less in terms of covering with an appropriate thickness, not excessively reducing the pore diameter, and preventing the decrease in Flux.
The dipping time of the base film into the coating liquid is preferably 8 to 24 hours.
In the step of removing the coating liquid, the substrate film immersed in the coating liquid for a predetermined period of time is centrifuged to remove the excess coating liquid adhering to the hollow portion and the outer periphery of the film. From the viewpoint of preventing adhesion of the dried films to each other due to the residual hydrophilic polymer, it is preferable to set the centrifugal force at the time of centrifugation to 10G or more and the centrifugation time to 30 minutes or more.
The virus-removing membrane of the present embodiment can be obtained by drying the membrane subjected to the liquid removal. The drying method is not particularly limited, and vacuum drying is preferable because it is most effective.
The inner diameter of the virus-removing film is preferably 200 to 400. Mu.m, and the film thickness is preferably 30 to 80. Mu.m, from the viewpoint of easiness of filter processing.
Examples (example)
The present invention will be described in detail with reference to the following examples, but the present invention is not limited to the following examples. The test methods shown in the examples are as follows.
(1) Measurement of inner diameter and film thickness and calculation of inner surface area
The inner diameter and the film thickness of the virus-removing film were obtained by photographing the vertical cut surface of the virus-removing film with a solid microscope. The vertical cut surface is produced by cutting hollow fibers forming a bundle with a razor blade. In addition, the cut section of the flat film is manufactured by vertically cutting the ice-embedded flat film.
The inner diameter is a value obtained by averaging the "inner diameters of 1 hollow fiber" of each of the 20 hollow fibers. For the inner diameter of 1 hollow fiber, an image of a vertical cross section of 1 hollow fiber was divided 90 times in the circumferential direction of the hollow fiber, and the inner diameter at 90 points was obtained and the average value was obtained.
The film thickness was set to a value obtained by averaging the "film thickness of 1 hollow fiber" of each of the 20 hollow fibers. For the film thickness of 1 hollow fiber, the image of the vertical cross section of 1 hollow fiber was divided 90 times in the circumferential direction of the hollow fiber, and the film thickness at 90 points was obtained and the average value was set.
In addition, the internal surface area is calculated from the internal diameter and the effective length of the membrane.
(2) Calculation of thickness unevenness ratio
The thickness unevenness ratio of 1 porous hollow fiber membrane was calculated by obtaining the thickness at 90 points with 1 hollow fiber, confirming the maximum value (Dmax) and the minimum value (Dmin), and dividing the maximum value by the minimum value. The thickness unevenness was obtained by averaging the "thickness unevenness ratio of 1 hollow fiber" of each of the 20 hollow fibers.
Thickness non-uniformity ratio = Dmax/Dmin
(3) Water permeability measurement
The filtration amount of pure water at 25℃obtained by constant pressure dead-end filtration at 1.0bar using a filter assembled so that the effective membrane area was 3.3cm 2 was measured at n=10, and the water permeation amount was calculated from the filtration time.
(4) Filtration assay for immunoglobulins (IgG)
The filter assembled in such a manner that the effective membrane area was formed to 3.3cm 2 was subjected to autoclaving at 122℃for 60 minutes. Using 5% intravenous injection (2.5G/50 mL) of hemoglobin IH, commercially available from Japanese blood preparation institution, the solution was diluted with water for injection in such a manner that the concentration of immunoglobulin G of the solution was 15G/L and the concentration of sodium chloride was 0.1M, pH to 4.5. The prepared solution was filtered in a dead-end manner at a constant pressure of 196kPa for 180 minutes. The cumulative immunoglobulin G permeation amount for 180 minutes was calculated from the filtrate recovery amount for 180 minutes and the membrane area of the filter.
In addition, the ratio (F60/F10) of immunoglobulin G Flux F60 from 50 minutes after the start of filtration to immunoglobulin G Flux F10 from the start of filtration to 10 minutes after the elapse of 60 minutes was measured.
(5) Aggregate filtration test
A5% intravenous injection (2.5 g/50 mL) of blood donated intravenous globulin was heated at 60℃for 3 hours, thereby preparing a heat denatured aggregate. Pretreatment was performed using a 0.22 μm sterilization filter, and aggregates were size-classified by size exclusion chromatography using AKTA AVANT (Cytiva). A Sephacryl300HR16/60 (Cytiva) column was used. As mobile phase, 10mM acetate buffer pH 4.5, 300mM ArgHCl was used. The flow rate was set at 1.0 ml/min. Fractions were collected at elution volumes of 41.2ml to 42.2 ml. The DLS measurement revealed that the polydispersity index was 0.097 and the Z-average particle diameter was 29.9nm. The resulting aggregate fraction was diluted to 5ug/ml with 10mM acetate buffer pH 4.5, 300mM ArgHCl.
The filter prepared by the same method as the above-mentioned "(4) filtration test for immunoglobulin (IgG)" was used, and 10mM acetate buffer pH 4.5, 300mM ArgHCl was filtered at a filtration pressure of 2.0bar for 15 minutes, and the average Flux at this time was set as initial Flux (F0). Then, 5ug/ml of the aggregate solution was filtered at a filtration pressure of 2.0bar for 3 hours, and kf/km was determined based on the above formula [ kf/km= (F0-Flux)/Flux ].
(6) Deviation of protein recovery efficiency
The same filtration as in the filtration test for "(4) immunoglobulin (IgG) was performed using a plurality of filters prepared by the same method as in the filtration test for" (4) immunoglobulin (IgG), and the average value obtained by dividing the standard deviation of the 1.5% immunoglobulin G throughput at 90 minutes after the start of filtration by the average of the 1.5% immunoglobulin G throughput at 90 minutes after the start of filtration was calculated as the deviation of the protein recovery efficiency.
(7) Determination of aggregate Capture Width
The filter after filtration in the above "(5) aggregate filtration test" was removed, and the membrane was taken out and immersed in 4% paraformaldehyde phosphate buffer I at 4℃to immobilize the captured aggregates. Then immersing in water. The water-replaced membrane was frozen and embedded in OCT complex, and 8 μm sections were prepared from the filtered membrane using Leica CM1950 (Leica).
The sections were washed 4 times with water, followed by 4 times with PBS-T. Proteostat (Enzo lifesience) diluted 1000 times with Dako Antibody Diluent was added and left to stand in the dark at room temperature for 40 minutes. Then washed 4 times with PBS-T and sectioned. The sectioned stained sections were observed using Leica DMi8 (Leica). The excitation wavelength was 552nm and the observation wavelength was 578nm. The exposure time was set to 10ms. The capture width of the aggregate was determined from the obtained image.
(8) Porcine parvovirus clearance assay
The solution prepared in the filtration test of immunoglobulin (IgG) of the above-mentioned "(4) was added with a labeled (spike) 0.5 vol% PPV solution as a filtration solution. PPV purified by the method described in non-patent document 2 is used as PPV. The prepared filtration solution was filtered in a dead-end manner at a constant pressure of 2.0bar for 120 minutes.
The titre (Titer) of the filtrate (TCID 50 value) was determined using virus analysis. The viral clearance of PPV was calculated by lrv=log (TCID 50)/mL (filtered solution) -Log (TCID 50)/mL (filtrate).
(Production example)
The polyhydroxyethyl methacrylate (PHEMA), the copolymer of 2-methacryloyloxyethyl phosphorylcholine and N-butyl methacrylate (PMPC-PBMA), and the copolymer of 2- (N-3-sulfopropyl-N, N-dimethylammonium) ethyl methacrylate and N-butyl methacrylate used in the examples were synthesized by conventional radical polymerization using α, α -azobisisobutyronitrile (manufactured by Kato chemical Co., ltd.) as an initiator.
In addition, polyethylene glycol-b-polystyrene (PEG-PSt) was synthesized according to Biomaterials, vol.20, p.963 (1999).
Example 1
24 Parts by mass of PES (ULTRASON (registered trademark) E6020P manufactured by BASF corporation), 36 parts by mass of NMP (KISHIDA CHEMICAL Co., ltd.) and 40 parts by mass of TriEG (manufactured by Kato chemical Co., ltd.) were mixed at 35℃and then vacuum defoamation was repeated 7 times under 2kPa to obtain a film-forming stock solution. From the annular portion of the double-tube nozzle, a film-forming stock solution was discharged by setting the spinneret temperature to 35 ℃, and a mixed solution of 75 parts by mass of NMP and 25 parts by mass of water was discharged from the center as a core solution. The sprayed film-forming stock solution and core solution were introduced into a coagulation bath containing 25 parts by mass of NMP and 75 parts by mass of a coagulation solution in which the solution was introduced at 18 ℃ through an air gap portion in which the solution was supplied in a flow direction opposite to the spraying direction.
The film drawn out of the coagulation bath was wound up in water using a winding shaft after the Nalson roll (NELSON ROLL) was moved in a water bath set at 55 ℃. The spinning speed was set at 5 m/min and the draft ratio was set at 2.
The wound film was cut at both ends of the winding shaft, formed into a bundle, held by the support body so as not to be loosened, immersed in hot water at 80 ℃, and washed for 60 minutes. The washed film was subjected to high-pressure hot water treatment at 128℃for 3 hours, and then vacuum-dried, whereby a hollow fiber-like base film was obtained.
The obtained hollow fibrous base material film was immersed in a coating liquid of 2.5 parts by mass of polyhydroxyethyl methacrylate (manufactured by Kanto chemical Co., ltd.) having a weight-average molecular weight of 80 kDa) and 97.5 parts by mass of methanol for 24 hours, and then subjected to centrifugal liquid removal at 12.5G for 30 minutes. After centrifugal liquid removal, the mixture was dried in vacuo for 18 hours to obtain a hollow fibrous porous membrane.
The spinning conditions of examples and comparative examples are shown in tables 1 and 2. The measurement results of (1) to (8) of the obtained porous films are shown in tables 3 and 4.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
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Claims (11)
1. A virus-removing film comprising a hydrophobic polymer and a hydrophilic polymer,
The ratio kf/km of the fouling resistance kf to the membrane resistance km of the virus-removing membrane obtained by filtering protein aggregates having an average particle diameter of 30nm and a polydispersity index of 0.10 at 2.0bar at 0.1g/m 2 is not less than 0.04 and not more than 1.0,
The capture width of the aggregate observed by Proteostat staining is 4 μm or more and 10 μm or less,
The logarithmic removal rate of the porcine parvovirus of the virus removal membrane is more than 4.0,
The thickness unevenness ratio of the virus-removing film is 1.2 or less.
2. The virus-removing membrane according to claim 1, wherein the hydrophobic polymer is at least one selected from the group consisting of polyolefin, polyamide, polyimide, polyester, polyketone, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone-based polymer.
3. The virus-removing film according to claim 1, wherein the hydrophilic polymer is at least one selected from the group consisting of a vinyl polymer, a polysaccharide, a block copolymer of ethylene glycol and a hydrophobic monomer, a random copolymer or a block copolymer of ethylene glycol and propylene glycol or phenyl ethylene glycol, a polymer in which one or both ends of polyethylene glycol are substituted with a hydrophobic group and are not water-dissolved, a polyethylene terephthalate which is hydrophilized, a polyether sulfone which is hydrophilized, and a hydrophilic polymer obtained by introducing a hydrophilic group into the hydrophobic polymer.
4. The virus-removal membrane of claim 1, wherein the membrane is hollow fiber in shape.
5. The virus-removal membrane of claim 1, wherein the membrane is in the shape of a flat membrane.
6. A method for producing a virus-removing film comprising a hydrophobic polymer and a hydrophilic polymer, the method comprising the steps of:
a step of ejecting a film-forming raw liquid containing a hydrophobic polymer to the air gap portion,
Vapor is supplied to the air gap portion in a flow direction opposite to the ejection direction of the film forming stock solution,
The absolute humidity of the steam is 3-34 g/m 3,
The air quantity of the steam is 0.5-3L/min,
The air gap distance of the air gap part is 5-40 cm.
7. The method according to claim 6, wherein the residence time of the film forming raw liquid in the air gap is 0.02 to 6.0 seconds.
8. The method according to claim 6, wherein the film-forming raw liquid discharged to the air gap is introduced into a solidification liquid.
9. The method according to claim 6, wherein the hydrophobic polymer is at least one selected from the group consisting of polyolefin, polyamide, polyimide, polyester, polyketone, polyvinylidene fluoride, polymethyl methacrylate, polyacrylonitrile, and polysulfone-based polymer.
10. The method according to claim 6, wherein the hydrophilic polymer is at least one selected from the group consisting of a vinyl polymer, a polysaccharide, a block copolymer of ethylene glycol and a hydrophobic monomer, a random copolymer or block copolymer of ethylene glycol and propylene glycol or phenyl ethylene glycol, a polymer in which one or both ends of polyethylene glycol are substituted with a hydrophobic group and are not water-dissolved, a hydrophilized polyethylene terephthalate or polyethersulfone, and a hydrophilic polymer obtained by introducing a hydrophilic group into the hydrophobic polymer.
11. A method for removing virus particles from a protein solution containing the virus particles using a virus-removing film,
The virus-removing film comprises a hydrophobic polymer and a hydrophilic polymer,
The ratio kf/km of the fouling resistance kf to the membrane resistance km of the virus-removing membrane obtained by filtering protein aggregates having an average particle diameter of 30nm and a polydispersity index of 0.10 at 2.0bar at 0.1g/m 2 is not less than 0.04 and not more than 1.0,
The capture width of the aggregate observed by Proteostat staining is 4 μm or more and 10 μm or less,
The logarithmic removal rate of the porcine parvovirus of the virus removal membrane is more than 4.0,
The thickness unevenness ratio of the virus-removing film is 1.2 or less.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022174344A JP2024065468A (en) | 2022-10-31 | 2022-10-31 | Virus removal membrane and its manufacturing method and use method |
| JP2022-174344 | 2022-10-31 |
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| Publication Number | Publication Date |
|---|---|
| CN117942791A true CN117942791A (en) | 2024-04-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311406115.6A Pending CN117942791A (en) | 2022-10-31 | 2023-10-27 | Virus removal membrane and methods of making and using the same |
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| Country | Link |
|---|---|
| JP (1) | JP2024065468A (en) |
| CN (1) | CN117942791A (en) |
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2022
- 2022-10-31 JP JP2022174344A patent/JP2024065468A/en active Pending
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- 2023-10-27 CN CN202311406115.6A patent/CN117942791A/en active Pending
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| Publication number | Publication date |
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| JP2024065468A (en) | 2024-05-15 |
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