Method for filtering cell culture using high-connectivity hollow fiber membrane

Abstract

[Problem] To provide a more stable filtration method capable of separating a culture containing cells from a product containing an enzyme and an antibody at a low tangential flow velocity without clogging. [Solution] A filtration method comprising a filtration step for passing a culture solution containing cells, a culture medium, a product, and optionally an antifoaming agent through a porous membrane formed from a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, in an SEM image of a membrane cross-section in a membrane thickness direction orthogonal to the inner surface of the porous membrane, in each region of four fields of view including a field of view including the inner surface, a field of view including the outer surface of the film, and two fields of view imaged between the fields of view at equal intervals, the sum of the areas of the resin parts having an area of 1 [mu] m2 or less is 70% or more with respect to the total area of the resin parts.

Description

This application claims the benefit of Japanese patent application No. 2023-182500, filed on October 24, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0001] The present invention relates generally to process filtration systems and, more particularly, to a method for filtering cell cultures using high connectivity hollow fiber membranes. Background Art

[0002] Filtration is used to separate, clarify, modify and / or concentrate fluid solutions, mixtures or suspensions. It is often a necessary step in the biotechnology, pharmaceutical and medical industries during the production, processing and analytical stages of drugs, diagnostics and chemicals. For example, filtration can be used to remove desired compounds from a solution, or to remove byproducts, leaving a more concentrated medium. These processes can be appropriately modified by selecting various filter materials, pore sizes and / or other filtration variables.

[0003] When producing cell culture, it is usually necessary to remove the waste in the developing culture. The progress of biomanufacturing process now allows large-scale production of cell culture, making it possible to produce recombinant proteins, virus-like particles (VLP), gene therapy particles and vaccines, usually using processing container devices. Cell retention devices that remove metabolic waste and update the culture with additional nutrients are widely available. Usually, this retention uses tangential flow filtration, which is carried out by perfusion filtration of hollow fiber membrane processing container culture. In addition, bioprocessing operations are being extended, and continuous culture has been adopted. Such operation may be extended to several days, weeks or months. Many typical components, such as filters, are expected to be free of scaling under such a long time, or fully operate when maintenance or replacement is not required.

[0004] Hollow fiber membrane is usually used for cell culture perfusion and separation of products, but such use may cause membrane fouling due to cell debris. And fouling may cause the expected product to remain in the membrane instead of passing it through. Hollow fiber membrane is usually made of polyethersulfone (PES), polysulfone, cellulose, polyvinylidene fluoride, polypropylene, polyethylene and other materials, and it is continuously needed to resist fouling and allow the membrane of the solution of high solid content to be filtered at the same time. Fluororesins such as polyethylene (PE) and polyvinylidene fluoride (PVDF) are hydrophobic materials that have been widely used in industrial filtration, and it is known that specific molecules are unlikely to be adsorbed. Specifically, it is found that the adsorption level of polyphenols, polysaccharides and tannins is low. On the other hand, the structure of the membrane is very different because of the different manufacturing methods, and even if the membrane made of the same material is used, the fouling phenomenon of the membrane is also very different because of the different manufacturing methods.

[0005] The following patent document 1 describes the relationship between the permeate filtration rate and the medium flow rate in tangential flow filtration. It also describes the use of a porous membrane containing a dense layer. In addition, as Figure 7 As shown, tangential flow filtration, also known as cross-flow filtration, is a method in which the sample of the solution to be filtered flows back in a direction parallel to the membrane surface, exerting a certain pressure on the membrane and allowing particles smaller than the membrane pore size to pass through. In contrast, common direct flow filtration methods, such as syringe filters and centrifugal filters, pass the liquid from the top to the bottom through the membrane surface. In this case, the particles contained in the sample will be trapped on the membrane surface and inside, accumulating to cause blockage and impose excessive pressure load on the sample. In contrast, the characteristic of tangential flow filtration is that because the sample continuously flows back in a direction parallel to the membrane surface, particles and molecules larger than the membrane pore diameter remain upstream of the membrane, the deposition of particles on the membrane is suppressed, and the load on the sample is low.

[0006] The following Patent Document 2 describes a cell culture method and apparatus that can remove cell growth inhibitory substances and return at least one component of serum, which is an expensive high-molecular component, to a culture tank during high-density culture to reduce running costs.

[0007] The following Patent Document 3 describes a method for increasing the yield of a recombinant protein in a mammalian cell culture expressing a recombinant protein, wherein the method comprises establishing a mammalian cell culture in a serum-free medium in a bioreactor and inducing cell growth arrest by perfusing with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less.

[0008] The following Patent Document 4 describes a study of monitoring and measuring the permeate flow rate from a filtration membrane during perfusion culture, and switching the filtration membrane when the permeate flow rate is at least 10% of the permeate flow rate at the start of tangential flow filtration.

[0009] The following Patent Document 5 describes a method for operating a tangential flow process in perfusion culture, which method can efficiently separate products. Prior art literature Patent Literature

[0010] [Patent Document 1] Japanese Patent Application Publication No. 2018-76291 [Patent Document 2] Japanese Patent Application Publication No. 2000-32977 [Patent Document 3] Japanese Patent Publication No. 2014-520534 [Patent Document 4] Japanese Patent Application Publication No. 2014-024824 [Patent Document 5] International Publication No. 2015 / 188009 Summary of the invention Problems to be solved by the invention

[0011] According to the technology described in Patent Documents 1-4, a method for recovering useful substances from a culture solution in a continuous culture of cells producing useful substances is described, the method comprising an effusion step of draining the culture solution from a cell culture tank and adding a fresh culture medium equal to the drained culture solution to the culture tank; and a filtration step of filtering the culture solution extracted from the culture tank using a porous membrane that does not substantially have a dense layer, wherein the filtration in the filtration step is tangential flow filtration. In addition, Patent Document 5 describes only a technology for efficiently separating products and impurities.

[0012] However, although all of Patent Documents 1-4 disclose research on membrane usage such as the speed of the permeate in the filtration process, there is no record of the most suitable membrane for tangential flow filtration, and it is not clear what specific structure and material can achieve stable tangential flow. In the continuous filtration process, membrane clogging is bound to occur, which must be solved. In addition, in terms of separation, it is also necessary to retain cells and microorganisms on the concentration side, while the product is not blocked by the membrane and permeates to the permeate side. In addition, when the culture object is animal cells, if the shear rate is too high, it will cause defects in cell growth, so there is a problem that the linear velocity of the tangential flow cannot be increased.

[0013] In view of the above-mentioned prior art level, the problem to be solved by the present invention is to provide a more stable filtration method, which can separate the culture from the product at a lower tangential streamline velocity and will not cause clogging. Solution to the problem

[0014] In order to solve the above-mentioned problems, the inventors of the present application repeatedly conducted in-depth research experiments and unexpectedly found that by using a porous filtration membrane with good pore permeability from the inner side of the membrane as the treated liquid side to the outer side of the membrane as the filtrate side, the membrane has good permeability to the culture product and is not easy to be clogged even when the tangential streamline velocity is low, thereby completing the present invention.

[0015] That is, the present invention is as follows. [1] A filtration method comprising a filtration step of passing a culture solution containing cells, a culture medium, a product and, optionally, a defoaming agent through a porous membrane formed of a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, Among them, in the SEM image of the film cross-section in the film thickness direction orthogonal to the inner surface of the porous film, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the film, and two fields of view taken at equal intervals between these fields of view, a total of four fields of view, there is a 1 μm 2 The sum of the areas of the resin portions with areas of 100 Å and below is 70% or more of the total area of ​​the resin portions. [2] A filtration method comprising a filtration step of passing a culture solution containing cells, a culture medium, a product and, optionally, a defoaming agent through a porous membrane formed of a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, Among them, in the SEM image of the film cross-section in the film thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the membrane, and two fields of view taken between these fields of view at equal intervals, a total of four fields of view, there is a 10 μm 2 The sum of the areas of the resin portions having an area of ​​100 mm or more is 15% or less with respect to the total area of ​​the resin portions. [3] A filtration method comprising a filtration step of passing a culture solution containing cells, a culture medium, a product and, optionally, a defoaming agent through a porous membrane formed of a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, Among them, in the SEM image of the film cross-section in the film thickness direction orthogonal to the inner surface of the porous film, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the film, and two fields of view taken at equal intervals between these fields of view, a total of four fields of view, there is a 1 μm 2 The sum of the areas of the resin portions with an area of ​​10 μm or less is 70% or more of the total area of ​​the resin portions, and 2 The sum of the areas of the resin portions having an area of ​​100 Å or more is 15% or less of the total area of ​​the resin portions. [4] A filtration method according to any one of the above [1] to [3], wherein in a SEM image of a membrane cross-section in a membrane thickness direction orthogonal to the inner surface of the porous membrane, in each of four fields of view, including a field of view including the inner surface, a field of view including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, there is a particle size of more than 1 μm. 2 And less than 10μm 2 The sum of the areas of the resin portions having an area of ​​1000 Å is 15% or less of the total area of ​​the resin portions. [5] The filtration method according to any one of [1] to [4] above, wherein the surface porosity of the porous membrane is 25 to 60%. [6] The filtration method according to any one of [1] to [5] above, wherein the flux of the porous membrane in the filtration step is 1 LMH or more. [7] The filtration method according to any one of [1] to [6] above, wherein the porous membrane is a hollow fiber membrane, and the filtration step is perfusion culture filtration. [8] The filtration method according to the above [7], wherein the linear velocity of the tangential flow in the hollow fiber membrane during the perfusion culture is 0.5 m / s or less. [9] The filtration method according to any one of [1] to [8] above, wherein the resin forming the porous membrane is a thermoplastic resin.

[10] The filtration method according to [9] above, wherein the thermoplastic resin is a fluororesin.

[11] The filtration method according to the above

[10] , wherein the fluororesin is selected from polyvinylidene fluoride resin (PVDF), chlorotrifluoroethylene resin, tetrafluoroethylene resin, ethylene-tetrafluoroethylene copolymer (ETFE), ethylene-monochlorotrifluoroethylene copolymer (ECTFE), hexafluoropropylene resin, and a mixture of these resins.

[12] The filtration method according to [9] above, wherein the thermoplastic resin is polyethylene (PE).

[13] The filtration method according to any one of [1] to

[12] above, wherein the product is selected from proteins including enzymes and antibodies, amino acids, nucleic acids, and organic matter. Effects of the Invention

[0016] The method for filtering culture fluid involved in the present invention uses a membrane with good connectivity of micropores from the inner side of the membrane on the treated fluid side as a porous filtration membrane to the outer side of the membrane on the filtrate side. Therefore, a culture containing cells and a product containing enzymes and antibodies can be separated at a lower tangential streamline velocity. The product has good permeability, cells are not likely to cause clogging, and the filtration performance is excellent. In addition, this method is a filtration method in which the membrane is not damaged during the filtration operation, so proteins such as antibodies can be separated efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

[0018] The preferred embodiments of the present invention (hereinafter referred to as "embodiments") are described in detail below. It should be noted that the embodiments shown below illustrate devices and methods for implementing the technical ideas of the present invention, and the combination of components and the like of the technical ideas of the present invention are not specific to the following contents. The technical ideas of the present invention can be modified in various ways in the patent claims.

[0019] In the following description, the upper limit or lower limit recorded in a certain numerical range in the numerical range recorded in stages can be replaced by the upper limit or lower limit of the numerical range in other staged records. In the present embodiment, the upper limit or lower limit recorded in a certain numerical range can also be replaced by the value recorded in the embodiment. In addition, about the term "process" in the following description, it goes without saying that an independent process, when it cannot be clearly distinguished from other processes, is also included in this term as long as the function of the "process" can be realized.

[0020] The filtration method of this embodiment includes a filtration step of passing a culture solution containing cells, a culture medium, a product, and a defoaming agent as an optional component as required through a porous membrane formed of a resin having a three-dimensional network structure, and separating the product from the culture solution. Among them, in the SEM image of the film cross-section in the film thickness direction orthogonal to the inner surface of the porous film, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the film, and two fields of view taken at equal intervals between these fields of view, a total of four fields of view, there is a 1 μm 2 The sum of the areas of the resin parts with an area of ​​less than 10 μm is 70% or more of the total area of ​​the resin parts. 2 The sum of the areas of the resin parts with an area of ​​100 μm or more is 15% or less relative to the total area of ​​the resin parts. In the porous membrane, in the SEM image of the membrane cross section in the membrane thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​the four fields of view including the field of view of the inner surface, the field of view including the outer surface of the membrane, and two fields of view taken between these fields of view at equal intervals, the number of the cross-sectional area of ​​the membrane preferably exceeds 1 μm. 2 And less than 10μm 2 The sum of the areas of the resin portions having an area of ​​1000 Å is 15% or less of the total area of ​​the resin portions.

[0021] The filtration method of this embodiment is characterized in that it includes a filtration step of passing a culture solution containing cells, a culture medium, a product, and a defoaming agent through a porous membrane formed of a resin with a three-dimensional network structure, and separating a filtrate containing the product and a culture solution containing cells from the culture. The above-mentioned products are not particularly limited and include proteins including enzymes and antibodies, amino acids, nucleic acids, organic substances, etc., and proteins including enzymes and antibodies that should be produced by cells, such as transgenic cells (such as CHO cells) that produce useful proteins. In addition, before, after, or before or after the above-mentioned filtration step, an additional treatment step such as centrifugal separation, diatomaceous earth filtration, other membrane separation, sieving treatment, etc. may be further included. The shape of the porous membrane is not particularly limited, and examples thereof include flat membranes, tubular membranes, and hollow fiber membranes. From the viewpoint of space saving of the filtration device, that is, because the membrane area per unit volume of the membrane module can be increased, a hollow fiber membrane is preferred.

[0022] As a filtration process in the filtration method of this embodiment, for example, it can be a so-called internal pressure filtration process in which a culture broth (treated liquid) containing cells, culture medium, useful substances and defoaming agents is supplied to the hollow part (inner surface) of the porous hollow fiber membrane, passed through the membrane thickness (wall thickness) part of the porous hollow fiber membrane, and the liquid seeping out from the outer surface of the porous hollow fiber membrane is taken out as the filtrate. It can also be a so-called external pressure filtration process in which a treated liquid is supplied from the outer surface of the porous hollow fiber membrane, and the filtrate seeping out from the inner surface of the porous hollow fiber membrane is taken out through the hollow part. In this specification, the term "interior of the porous film" refers to a film thickness (wall thickness) portion where numerous micropores are formed.

[0023] The above-mentioned product can be an antibody protein, as described in the general definition in biochemistry, and can be listed as a glycoprotein molecule (also called gamma globulin or immunoglobulin) produced by B lymphocytes as a defense mechanism against infection in vertebrates. For example, in the embodiment, the antibody purified by the porous membrane is used as a human drug, and has substantially the same structure as the antibody in the human body as the subject of administration.

[0024] The above products can be various antibody vaccines, vector viruses, plasmid DNA or industrial enzymes such as amylase and protease. Different culture objects can be selected for different products. For example, when the product is an enzyme, the culture object can be bacteria such as genetically modified Escherichia coli; when the product is a protein such as albumin, the culture object can be genetically modified Pichia pastoris.

[0025] Hereinafter, the structure, material (materials), and production method of the porous membrane used in the method for filtering a culture solution according to the present embodiment will be described in detail. <Porous membrane> The porous membrane used in the method for filtering the culture broth of the present embodiment is any porous membrane selected from the following: in an SEM image of a membrane cross section in a membrane thickness direction perpendicular to the inner surface of the porous membrane, in each of four fields of view including the inner surface, the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, there is a 1 μm 2 The total area of ​​the resin part with an area of ​​10 μm or less is 70% or more of the total area of ​​the resin part; in each of the above regions, there is a porous membrane with a 2The total area of ​​the resin portion of the porous membrane with an area of ​​1 μm or more is 15% or less relative to the total area of ​​the resin portion; in each of the above regions, there is a 1 μm 2 The sum of the areas of the resin parts with an area of ​​less than 10 μm is 70% or more of the total area of ​​the resin parts. 2 The total area of ​​the resin parts with an area of ​​1 μm or more is 15% or less of the total area of ​​the resin parts. 2 The sum of the areas of the resin parts with an area of ​​less than 1 μm is 70% or more of the total area of ​​the resin parts. 2 And less than 10μm 2 The sum of the areas of the resin parts with an area of ​​10 μm is 15% or less of the total area of ​​the resin parts. 2 A porous membrane in which the sum of the areas of the resin portions having an area of ​​1000 Å or more is 15% or less of the total area of ​​the resin portions.

[0026] Figure 1 An example of a SEM image of a cross section of a porous membrane used in the filtering method of the present embodiment. The SEM image is an image obtained by photographing a given field of view in the following region and binarizing the obtained SEM image photograph, wherein the region is a SEM image of a membrane cross section in a film thickness direction orthogonal to the inner surface of a hollow fiber porous membrane, in a total of four fields of view, including a field of view of the inner surface, a field of view of the outer surface of the membrane, and two fields of view photographed between these fields of view at equal intervals, in a region closest to the inner side, and in a region closest to the inner side. It should be noted that in the above-mentioned regions, the difference in the distribution of the resin part, that is, the anisotropy of the connectivity of the pores, between the membrane cross-section in the membrane thickness direction orthogonal to the inner surface of the hollow fiber porous membrane and the cross-section parallel to the inner surface, is actually negligible. In the present specification, the term "resin portion" refers to a tree-like skeleton portion of a three-dimensional network structure formed of a resin that forms numerous pores in a porous film. Figure 1 The black part is the resin part, and the white part is the hole. In the porous membrane, there are connected pores that are bent and connected from the inside to the outside of the membrane. In the SEM image of the membrane cross-section in the membrane thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​the four fields of view including the field of view of the inner surface, the field of view including the outer surface of the membrane, and the two fields of view taken between these fields of view at equal intervals, if there is a 1 μm 2If the sum of the areas of the resin parts with an area of ​​1 μm or less is 70% or more relative to the total area of ​​the resin parts, the pore connectivity is high (i.e., the proportion of the interconnected pores inside the membrane is high), the flux of the treated liquid (water permeability, water permeability), and the water permeability retention rate after washing are high, and the damage to the membrane after washing with the tensile elongation at break as an indicator is also reduced. However, if the membrane has a 1 μm 2 If the ratio of the sum of the areas of the resin parts with an area of ​​1 μm or less to the total area of ​​the resin parts is too high, the dendritic skeleton part of the three-dimensional network structure formed by the resin forming numerous pores in the porous membrane becomes too thin. 2 The sum of the areas of the resin parts with an area of ​​less than 1 μm is maintained at 70% or more relative to the total area of ​​the resin parts, and the 2 And less than 10μm 2 The sum of the areas of the resin portions with a surface area of ​​2 μm or more and 30% or less relative to the total area of ​​the resin portions is present, and more preferably 10 μm or more. 2 The total area of ​​the resin parts with an area of ​​more than 1 μm is 15% or less relative to the total area of ​​the resin parts, and it is more preferable to have a total area of ​​more than 1 μm. 2 And less than 10μm 2 The sum of the areas of the resin parts with an area of ​​10 μm is 15% or less of the total area of ​​the resin parts. 2 The sum of the areas of the resin portions having an area of ​​more than 1 μm is 2% or more and 15% or less relative to the total area of ​​the resin portion. 2 And less than 10μm 2 If the sum of the areas of the resin parts is 2% or more and 30% or less, preferably 15% or less, relative to the total area of ​​the resin parts, the tree-like skeleton part of the three-dimensional network structure formed by the resin will not be too thin, so the strength and tensile elongation at break of the porous membrane can be properly maintained.

[0027] Figures 2 to 6 The following are bar graphs showing the ratio (%) of the sum of the areas of resin portions having a given area to the total area of ​​the resin portions in each region (circle 1 to circle 4), including a field of view including the inner surface, a field of view including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, in the SEM images of the membrane cross-section in the membrane thickness direction orthogonal to the inner surface of the porous membrane used in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1. Figure 1 In the binary SEM image in , the resin portion appears granular. Figures 2 to 6In the figure, the area of ​​each granular resin portion is measured, and the area ratio of each granular resin portion relative to the total area of ​​the entire resin portion in a field of view of a given size in each region is shown in the form of a bar graph. Figures 2 to 6 Circle 1 in the figure is the number of the area closest to the inside in the SEM image of the membrane cross section in the membrane thickness direction orthogonal to the inner surface of the porous membrane, and circle 4 is the number of the area closest to the inside in the area of ​​four fields of view, including the field of view of the inner surface, the field of view of the outer surface of the membrane, and two fields of view taken between these fields of view at equal intervals. For example, circle 1 of Example 1 is a bar graph when a field of view of a given size in the innermost area of ​​the porous hollow fiber membrane of Example 1 is taken. The method for measuring the area distribution of the resin portion in each area of ​​the porous hollow fiber membrane will be described later.

[0028] The surface porosity of the porous membrane is preferably 25 to 60%, more preferably 25 to 50%, and further preferably 25 to 45%. If the surface porosity on the side in contact with the treatment object liquid is 25% or more, the deterioration of the water permeability caused by clogging and rubbing of the membrane surface is reduced, so the filtration stability can be improved. On the other hand, if the surface porosity becomes high and the pore size is too large, there is a hidden danger that the required separation performance cannot be exerted. Therefore, the average micropore diameter of the porous membrane is preferably 100 to 700nm, more preferably 20 to 600nm. If the average micropore diameter is 30 to 400nm, the separation performance is sufficient and the connectivity of the pores can also be ensured. The determination methods of the surface porosity and the average micropore diameter are described below.

[0029] The thickness of the porous membrane is preferably 80 to 1,000 μm, more preferably 100 to 300 μm. If the membrane thickness is 80 μm or more, the membrane strength can be ensured, while if the membrane thickness is 1,000 μm or less, the pressure loss due to membrane resistance becomes small.

[0030] The shape of the porous hollow fiber membrane may be a toroidal single-layer membrane, or a multilayer membrane in which the separation layer and the support layer supporting the separation layer have different pore sizes. In addition, the membrane may have a profiled cross-sectional structure with protrusions on the inner and outer surfaces.

[0031] (Material of porous membrane) The resin constituting the porous membrane is preferably a thermoplastic resin such as polyethylene, and more preferably a fluororesin. As the fluororesin, there can be mentioned those selected from vinylidene fluoride resin (PVDF), chlorotrifluoroethylene resin, tetrafluoroethylene resin, ethylene-tetrafluoroethylene copolymer (ETFE), ethylene-monochlorotrifluoroethylene copolymer (ECTFE), hexafluoropropylene resin, and mixtures of these resins. As thermoplastic resins, there can be cited: copolymers of polyolefins, olefins and halogenated olefins, halogenated polyolefins, and mixtures thereof. As thermoplastic resins, there can be cited, for example: polyethylene (PE), polypropylene, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, ethylene-tetrafluoroethylene copolymer, polyvinylidene fluoride (which may also contain the domain of hexafluoropropylene), and mixtures thereof. These resins are thermoplastic, so they are excellent in operability and tough, and therefore excellent as membrane materials. Among these, homopolymers or copolymers of vinylidene fluoride resins, tetrafluoroethylene resins, hexafluoropropylene resins or mixtures thereof, ethylene, tetrafluoroethylene, chlorotrifluoroethylene, or mixtures of homopolymers and copolymers are preferred because they have excellent mechanical strength, chemical strength (chemical resistance), and good formability. More specifically, fluororesins such as polyvinylidene fluoride, vinylidene fluoride-hexafluoropropylene copolymers, ethylene-tetrafluoroethylene copolymers, and ethylene-chlorotrifluoroethylene copolymers can be cited.

[0032] The porous film may contain components (impurities, etc.) other than the thermoplastic resin within about 5% by mass. For example, the porous film may contain a solvent used when the porous film is manufactured. As described later, the porous film may contain the first solvent (hereinafter also referred to as a non-solvent), the second solvent (hereinafter also referred to as a good solvent or a poor solvent), or both of them used as a solvent when the porous film is manufactured. These solvents can be detected by pyrolysis GC-MS (gas chromatography mass spectrometry).

[0033] The first solvent may be at least one selected from sebacate, citrate, acetylcitrate, adipate, trimellitate, oleate, palmitate, stearate, phosphate, fatty acid having 6 or more and 30 or less carbon atoms, and epoxidized vegetable oil. In addition, the second solvent is different from the first solvent and can be at least one selected from sebacate, citrate, acetylcitrate, adipate, trimellitate, oleate, palmitate, stearate, phosphate, fatty acid having 6 or more and 30 or less carbon atoms, and epoxidized vegetable oil. As the fatty acid having 6 or more and 30 or less carbon atoms, capric acid, lauric acid, oleic acid, etc. can be listed. In addition, as the epoxidized vegetable oil, epoxy soybean oil, epoxidized linseed oil, etc. can be listed. The first solvent is preferably a non-solvent in which the thermoplastic resin does not uniformly dissolve in the first solvent even when the temperature of the first mixed liquid is increased to the boiling point of the first solvent in the first mixed liquid having a ratio of the thermoplastic resin to the first solvent of 20:80. The second solvent is preferably a good solvent in which the thermoplastic resin is uniformly dissolved in the second solvent at any temperature of the second mixed liquid being higher than 25° C. and below the boiling point of the second solvent in a second mixed liquid having a ratio of the thermoplastic resin to the second solvent of 20:80. The second solvent is more preferably a poor solvent in which, in the second mixed liquid having a ratio of thermoplastic resin to the second solvent of 20:80, the thermoplastic resin is not uniformly dissolved in the second solvent when the temperature of the second mixed liquid is 25°C, and the thermoplastic resin is uniformly dissolved in the second solvent at any temperature where the temperature of the second mixed liquid is higher than 100°C and below the boiling point of the second solvent. Alternatively, only the first solvent may be used.

[0034] In the method for filtering a culture solution of the present embodiment, a porous hollow fiber membrane using polyvinylidene fluoride (PVDF) as a thermoplastic resin and containing a first solvent (non-solvent) can be used. In this case, the first solvent may be at least one selected from sebacate, citrate, acetylcitrate, adipate, trimellitate, oleate, palmitate, stearate, phosphate, fatty acid having 6 or more and 30 or less carbon atoms, and epoxidized vegetable oil, and in the first mixed solution having a ratio of polyvinylidene fluoride to the first solvent of 20:80, the first solvent may be a non-solvent in which polyvinylidene fluoride does not uniformly dissolve in the first solvent even when the temperature of the first mixed solution is raised to the boiling point of the first solvent. As the non-solvent, bis(2-ethylhexyl) adipate (DOA) is preferred. In addition, the porous hollow fiber membrane may also contain a second solvent different from the first solvent. In this case, the second solvent is preferably selected from at least one of sebacate, citrate, acetylcitrate, adipate, trimellitate, oleate, palmitate, stearate, phosphate, fatty acid with 6 or more carbon atoms and 30 or less carbon atoms, and epoxidized vegetable oil, and in the second mixed solution where the ratio of polyvinylidene fluoride to the second solvent is 20:80, it is a good solvent in which polyvinylidene fluoride is uniformly dissolved in the second solvent at any temperature above 25°C and below the boiling point of the second solvent. In addition, the second solvent is more preferably a poor solvent in which polyvinylidene fluoride is not uniformly dissolved in the second solvent when the temperature of the second mixed solution is 25°C, and polyvinylidene fluoride is uniformly dissolved in the second solvent at any temperature above 100°C and below the boiling point of the second solvent. As a poor solvent, acetyl tributyl citrate (ATBC) is preferred.

[0035] (Physical properties of porous membrane) When the porous membrane is washed, it is preferred that the relationship between the tensile elongation at break E0 of the porous membrane before the washing process and the tensile elongation at break E1 of the porous membrane after the washing process is E1 / E0×100≥98% of the porous membrane. In addition, it is preferred that the relationship between the tensile elongation at break E0 of the porous membrane before the washing process and the tensile elongation at break EX of the porous membrane after repeating the washing process X times (wherein X is an integer of 2 to 10) is: EX / E0×100≥97%. The initial value of the tensile elongation at break is preferably 60% or more, more preferably 80% or more, further preferably 100% or more, and particularly preferably 120% or more. The method for measuring the tensile elongation at break will be described later. When only an aqueous solution containing a surfactant is used as the cleaning liquid (chemical solution), for example, when only an aqueous solution containing 1 weight % of sodium dodecyl sulfate is used, there will be no particular problem with the chemical solution resistance of the membrane. However, when an aqueous solution containing 0.1 weight % or more and 4 weight % or less of sodium hydroxide and 0.01 weight % or more and 0.5 weight % or less of sodium hypochlorite in addition to the aqueous solution containing a surfactant is used as the cleaning liquid, the chemical solution resistance (the degree to which damage to the membrane is unlikely to occur) becomes a problem. In this case, for the drug solution resistance of the membrane, an aqueous solution containing 4 wt% sodium hydroxide and 0.5 wt% sodium hypochlorite can be used as the resistance test solution, and the retention rate of the tensile elongation at break before and after the drug solution circulation filtration and washing (elongation retention rate after drug solution circulation filtration) is used as an indicator. Specifically, after a series of steps of real liquid filtration followed by drug solution circulation washing, the tensile elongation at break (equivalent to the tensile elongation at break E1 of the porous hollow fiber membrane after the washing step) is preferably maintained at 98% or above relative to the initial value (equivalent to the tensile elongation at break E0 of the membrane before the washing step). The relationship between the initial value E0 and the tensile elongation at break EX of the membrane after a series of steps of real liquid filtration followed by chemical solution circulation washing are repeated X times (X is an integer of 2 to 10) is preferably: EX / E0 ≥ 97%.

[0036] From a practical point of view, the compressive strength of the porous membrane is preferably 0.1 MPa or more, more preferably 0.15 to 1.0 MPa, and even more preferably 0.2 to 1.0 MPa.

[0037] (Water permeability of porous membrane) As for the porous membrane, the relationship between the pure water flux L0 of the porous membrane before the filtration step and the pure water flux L1 of the porous membrane after the washing step is preferably L1 / L0×100≥80%. In addition, as a porous membrane, the relationship between the flux L0 of the porous membrane before the filtration step and the flux LX of the porous membrane after repeating the washing step X times (where X is an integer of 2 to 10) is preferably: LX / L0×100≥80%.

[0038] <Method for producing porous film> Hereinafter, a method for producing a porous hollow fiber membrane will be described. However, the method for producing a porous hollow fiber membrane used in the filtration method of the present embodiment is not limited to the following production method. The method for producing the porous hollow fiber membrane used in the filtration method of the present embodiment may include (a) a step of preparing a molten kneaded product, (b) a step of supplying the molten kneaded product to a spinning nozzle of a multilayer structure and extruding the molten kneaded product from the spinning nozzle to obtain a hollow fiber membrane, and (c) a step of extracting a plasticizer from the hollow fiber membrane. When the molten kneaded product contains an additive, the step of further extracting the additive from the hollow fiber membrane may be included after step (c).

[0039] The concentration of the thermoplastic resin in the melt-kneaded product is preferably 20 to 60% by mass, more preferably 25 to 45% by mass, and further preferably 30 to 45% by mass. If this value is 20% by mass or more, the mechanical strength can be improved, while if it is 60% by mass or less, the water permeability can be improved. The melt-kneaded product may contain additives. The melt-kneaded product may contain two components, namely, a thermoplastic resin and a solvent, or three components, namely, a thermoplastic resin, an additive, and a solvent. The solvent includes at least a non-solvent as described below. As the extractant used in step (c), it is preferable to use a liquid which does not dissolve the thermoplastic resin but has a high affinity with the plasticizer, such as dichloromethane and various alcohols. In the case of using a melt-kneaded product that does not contain additives, the hollow fiber membrane obtained by step (c) can also be used as a porous hollow fiber membrane. In the case of using a melt-kneaded product containing additives to manufacture a porous hollow fiber membrane, it is preferred to further extract and remove the additives from the hollow fiber membrane (d) after step (c) to obtain a porous hollow fiber membrane. In the extractant in step (d), hot water, or a liquid such as an acid or alkali that can dissolve the additive used but does not dissolve the thermoplastic resin is preferably used.

[0040] Inorganic substances can also be used as additives. The inorganic substance is preferably an inorganic fine powder. The primary particle size of the inorganic fine powder contained in the molten kneaded material is preferably 50 nm or less, more preferably 5 nm and above and less than 30 nm. Specific examples of inorganic fine powders include silicon dioxide (including fine powder silicon dioxide), titanium oxide, lithium chloride, calcium chloride, organic clay, etc. Among these, fine powder silicon dioxide is preferred from the perspective of cost. The above-mentioned "primary particle size of inorganic fine powder" represents the value obtained by analyzing the electron microscope photograph. That is, first, a group of inorganic fine powders are pre-treated using the method of ASTM D3849. Afterwards, the diameters of 3000 to 5000 particles photographed on the transmission electron microscope photograph can be measured, and the primary particle size of the inorganic fine powder can be calculated by arithmetically averaging these values. The inorganic fine powder inside the porous hollow fiber membrane can be identified by identifying the elements present using fluorescent X-rays or the like, thereby identifying the material of the inorganic fine powder present. When an organic substance is used as an additive, a hydrophilic polymer such as polyvinyl pyrrolidone or polyethylene glycol can be used to impart hydrophilicity to the hollow fiber membrane. In addition, a high viscosity additive such as glycerol or ethylene glycol can be used to control the viscosity of the melt-kneaded product.

[0041] Next, the step (a) of preparing the melt-kneaded product in the method for producing the porous hollow fiber membrane according to the present embodiment will be described in detail. In the manufacturing method of the porous hollow fiber membrane of the present embodiment, a non-solvent of a thermoplastic resin is mixed with a good solvent or a poor solvent. The mixed solvent is a non-solvent of the thermoplastic resin used. If a non-solvent is used as a raw material of the membrane in this way, a porous hollow fiber membrane with a three-dimensional network structure can be obtained. Although its mechanism of action is not necessarily clear, it can be considered that when a solvent mixed with a non-solvent is used to make the solubility lower, the crystallization of the polymer will be appropriately hindered and a three-dimensional network structure will be easily formed. For example, with respect to non-solvents, and poor solvents or good solvents, various esters such as phthalates, sebacates, citrates, acetylcitrates, adipic acid esters, trimellitates, oleates, palmitates, stearates, phosphates, fatty acids with 6 or more carbon atoms and 30 or less carbon atoms, and epoxidized vegetable oils can be selected. A solvent that can dissolve a thermoplastic resin at room temperature is called a good solvent, a solvent that cannot dissolve a thermoplastic resin at room temperature but can dissolve a thermoplastic resin at a high temperature is called a poor solvent for the thermoplastic resin, and a solvent that cannot dissolve a thermoplastic resin even at a high temperature is called a non-solvent. Good solvents, poor solvents, and non-solvents can be determined as follows. About 2 g of thermoplastic resin and about 8 g of solvent are added to a test tube, and the temperature is increased by 10°C intervals using a block heater for the test tube until the boiling point of the solvent is reached. The contents of the test tube are mixed with a spatula or the like. The solvent in which the thermoplastic resin is dissolved is a good solvent or a poor solvent, and the solvent in which the thermoplastic resin is not dissolved is a non-solvent. A solvent that is dissolved at a relatively low temperature of 100°C or less is judged as a good solvent, and a solvent that is not dissolved unless a high temperature of 100°C or more and below the boiling point is reached is judged as a poor solvent. For example, if polyvinylidene fluoride (PVDF) is used as a thermoplastic resin and acetyl tributyl citrate (ATBC), dibutyl sebacate or dibutyl adipate is used as a solvent, PVDF is uniformly mixed and dissolved in these solvents at around 200° C. On the other hand, if bis(2-ethylhexyl) adipate (DOA), diisononyl adipate or bis(2-ethylhexyl) sebacate is used as a solvent, PVDF does not dissolve in these solvents even if the temperature is raised to 250° C. When ethylene-tetrafluoroethylene copolymer (ETFE) is used as the thermoplastic resin and diethyl adipate is used as the solvent, ETFE is uniformly mixed and dissolved at about 200° C. On the other hand, when bis(2-ethylhexyl) adipate (DOA) is used as the solvent, no dissolution occurs. In addition, when ethylene-monochlorotrifluoroethylene copolymer (ECTFE) is used as the thermoplastic resin and triethyl citrate is used as the solvent, it is uniformly dissolved at about 200° C., but when triphenyl phosphite (TPP) is used, it is not dissolved. When polyethylene (PE) is used as the thermoplastic resin and bis(2-ethylhexyl) adipate (DOA) is used as the solvent, PE is uniformly mixed and dissolved at about 200° C. On the other hand, when dibutyl adipate is used as the solvent, it does not dissolve even at a temperature near the boiling point of 300° C. [Example]

[0042] The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these Examples. The physical property values ​​in Examples and Comparative Examples were determined by the following methods.

[0043] (1) Outer and inner diameters of porous hollow fiber membranes The porous hollow fiber membrane was cut into thin slices along a cross section perpendicular to the length direction using a razor, and the outer diameter and inner diameter were measured using a 100-fold magnifying glass. For one sample, the cut surface was measured at 60 locations at intervals of 30 mm in the length direction, and the average value was used as the outer diameter and inner diameter of the hollow fiber membrane.

[0044] (2) Electron microscope photography The porous hollow fiber membrane was cut into a ring shape along a section perpendicular to the length direction, stained with 10% phosphotungstic acid + osmium tetroxide, and embedded in epoxy resin. Then, after cutting, the sample section was subjected to BIB processing to produce a smooth section, and a conductive treatment was performed to produce a microscopic sample. For the prepared microscopic sample, an electron microscope SU8000 series manufactured by HITACHI was used with an accelerating voltage of 1 kV and 5,000 to 30,000 times to examine the fields of view including the inner surface of the membrane thickness (thick wall part) section, the field of view including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, totaling four fields of view ( Figures 2 to 5 In the circle 1 to circle 4 in the figure, an electron microscope (SEM) image of a cross section of the membrane is taken with a given field of view. The magnification can be changed according to the average pore size for measurement. Specifically, it is set to 5000 times when the average pore size is 0.1 μm or more, 10,000 times when the average pore size is 0.05 μm or more and less than 0.1 μm, and 30,000 times when the average pore size is less than 0.05 μm. In addition, the size of the field of view is set to 2560×1920 pixels. In image processing, the captured SEM image was subjected to threshold processing (Image-Adjust-Treshold: Otsu method (selected Otsu)) using ImageJ, thereby binarizing the hole portion and the resin portion. Surface porosity: The surface porosity was measured by calculating the ratio of the resin portion to the pore portion in the binary image. Area distribution of resin part: The size of each binary granular resin part included in the captured SEM image was measured using the "Analyze Particle" command (Analyz Particle: Size 0.10-Infinity) of ImageJ. The total area of ​​all resin parts included in the SEM image was ΣS, and 1 μm 2 The area of ​​the resin portion below is set to ΣS (<1μm 2 ) is calculated by 2 ) / ΣS and calculated the 2 Likewise, the area ratio of the resin portion having an area within a given range was calculated. In addition, the noise reduction during binarization is less than 0.1 μm. 2 The resin part of the area is removed as noise, and the 0.1μm 2The resin part with an area of ​​1 μm or more was selected as the analysis object. In addition, the noise was removed by performing median filtering (Process-Filters-Median: Radius: 3.0 pixels). 2 The area of ​​the resin part below is exactly 0.1μm 2 and above and 1μm 2 The area of ​​the resin portion below. In addition, the granular resin part cut at the edge of the SEM image was also measured. In addition, no "Incude Holes" processing was performed. In addition, no correction processing such as correcting the "snowman" shape to a "flat" shape was performed. Average pore size: measured using the "Plugins-Bone J-Thickness" command of ImageJ. In addition, the space size is defined as the maximum circle size that can enter the space.

[0045] (3) Flux (water permeability, initial pure water flux (LMH)) After the porous hollow fiber membrane is immersed in ethanol, it is repeatedly immersed in pure water. Then, an injection needle is inserted at both ends of the wet hollow fiber membrane of about 10 cm in length. Pure water at 25°C is circulated and filtered at a transmembrane pressure of 0.03 MPa. The amount of pure water permeating from the inner surface of the membrane is measured by the following formula: Initial pure water flux [L / m 2 / h] = 60 × (permeate volume [L]) / {π × (membrane inner diameter [m]) × (membrane effective length [m]) × (measurement time [min])} The pure water flux was determined and the water permeability was evaluated. In addition, the "effective membrane length" refers to the net membrane length excluding the portion where the injection needle is inserted.

[0046] (4) Tensile elongation at break (%) A porous hollow fiber membrane was used as a sample, and the tensile elongation at break was calculated in accordance with JIS K7161. The load and displacement at the tensile break were measured under the following conditions. Measuring instrument: Instron tensile testing machine (AGS-5D manufactured by Shimadzu Corporation) Distance between fixtures: 5cm Stretching speed: 20cm / min

[0047] (5) Chemical resistance test After the actual liquid was filtered, a series of steps of circulating washing with the chemical solution were repeated 10 times. Next, the initial value of the tensile elongation at break (tensile elongation at break before immersion) was set as E0, and the values ​​of the tensile strength at break of the porous hollow fiber membrane after repeating the washing step once and 10 times were set as E1 and E10, respectively, and E10 / E0 was calculated as the "tensile elongation at break retention rate (%) after 10 cycles of chemical solution washing" to evaluate the chemical solution resistance. In addition, the initial pure water permeability is set to L0 (flux L0), and a series of steps based on circulating washing with chemical solution after filtration of the actual liquid are repeated 1 time and 10 times, and the water permeability after the washing step is set to L1 and L10 (flux L10), respectively, and L10 / L0 is calculated as the "water permeability retention rate (%) after 10 cycles of chemical solution washing".

[0048] (6) Transmission pressure (kPa) The permeation pressure of the membrane was calculated as follows: That is, when the inlet pressure of the hollow fiber membrane module is Pi, the outlet pressure is Po, and the permeate side outlet pressure (back pressure) is Pf, the permeation pressure TMP is calculated as follows. TMP=(Pi+Po) / 2-Pf

[0049] (7) Tangential streamline velocity (m / s) The tangential streamline velocity Lv is calculated as follows: That is, when the number of hollow fibers introduced into the hollow fiber membrane module is n (fibers), the inner diameter of the hollow fiber membrane is Di (m), and the flow rate of the raw liquid supplied to the hollow fiber membrane module is V (m3 / s), the tangential streamline velocity is calculated as follows. Lv=V / (nx(πx(Di / 2)^2))

[0050] (8) Minimum antibody permeability The minimum antibody permeability is calculated as follows. That is, after the start of the culture, the antibody concentrations in the culture fluid and the permeate are measured every day, and are set as Cm and Cf respectively, and their ratio (Cf / Cm) is the antibody permeability. The lowest value of the antibody permeability during the entire operation is taken as the minimum antibody permeability. The antibody concentration can be determined by known methods such as ELISA and LC-MS. The method in this embodiment is to use the xP workstation automated immunoassay system manufactured by Gyrolab for quantitative analysis.

[0051] [Example 1] A melt-kneaded product was prepared using 40% by mass of PVDF resin (Kureha, KF-W#1000) as a thermoplastic resin, 23% by mass of fine powder silica (primary particle size: 16 nm), 32.9% by mass of bis(2-ethylhexyl) adipate (DOA) as a non-solvent, and 4.1% by mass of acetyl tributyl citrate (ATBC, boiling point 343°C) as a poor solvent. The temperature of the obtained melt-kneaded product was 240°C. The obtained melt-kneaded product was solidified in water at 30°C after passing a hollow fiber-like extrudate through an empty distance of 120 mm using a spinning nozzle with a double-layer tube structure, and a porous structure was grown using a thermally induced phase separation method. The obtained hollow fiber-like extrudate was taken at a speed of 5m / min and wound on a reel. The wound hollow fiber extrudate was immersed in isopropanol to extract and remove DOA and ATBC, then immersed in water for 30 minutes to replace the water in the hollow fiber membrane, and then immersed in a 20 mass % NaOH aqueous solution at 70°C for 1 hour, and further repeatedly washed with water to extract and remove fine powder silica, thereby producing a porous hollow fiber membrane. The blending composition and production conditions of the obtained porous membrane, as well as various physical properties are shown in the following Table 1. The obtained porous hollow fiber membrane has a three-dimensional network structure.

[0052] The hollow fiber membrane was placed in a module housing with an effective length of 20 cm, so that the membrane area (converted to the inner surface) was 1.9 m 2 The two ends are sealed with epoxy resin to form a hollow fiber filter membrane micromodule with openings at both ends.

[0053] like Figure 8 As shown, the hollow fiber filtration membrane micromodule and KML-100TFF perfusion system (manufactured by Repligen) were used to introduce CHO-K1 cells into a 15L (effective volume: 8.5L) glass bioreactor (manufactured by Repligen) to perform a continuous culture experiment. The basic medium containing glucose and defoaming agent was continuously added and adjusted to keep the cell concentration at 75±10×10 6 cells / mL. At this point, 3 / 4 of the effective volume is replaced within one day. The permeation flux of the hollow fiber membrane micromodule is set to: 1L / m 2 / h, permeating from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow velocity of the culture fluid through the hollow part was set to 0.15m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was stabilized at about 10kPa.

[0054] The permeation rate of the antibody in the permeated liquid was confirmed to be 85% or more in 40 days.

[0055] [Example 2] A melt-kneaded product was prepared using 40% by mass of ETFE resin (manufactured by Asahi Glass, TL-081) as a thermoplastic resin, 23% by mass of fine powder silica (primary particle size: 16 nm), 32.9% by mass of bis(2-ethylhexyl) adipate (DOA) as a non-solvent, and 4.1% by mass of diisobutyl adipate (DIBA) as a poor solvent. The temperature of the obtained melt-kneaded product was 240°C. The obtained melt-kneaded product was solidified in water at 30°C after passing a hollow fiber-like extrudate through a 120mm empty distance using a spinning nozzle with a double-layer tube structure, and a porous structure was grown using a thermally induced phase separation method. The obtained hollow fiber-like extrudate was taken at a speed of 5m / min and wound on a reel. The wound hollow fiber extrudate was immersed in isopropanol to extract and remove DOA and DIBA, then immersed in water for 30 minutes to replace the water in the hollow fiber membrane, and then immersed in a 20 mass % NaOH aqueous solution at 70°C for 1 hour, and further repeatedly washed with water to extract and remove fine powder silica, thereby producing a porous hollow fiber membrane. The blending composition, production conditions, and various physical properties of the obtained porous membrane are shown in Table 1 below.

[0056] The hollow fiber membrane was placed in a module housing with an effective length of 20 cm, so that the membrane area (converted to the inner surface) was 1.9 m 2 The two ends are sealed with epoxy resin to form a hollow fiber filter membrane micromodule with openings at both ends.

[0057] like Figure 8 As shown, the hollow fiber filtration membrane micromodule and KML-100TFF perfusion system (manufactured by Repligen) were used to introduce CHO-K1 cells into a 15L (effective volume: 8.5L) glass bioreactor (manufactured by Repligen) to perform a continuous culture experiment. The basic medium containing glucose and defoaming agent was continuously added and adjusted to keep the cell concentration at 75±10×10 6 cells / mL. At this point, 3 / 4 of the effective volume is replaced within one day. The permeation flux of the hollow fiber membrane micromodule is set to: 1L / m 2 / h, permeating from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow velocity of the culture fluid through the hollow part was set to 0.3m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was stabilized at about 11kPa.

[0058] It was confirmed that the permeation rate of the antibody in the permeated liquid was 80% or more in 40 days.

[0059] [Example 3] A melt-kneaded product was prepared using 40% by mass of ECTFE resin (Halar901, manufactured by Solvay Specialty Polymers) as a thermoplastic resin, 23% by mass of fine powder silica (primary particle size: 16 nm), 32.9% by mass of triphenyl phosphite (TPP) as a non-solvent, and 4.1% by mass of bis(2-ethylhexyl) adipate (DOA) as a poor solvent. The temperature of the obtained melt-kneaded product was 240°C. The obtained melt-kneaded product was solidified in water at 30°C after passing a hollow fiber extrudate through a 120 mm air distance using a spinning nozzle with a double-layer tube structure, and a porous structure was grown using a thermally induced phase separation method. The obtained hollow fiber extrudate was taken at a speed of 5 m / min and wound on a reel. The wound hollow fiber extrudate was immersed in isopropanol to extract and remove TPP and DOA, then immersed in water for 30 minutes to replace the water in the hollow fiber membrane, and then immersed in a 20 mass % NaOH aqueous solution at 70°C for 1 hour, and further repeatedly washed with water to extract and remove fine powder silica, thereby producing a porous hollow fiber membrane. The blending composition, production conditions, and various physical properties of the obtained porous membrane are shown in Table 1 below.

[0060] The hollow fiber membrane was placed in a module housing with an effective length of 20 cm, so that the membrane area (converted to the inner surface) was 1.9 m 2 The two ends are sealed with epoxy resin to form a hollow fiber filter membrane micromodule with openings at both ends.

[0061] like Figure 8 As shown, the hollow fiber filtration membrane micromodule and KML-100TFF perfusion system (manufactured by Repligen) were used to introduce CHO-K1 cells into a 15L (effective volume: 8.5L) glass bioreactor (manufactured by Repligen) to perform a continuous culture experiment. The basic medium containing glucose and defoaming agent was continuously added and adjusted to keep the cell concentration at 75±10×10 6 cells / mL. At this point, 3 / 4 of the effective volume is replaced within one day. The permeation flux of the hollow fiber membrane micromodule is set to: 1L / m 2 / h, permeating from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow velocity of the culture fluid through the hollow part was set to 0.2m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was stabilized at about 10kPa.

[0062] The permeation rate of the antibody in the permeated liquid was confirmed to be 83% or more in 40 days.

[0063] [Example 4] A melt-kneaded product was prepared using 18.5% by mass of PE resin (manufactured by Asahi Chemical Industry Co., Ltd., SH800) as a thermoplastic resin, 27.2% by mass of fine powder silica (primary particle size: 16 nm), and 54.3% by mass of dibutyl adipate (DBA) as a non-solvent. The temperature of the obtained melt-kneaded product was 240°C. The obtained melt-kneaded product was solidified in water at 30°C after passing a hollow fiber extrudate through a 120 mm air distance using a spinning nozzle with a double-layer tube structure, and a porous structure was grown using a thermally induced phase separation method. The obtained hollow fiber extrudate was taken at a speed of 5 m / min and wound on a reel. The wound hollow fiber extrudate was immersed in isopropanol to extract and remove DBA, then immersed in water for 30 minutes to replace the water in the hollow fiber membrane, and then immersed in a 20 mass % NaOH aqueous solution at 70°C for 1 hour, and further repeatedly washed with water to extract and remove fine powder silica, thereby producing a porous hollow fiber membrane. The blending composition, production conditions, and various physical properties of the obtained porous membrane are shown in Table 1 below.

[0064] The hollow fiber membrane was placed in a module housing with an effective length of 20 cm, so that the membrane area (converted to the inner surface) was 1.9 m 2 The two ends are sealed with epoxy resin to form a hollow fiber filter membrane micromodule with openings at both ends.

[0065] like Figure 8 As shown, the hollow fiber filtration membrane micromodule and KML-100TFF perfusion system (manufactured by Repligen) were used to introduce CHO-K1 cells into a 15L (effective volume: 8.5L) glass bioreactor (manufactured by Repligen) to perform a continuous culture experiment. The basic medium containing glucose and defoaming agent was continuously added and adjusted to keep the cell concentration at 75±10×10 6 cells / mL. At this point, 3 / 4 of the effective volume is replaced within one day. The permeation flux of the hollow fiber membrane micromodule is set to: 1L / m 2 / h, permeating from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow velocity of the culture fluid through the hollow part was set to 0.25m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was stabilized at about 12kPa.

[0066] The permeation rate of the antibody in the permeated liquid was confirmed to be 88% or more in 40 days.

[0067] [Comparative Example 1] The membrane was formed in the same manner as in Example 1 except that ATBC was used as the solvent, thereby obtaining a hollow fiber membrane of Comparative Example 1. The blending composition and production conditions of the obtained porous membrane, as well as various physical properties, are shown in Table 1 below.

[0068] The hollow fiber membrane was placed in a module housing with an effective length of 20 cm, so that the membrane area (converted to the inner surface) was 1.9 m 2 The two ends are sealed with epoxy resin to form a hollow fiber filter membrane micromodule with openings at both ends.

[0069] like Figure 8 As shown in the figure, the hollow fiber filtration membrane micromodule and KML-100TF perfusion system (manufactured by Repligen) were used to introduce CHO-K1 cells into which the gene expressing the monoclonal antibody was transferred into a 15L (effective volume: 8.5L) glass bioreactor (manufactured by Repligen) for continuous culture experiments. The basic medium supplemented with glucose and defoaming agent was continuously added and adjusted to keep the cell concentration at 75±10×10 6 cells / mL. At this point, 3 / 4 of the effective volume is replaced within one day. The permeation flux of the hollow fiber membrane micromodule is set to: 1L / m 2 / h, permeating from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow velocity of the culture solution through the hollow part was set to 0.3m / s. After about 20 days of cultivation, the permeation pressure of the hollow fiber membrane rose to about 100kPa, and stable operation could not be achieved.

[0070] It was confirmed that the permeation rate of the antibody in the permeated liquid had dropped to 50% on the 20th day.

[0071]

Table 1

[0072] The method for filtering culture fluid involved in the present invention uses a membrane with good connectivity of micropores from the inner side of the membrane on the treated fluid side as a porous filtration membrane to the outer side of the membrane on the filtrate side. Therefore, a culture containing cells and a product containing enzymes and antibodies can be separated at a lower tangential streamline velocity. The product has good permeability, cells are not likely to cause clogging, and the filtration performance is excellent. In addition, this method is a filtration method in which the membrane is not damaged during the filtration operation, so proteins such as antibodies can be separated efficiently.

Claims

1. A filtration method comprising a filtration step of passing a culture solution containing cells, a culture medium, a product and an optional defoaming agent through a porous membrane formed of a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, in, In the SEM image of the film cross section in the film thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, a total of four fields of view, there is a 1 μm 2 The sum of the areas of the resin portions with areas of 100 Å and below is 70% or more of the total area of ​​the resin portions.

2. A filtration method comprising a filtration step of passing a culture solution containing cells, a culture medium, a product and an optional defoaming agent through a porous membrane formed of a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, in, In the SEM image of the film cross section in the film thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, a total of four fields of view, there is a 10 μm 2 The sum of the areas of the resin portions having an area of ​​100 Å or more is 15% or less of the total area of ​​the resin portions.

3. A filtration method comprising a filtration step of passing a culture solution containing cells, a culture medium, a product and an optional defoaming agent through a porous membrane formed of a resin having a three-dimensional network structure, and separating a filtrate from the culture solution, in, In the SEM image of the film cross section in the film thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​the field of view including the inner surface, the field of view including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields of view, a total of four fields of view, there is a 1 μm 2 The sum of the areas of the resin portions with an area of ​​10 μm or less is 70% or more of the total area of ​​the resin portions, and 2 The sum of the areas of the resin portions having an area of ​​100 Å or more is 15% or less of the total area of ​​the resin portions.

4. The filtering method according to any one of claims 1 to 3, wherein: In the SEM image of the film cross section of the porous membrane in the film thickness direction orthogonal to the inner surface of the porous membrane, in each area of ​​a total of four fields of view including the field of view of the inner surface, the field of view including the outer surface of the membrane, and two fields of view taken between these fields of view at equal intervals, there is a thickness of more than 1 μm 2 And less than 10μm 2 The sum of the areas of the resin portions having an area of ​​1000 Å is 15% or less of the total area of ​​the resin portions. 5 . The filtration method according to claim 1 , wherein the surface porosity of the porous membrane is 25 to 60%.

6. The filtration method according to any one of claims 1 to 3, wherein the flux of the porous membrane in the filtration step is 1 LMH or more. 7 . The filtration method according to claim 1 , wherein the porous membrane is a hollow fiber membrane, and the filtration step is perfusion culture filtration. 8 . The filtration method according to claim 7 , wherein the linear velocity of the tangential flow in the hollow fiber membrane during the perfusion culture is 0.3 m / s or less.

9. The filtering method according to any one of claims 1 to 3, wherein: The resin forming the porous film is a thermoplastic resin.

10. The filtering method according to claim 9, wherein: The thermoplastic resin is a fluororesin.

11. The filtering method according to claim 10, wherein: The fluororesin is selected from the group consisting of vinylidene fluoride resin (PVDF), chlorotrifluoroethylene resin, tetrafluoroethylene resin, ethylene-tetrafluoroethylene copolymer (ETFE), ethylene-monochlorotrifluoroethylene copolymer (ECTFE), hexafluoropropylene resin, and mixtures of these resins.

12. The filtering method according to claim 9, wherein: The thermoplastic resin is polyethylene (PE).

13. The filtering method according to any one of claims 1 to 3, wherein: The product is selected from proteins, amino acids, nucleic acids and organic substances including enzymes and antibodies.

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