Method for filtering cell culture through high communication film

Abstract

To provide a more stable filtration method capable of separating a culture containing cells and a product containing an enzyme and an antibody without clogging at a lower tangential flow linear velocity.SOLUTION: This filtration method comprises: a filtration step of passing a culture liquid containing a cell, a medium, a product, and an optional antifoam through a porous film composed of a three-dimensional mesh structure, and separating a filtrate containing the liquid from the culture liquid. In each region of totally four fields of views, that is, a field of view including an inner surface of the porous film, a field of view including the outer surface of the porous film, and two fields of views obtained by photographing these fields at equal intervals, in a SEM image of a film section in a film thickness direction orthogonal to the inner surface of the porous film, a total area of the resin part having an area of 1 μm2 or less is 70% or more of a gross area of the resin part.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present invention relates generally to process filtration systems, and more specifically to a method for filtration of cell cultures utilizing high-opening hollow fiber membranes. [Background technology]

[0002] Filtration is utilized to separate, purify, modify, and / or concentrate fluid solutions, mixtures, or suspensions. It is often a necessary step in the production, processing, and analysis stages of drugs, diagnostics, and chemicals by the biotechnology, pharmaceutical, and medical industries. Filtration may be used, for example, to remove desired compounds from a solution or to remove by-products, leaving only a more concentrated medium. Such processes may be modified as needed by selecting various filter materials, pore sizes, and / or other filter variables.

[0003] When producing cell cultures, it is often necessary to remove waste products from the growth culture. Advances in biological manufacturing processes now allow for the large-scale production of cell cultures, often using process vessel devices to produce recombinant proteins, virus-like particles (VLPs), gene therapy particles, and vaccines. Cell retention devices are widely available that remove metabolic waste products and add nutrients to renew the culture. Typically, this retention is performed by perfusion filtration of the process vessel culture with hollow fiber membranes using tangential flow filtration. In addition, bioprocessing runs are long term and continuous cultures are utilized. Such runs can last for days, weeks, or months. It is desirable for many typical components, such as filters, to function satisfactorily for such periods without fouling or otherwise requiring maintenance or replacement.

[0004] Hollow fiber membranes are often used for cell culture perfusion and product separation, but such use can result in membrane fouling by cell debris and the like. Fouling can then cause the desired product to be retained within the membrane rather than passing through. Hollow fiber membranes are commonly made of polyethersulfone (PES), polysulfone, cellulose, polyvinylidene fluoride, polypropylene, polyethylene and other materials, and there is a continuing demand for membranes that are resistant to fouling and allow for the filtration of solutions containing high solids. Fluororesins such as polyethylene (PE) and polyvinylidene fluoride (PVDF) are hydrophobic materials that have been commonly used in industrial filtration, and are known to adsorb certain molecules less. Specifically, polyphenols, polysaccharides, and tannins are adsorbed at lower levels. On the other hand, the structure of the membrane varies greatly depending on the manufacturing method, and even if the membrane is made of the same material, the fouling phenomenon on the membrane varies greatly depending on the manufacturing method.

[0005] The following Patent Document 1 describes the relationship between the filtration rate of the permeate in tangential flow filtration and the flow rate of the culture solution. It also describes the use of a porous membrane containing a dense layer. As shown in FIG. 7, tangential flow filtration is also known as cross-flow filtration, and is a method in which a solution sample to be filtered is refluxed in a direction parallel to the membrane surface, generating a certain pressure on the membrane and allowing particles smaller than the membrane pore size to pass through. In contrast, general direct flow filtration is a method in which a liquid is passed from top to bottom on a membrane surface such as a syringe filter or centrifugal filter, in which case, fine particles contained in the sample are captured on the membrane surface or inside and accumulate, causing clogging and generating excessive pressure load on the sample. In contrast, in tangential flow filtration, the sample is constantly refluxed in a direction parallel to the membrane surface, so particles and molecules larger than the membrane pore size remain upstream of the membrane, and the accumulation of fine particles on the membrane is suppressed, resulting in a low load on the sample.

[0006] The following Patent Document 2 describes a cell culture method and device that can remove cell growth inhibitors, perform high-density culture, and return at least one of the components of serum, which is an expensive polymeric medium component, to the culture tank, thereby reducing running costs.

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

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

[0009] The following Patent Document 5 describes a method for efficiently separating a product using a tangential flow filtration method in perfusion culture. [Prior art documents] [Patent documents]

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

[0011] According to the techniques described in Patent Documents 1 to 4, there is described a method for recovering a useful substance from a culture solution in continuous culture of cells that produce a useful substance, the method comprising: a bleeding step of discharging the culture solution from a cell culture tank and adding to the culture tank an amount of fresh medium equal to the amount of the discharged culture solution; 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, the filtration step being tangential flow filtration, and recovering the useful substance. Furthermore, Patent Document 5 merely describes a technique for efficiently separating the product from impurities.

[0012] However, although Patent Documents 1 to 4 disclose ideas on how to use the membrane, such as the speed of the permeate in the filtration process, there is no description of the membrane that is optimal for tangential flow filtration, and it is not clear what specific structure or material of the membrane should be used to achieve stable tangential flow. When continuing filtration, clogging of the membrane inevitably occurs and it is necessary to deal with it, and in terms of separation, a basic separation function is required in which cells and microorganisms are retained on the concentrated side and the product is permeated to the permeation side without being blocked by the membrane. In addition, when the culture target is animal cells, there is a problem that the linear velocity of the tangential flow cannot be increased because a too high shear rate causes problems in cell growth.

[0013] In view of the above-mentioned state of the art, the problem to be solved by the present invention is to provide a more stable filtration method capable of separating a culture product from a product at a lower linear tangential flow velocity without clogging. [Means for solving the problem]

[0014] As a result of intensive research and repeated experiments to solve the above-mentioned problems, the inventors of the present application unexpectedly discovered that by using a porous filtration membrane with good pore connectivity from the inside of the membrane, which is the treated liquid side, to the outside of the membrane, which is the filtrate side, the permeability of the culture product is good and furthermore the membrane is less likely to clog even when the linear velocity of the tangential flow is low, which led to the completion of 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 medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure to separate a filtrate containing the product from the culture solution, In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the membrane, a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view, each region was measured at 1 μm 2 The above filtration method, characterized in that the sum of the areas of the resin parts having the following areas is 70% or more of the total area of ​​the resin parts: [2] A filtration method comprising a filtration step of passing a culture solution containing cells, a medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure to separate a filtrate containing the product from the culture solution, In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the membrane, a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view, each region was measured at 10 μm 2 The above filtration method, characterized in that the sum of the areas of the resin parts having the above area is 15% or less of the total area of ​​the resin parts. [3] A filtration method comprising a filtration step of passing a culture solution containing cells, a medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure to separate a filtrate containing the product from the culture solution, In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the membrane, a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view, each region was measured at 1 μm 2 The total area of ​​the resin parts having an area of ​​10 μm or less is 70% or more of the total area of ​​the resin parts, and 2 The above filtration method, characterized in that the sum of the areas of the resin parts having the above area is 15% or less of the total area of ​​the resin parts. [4] In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the porous membrane, the field of view including the inner surface, the field of view including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view, each of the four fields of view has a resolution of 1 μm or less. 2 Ultra 10μm 2 The filtration method according to any one of the above [1] to [3], wherein the sum of the areas of the resin parts having an area of ​​less than 15% of the total area of ​​the resin parts. [5] The filtration method according to any one of [1] to [4] above, wherein the surface opening ratio of the porous membrane is 25 to 60%. [6] The filtration method according to any one of [1] to [5], 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 [7], wherein the intramembrane linear velocity of the tangential flow of the hollow fiber membrane during the perfusion culture is 0.5 m / s or less. [9] The filtration method according to any one of the above [1] to [8], wherein the resin constituting the porous membrane is a thermoplastic resin.

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

[11] The filtration method according to

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

[12] The filtration method described in [9], 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 the group consisting of proteins including enzymes and antibodies, amino acids, nucleic acids, and organic substances. Effect of the Invention

[0016] The method for filtering a culture solution according to the present invention uses a porous filtration membrane having pores with good interconnectivity extending from the inside of the membrane, which is the treated liquid side, to the outside of the membrane, which is the filtrate side. This allows for good product permeability and is less likely to cause clogging of the cells at a slower linear tangential flow velocity, making it possible to separate a culture containing cells from a product containing enzymes and antibodies. This method has excellent filtration performance, and furthermore does not damage the membrane during filtration operation, allowing efficient separation of proteins such as antibodies. [Brief description of the drawings]

[0017] [Figure 1] 1 is an example of an SEM image of a cross section of a porous membrane used in the filtration method of the present embodiment (black parts indicate resin, and white parts indicate pores (open holes)). [Diagram 2] This is a histogram showing the percentage (%) of the total area of ​​resin parts having a given area relative to the total area of ​​resin parts in each region (circle 1 to circle 4) of a total of four visual fields in an SEM image of a membrane cross section in the film thickness direction perpendicular to the inner surface of the porous membrane used in Example 1, including a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields taken at equal intervals between these visual fields. [Diagram 3]This is a histogram showing the percentage (%) of the total area of ​​resin parts having a given area relative to the total area of ​​resin parts in each region (circle 1 to circle 4) of a total of four visual fields in an SEM image of a membrane cross section in the film thickness direction perpendicular to the inner surface of the porous membrane used in Example 2, including a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields taken at equal intervals between these visual fields. [Figure 4] This is a histogram showing the percentage (%) of the total area of ​​resin parts having a given area relative to the total area of ​​resin parts in each region (circle 1 to circle 4) of a total of four visual fields in an SEM image of a membrane cross section in the film thickness direction perpendicular to the inner surface of the porous membrane used in Example 3, including a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields taken at equal intervals between these visual fields. [Diagram 5] This is a histogram showing the percentage (%) of the total area of ​​resin parts having a given area relative to the total area of ​​resin parts in each region (circle 1 to circle 4) of a total of four visual fields in an SEM image of a membrane cross section in the film thickness direction perpendicular to the inner surface of the porous membrane used in Example 4, including a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields taken at equal intervals between these visual fields. [Figure 6] This is a histogram showing the percentage (%) of the total area of ​​resin parts having a given area relative to the total area of ​​resin parts in each region (circle 1 to circle 4) of a total of four visual fields in an SEM image of a membrane cross section in the film thickness direction perpendicular to the inner surface of the porous membrane used in Comparative Example 1, including a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields taken at equal intervals between these visual fields. [Figure 7] FIG. 1 is an explanatory diagram of tangential flow filtration. [Figure 8] FIG. 2 is an explanatory diagram of the perfusion culture filtration process in the continuous culture used in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] A preferred embodiment of the present invention (hereinafter referred to as "embodiment") will be described in detail below. Note that the embodiment shown below is an example of an apparatus and method for embodying the technical idea of ​​the present invention, and the technical idea of ​​the present invention is not limited to the combination of components described below. The technical idea of ​​the present invention can be modified in various ways within the scope of the claims.

[0019] In the following description, the upper or lower limit of a stepwise described numerical range may be replaced with the upper or lower limit of another stepwise described numerical range. In addition, in the following description, the upper or lower limit of a certain numerical range may be replaced with a value described in the examples. Furthermore, with respect to the term "step" in the following description, not only an independent step, but also a step that cannot be clearly distinguished from other steps may be included in this term as long as the function of the "step" is achieved.

[0020] The filtration method of the present embodiment is a filtration method including a filtration step of passing a culture solution containing cells, a medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure, and separating a filtrate containing the product from the culture solution, In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the membrane, a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view, each region was measured at 1 μm 2 The sum of the areas of the resin parts having an area of ​​10 μm or less is 70% or more of the total area of ​​the resin parts, and / or 2 In the above-mentioned filtration method, the sum of the areas of the resin parts having an area of ​​1 μm or more is 15% or less of the total area of ​​the resin parts. In the above-mentioned porous membrane, in an SEM image of a cross section of the membrane in a thickness direction perpendicular to the inner surface of the porous membrane, a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields photographed at equal intervals between these visual fields, a total of four visual fields, 2 Ultra 10μm 2It is preferable that the sum of the areas of the resin parts having an area of ​​less than 15% of the total area of ​​the resin parts.

[0021] The filtration method of the present embodiment is characterized by including a filtration step in which a culture solution containing cells, medium, product, antifoaming agent, etc. is passed through a porous membrane composed of a resin with a three-dimensional mesh structure to separate a filtrate containing the product and a culture solution containing cells from the culture solution. The product is not particularly limited, but examples thereof include proteins including enzymes and antibodies, amino acids, nucleic acids, organic substances, and the like, and examples thereof include proteins including enzymes and antibodies to be produced by cells, for example, genetically modified cells that produce useful proteins, for example, CHO cells. The method may also include additional processing steps, such as centrifugation, diatomaceous earth filtration, other membrane separation, sieving, or the like, before, after, or both before and after the filtration step. The shape of the porous membrane is not particularly limited, and examples thereof include a flat membrane, a tubular membrane, and a hollow fiber membrane. From the viewpoint of space saving of the filtration device, i.e., because it is possible to increase the membrane area per unit volume of the membrane module, a hollow fiber membrane is preferred.

[0022] The filtration step in the filtration method of this embodiment may be, for example, a so-called internal pressure filtration step in which a culture broth (liquid to be treated) containing cells, a culture medium, useful substances, an antifoaming agent, etc. is supplied to the hollow part (inner surface) of a porous hollow fiber membrane, passed through the membrane thickness (thick wall) part of the porous hollow fiber membrane, and the liquid that seeps out from the outer surface of the porous hollow fiber membrane is taken out as filtrate, or a so-called external pressure filtration step in which the liquid to be treated is supplied from the outer surface of the porous hollow fiber membrane, and the filtrate that seeps out from the inner surface of the porous hollow fiber membrane is taken out via the hollow part. In this specification, the term "inside the porous membrane" refers to the membrane thickness (thick wall) portion where numerous pores are formed.

[0023] The product may be an antibody protein, or as generally defined in biochemistry, a glycoprotein molecule (also called gamma globulin or immunoglobulin) produced by B lymphocytes as a defense mechanism against infection in vertebrates. For example, the antibody protein separated by the porous membrane according to the embodiment is used as a human pharmaceutical and has a structure substantially identical to that of an antibody protein present in the human body to which it is administered.

[0024] The product may be various antibody vaccines, vector viruses, plasmid DNA, or industrial enzymes such as amylase and protease. Various culture objects are selected according to each product. For example, when the product is an enzyme, the culture object may be bacteria such as genetically modified Escherichia coli, and when the product is a protein such as albumin, the culture object may be genetically modified Pichia yeast.

[0025] The structure, material, and manufacturing method of the porous membrane used in the culture solution filtration method of this embodiment will be described in detail below. <Porous membrane> The porous membrane used in the method for filtering a culture broth according to the present embodiment has a cross section in a thickness direction perpendicular to the inner surface of the porous membrane, and has a cross section of 1 μm or less in each of four fields of view, including a field including the inner surface, a field including the outer surface of the membrane, and two fields of view taken at equal intervals between the two fields of view. 2 The sum of the areas of the resin parts having the following areas is 70% or more of the total area of ​​the resin parts; 2 The sum of the areas of the resin parts having an area of ​​1 μm or more is 15% or less of the total area of ​​the resin parts; or 2 The total area of ​​the resin parts having an area of ​​10 μm or less is 70% or more of the total area of ​​the resin parts, and 2 or more, the sum of the areas of the resin parts having an area of ​​1 μm or less relative to the total area of ​​the resin parts. 2 The total area of ​​the resin parts having an area of ​​1 μm or less is 70% or more of the total area of ​​the resin parts.2 Ultra 10μm 2 The total area of ​​the resin parts having an area of ​​less than 10 μm is 15% or less of the total area of ​​the resin parts, and 2 The sum of the areas of the resin parts having the above area is 15% or less of the total area of ​​the resin parts.

[0026] Fig. 1 is an example of an SEM image of a cross section of a porous membrane used in the filtration method of this embodiment. This SEM image is an image obtained by binarizing a SEM image photograph obtained by photographing a predetermined field of view in the area closest to the inside, among a total of four fields of view, including a field of view including the inner surface of the hollow fiber porous membrane, a field of view including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view, in an SEM image of a membrane cross section in a thickness direction perpendicular to the inner surface of the hollow fiber porous membrane. Furthermore, within each of the above-mentioned regions, the difference in the distribution of the resin parts, i.e., the anisotropy of the pore interconnectivity, can be virtually ignored between the membrane cross section in the thickness direction perpendicular to the inner surface of the hollow fiber porous membrane and the cross section parallel to the inner surface. In this specification, the term "resin portion" refers to a dendritic skeleton portion of a three-dimensional network structure composed of resin that forms a large number of pores in a porous membrane. In Figure 1, the black parts are resin portions, and the white parts are pores. The porous membrane has holes inside that are connected to the outside of the membrane while bending. In the SEM image of the membrane cross section in the thickness direction perpendicular to the inner surface of the porous membrane, the field including the inner surface, the field including the outer surface of the membrane, and two fields of view taken at equal intervals between these fields, a total of four fields of view, are shown. 2 If the sum of the areas of the resin parts having the following areas is 70% or more of the total area of ​​the resin parts, the interconnectivity of the pores is high (i.e., the proportion of interconnected pores inside the membrane is high), the flux (water permeability, permeability) of the treated liquid and the water permeability retention rate after cleaning are high, and damage to the membrane after cleaning with a chemical solution, as indicated by the tensile breaking elongation, is also reduced. However, if the sum of the areas of the resin parts having the following areas is 70% or more of the total area of ​​the resin parts, the interconnectivity of the pores is high (i.e., the proportion of interconnected pores inside the membrane is high), the flux (water permeability, permeability) of the treated liquid and the water permeability retention rate after cleaning are high, and damage to the membrane after cleaning with a chemical solution, as indicated by the tensile breaking elongation, is also reduced. 2If the ratio of the total area of ​​the resin portion having an area of ​​less than 1 μm is too high, the dendritic skeleton of the three-dimensional network structure made of resin, which forms a large number of pores in the porous membrane, becomes too thin. 2 The total area of ​​the resin parts having the area below 1 μm is maintained at 70% or more of the total area of ​​the resin parts. 2 Ultra 10μm 2 The total area of ​​the resin parts having an area of ​​less than 10 μm is preferably 2% or more and 30% or less of the total area of ​​the resin parts. 2 More preferably, the total area of ​​the resin parts having an area of ​​1 μm or more accounts for 15% or less of the total area of ​​the resin parts. 2 Ultra 10μm 2 The total area of ​​the resin parts having an area of ​​less than 10 μm is 15% or less of the total area of ​​the resin parts, and 2 It is more preferable that the total area of ​​the resin parts having an area of ​​1 μm or more is 2% or more and 15% or less of the total area of ​​the resin parts. 2 Ultra 10μm 2 When the sum of the areas of the resin parts having an area of ​​less than 2% is 2% or more and 30% or less, preferably 15% or less, of the total area of ​​the resin parts, the dendritic skeleton part of the three-dimensional mesh structure composed of the resin is not too thin, so that the strength and tensile breaking elongation of the porous membrane can be appropriately maintained.

[0027] 2 to 6 are histograms showing the ratio (%) of the total area of ​​resin parts having a predetermined area to the total area of ​​resin parts in each region (circle 1 to circle 4) of a total of four visual fields in the SEM images of the membrane cross section in the thickness direction perpendicular to the inner surface of the porous membrane used in Example 1, Example 2, Example 3, Example 4, and Comparative Example 1, including a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields photographed at equal intervals between these visual fields. In the binarized SEM image of FIG. 1, the resin parts appear as granules. In FIGS. 2 to 6, the area of ​​each of these granular resin parts is measured, and the area ratio of each of the granular resin parts to the total area of ​​all resin parts in a visual field of a predetermined size in each region is shown as a histogram. In Figures 2 to 6, circle 1 is the number of the area closest to the inside of the four fields of view in the SEM image of the membrane cross section in the thickness direction perpendicular to the inner surface of the porous membrane, including the field of view including the inner surface, the field of view including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields, and circle 4 is the number of the area closest to the inside. For example, circle 1 in Example 1 is a histogram when a field of view of a predetermined size in the innermost area of ​​the porous hollow fiber membrane of Example 1 is photographed. The method for measuring the area distribution of the resin part in each area of ​​the porous hollow fiber membrane will be described later.

[0028] The surface opening ratio of the porous membrane is preferably 25 to 60%, more preferably 25 to 50%, and even more preferably 25 to 45%. If the surface opening ratio on the side in contact with the liquid to be treated is 25% or more, the deterioration of the water permeability due to clogging and abrasion of the membrane surface is reduced, so that the filtration stability can be improved. On the other hand, if the surface opening ratio is high and the pore size is too large, the required separation performance may not be exhibited. Therefore, the average pore size of the porous membrane is preferably 100 to 700 nm, more preferably 20 to 600 nm. If the average pore size is 30 to 400 nm, the separation performance is sufficient and the interconnection of the pores can be ensured. The methods for measuring the surface opening ratio and the average pore size will be described later.

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

[0030] The shape of the porous hollow fiber membrane can be a circular single layer membrane, but it may also be a multi-layer 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 non-circular cross-sectional structure, such as having 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, more preferably a fluororesin, such as polyvinylidene fluoride resin (PVDF), chlorotrifluoroethylene resin, tetrafluoroethylene resin, ethylene-tetrafluoroethylene copolymer (ETFE), ethylene-monochlorotrifluoroethylene copolymer (ECTFE), hexafluoropropylene resin, and mixtures of these resins. Examples of the thermoplastic resin include polyolefin, copolymers of olefin and halogenated olefin, halogenated polyolefin, and mixtures thereof. Examples of the thermoplastic resin include polyethylene (PE), polypropylene, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, ethylene-tetrafluoroethylene copolymer, polyvinylidene fluoride (which may contain a domain of hexafluoropropylene), and mixtures thereof. These resins are excellent as membrane materials because they are thermoplastic and therefore easy to handle and tough. Among these, vinylidene fluoride resin, tetrafluoroethylene resin, hexafluoropropylene resin, or mixtures thereof, homopolymers or copolymers of ethylene, tetrafluoroethylene, and chlorotrifluoroethylene, or mixtures of homopolymers and copolymers are preferred because they have excellent mechanical strength, chemical strength (chemical resistance), and good moldability. More specifically, fluororesins such as polyvinylidene fluoride, vinylidene fluoride-hexafluoropropylene copolymer, ethylene-tetrafluoroethylene copolymer, and ethylene-chlorotrifluoroethylene copolymer are examples of the fluororesins.

[0032] The porous membrane may contain up to about 5% by mass of components (impurities, etc.) other than the thermoplastic resin. For example, the solvent used in the production of the porous membrane is included. As described later, 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, used as the solvent in the production of the porous membrane are included. These solvents can be detected by pyrolysis GC-MS (gas chromatography mass spectrometry).

[0033] The first solvent may be at least one selected from the group consisting of sebacic acid esters, citrate esters, acetyl citrate esters, adipate esters, trimellitate esters, oleic acid esters, palmitic acid esters, stearic acid esters, phosphate esters, fatty acids having 6 to 30 carbon atoms, and epoxidized vegetable oils. The second solvent, unlike the first solvent, may be at least one selected from the group consisting of sebacic acid esters, citrate esters, acetyl citrate esters, adipate esters, trimellitate esters, oleate esters, palmitate esters, stearates, phosphate esters, fatty acids having 6 to 30 carbon atoms, and epoxidized vegetable oils. Examples of fatty acids having 6 to 30 carbon atoms include capric acid, lauric acid, and oleic acid. Examples of epoxidized vegetable oils include epoxy soybean oil and epoxidized linseed oil. The first solvent is preferably a non-solvent in which the thermoplastic resin does not dissolve uniformly in the first solvent, even when the temperature of the first mixed liquid is raised to the boiling point of the first solvent in a first mixed liquid having a ratio of thermoplastic resin to first solvent of 20:80. The second solvent is preferably a good solvent in which the thermoplastic resin dissolves uniformly in the second solvent when the temperature of the second mixed liquid is higher than 25°C and lower than the boiling point of the second solvent in a second mixed liquid having a ratio of thermoplastic resin to second solvent of 20:80. It is more preferable that the second solvent is a poor solvent in which, in a second mixed liquid having a ratio of thermoplastic resin to second solvent of 20:80, the thermoplastic resin does not dissolve uniformly in the second solvent when the temperature of the second mixed liquid is 25°C, but the thermoplastic resin dissolves uniformly in the second solvent when the temperature of the second mixed liquid is any temperature higher than 100°C and lower than the boiling point of the second solvent. Alternatively, it may be the first solvent alone.

[0034] Furthermore, in the culture solution filtration method 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 the group consisting of sebacic acid esters, citrate esters, acetyl citrate esters, adipate esters, trimellitic acid esters, oleate esters, palmitate esters, stearic acid esters, phosphate esters, fatty acids having 6 to 30 carbon atoms, and epoxidized vegetable oils, and may be a non-solvent that does not uniformly dissolve polyvinylidene fluoride in the first solvent even when the temperature of the first mixed liquid is raised to the boiling point of the first solvent in a first mixed liquid having a ratio of polyvinylidene fluoride to the first solvent of 20:80. As the non-solvent, bis(2-ethylhexyl) adipate (DOA) is preferred. The porous hollow fiber membrane may contain a second solvent different from the first solvent. In this case, the second solvent is preferably at least one selected from the group consisting of sebacic acid esters, citrate esters, acetyl citrate esters, adipate esters, trimellitic acid esters, oleic acid esters, palmitic acid esters, stearic acid esters, phosphate esters, fatty acids having 6 to 30 carbon atoms, and epoxidized vegetable oils, and is a good solvent in which polyvinylidene fluoride dissolves uniformly in a second mixed liquid having a ratio of polyvinylidene fluoride to the second solvent of 20:80 at a temperature higher than 25° C. and lower than the boiling point of the second solvent. Moreover, the second solvent is more preferably a poor solvent in which polyvinylidene fluoride does not dissolve uniformly in the second solvent when the temperature of the second mixed liquid is 25° C., but polyvinylidene fluoride dissolves uniformly in the second solvent when the temperature of the second mixed liquid is any temperature higher than 100° C. and lower than the boiling point of the second solvent. As the poor solvent, acetyl tributyl citrate (ATBC) is preferable.

[0035] (Properties of porous membrane) When the porous membrane is washed, the relationship between the tensile breaking elongation E0 of the porous membrane before the washing step and the tensile breaking elongation E1 of the porous membrane after the washing step is preferably E1 / E0×100≧98%. Also, the relationship between the tensile breaking elongation E0 of the porous membrane before the washing step and the tensile breaking elongation EX of the porous membrane after the washing step is repeated X times (wherein X is an integer of 2 to 10) is preferably 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 a surfactant-containing aqueous solution is used as the cleaning solution (chemical solution), for example, when only one containing 1 wt % sodium dodecyl sulfate is used, the chemical solution resistance of the membrane does not pose a particular problem. However, when a hypocaustic aqueous solution containing 0.1 wt % to 4 wt % sodium hydroxide and 0.01 wt % to 0.5 wt % sodium hypochlorite is used as the cleaning solution in addition to the surfactant-containing aqueous solution, chemical solution resistance (the likelihood of damage to the membrane) becomes an issue. In this case, the chemical resistance of the membrane can be indexed by the retention of tensile breaking elongation before and after filtration and cleaning with circulating chemical solution (elongation retention after filtration and cleaning with circulating chemical solution) using a hypocaustic aqueous solution containing 4 wt. % sodium hydroxide and 0.5 wt. % sodium hypochlorite as the resistance test chemical solution. Specifically, it is preferable that the tensile breaking elongation after a series of steps of filtration of the actual solution followed by cleaning with circulating chemical solution (corresponding to the tensile breaking elongation E1 of the porous hollow fiber membrane after the cleaning step) is maintained at 98% or more of the initial value (corresponding to the tensile breaking elongation E0 of the membrane before the cleaning step). In addition, the relationship between the initial value E0 and the tensile breaking elongation EX of the membrane after a series of steps of filtration of the actual liquid followed by chemical circulation cleaning is repeated X times (X is an integer from 2 to 10) is preferably EX / E0≧97%.

[0036] From a practical standpoint, 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) The porous membrane is preferably one in which 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 satisfies L1 / L0×100≧80%. In addition, the porous membrane is preferably one in which the relationship between the flux L0 of the porous membrane before the filtration step and the flux LX of the porous membrane after the cleaning step is repeated X times (wherein X is an integer of 2 to 10) is LX / L0×100≧80%.

[0038] <Method of manufacturing porous membrane> A method for producing the porous hollow fiber membrane will be described below, although the method for producing the porous hollow fiber membrane used in the filtration method of the present embodiment is not limited to the following method. The method for producing the porous hollow fiber membrane used in the filtration method of this embodiment can include the steps of (a) preparing a molten mixture, (b) supplying the molten mixture to a multi-structure spinning nozzle and extruding the molten mixture from the spinning nozzle to obtain a hollow fiber membrane, and (c) extracting the plasticizer from the hollow fiber membrane. If the molten mixture contains an additive, the method may further include, after step (c), a step (d) of extracting the additive from the hollow fiber membrane.

[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 even more preferably 30 to 45% by mass. If this value is 20% by mass or more, the mechanical strength can be increased, while if it is 60% by mass or less, the water permeability can be increased. The melt-kneaded product may contain an additive. The melt-kneaded product may be composed of two components, a thermoplastic resin and a solvent, or may be composed of three components, a thermoplastic resin, an additive, and a solvent. The solvent contains at least a non-solvent, as described later. As the extractant used in step (c), it is preferable to use a liquid such as methylene chloride or various alcohols that does not dissolve the thermoplastic resin but has a high affinity for the plasticizer. When a melt-kneaded product containing no additives is used, the hollow fiber membrane obtained through step (c) may be used as a porous hollow fiber membrane. When a melt-kneaded product containing additives is used to produce a porous hollow fiber membrane, it is preferable to further carry out a step (d) of extracting and removing the additives from the hollow fiber membrane to obtain a porous hollow fiber membrane after step (c). As the extractant in step (d), it is preferable to use hot water or a liquid such as an acid or alkali that can dissolve the additives used but does not dissolve the thermoplastic resin.

[0040] An inorganic substance may be used as an additive. The inorganic substance is preferably an inorganic fine powder. The primary particle size of the inorganic fine powder contained in the molten kneaded product is preferably 50 nm or less, more preferably 5 nm or more and less than 30 nm. Specific examples of the inorganic fine powder include silica (including fine silica), titanium oxide, lithium chloride, calcium chloride, and organic clay, and among these, fine silica is preferred from the viewpoint of cost. The above-mentioned "primary particle size of the inorganic fine powder" means a value obtained by analyzing an electron microscope photograph. That is, first, a group of inorganic fine powders is pretreated by the method of ASTM D3849. Thereafter, the particle diameters of 3000 to 5000 particles photographed in a transmission electron microscope photograph are measured, and the primary particle size of the inorganic fine powder can be calculated by arithmetically averaging these values. By identifying the elements present in the inorganic fine powder inside the porous hollow fiber membrane using fluorescent X-rays or other methods, the material of the inorganic fine powder present can be identified. When using organic substances as additives, hydrophilic polymers such as polyvinylpyrrolidone and polyethylene glycol can impart hydrophilicity to the hollow fiber membrane. Also, the viscosity of the molten mixture can be controlled by using highly viscous additives such as glycerin and ethylene glycol.

[0041] Next, the step (a) of preparing a molten mixture in the method for producing a porous hollow fiber membrane of this embodiment will be described in detail. In the method for producing a porous hollow fiber membrane of this embodiment, a non-solvent for a thermoplastic resin is mixed with a good solvent or a poor solvent. The mixed solvent after mixing is a non-solvent for the thermoplastic resin used. When a non-solvent is used as a raw material for the membrane in this way, a porous hollow fiber membrane having a three-dimensional mesh structure is obtained. Although the mechanism of action is not necessarily clear, it is considered that the crystallization of the polymer is appropriately inhibited and a three-dimensional mesh structure is easily formed by using a solvent with a lower solubility by mixing a non-solvent. For example, the non-solvent and the poor solvent or good solvent are selected from the group consisting of various esters such as phthalic acid esters, sebacic acid esters, citric acid esters, acetyl citrate esters, adipic acid esters, trimellitic acid esters, oleic acid esters, palmitic acid esters, stearic acid esters, phosphate esters, fatty acids having 6 to 30 carbon atoms, and epoxidized vegetable oils. 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 it at high temperatures is called a poor solvent for that thermoplastic resin, and a solvent that cannot dissolve the thermoplastic resin even at high temperatures is called a non-solvent. Good solvents, poor solvents, and non-solvents can be determined as follows. Put about 2g of thermoplastic resin and about 8g of solvent into a test tube, heat it to the boiling point of the solvent in 10℃ increments using a test tube block heater, and mix the inside of the test tube with a spatula, etc. If the thermoplastic resin dissolves, it is a good or poor solvent, and if it does not dissolve, it is a non-solvent. If it dissolves at a relatively low temperature below 100℃, it is judged to be a good solvent, and if it does not dissolve unless it is heated to a high temperature above 100℃ but below the boiling point, it is judged to be a poor solvent. For example, if polyvinylidene fluoride (PVDF) is used as the thermoplastic resin and acetyl tributyl citrate (ATBC), dibutyl sebacate, or dibutyl adipate is used as the solvent, PVDF will mix uniformly and dissolve in these solvents at about 200° C. On the other hand, if bis 2-ethylhexyl adipate (DOA), diisononyl adipate, or bis 2-ethylhexyl sebacate is used as the solvent, PVDF will not dissolve in these solvents even if the temperature is raised to 250° C. In addition, when ethylene-tetrafluoroethylene copolymer (ETFE) is used as the thermoplastic resin and diethyl adipate is used as the solvent, ETFE is homogeneously mixed and dissolved at about 200°C. On the other hand, when bis-2-ethylhexyl adipate (DOA) is used as the solvent, ETFE does not dissolve. In addition, when ethylene-monochlorotrifluoroethylene copolymer (ECTFE) is used as the thermoplastic resin and triethyl citrate is used as the solvent, it dissolves uniformly at about 200°C, but does not dissolve when triphenyl phosphite (TPP) is used. In addition, when polyethylene (PE) is used as the thermoplastic resin and bis(2-ethylhexyl) adipate (DOA) is used as the solvent, PE is homogeneously mixed and dissolved at 200°C. On the other hand, when dibutyl adipate is used as the solvent, it does not dissolve even when the temperature, which is near the boiling point, is raised to 300°C. EXAMPLES

[0042] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. The physical properties in the examples and comparative examples were determined by the following methods.

[0043] (1) Outer and inner diameters of porous hollow fiber membrane The porous hollow fiber membrane was thinly sliced ​​using a razor at a cross section perpendicular to the length direction, and the outer and inner diameters were measured using a 100x magnifying glass. For one sample, measurements were taken at 60 cut surfaces at 30 mm intervals along the length direction, and the average values ​​were taken as the outer and inner diameters of the hollow fiber membrane.

[0044] (2) Electron microscope photography The porous hollow fiber membrane was cut into a circular shape at a cross section perpendicular to the length direction, stained with 10% phosphotungstic acid + osmium tetroxide, and embedded in epoxy resin. Next, after trimming, the cross section of the sample was subjected to BIB processing to prepare a smooth cross section, and conductive treatment was performed to prepare a microscopic specimen. The prepared microscopic specimen was photographed at a magnification of 5,000 to 30,000 times using a HITACHI electron microscope SU8000 series at an accelerating voltage of 1 kV, and electron microscope (SEM) images of the cross section of the membrane were photographed at a predetermined field of view within each area (circle 1 to circle 4 in Figures 2 to 5) of a total of four fields of view, including a field including the inner surface of the cross section of the membrane (thick part), a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields of view. The magnification can be changed according to the average pore size, specifically, 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 but less than 0.1 μm, and 30,000 times when the average pore size is less than 0.05 μm. The size of the field of view was 2560 × 1920 pixels. Image processing was performed using ImageJ, and the captured SEM images were subjected to threshold processing (Image-Adjust-Threshold: Otsu method (Otsu selected)) to binarize the hole and resin parts. Surface opening ratio: The surface opening ratio was measured by calculating the ratio of resin parts to pore parts in the binarized image. Resin area distribution: Using the "Analyze Particle" command of ImageJ (Analyze Particle:Size0.10-Infinity), the size of the binarized granular resin parts contained in the captured SEM images was measured. The total area of ​​all resin parts contained in the SEM images was taken as ΣS, and 1 μm 2 The area of ​​the following resin part is ΣS(<1μm 2 ), ΣS(<1μm 2 ) / ΣS was calculated to calculate the area ratio of the resin portion having an area of ​​1 μm2 or less. Similarly, the area ratio of the resin portion having an area in a predetermined range was calculated. In addition, noise removal during binarization is performed at 0.1 μm 2 Resin parts with an area of ​​less than 0.1 μm are removed as noise. 2The resin part with an area of ​​1 μm or more was analyzed. Noise removal was performed by applying a median filter (Process-Filters-Median:Radius:3.0 pixels). 2 The area of ​​the following resin parts is 0.1μm 2 More than 1μm 2 The area of ​​the resin part is as follows. In addition, the granular resin parts cut off at the edge of the SEM image were also included in the measurement. In addition, "include holes" processing was not performed. In addition, no processing was performed to correct the shape from a "snowman" type to a "flat" type, etc. Average pore size: Measured using the "Plugins-Bone J-Thickness" command in ImageJ. The space size was defined as the maximum circle size that fits into the void.

[0045] (3) Flux (permeability, initial pure water flux (LMH)) After immersing the porous hollow fiber membrane in ethanol, it was repeatedly immersed in pure water several times, and then syringe needles were inserted into both ends of a wet hollow fiber membrane of about 10 cm in length, and pure water at 25°C was circulated and filtered at a transmembrane pressure of 0.03 MPa. The amount of pure water permeating from the inner surface of the membrane was measured, and the amount of pure water permeating from the inner surface of the membrane was calculated using the following formula: Initial pure water flux [L / m 2 / h] = 60 × (permeation rate [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. 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 (%) The porous hollow fiber membrane was used as a sample, and the tensile breaking elongation was calculated according to JIS K7161. The load and displacement at tensile breaking were measured under the following conditions. Measuring equipment: Instron type tensile testing machine (Shimadzu Corporation AGS-5D) Chuck distance: 5cm Pulling speed: 20cm / min

[0047] (5) Chemical resistance test The series of steps, which consisted of filtering the actual solution and then circulating and washing with the chemical solution, was repeated 10 times. The initial value of the tensile breaking elongation (the tensile breaking elongation before immersion) was designated as E0, and the tensile breaking strength values ​​of the porous hollow fiber membrane after the washing steps were repeated once and 10 times were designated as E1 and E10, respectively. The chemical resistance was evaluated by calculating E10 / E0 as the "tensile breaking elongation retention rate (%) after 10 cycles of repeated chemical washing." The initial pure water permeability was defined as L0 (flux L0), and the series of processes consisting of filtration of the actual liquid followed by circulating cleaning with chemical solution was repeated once and ten times. The permeability after the cleaning process was defined as L1 and L10 (flux L10), respectively, and L10 / L0 was calculated as the "permeability retention rate (%) after 10 cycles of repeated chemical cleaning."

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

[0049] (7) Tangential flow linear velocity (m / s) The tangential flow linear velocity Lv was calculated as follows: n (number of hollow fibers) is the number of hollow fibers in the hollow fiber membrane module, Di (m) is the inner diameter of the hollow fiber membrane, and V (m) is the flow rate of the raw solution supplied to the hollow fiber membrane module. 3 / s), the tangential flow linear velocity was calculated using the following formula: Lv=V / (n×(π×(Di / 2)^2))

[0050] (8) Minimum antibody penetration rate The minimum antibody permeability was determined as follows. That is, after the start of culture, the antibody concentrations in the culture medium and the permeate were measured every day and were designated as Cm and Cf, respectively, and the ratio (Cf / Cm) was designated as the antibody permeability. The minimum antibody permeability during the entire operation period was designated as the minimum antibody permeability. The antibody concentration can be determined by known methods such as ELISA and LC-MS. In the method of this embodiment, the antibody concentration was quantified using an automated immunoassay system of xP workstation manufactured by Gyrolab.

[0051] [Example 1] A melt-kneaded material was prepared using 40% by mass of PVDF resin (Kureha Corporation, KF-W#1000) as a thermoplastic resin, 23% by mass of fine 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 material was 240°C. The obtained melt-kneaded material was passed through a spinning nozzle with a double tube structure, and the hollow fiber-like extrudate was passed through an idle running distance of 120 mm, and then solidified in water at 30°C, and a porous structure was developed by a thermally induced phase separation method. The obtained hollow fiber-like extrudate was taken up at a speed of 5 m / min and wound into a skein. The wound hollow fiber extrudate was immersed in isopropyl alcohol 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% by mass aqueous NaOH solution at 70°C for 1 hour. The hollow fiber membrane was then repeatedly washed with water to extract and remove the fine silica, thereby producing a porous hollow fiber membrane. The 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 had a three-dimensional network structure.

[0052] The membrane area (calculated as the inner surface) of this hollow fiber membrane is 1.9 m 2 The membrane was placed in a module case with an effective length of 20 cm so that the effective length was 20 cm, and both ends were potted with epoxy resin to prepare a hollow fiber filtration membrane mini module with open ends.

[0053] As shown in Figure 8, using the prepared hollow fiber filtration membrane mini-module, CHO-K1 cells incorporating a gene expressing a monoclonal antibody were introduced into a 15 L (effective volume: 8.5 L) glass bioreactor (Repligen) using a KML-100TFF perfusion system (Repligen), and a continuous culture experiment was performed. A basal medium containing glucose and an antifoaming agent was continuously added until the cell concentration reached 75 ± 10 × 10 6 The flow rate of the hollow fiber membrane mini module was adjusted to maintain the flow rate at 1 L / m. At this time, 3 / 4 of the effective volume was replaced in one day. 2 The flow rate was set to 0.15 m / s / h, and the culture solution was allowed to permeate from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow linear velocity of the culture solution flowing through the hollow was set to 0.15 m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was about 10 kPa, and stable operation was achieved.

[0054] When the antibody transmittance in the permeate was checked, it was found to be 85% or more over a period of 40 days.

[0055] [Example 2] A melt-kneaded material was prepared using 40% by mass of ETFE resin (Asahi Glass Co., Ltd., TL-081) as a thermoplastic resin, 23% by mass of fine 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 material was 240°C. The obtained melt-kneaded material was passed through a spinning nozzle with a double-tube structure, and the hollow fiber-like extrudate was passed through an idle running distance of 120 mm, and then solidified in water at 30°C, and a porous structure was developed by a thermally induced phase separation method. The obtained hollow fiber-like extrudate was taken up at a speed of 5 m / min and wound into a skein. The wound hollow fiber extrudate was immersed in isopropyl alcohol to extract and remove the 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% by mass aqueous NaOH solution at 70°C for 1 hour.Furthermore, washing with water was repeated to extract and remove the fine silica, thereby producing a porous hollow fiber membrane. The composition and production conditions of the resulting porous membrane, as well as various physical properties, are shown in Table 1 below.

[0056] The membrane area (calculated as the inner surface) of this hollow fiber membrane is 1.9 m 2 The membrane was placed in a module case with an effective length of 20 cm so that the effective length was 20 cm, and both ends were potted with epoxy resin to prepare a hollow fiber filtration membrane mini module with open ends.

[0057] As shown in Figure 8, using the prepared hollow fiber filtration membrane mini-module, CHO-K1 cells incorporating a gene expressing a monoclonal antibody were introduced into a 15 L (effective volume: 8.5 L) glass bioreactor (Repligen) using a KML-100TFF perfusion system (Repligen), and a continuous culture experiment was performed. A basal medium containing glucose and an antifoaming agent was continuously added until the cell concentration reached 75 ± 10 × 10 6 The flow rate of the hollow fiber membrane mini module was adjusted to maintain the flow rate at 1 L / m. At this time, 3 / 4 of the effective volume was replaced in one day. 2 The flow rate was set to 0.3 m / s / h, and the culture solution was allowed to permeate from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow linear velocity of the culture solution flowing through the hollow was set to 0.3 m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was about 11 kPa, and stable operation was achieved.

[0058] When the antibody transmittance in the permeate was checked, it was found to be 80% or more for 40 days.

[0059] [Example 3] A melt-kneaded material was prepared using 40% by mass of ECTFE resin (Solvay Specialty Polymers, Halar 901) as a thermoplastic resin, 23% by mass of fine 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 material was 240°C. The obtained melt-kneaded material was passed through a spinning nozzle with a double tube structure, and the hollow fiber-like extrudate was passed through an idle running distance of 120 mm, and then solidified in water at 30°C, and a porous structure was developed by a thermally induced phase separation method. The obtained hollow fiber-like extrudate was taken up at a speed of 5 m / min and wound into a skein. The wound hollow fiber extrudate was immersed in isopropyl alcohol 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% by mass aqueous NaOH solution at 70°C for 1 hour.Furthermore, the hollow fiber membrane was repeatedly washed with water to extract and remove the fine silica, thereby producing a porous hollow fiber membrane. The composition and production conditions of the resulting porous membrane, as well as various physical properties, are shown in Table 1 below.

[0060] The membrane area (converted into the inner surface) of this hollow fiber membrane is 1.9 m 2 The membrane was placed in a module case with an effective length of 20 cm so that the effective length was 20 cm, and both ends were potted with epoxy resin to prepare a hollow fiber filtration membrane mini module with open ends.

[0061] As shown in Figure 8, using the prepared hollow fiber filtration membrane mini-module, CHO-K1 cells incorporating a gene expressing a monoclonal antibody were introduced into a 15 L (effective volume: 8.5 L) glass bioreactor (Repligen) using a KML-100TFF perfusion system (Repligen), and a continuous culture experiment was performed. A basal medium containing glucose and an antifoaming agent was continuously added until the cell concentration reached 75 ± 10 × 10 6 The flow rate of the hollow fiber membrane mini module was adjusted to maintain the flow rate at 1 L / m. At this time, 3 / 4 of the effective volume was replaced in one day. 2The flow rate was set to 0.1 / h, and the culture solution was allowed to permeate from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow linear velocity of the culture solution flowing through the hollow was set to 0.2 m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was about 10 kPa, and stable operation was achieved.

[0062] When the antibody transmittance in the permeate was checked, it was found to be 83% or more over a period of 40 days.

[0063] [Example 4] A melt-kneaded material was prepared using 18.5% by mass of PE resin (manufactured by Asahi Kasei Corporation, SH800) as a thermoplastic resin, 27.2% by mass of fine 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 material was 240°C. The obtained melt-kneaded material was passed through a spinning nozzle with a double-tube structure, and the hollow fiber-like extrudate was passed through an idle distance of 120 mm, and then solidified in water at 30°C, and a porous structure was developed by a thermally induced phase separation method. The obtained hollow fiber-like extrudate was taken up at a speed of 5 m / min and wound into a skein. The wound hollow fiber extrudate was immersed in isopropyl alcohol to extract and remove the DBA, then immersed in water for 30 minutes to replace the water in the hollow fiber membrane, and then immersed in a 20% by mass aqueous NaOH solution at 70°C for 1 hour.Furthermore, the hollow fiber membrane was repeatedly washed with water to extract and remove the fine silica, thereby producing a porous hollow fiber membrane. The composition and production conditions of the resulting porous membrane, as well as various physical properties, are shown in Table 1 below.

[0064] The membrane area (calculated as the inner surface) of this hollow fiber membrane is 1.9 m 2 The membrane was placed in a module case with an effective length of 20 cm so that the effective length was 20 cm, and both ends were potted with epoxy resin to prepare a hollow fiber filtration membrane mini module with open ends.

[0065] As shown in Figure 8, using the prepared hollow fiber filtration membrane mini-module, CHO-K1 cells incorporating a gene expressing a monoclonal antibody were introduced into a 15 L (effective volume: 8.5 L) glass bioreactor (Repligen) using a KML-100TFF perfusion system (Repligen), and a continuous culture experiment was performed. A basal medium containing glucose and an antifoaming agent was continuously added until the cell concentration reached 75 ± 10 × 10 6 The flow rate of the hollow fiber membrane mini module was adjusted to maintain the flow rate at 1 L / m. At this time, 3 / 4 of the effective volume was replaced in one day. 2 The flow rate was set to 0.5 / h, and the culture solution was allowed to permeate from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow linear velocity of the culture solution flowing through the hollow was set to 0.25 m / s. The culture was carried out for about 40 days, during which time the permeation pressure of the hollow fiber membrane was about 12 kPa, and stable operation was achieved.

[0066] When the antibody transmittance in the permeate was checked, it was found to be 88% or more over a period of 40 days.

[0067] [Comparative Example 1] Except for using only ATBC as the solvent, the membrane was produced in the same manner as in Example 1 to obtain a hollow fiber membrane of Comparative Example 1. The blend composition, production conditions, and various physical properties of the obtained porous membrane are shown in Table 1 below.

[0068] The membrane area (calculated as the inner surface) of this hollow fiber membrane is 1.9 m 2 The membrane was placed in a module case with an effective length of 20 cm so that the effective length was 20 cm, and both ends were potted with epoxy resin to prepare a hollow fiber filtration membrane mini module with open ends.

[0069] As shown in Figure 8, using the prepared hollow fiber filtration membrane mini-module, CHO-K1 cells incorporating a gene expressing a monoclonal antibody were introduced into a 15 L (effective volume: 8.5 L) glass bioreactor (Repligen) using a KML-100TFF perfusion system (Repligen), and a continuous culture experiment was performed. A basal medium containing glucose and an antifoaming agent was continuously added until the cell concentration reached 75 ± 10 × 106 The flow rate of the hollow fiber membrane mini module was adjusted to maintain the flow rate at 1 L / m. At this time, 3 / 4 of the effective volume was replaced in one day. 2 The flow rate was set to 0.3 m / s / h, and the culture solution was allowed to permeate from the inner surface to the outer surface of the hollow fiber membrane, and the tangential flow linear velocity of the culture solution flowing through the hollow was set to 0.3 m / s. After about 20 days of culture, the permeation pressure of the hollow fiber membrane rose to about 100 kPa, and stable operation was not possible.

[0070] When the antibody transmittance in the permeate was checked, it was found to have decreased to 50% on the 20th day.

[0071] [Table 1] [Industrial Applicability]

[0072] The method for filtering a culture solution according to the present invention uses a porous filtration membrane having pores with good interconnectivity extending from the inside of the membrane, which is the treated liquid side, to the outside of the membrane, which is the filtrate side. This allows for good product permeability and is less susceptible to clogging by cells at a slower linear tangential flow velocity, making it possible to separate a culture containing cells from a product containing enzymes and antibodies. This method has excellent filtration performance, and furthermore does not damage the membrane during filtration operation, allowing efficient separation of proteins such as antibodies.

Claims

1. A filtration method comprising a filtration step of passing a culture solution containing cells, a medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure to separate a filtrate containing the product from the culture solution, In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the membrane, a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields, a total of four fields of view, each of which has a resolution of 1 μm ２ The above filtration method, characterized in that the sum of the areas of the resin parts having the following areas is 70% or more of the total area of ​​the resin parts.

2. A filtration method comprising a filtration step of passing a culture solution containing cells, a medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure to separate a filtrate containing the product from the culture solution, In the SEM image of the membrane cross section in the thickness direction perpendicular to the inner surface of the porous membrane, a visual field including the inner surface, a visual field including the outer surface of the membrane, and two visual fields photographed at equal intervals between these visual fields, a total of four visual fields, ２ The above filtration method, characterized in that the sum of the areas of the resin parts having the above area is 15% or less of the total area of ​​the resin parts.

3. A filtration method comprising a filtration step of passing a culture solution containing cells, a medium, a product, and an optional antifoaming agent through a porous membrane made of a resin having a three-dimensional mesh structure to separate a filtrate containing the product from the culture solution, In the SEM image of the cross section of the porous membrane in the thickness direction perpendicular to the inner surface of the membrane, a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between these fields, a total of four fields of view, each of which has a resolution of 1 μm ２ The total area of ​​the resin parts having the following area is 70% or more of the total area of ​​the resin parts, and ２ The above filtration method, characterized in that the sum of the areas of the resin parts having the above area is 15% or less of the total area of ​​the resin parts.

4. The porous membrane is a porous membrane having a cross section in a thickness direction perpendicular to the inner surface of the porous membrane, and has a thickness of 1 μm or less in each of four fields of view, including a field including the inner surface, a field including the outer surface of the membrane, and two fields of view photographed at equal intervals between the two fields of view. ２ Super 10μm ２ The filtration method according to any one of claims 1 to 3, wherein the sum of the areas of the resin parts having an area of ​​less than 15% of the total area of ​​the resin parts.

5. The filtration method according to any one of claims 1 to 3, wherein the surface opening ratio 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 any one of claims 1 to 3, 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 hollow fiber membrane has an intramembrane linear velocity of tangential flow during the perfusion culture of 0.3 m / s or less.

9. The filtration method according to any one of claims 1 to 3, wherein the resin constituting the porous membrane is a thermoplastic resin.

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

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

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

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

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