Evaluation method of filtration condition and production method of cell product

Abstract

A method for evaluating filtration condition(s), the method including: under conditions where cell growth is suppressed, feeding a culture fluid containing cells from a culture tank 11 containing the culture fluid to a porous membrane 12, returning the culture fluid, which has passed through the porous membrane 12 without being filtered, back to the culture tank 11 so as to circulate the culture fluid between the culture tank 11 and the porous membrane 12; and evaluating one or more filtration conditions in the porous membrane 12.

Description

Method for evaluating filtration conditions and method for producing cell products

[0001] The present invention relates to filtration technology, a method for evaluating filtration conditions, and a method for producing cell products.

[0002] Cell culture technology is essential for the production of various biopharmaceuticals, such as antibodies, growth hormones, and insulin, and has contributed greatly to the advancement of medicine in recent years. Among biopharmaceuticals, antibody drugs in particular have attracted attention. Highly efficient and stable production of monoclonal antibodies by culturing antibody-producing cells is one of the most important industrial themes.

[0003] Industrial cell culture methods for producing useful cellular products such as antibodies can be broadly classified into two types: adherent culture and suspension culture. In adherent culture, cells adhere to the inner surface of a culture vessel. In suspension culture, cells are suspended in a culture solution. Of these, suspension culture is the mainstream due to its ease of scale-up and control on a large scale.

[0004] In suspension culture, a method has been proposed for culturing cells at high density and high efficiency in a continuous manner to continuously produce cell products. This method involves supplying fresh culture medium to a culture vessel at a constant rate while filtering and discharging old culture medium containing cell products from the vessel at a constant rate. This type of culture is generally referred to as continuous culture or perfusion culture (see, for example, Patent Documents 1 to 5). In continuous culture, the amount of culture medium supplied to the culture vessel can be controlled to be equal to the amount of culture medium discharged from the vessel. The key to continuous culture is to efficiently separate the cells in the culture medium from the old culture medium and cell products over a long period of time, remove the old culture medium and cell products from the vessel, and maintain the cell growth environment in the vessel under optimal conditions for a long period of time.

[0005] Japanese Patent Application Laid-Open No. 2018-76291 Japanese Patent Application Laid-Open No. 2009-45019 Japanese Patent Application Laid-Open No. 2021-48776 Japanese Patent No. 5696479 Japanese Patent Application Laid-Open No. 2022-516516

[0006] In continuous culture, a porous membrane is used to separate cells in a culture medium in a culture tank from old culture medium and cell products. A method for appropriately evaluating the filtration conditions for a porous membrane is desired. Therefore, one of the objectives of the present invention is to provide a filtration condition evaluation method that can appropriately evaluate the filtration conditions of a cell culture medium and a method for producing a cell product.

[0007] [1] According to an aspect of the present invention, there is provided a method for evaluating filtration conditions, comprising: feeding a culture medium containing cells from a culture tank containing the culture medium to a porous membrane under conditions in which cell growth is suppressed; returning the culture medium that passes through the porous membrane without being filtered back to the culture tank; and circulating the culture medium between the culture tank and the porous membrane; and evaluating one or more filtration conditions for the porous membrane.

[0008] [2] In the method for evaluating filtration conditions described in [1] above, when returning the culture solution that passed through the porous membrane without being filtered, the culture solution that passed through the porous membrane without being filtered and the culture solution that was filtered through the porous membrane may be returned to the culture tank.

[0009] [3] The method for evaluating filtration conditions according to [1] or [2] above may further include evaluating the filtration performance of the porous membrane under one or more filtration conditions, and may evaluate the one or more filtration conditions for the porous membrane based on the filtration performance of the porous membrane.

[0010] [4] In the method for evaluating filtration conditions according to any one of [1] to [3] above, the conditions under which cell proliferation is inhibited may be conditions under which progression of the cell cycle is inhibited.

[0011] [5] In the method for evaluating filtration conditions according to any one of [1] to [4] above, the condition under which cell growth is inhibited may be at least one of an atmospheric temperature and a culture solution temperature of 15°C or less.

[0012] [6] In the method for evaluating filtration conditions according to any one of [1] to [5] above, at least one of the ambient temperature and the culture solution temperature of 15°C or less may be at least one of the ambient temperature and the culture solution temperature of 10°C or less or 5°C or less.

[0013] [7] In the method for evaluating filtration conditions according to any one of [1] to [6] above, the conditions under which cell growth is inhibited may be conditions under which enzymatic activity of the cells is inhibited.

[0014] [8] In the method for evaluating filtration conditions according to [7] above, the enzyme may be a cyclin-dependent kinase.

[0015] [9] In the method for evaluating filtration conditions according to any one of [1] to [8] above, the condition that inhibits cell proliferation may be the presence of a cell cycle inhibitor in the culture medium.

[0016]

[10] In the method for evaluating filtration conditions according to any one of [1] to [9] above, one or more filtration conditions for a porous membrane may be evaluated based on the permeability of a cell product through the porous membrane.

[0017]

[11] In the method for evaluating filtration conditions according to any one of [1] to

[10] above, one or more filtration conditions for a porous membrane may be evaluated based on the permeation flux through the porous membrane.

[0018]

[12] In the method for evaluating filtration conditions according to any one of [1] to

[11] above, one or more filtration conditions for a porous membrane may be evaluated based on the transmembrane pressure difference across the porous membrane.

[0019]

[13] In the method for evaluating filtration conditions according to any one of [1] to

[12] above, one or more filtration conditions for a porous membrane may be evaluated based on the turbidity of the filtrate.

[0020]

[14] In the method for evaluating filtration conditions according to any one of [1] to

[13] above, the one or more filtration conditions may be one or more conditions relating to the structure, material, or physical properties of the porous membrane.

[0021]

[15] In the method for evaluating filtration conditions according to any one of [1] to

[14] above, the one or more filtration conditions may be one or more conditions for the culture medium.

[0022]

[16] In the method for evaluating filtration conditions according to any one of [1] to

[15] above, the one or more filtration conditions may be one or more conditions relating to cell density in the culture medium.

[0023]

[17] In any of the methods for evaluating filtration conditions [1] to

[16] above, the one or more filtration conditions may be one or more conditions of the flow rate of the culture solution sent to the porous membrane.

[0024]

[18] In the method for evaluating filtration conditions according to any one of [1] to

[17] above, the one or more filtration conditions may be one or more conditions of the flow rate of the culture solution filtered through the porous membrane.

[0025]

[19] In the method for evaluating filtration conditions according to any one of [1] to

[18] above, the one or more filtration conditions may be one or more conditions of shear stress on the liquid-contacting surface of the porous membrane due to the flow of the culture medium sent to the porous membrane.

[0026]

[20] In the method for evaluating filtration conditions according to any one of [1] to

[19] above, the culture vessel and the path through which the culture solution containing the porous membrane circulates may be disposed in a temperature-controlled vessel.

[0027]

[21] In the method for evaluating filtration conditions according to any one of [1] to

[20] above, when the culture solution is circulated between the culture tank and the porous membrane, no active operation is required to maintain the composition of the culture solution.

[0028]

[22] In the method for evaluating filtration conditions according to any one of [1] to

[21] above, when the culture solution is circulated between the culture tank and the porous membrane, it is not necessary to add a culture medium from the outside to the path through which the culture solution circulates, including the culture tank and the porous membrane.

[0029]

[23] In the method for evaluating filtration conditions according to any one of [1] to

[22] above, when the culture solution is circulated between the culture tank and the porous membrane, it is not necessary to control at least one of the dissolved oxygen and the pH of the culture solution.

[0030]

[24] In the method for evaluating filtration conditions according to any one of [1] to

[23] above, when the culture medium is circulated between the culture tank and the porous membrane, it is not necessary to bleed cells from the path through which the culture medium circulates, including the culture tank and the porous membrane.

[0031]

[25] In the method for evaluating filtration conditions according to any one of [1] to

[24] above, when the culture solution is circulated between the culture tank and the porous membrane, the culture solution may be collected for sampling from a path through which the culture solution circulates, including the culture tank and the porous membrane.

[0032]

[26] The method for evaluating filtration conditions according to

[25] above may further comprise measuring the cell density in the sampled culture medium.

[0033]

[27] The method for evaluating filtration conditions according to

[25] or

[26] above may further comprise measuring the viability of cells in the sampled culture medium.

[0034]

[28] The method for evaluating filtration conditions according to any one of

[25] to

[27] above may further comprise measuring the concentration of a cell product in the sampled culture medium.

[0035]

[29] The method for evaluating filtration conditions according to any one of

[25] to

[28] above may further comprise measuring the turbidity of the sampled culture medium.

[0036]

[30] In the method for evaluating filtration conditions according to any one of the above [1] to

[29] , the porous membrane may be a hollow fiber membrane.

[0037]

[31] In the method for evaluating filtration conditions according to any one of the above [1] to

[30] , the porous membrane may be a microfiltration membrane.

[0038]

[32] According to an aspect of the present invention, there is provided a method for producing a cell product, comprising: using filtration conditions under conditions in which cells grow; sending a culture medium from a culture tank containing a cell-containing culture medium to a porous membrane; returning the culture medium that passes through without being filtered by the porous membrane to the culture tank; recovering the culture medium containing the cell product that is filtered through the porous membrane; and circulating at least a portion of the culture medium between the culture tank and the porous membrane; wherein the filtration conditions are filtration conditions obtained by evaluating one or more filtration conditions for the porous membrane under conditions in which cell growth is inhibited; sending a culture medium from a culture tank containing a cell-containing culture medium to the porous membrane; returning the culture medium that passes through without being filtered by the porous membrane to the culture tank; and circulating the culture medium between the culture tank and the porous membrane.

[0039]

[33] In the method for producing a cell product according to

[32] above, when evaluating one or more filtration conditions for the porous membrane, the culture solution that passes through the porous membrane without being filtered may be returned to the culture tank, and the culture solution that passes through the porous membrane without being filtered may also be returned to the culture tank.

[0040]

[34] In the method for producing a cell product according to

[32] or

[33] above, the conditions for cell growth may be conditions for progression of the cell cycle.

[0041]

[35] In any of the methods for producing a cell product described above in

[32] to

[34] , the conditions for cell growth may be at least one of an atmospheric temperature and a culture solution temperature of 15°C or higher.

[0042]

[36] In the method for producing a cell product according to

[35] above, at least one of the atmospheric temperature and the culture solution temperature higher than 15°C may be at least one of the atmospheric temperature and the culture solution temperature higher than 20°C, 25°C, 30°C, or 35°C.

[0043]

[37] In any of the methods for producing a cell product described above in

[32] to

[36] , the conditions under which the cells grow may be conditions under which the enzymes in the cells are activated.

[0044]

[38] In the method for producing a cell product according to

[37] above, the enzyme may be a cyclin-dependent kinase.

[0045]

[39] In the method for producing a cell product according to any one of

[32] to

[38] above, the conditions for cell growth may be the absence of cell cycle inhibitors in the culture medium.

[0046]

[40] In the method for producing a cell product according to any one of

[32] to

[39] above, the conditions under which cell proliferation is inhibited may be conditions under which progression of the cell cycle is inhibited.

[0047]

[41] In any of the methods for producing a cell product described above in

[32] to

[40] , the conditions for inhibiting cell growth may be at least one of an atmospheric temperature and a culture solution temperature of 15°C or less.

[0048]

[42] In the method for producing a cell product according to

[41] above, at least one of the atmospheric temperature and the culture solution temperature of 15°C or less may be at least one of the atmospheric temperature and the culture solution temperature of 10°C or less or 5°C or less.

[0049]

[43] In the method for producing a cell product according to any one of

[32] to

[42] above, the conditions under which cell growth is inhibited may be conditions under which the enzymatic activity of the cells is inhibited.

[0050]

[44] In the method for producing a cell product according to

[43] above, the enzyme may be a cyclin-dependent kinase.

[0051]

[45] In any of the methods for producing a cell product described above in

[32] to

[44] , the condition for inhibiting cell proliferation may be the presence of a cell cycle inhibitor in the culture medium.

[0052]

[46] In any of the methods for producing a cell product described above in

[32] to

[45] , one or more filtration conditions for the porous membrane may be evaluated based on the permeability of the cell product through the porous membrane.

[0053]

[47] In any of the methods for producing a cell product described above in

[32] to

[46] , one or more filtration conditions for the porous membrane may be evaluated based on the permeation flux through the porous membrane.

[0054]

[48] ​​In any of the methods for producing a cell product described above in

[32] to

[47] , one or more filtration conditions for the porous membrane may be evaluated based on the transmembrane pressure difference across the porous membrane.

[0055]

[49] In any of the cell product production methods

[32] to

[48] above, one or more filtration conditions for the porous membrane may be evaluated based on the turbidity of the filtrate.

[0056]

[50] In any of the cell product production methods

[32] to

[49] above, the one or more filtration conditions may be one or more conditions regarding the structure, material, or physical properties of the porous membrane.

[0057]

[51] In any of the methods for producing a cell product described above in

[32] to

[50] , the one or more filtration conditions may be one or more conditions for the culture medium.

[0058]

[52] In any of the methods for producing a cell product described above in

[32] to

[51] , the one or more filtration conditions may be one or more conditions relating to the density of cells in the culture medium.

[0059]

[53] In any of the methods for producing a cell product described above in

[32] to

[52] , the one or more filtration conditions may be one or more conditions for the flow rate of the culture medium fed to the porous membrane.

[0060]

[54] In any of the cell product production methods

[32] to

[53] above, the one or more filtration conditions may be one or more conditions of the flow rate of the culture solution filtered through the porous membrane.

[0061]

[55] In any of the methods for producing a cell product described above in

[32] to

[54] , the one or more filtration conditions may be one or more conditions of shear stress on the liquid-contacting surface of the porous membrane due to the flow of the culture medium sent to the porous membrane.

[0062]

[56] In the method for producing a cell product according to any one of

[32] to

[55] above, when evaluating one or more filtration conditions for a porous membrane, a path through which the culture medium circulates, including the culture vessel and the porous membrane, may be placed in a temperature-controlled vessel.

[0063]

[57] In the cell product production method according to any one of

[32] to

[56] above, when evaluating multiple filtration conditions for a porous membrane, active manipulation to maintain the composition of the culture solution may be omitted when circulating the culture solution between the culture tank and the porous membrane.

[0064]

[58] In the method for producing a cell product according to any one of

[32] to

[57] above, when evaluating multiple filtration conditions for a porous membrane, in circulating the culture medium between the culture tank and the porous membrane, it is not necessary to add a culture medium from the outside to the path through which the culture medium circulates, including the culture tank and the porous membrane.

[0065]

[59] In the method for producing a cell product according to any one of

[32] to

[58] above, when evaluating multiple filtration conditions for a porous membrane, it is not necessary to control at least one of the dissolved oxygen and pH of the culture solution when circulating the culture solution between the culture tank and the porous membrane.

[0066]

[60] In any of the cell product production methods

[32] to

[59] above, when evaluating multiple filtration conditions for a porous membrane, when the culture medium is circulated between the culture tank and the porous membrane, it is not necessary to bleed cells from the path through which the culture medium circulates, including the culture tank and the porous membrane.

[0067]

[61] In any of the cell product production methods

[32] to

[60] above, when evaluating one or more filtration conditions for the porous membrane, the culture medium may be circulated between the culture tank and the porous membrane, and the culture medium may be collected for sampling from a path through which the culture medium circulates, including the culture tank and the porous membrane.

[0068]

[62] The method for producing a cell product according to

[61] above may further comprise measuring the density of cells in the sampled culture medium.

[0069]

[63] The method for producing a cell product according to

[61] or

[62] above may further comprise measuring the survival rate of cells in the sampled culture medium.

[0070]

[64] The method for producing a cell product according to any one of

[61] to

[63] above may further comprise measuring the concentration of the cell product in the sampled culture medium.

[0071]

[65] The method for producing a cell product according to any one of

[61] to

[64] above may further comprise measuring the turbidity of the sampled culture medium.

[0072]

[66] In any of the methods for producing a cell product described above in

[32] to

[65] , the porous membrane may be a hollow fiber membrane.

[0073]

[67] In any of the methods for producing a cell product described above in

[32] to

[66] , the porous membrane may be a microfiltration membrane.

[0074]

[68] In any of the methods for producing a cell product described above in

[32] to

[67] , the cells may be perfusion cultured.

[0075] According to the present invention, it is possible to provide a method for evaluating filtration conditions that can appropriately evaluate the filtration conditions for a cell culture medium, and a method for producing a cell product.

[0076] FIG. 1 is a schematic diagram showing a system for evaluating filtration conditions for hollow fiber membranes according to an embodiment. FIG. 2 is a schematic diagram showing a system for producing a cell product according to an embodiment. FIG. 3 is a graph showing the change over time in cell density in a culture medium circulating between a culture tank and a hollow fiber membrane according to an example. FIG. 4 is a graph showing the change over time in cell density in a culture medium circulating between a culture tank and a hollow fiber membrane according to an example. FIG. 5 is a graph showing the change over time in cell viability in a culture medium circulating between a culture tank and a hollow fiber membrane according to an example. FIG. 6 is a graph showing the change over time in cell viability in a culture medium circulating between a culture tank and a hollow fiber membrane according to an example. FIG. 7 is a graph showing the change over time in antibody concentration in a culture medium circulating between a culture tank and a hollow fiber membrane according to an example. FIG. 8 is a graph showing the change over time in antibody concentration in a culture medium circulating between a culture tank and a hollow fiber membrane according to an example. FIG. 9 is a graph showing the change over time in cell density in a culture medium circulating between a culture tank and a hollow fiber membrane according to a comparative example. FIG. 10 is a graph showing the change over time in cell viability in a culture medium circulating between a culture tank and a hollow fiber membrane according to a comparative example. 1 is a graph showing the change over time in the concentration of an antibody in a culture medium circulating between a culture tank and a hollow fiber membrane according to a comparative example. FIG. 2 is a graph showing the change over time in the density of cells in the culture medium in a culture tank according to a reference example. FIG. 3 is a graph showing the change over time in the viability of cells in the culture medium in a culture tank according to a reference example. FIG. 4 is a graph showing the change over time in the concentration of an antibody in the culture medium in a culture tank according to a reference example. FIG. 5 is a graph showing the change over time in the density of cells in the culture medium in a culture tank according to a reference example. FIG. 6 is a graph showing the change over time in the viability of cells in the culture medium in a culture tank according to a reference example. FIG. 7 is a graph showing the change over time in the concentration of an antibody in the culture medium in a culture tank according to a reference example.

[0077] Hereinafter, an embodiment for carrying out the present invention (hereinafter referred to as "the present embodiment") will be described in detail. Note that the present embodiment is intended to facilitate understanding of the present invention and is not intended to limit the present invention. The present invention is not limited to the present embodiment, and can be carried out in various modifications within the scope of the gist thereof.

[0078] Referring to FIG. 1 , the method for evaluating filtration conditions according to this embodiment includes: feeding a culture solution containing cells from a culture tank 11 to a porous membrane 12 under conditions that inhibit cell growth; returning the culture solution that passes through the porous membrane 12 without being filtered back to the culture tank 11; and circulating the culture solution between the culture tank 11 and the porous membrane 12; evaluating the filtration performance of the porous membrane 12 under one or more filtration conditions; and evaluating one or more filtration conditions for the porous membrane 12 based on the filtration performance of the porous membrane 12. When returning the culture solution that passes through the porous membrane 12 without being filtered back to the culture tank 11, the culture solution that passes through the porous membrane 12 without being filtered and the culture solution filtered through the porous membrane 12 may be returned to the culture tank 11. However, this does not prevent the culture solution from being recovered without returning the entire amount of the culture solution to the culture tank 11, as long as the culture solution in the culture tank 11 is supplemented with an equal amount of medium as the recovered culture solution. The culture solution may be recovered by recovering all or part of the culture solution filtered through the porous membrane 12 without returning it to the culture tank 11. The amount of culture medium to be collected is not limited, and by supplementing the system with the same amount of medium, fluctuations in the cell density and product concentration contained in the culture medium in the culture tank 11 are suppressed. Note that this collection is to be distinguished from sampling, which will be described later, for the purpose of analyzing the culture medium.

[0079] The culture tank 11 is, for example, a container having an internal space that is closed off from the outside air. However, the culture tank 11 may be provided with a vent or a filter to maintain a constant internal pressure. The vent or filter preferably has a pore size of 0.2 μm or less to prevent external bacteria from entering the culture tank 11. The culture tank 11 may be provided with an agitator 16 for agitating the culture medium in the culture tank 11. Cells are cultured in suspension in the culture medium in the culture tank 11.

[0080] The cells cultured in the culture tank 11 are not particularly limited. The cells may be derived from animals, including humans, or from microorganisms. The cells may be eukaryotic or prokaryotic. Examples of animals include mammals, reptiles, birds, amphibians, fish, and insects. The cells may be genetically modified cells. Examples of cells include CHO (Chinese Hamster Ovary) cells, HEK cells, BHK-21 cells, Sp2 / 0 cells, SP2 / 0-Ag14 cells, NS0 cells, Vero cells, PER.C6 cells, yeast, Bacillus subtilis, and Escherichia coli.

[0081] The cells may, for example, produce and release into the culture medium a pharmaceutically usable product. Examples of pharmaceutically usable products include peptides, proteins, and viruses (including virus-like particles). Examples of proteins include antibodies, hormones, cytokines, growth factors, enzymes, and plasma proteins. The protein may be a recombinant protein.

[0082] The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be a human antibody or an antibody protein derived from a non-human mammal, such as a bovine or a mouse. Alternatively, the antibody may be a chimeric antibody protein with human IgG, or a humanized antibody. A chimeric antibody with human IgG is an antibody in which the variable region is derived from a non-human organism, such as a mouse, but the other constant regions are substituted with immunoglobulins derived from humans. A humanized antibody is an antibody in which the complementarity-determining region (CDR) of the variable region is derived from a non-human organism, but the other framework region (FR) is derived from humans. Humanization further reduces immunogenicity compared to chimeric antibodies.

[0083] The shape of the porous membrane 12 is not particularly limited. Examples of porous membranes include hollow fiber membranes, flat membranes, and tubular membranes. Below, an example will be described in which the porous membrane 12 is a hollow fiber membrane. In the porous membrane 12, the surface to which the culture solution is supplied is referred to as the primary side of the hollow fiber membrane. Furthermore, the surface from which the permeated liquid that has permeated the hollow fiber membrane flows out is referred to as the secondary side of the hollow fiber membrane. In an embodiment in which the culture solution to be filtered is supplied to the inner circumferential surface, the inner circumferential surface of the hollow fiber membrane is the primary side, and the outer circumferential surface of the hollow fiber membrane is the secondary side. In an embodiment in which the culture solution to be filtered is supplied to the outer circumferential surface, the outer circumferential surface of the hollow fiber membrane is the primary side, and the inner circumferential surface of the hollow fiber membrane is the secondary side.

[0084] There is no particular limitation on the type of porous membrane 12. Examples of porous membranes include coarse filtration membranes, microfiltration membranes, ultrafiltration membranes, dialysis membranes, nanofiltration membranes, reverse osmosis membranes, and forward osmosis membranes.

[0085] The filtration method in the porous membrane 12 may be a tangential flow filtration (TFF) method. Tangential flow filtration is a filtration method in which a culture solution is flowed in a direction parallel to the primary surface of the hollow fiber membrane on the primary surface of the hollow fiber membrane. Tangential flow filtration includes alternating tangential flow filtration (ATF) method. In this embodiment, the term "tangential flow filtration (TFF)" may refer to a filtration method in which a culture solution is flowed in one direction on the primary surface of the hollow fiber membrane. Alternating tangential flow filtration (ATF) refers to a filtration method in which a culture solution is flowed back and forth on the primary surface of the hollow fiber membrane.

[0086] In the example shown in Figure 1, between the culture tank 11 and the porous membrane 12, there are arranged a flow path 13 for sending the culture solution in the culture tank 11 to the porous membrane 12, a flow path 14 for returning the culture solution that has passed through the porous membrane 12 without being filtered by the porous membrane 12 to the culture tank 11, and optionally a flow path 15 for returning the culture solution filtered by the porous membrane 12 to the culture tank 11. The culture solution flowing through the flow path 13 may contain cells and products of the cells. The culture solution that has passed through the porous membrane 12 without being filtered and that flows through the flow path 14 may contain cells and products of the cells. The culture solution that has been filtered through the porous membrane 12 and that flows through the flow path 15 may contain products of the cells.

[0087] The flow path 13 is provided with, for example, a pump 23 for sending the culture solution in the culture tank 11 to the porous membrane 12. Examples of the pump include, but are not limited to, a diaphragm pump, a tube pump, a centrifugal pump, and a rotary pump. The flow path 13 may be provided with a pressure gauge 33 for measuring the pressure of the culture solution supplied to the porous membrane 12. The flow path 13 may be provided with a flow meter for measuring at least one of the flow rate and flow rate of the culture solution flowing in the flow path 13. The flow path 13 may be provided with a thermometer for measuring the temperature of the culture solution flowing in the flow path 13. The flow path 13 may be provided with a sampling unit for sampling the culture solution flowing in the flow path 13. The sampling unit is closed except during sampling.

[0088] The flow path 14 may be provided with a pump for sending the culture solution that has passed through the hollow portion without passing through the pores of the porous membrane 12 and has not been filtered by the porous membrane 12 to the culture tank 11. The pump may be provided in both the flow path 13 and the flow path 14, or in either one of them. The flow path 14 may be provided with a pressure gauge 34 that measures the pressure of the culture solution that has passed through the porous membrane 12. The flow path 14 may be provided with a flow meter that measures at least one of the flow rate and flow rate of the culture solution flowing in the flow path 14. The flow path 14 may be provided with a thermometer that measures the temperature of the culture solution flowing in the flow path 14. The flow path 14 may be provided with a sampling unit for sampling the culture solution flowing in the flow path 14. The sampling unit is closed except during sampling.

[0089] The flow path 15 is provided with, for example, a pump 25 for sending the culture solution that has passed through the pores of the porous membrane 12 and been filtered by the porous membrane 12 to the culture tank 11. The flow path 15 may be provided with a pressure gauge 35 that measures the pressure of the culture solution that has been filtered by the porous membrane 12. The flow path 15 may be provided with a flow meter that measures at least one of the flow velocity and flow rate of the culture solution flowing within the flow path 15. The flow path 15 may be provided with a sampling unit for sampling the culture solution flowing within the flow path 15. The sampling unit is closed except when sampling is being performed.

[0090] The culture tank 11 and the porous membrane 12 form at least a part of a path through which the culture solution circulates. The flow paths 13, 14, and 15 also form at least a part of the path through which the culture solution circulates. The path through which the culture solution circulates may be closed from the outside, but as described above, the culture tank 11 may be provided with a vent or filter to maintain a constant internal pressure.

[0091] The culture tank 11 and the porous membrane 12 may be placed in a temperature-controlled tank 50 that controls the internal temperature. The flow paths 13, 14, and 15 may be placed in the temperature-controlled tank 50. The temperature-controlled tank 50 controls the ambient temperature of the culture tank 11, the porous membrane 12, and the flow paths 13, 14, and 15. The temperature-controlled tank 50 may be a thermostatic tank or a refrigerator, or may be a booth, room, or building that is maintained at a constant temperature.

[0092] The conditions under which cell proliferation is inhibited are not particularly limited, and include, for example, conditions under which progression through the cell cycle is inhibited. Conditions under which progression through the cell cycle is inhibited include, for example, a low atmospheric temperature of 15°C or less in the culture vessel 11 and the porous membrane 12, or a temperature of 15°C or less in the culture solution. The atmospheric temperature or the temperature of the culture solution is preferably 10°C or less or 5°C or less. From the viewpoint of inhibiting cell death, the atmospheric temperature of the culture vessel 11 and the porous membrane 12 or the temperature of the culture solution is preferably 0°C or higher, 1°C or higher, 2°C or higher, or 3°C or higher. The temperature of the culture solution may be measured directly, or may be estimated from the atmospheric temperatures of the culture vessel 11 and the porous membrane 12. Based on the measured temperature of the culture solution, the temperature of the culture solution may be directly controlled using a temperature control device, or the atmospheric temperatures of the culture vessel 11 and the porous membrane 12 may be controlled using a temperature control device. Conditions under which progression through the cell cycle is inhibited include, for example, conditions under which enzyme activity such as cyclin-dependent kinase in cells is inhibited. The condition under which cell cycle progression is inhibited may be the presence of a cell cycle inhibitor in the culture medium. Under the condition under which cell proliferation is inhibited, cell activity is reduced, cell cycle progression is inhibited, cell division is inhibited, and product production is inhibited.

[0093] In this embodiment, both the culture solution that passes through the porous membrane 12 without being filtered and the culture solution that has been filtered through the porous membrane 12 may be returned to the culture tank 11. In this case, under conditions in which cell growth is suppressed, the total amount of cells and the total amount of products in the system are maintained, thereby suppressing fluctuations in the cell density and product concentration contained in the culture solution circulating between the culture tank 11 and the porous membrane 12.

[0094] Under conditions where cell growth is inhibited, cell activity decreases and the amount of culture medium components required by the cells also decreases, making it unnecessary to add culture medium from outside to the circulation path of the culture medium. Furthermore, not adding culture medium suppresses fluctuations in cell density and product concentration contained in the culture medium. Furthermore, under conditions where cell growth is inhibited, cell activity decreases and fluctuations in the dissolved oxygen concentration (DO) and pH of the culture medium are suppressed. Therefore, it is unnecessary to add gases such as oxygen or carbon dioxide from outside to the circulation path of the culture medium to control the dissolved oxygen concentration and pH. Furthermore, under conditions where cell growth is inhibited, the cell density in the culture medium becomes almost constant, making it unnecessary to bleed cells from the circulation path of the culture medium. Therefore, the filtration condition evaluation method according to this embodiment can simplify cell culture operations. However, a portion of the culture medium may be sampled, for example, to monitor cell density, cell viability, and cell product concentration. Sampling is distinguished from bleeding because it is not intended to adjust cell density.

[0095] One or more filtration conditions for the porous membrane 12 are evaluated based on the filtration performance of the porous membrane 12. The filtration performance of the porous membrane 12 may be expressed, for example, by at least one of a high permeability of the cell product through the porous membrane 12, a high shielding rate of impurities through the porous membrane 12, a low transmembrane pressure difference through the porous membrane 12, a large amount of liquid that can be filtered before the transmembrane pressure difference through the porous membrane 12 increases, a large amount of liquid that can be filtered before the permeation flux through the porous membrane 12 begins to decrease, a low pressure drop through the porous membrane 12, a large flow rate through the porous membrane 12, a high flow velocity through the porous membrane 12, and a high permeation flux rate through the porous membrane 12.

[0096] The one or more filtration conditions for the porous membrane 12 are evaluated based on whether the filtration performance of the porous membrane 12 satisfies a predetermined standard or is relatively superior under the one or more filtration conditions for the porous membrane 12. Alternatively, at least some of the filtration conditions may be extracted from the multiple filtration conditions for the porous membrane 12 based on whether the filtration performance of the porous membrane 12 satisfies a predetermined standard or is relatively superior.

[0097] Fluctuations in the cell density and product concentration contained in the culture solution supplied to the porous membrane 12 can make it difficult to accurately evaluate the filtration performance of the porous membrane 12. In contrast, as described above, in the present embodiment, cell proliferation is suppressed, thereby suppressing fluctuations in the cell density and product concentration contained in the culture solution. Returning the culture solution filtered through the porous membrane 12 to the culture tank 11 further suppresses fluctuations in the cell density and product concentration contained in the culture solution. Therefore, according to the present embodiment, it is possible to evaluate the filtration performance of the porous membrane 12 while maintaining the cell density and product concentration contained in the culture solution substantially constant. Furthermore, returning the culture solution filtered through the porous membrane 12 to the culture tank 11 may eliminate the need to replenish the culture medium in the culture tank 11.

[0098] The one or more filtration conditions are, for example, one or more conditions of the structure of the porous membrane 12. The structural conditions of the porous membrane 12 include, for example, membrane thickness, inner diameter, outer diameter, membrane surface aperture ratio, porosity, pore size, average pore size, pore size variation, blocking pore size, pore size to fiber diameter ratio, membrane surface anisotropy, membrane pore anisotropy, membrane surface roughness, and pore size change from the primary side to the secondary side. For example, multiple porous membranes 12 having various structures are prepared, and the filtration performance of each porous membrane 12 is evaluated. Furthermore, it is evaluated whether the one or more structural conditions of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether it is relatively superior.

[0099] The one or more filtration conditions are, for example, one or more conditions for the material of the porous membrane 12. For example, a plurality of porous membranes 12 made of various materials are prepared, and the filtration performance of each porous membrane 12 is evaluated. Furthermore, it is evaluated whether the one or more conditions for the material of the porous membrane 12 provide filtration performance that satisfies a predetermined standard, or whether the filtration performance is relatively superior.

[0100] The one or more filtration conditions are, for example, one or more conditions for the physical properties of the porous membrane 12. The conditions for the physical properties of the porous membrane 12 are, for example, hydrophilicity, hydrophobicity, cationicity, anionicity, elastic limit pressure, and bubble point. For example, a plurality of porous membranes 12 having various physical properties are prepared, and the filtration performance of each porous membrane 12 is evaluated. Furthermore, it is evaluated whether the one or more conditions for the physical properties of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether the filtration performance is relatively superior.

[0101] The one or more filtration conditions are, for example, one or more conditions of the culture solution. The culture solution conditions include, for example, the contents of the culture solution, the concentration of each of the contents in the culture solution, the viscosity of the culture solution, the pH of the culture solution, the electrical conductivity of the culture solution, and the turbidity of the culture solution. Examples of the contents of the culture solution include the composition of the culture solution, antifoaming agents, salts, nucleic acids such as DNA and RNA, host cell-derived proteins (HCPs), lipids, and polysaccharides. For example, multiple culture solutions with different conditions are prepared, and the filtration performance of the porous membrane 12 is evaluated when each culture solution is used. Furthermore, it is evaluated whether one or more conditions of the culture solution provide filtration performance that meets a predetermined standard, or whether they are relatively superior.

[0102] The one or more filtration conditions are, for example, multiple cell density conditions in the culture solution. For example, multiple culture solutions with different cell densities are prepared, and the filtration performance of the porous membrane 12 is evaluated when each culture solution is used. Furthermore, it is evaluated whether the one or more cell density conditions provide filtration performance that meets a predetermined standard, or whether they are relatively superior.

[0103] The one or more filtration conditions are, for example, one or more conditions for the flow rate of the culture solution fed to the porous membrane 12. For example, the culture solution is fed to the porous membrane 12 at different flow rates, and the filtration performance of the porous membrane 12 is evaluated when each flow rate is used. Furthermore, it is evaluated whether the one or more conditions for the flow rate of the culture solution fed to the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether they are relatively superior. The flow rate may be expressed as a volumetric flow rate, a mass flow rate, a linear velocity, or a shear rate at the surface of the porous membrane 12.

[0104] The one or more filtration conditions are, for example, one or more conditions for the flow rate of the culture solution filtered through the porous membrane 12. For example, the culture solution filtered through the porous membrane 12 is fed at different flow rates, and the filtration performance of the porous membrane 12 is evaluated when each flow rate is used. Furthermore, it is evaluated whether the one or more conditions for the flow rate of the culture solution filtered through the porous membrane 12 provide filtration performance that satisfies a predetermined standard, or whether they are relatively superior.

[0105] The one or more filtration conditions are, for example, one or more conditions of shear stress at the liquid-contacting surface of the porous membrane 12. The liquid-contacting surface is the surface of the porous membrane 12 that comes into contact with the liquid to be filtered. If the porous membrane 12 is a hollow fiber membrane, it is the primary side surface. For example, the shear stress at the liquid-contacting surface of the porous membrane 12 is changed, and the filtration performance of the porous membrane 12 is evaluated when each shear stress is used. Furthermore, it is evaluated whether the one or more conditions of shear stress at the liquid-contacting surface of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether it is relatively superior.

[0106] According to the method for evaluating filtration conditions of this embodiment, it is possible to select one or more filtration conditions that allow the porous membrane 12 to exhibit filtration performance that meets predetermined standards while suppressing fluctuations in the cell density and product concentration contained in the culture solution circulating between the culture tank 11 and the porous membrane 12.

[0107] Next, referring to Figure 2, the method for producing cell products according to this embodiment includes, under conditions in which cells grow, using the filtration conditions obtained by the above-mentioned method for evaluating filtration conditions, sending a culture solution from a culture tank 111 containing a culture solution containing cells to a porous membrane 112, returning the culture solution that passes through the porous membrane 112 without being filtered back to the culture tank 111, recovering the culture solution containing the cell products that has been filtered by the porous membrane 112, and circulating at least a portion of the culture solution between the culture tank 111 and the porous membrane 112.

[0108] The conditions for cell growth are not particularly limited as long as the cells produce a product and the product is continuously supplied to the culture medium, but include, for example, conditions under which the cell cycle progresses. Conditions under which the cell cycle progresses include, for example, an ambient temperature of 15°C or higher in the culture vessel 111 and porous membrane 112, or a temperature of 15°C or higher in the culture medium. The ambient temperature or the temperature of the culture medium is preferably higher than 20°C, 25°C, 30°C, or 35°C. Furthermore, from the viewpoint of suppressing cell death, the ambient temperature of the culture vessel 111 and porous membrane 112 or the temperature of the culture medium is preferably 60°C or lower, 50°C or lower, or 40°C or lower. The temperature of the culture medium may be measured directly, or may be estimated from the ambient temperatures of the culture vessel 111 and porous membrane 112. Based on the measured temperature of the culture medium, the temperature of the culture medium may be directly controlled by a temperature control device, or the ambient temperature of the culture vessel 111 and porous membrane 112 may be controlled by a temperature control device. Conditions under which the cell cycle progresses may be, for example, conditions under which an enzyme such as a cyclin-dependent kinase is activated in the cells, or conditions under which the cell cycle progresses may be the absence of a cell cycle inhibitor in the culture medium.

[0109] The cells cultured in the culture tank 111 are preferably the same as the cells used in the filtration condition evaluation method, but may be different from the cells used in the filtration condition evaluation method. Also, the product of the cells filtered through the porous membrane 112 is preferably the same as the product of the cells filtered in the filtration condition evaluation method, but may be different from the product of the cells filtered in the filtration condition evaluation method.

[0110] Between the culture tank 111 and the porous membrane 112, there are arranged a flow path 113 for sending the culture solution in the culture tank 111 to the porous membrane 112, and a flow path 114 for returning the culture solution that has passed through the porous membrane 112 without being filtered by the porous membrane 112 to the culture tank 111. The culture solution flowing through the flow path 113 may contain cells and cell products. The culture solution flowing through the flow path 114, which passes through the hollow portion of the porous membrane 112 without passing through the pores and has not been filtered by the porous membrane 112, may contain cells and cell products. In addition, a flow path 115 for recovering the culture solution filtered by the porous membrane 112 is connected to the porous membrane 112. The culture solution flowing through the flow path 115, which has passed through the pores of the porous membrane 112 and been filtered by the porous membrane 112, may contain cell products. The culture solution filtered by the porous membrane 112 is recovered, for example, in a container 201. The container 201 may be aseptically connected to the flow path 115. The flow path 115, through which the culture solution filtered through the porous membrane 112 flows, may be directly connected to, for example, a column used for the next purification step. The culture solution filtered through the porous membrane 112 is not returned to the culture tank 111.

[0111] The flow path 113 may be provided with, for example, a pump 123 for sending the culture solution in the culture tank 111 to the porous membrane 112. The flow path 113 may be provided with a pressure gauge 133 for measuring the pressure of the culture solution supplied to the porous membrane 112. The flow path 113 may be provided with a flow meter for measuring at least one of the flow velocity and flow rate of the culture solution flowing in the flow path 113. The flow path 113 may be provided with a thermometer for measuring the temperature of the culture solution flowing in the flow path 113. The flow path 113 may be provided with a sampling unit for sampling the culture solution flowing in the flow path 113. The sampling unit is closed except during sampling.

[0112] The flow path 114 may be provided with a pump for sending the culture solution that has passed through the hollow portion without passing through the pores of the porous membrane 112 and has not been filtered by the porous membrane 112 to the culture tank 111. Note that pumps may be provided in both the flow path 113 and the flow path 114, or in either one. The flow path 114 may be provided with a pressure gauge 134 that measures the pressure of the culture solution that has passed through the porous membrane 112. The flow path 114 may be provided with a flow meter that measures at least one of the flow rate and flow rate of the culture solution flowing through the flow path 114. The flow path 114 may be provided with a thermometer that measures the temperature of the culture solution flowing through the flow path 114. The flow path 114 may be provided with a sampling unit for sampling the culture solution flowing through the flow path 114. The sampling unit is closed except during sampling.

[0113] The flow path 115 is provided with, for example, a pump 125 for sending the culture solution filtered through the porous membrane 112. The flow path 115 may be provided with a pressure gauge 135 for measuring the pressure of the culture solution filtered through the porous membrane 112. The flow path 115 may be provided with a flow meter for measuring at least one of the flow velocity and flow rate of the culture solution flowing through the flow path 115. The flow path 115 may be provided with a sampling unit for sampling the culture solution flowing through the flow path 115. The sampling unit is closed except when sampling is being performed.

[0114] The culture vessel 111 and the porous membrane 112 form at least a part of a path through which the culture medium circulates. The flow paths 113 and 114 also form at least a part of a path through which the culture medium circulates.

[0115] A flow path 116 for supplying a culture medium to the culture tank 111 may be connected to the culture tank 111. The flow path 116 is connected to, for example, a culture medium tank 216 that stores the culture medium. The flow path 116 is provided with, for example, a pump 126 for sending the culture medium to the culture tank 111. For example, the pumps 125 and 126 are controlled so that the amount of culture medium that is filtered through the porous membrane 112 and does not return to the culture tank 111 is the same as the amount of culture medium supplied to the culture tank 111. This control may be performed by observing the liquid level using a liquid level sensor (level sensor) installed in the culture tank 111 and maintaining a constant liquid level, or by measuring the weight of the entire culture tank 111 containing the culture medium and maintaining a constant weight.

[0116] A flow path 117 for supplying air containing carbon dioxide to the culture tank 111 may be connected to the culture tank 111. The flow path 117 is connected to, for example, a container 217 that stores air containing carbon dioxide. In addition, a flow path 118 for supplying oxygen to the culture tank 111 may be connected to the culture tank 111. The flow path 118 is connected to, for example, a container 218 that stores oxygen.

[0117] A flow path 119 for discharging at least a portion of the cells in the culture tank 111 may be connected to the culture tank 111. For example, by discharging at least a portion of the cells in the culture tank 111 using the flow path 119, the cell density in the culture solution in the culture tank 111 is maintained constant. This prevents an increase in cell density, which could lead to a shortage of oxygen and culture solution components in the culture solution, or an increase in the concentration of impurities. Discharging at least a portion of the cells in the culture tank 111 is called bleeding.

[0118] The culture tank 111 may be connected to a thermometer for measuring the temperature of the culture solution in the culture tank 111, a DO meter for measuring DO (dissolved oxygen), or a pH meter for measuring pH.

[0119] According to the method for producing a cell product of this embodiment, the cell product is filtered using filtration conditions that have been acquired in advance, so that the cell product can be obtained efficiently.

[0120] Example 1 A monoclonal antibody-producing Chinese hamster ovary (CHO) cell line (ATCC CRL-12445) was thawed after selecting cells that had been adapted to serum-free medium and suspended, and then frozen. The cells were placed in a 125 mL Erlenmeyer flask containing 10 mL of serum-free medium having the composition shown in Table 1, and the cells and medium were mixed in the Erlenmeyer flask. The cell count was confirmed using a viable cell autoanalyzer (Vi-CELL XR, Beckman), and the cell density was determined to be 3.5 x 10 ５ The cells were diluted with the medium to 100 cells / mL. Then, the cells were incubated in an incubator at 37°C and 5% CO ２ The cells were cultured with shaking in an atmosphere for 4 days.

[0121] Four days after thawing the cells, 3.5 × 10 ５ 50 mL of culture medium containing cells at a density of 1000 cells / mL was placed into two new 125 mL Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ The cells were cultured with shaking under atmospheric conditions for 3 days. After 3 days, 3.5 × 10 ５ 120 to 130 mL of culture medium containing cells at a density of 1000 cells / mL was placed into two new 250 mL Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ The cells were cultured with shaking under atmospheric conditions for 3 days. After 3 days, 3.5 × 10 ５ 500 mL of culture medium containing cells at a density of 1000 cells / mL was placed into three new 1 L Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ The cells were cultured with shaking in an atmosphere for 3 days.

[0122] A 12 L culture vessel that had been autoclaved in advance was charged with 5.5 x 10 ５ 6 L of culture medium containing cells at a density of 1000 cells / mL was aseptically placed in the wells. The medium was incubated at 37°C, 5% CO ２ The cells were cultured under stirring for 3 days while blowing oxygen into the atmosphere so that the DO did not fall below 70%. After 3 days, a serum-free medium having the composition shown in Table 2 was added to the culture vessel at a flow rate of 0.28 mL / min for 3 days.

[0123] Next, 1.5 x 10 ７ The culture solution containing cells at a density of 1000 cells / mL was aseptically collected, and 600 mL of the culture solution was transferred into a spinner flask as a culture vessel that had been pre-sterilized by autoclaving. ２ A mini-module of porous hollow fiber membranes (manufactured by Asahi Kasei Medical, BioOptimal MF-SL, blocking pore size 0.4 μm) was prepared, with the effective length adjusted to satisfy the following: A first opening in the hollow portion, which is the primary side of the porous hollow fiber membrane, was connected to the spinner flask via a first flow path, a second opening in the hollow portion of the porous hollow fiber membrane was connected to the spinner flask via a second flow path, and a third flow path was connected to the secondary side of the porous hollow fiber membrane and the spinner flask.

[0124] The ambient temperature of the spinner flask and the porous hollow fiber membrane was set to 4°C, and the culture solution was stirred in the spinner flask. The culture solution was sent from the spinner flask to the porous hollow fiber membrane via the first flow path using a magnetic levitation centrifugal pump (Levitronix PuraLevi i30SU), and tangential flow filtration was performed. The culture solution that passed through the hollow part of the porous hollow fiber membrane without being filtered by the porous hollow fiber membrane was returned to the spinner flask via the second flow path. The culture solution filtered by the porous hollow fiber membrane was returned to the spinner flask via the third flow path. The first and third flow paths had a structure that allowed sampling of the culture solution inside.

[0125] Single-use pressure gauges (PREPS-N-000 or PREPS-N-012, manufactured by PendoTECH) were installed on the primary and secondary sides of the porous hollow fiber membrane, and the transmembrane pressure (TMP) was measured over time. The viscosity of the culture medium was measured with an EMS viscometer (EMS-1000S). The amount of culture medium transferred from the spinner flask to the porous hollow fiber membrane was determined when the shear stress on the inner surface of the porous hollow fiber membrane was 4.58 N / m. ２ The filtration flow rate in the porous hollow fiber membrane was set to 5 μL / min by a pump so as to be constant (1 LMH).

[0126] The culture medium circulating between the spinner flask and the porous hollow fiber membrane was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant. The culture medium was circulated at a flow rate of 300 L / m using BioOptimal MF-SL. ２ At the time of filtration, the permeability of the monoclonal antibody through BioOptimal MF-SL was 78.8%, and the transmembrane pressure was 43.4 kPa.

[0127] (Example 2) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 1 was carried out, except that a porous hollow fiber membrane (Microza UMP, manufactured by Asahi Kasei, with a blocking pore size of 0.2 μm) was used as a mini-module of porous hollow fiber membranes with an effective length adjusted to 300 L / m. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant. ２ At the time of filtration, the permeability of the monoclonal antibody through MICROZA UMP was 80.2%, and the transmembrane pressure was 81.0 kPa.

[0128] (Example 3) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 1 was carried out, except that a porous hollow fiber membrane with a blocking pore size of 0.65 μm, manufactured by Asahi Kasei Corporation, was used as a mini-module of porous hollow fiber membranes with an effective length adjusted to 300 L / m. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant. ２ At the time of filtration, the permeability of the monoclonal antibody through MICROZA UJP was 99.2%, and the transmembrane pressure was 1.3 kPa.

[0129] (Example 4) The membrane area of ​​the liquid-contacting part is 3 cm ２The same method as in Example 1 was carried out, except that a porous hollow fiber membrane manufactured by Repligen with a blocking pore size of 0.2 μm was used as a mini-module of porous hollow fiber membranes with an effective length adjusted to 300 L / m. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant. ２ At the time of filtration, the permeability of the monoclonal antibody through the hollow fiber membrane manufactured by Repligen was 86.4%, and the transmembrane pressure difference was 1.0 kPa.

[0130] (Example 5) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 1 was carried out, except that a porous hollow fiber membrane manufactured by Cytiva and having a blocking pore size of 0.45 μm was used as a mini-module of porous hollow fiber membranes whose effective length was adjusted to 300 L / m. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant. ２ At the time of filtration, the permeability of the monoclonal antibody through the hollow fiber manufactured by Cytiva was 80.6%, and the transmembrane pressure difference was 2.0 kPa.

[0131] Example 6 A monoclonal antibody-producing Chinese hamster ovary (CHO) cell line (ATCC CRL-12445) was thawed after selecting cells that had been adapted to serum-free medium and suspended, and then frozen. The cells were placed in a 125 mL Erlenmeyer flask containing 10 mL of serum-free medium having the composition shown in Table 3, and the cells and medium were mixed in the Erlenmeyer flask. The cell count was confirmed using a viable cell autoanalyzer (Vi-CELL XR, Beckman), and the cell density was determined to be 3.5 x 10 ５ The cells were diluted with the medium to 100 cells / mL. Then, the cells were incubated in an incubator at 37°C and 5% CO ２ The cells were cultured with shaking in an atmosphere for 4 days.

[0132] Four days after thawing the cells, 3.5 × 10 ５50 mL of culture medium containing cells at a density of 1000 cells / mL was placed into two new 125 mL Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ The cells were cultured with shaking under atmospheric conditions for 3 days. After 3 days, 3.5 × 10 ５ 120 to 130 mL of culture medium containing cells at a density of 1000 cells / mL was placed into two new 250 mL Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ The cells were cultured with shaking under atmospheric conditions for 3 days. After 3 days, 3.5 × 10 ５ 500 mL of culture medium containing cells at a density of 1000 cells / mL was placed into three new 1 L Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ The cells were cultured with shaking in an atmosphere for 3 days.

[0133] A 12 L culture vessel that had been autoclaved in advance was charged with 5.5 x 10 ５ 5 L of culture medium containing cells at a density of 1000 cells / mL was aseptically placed in the wells. The medium was maintained at 37°C, 5% CO ２ The cells were cultured under stirring for 3 days while blowing oxygen into the atmosphere so that the DO did not fall below 70%. After 3 days, continuous culture was initiated, involving filtration through a porous hollow fiber membrane (Asahi Kasei Medical, BioOptimal MF-SL0190, pore size 0.4 μm) and the addition of culture medium to the culture tank.

[0134] The same amount of fresh medium as the amount of culture solution extracted from the culture tank by filtration was aseptically transferred from a 50 L bag (Thermo Fisher, Productainer BioProcess Container (BPC), 50 L) to the culture tank, and the amount of culture solution in the culture tank was controlled to be constant. The culture medium exchange rate was 1 vvd －１ (vessel volumes per day), and when the glucose concentration became 1 g / L or less or the glutamine concentration became 1 mmol / L or less, the medium exchange rate of the culture solution was reduced to 0.5 vvd －１ minutes.

[0135] After 7 days from the start of continuous culture, 1 × 10 ８The culture solution containing cells at a density of 1000 cells / mL was aseptically collected, and 600 mL of the culture solution was transferred into a spinner flask as a culture vessel that had been pre-sterilized by autoclaving. ２ A mini-module of porous hollow fiber membranes (manufactured by Asahi Kasei Medical, BioOptimal MF-SL, blocking pore size 0.4 μm) was prepared, with the effective length adjusted to satisfy the following: A first opening in the hollow portion, which is the primary side of the porous hollow fiber membrane, was connected to the spinner flask via a first flow path, a second opening in the hollow portion of the porous hollow fiber membrane was connected to the spinner flask via a second flow path, and a third flow path was connected to the secondary side of the porous hollow fiber membrane and the spinner flask.

[0136] The ambient temperature of the spinner flask and the porous hollow fiber membrane was set to 4°C, and the culture solution was stirred in the spinner flask. The culture solution was sent from the spinner flask to the porous hollow fiber membrane via the first flow path using a magnetic levitation centrifugal pump (Levitronix PuraLevi i30SU), and tangential flow filtration was performed. The culture solution that passed through the hollow part of the porous hollow fiber membrane without being filtered by the porous hollow fiber membrane was returned to the spinner flask via the second flow path. The culture solution filtered by the porous hollow fiber membrane was returned to the spinner flask via the third flow path. The first and third flow paths had a structure that allowed sampling of the culture solution inside.

[0137] Single-use pressure gauges (PREPS-N-000 or PREPS-N-012, manufactured by PendoTECH) were installed on the primary and secondary sides of the porous hollow fiber membrane, and the transmembrane pressure (TMP) was measured over time. The viscosity of the culture medium was measured with an EMS viscometer (EMS-1000S). The amount of culture medium transferred from the spinner flask to the porous hollow fiber membrane was determined when the shear stress on the inner surface of the porous hollow fiber membrane was 1.69 N / m. ２ The filtration flow rate in the porous hollow fiber membrane was set to 5 μL / min by a pump so as to be constant (1 LMH).

[0138] The culture medium circulating between the spinner flask and the porous hollow fiber membrane was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant.

[0139] (Example 7) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 6 was carried out, except that a Microza UMP porous hollow fiber membrane manufactured by Asahi Kasei Corporation, with a blocking pore size of 0.2 μm, was used as a mini-module of porous hollow fiber membranes whose effective length was adjusted to be 1 / 2 μm. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant.

[0140] (Example 8) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 6 was carried out, except that a Microza UJP porous hollow fiber membrane manufactured by Asahi Kasei Corporation, with a blocking pore size of 0.65 μm, was used as the mini-module of porous hollow fiber membranes whose effective length was adjusted to be 1 / 2. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant.

[0141] (Example 9) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 6 was carried out, except that a porous hollow fiber membrane manufactured by Repligen and having a blocking pore size of 0.2 μm was used as the mini-module of porous hollow fiber membranes whose effective length was adjusted so that the effective length was 1 / 2 μm. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant.

[0142] (Example 10) The membrane area of ​​the liquid-contacting part is 3 cm ２ The same method as in Example 6 was carried out, except that a porous hollow fiber membrane manufactured by Cytiva and having a blocking pore size of 0.45 μm was used as the mini-module of porous hollow fiber membranes whose effective length was adjusted so that the effective length was 0.45 μm. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium were all approximately constant.

[0143] (Comparative Example 1) 3.5 × 10 ５ 120 to 130 mL of culture medium containing cells at a density of 1000 cells / mL was placed into two new 250 mL Erlenmeyer flasks and incubated in an incubator at 37°C, 5% CO ２ After the cells were cultured under shaking in an atmosphere for 3 days, 5.5 × 10 cells were added to a 3 L culture vessel that had been autoclaved in advance. ５ 1.28 L of culture medium containing cells at a density of 1.28 cells / mL was aseptically added. The cells were incubated at 37°C, 5% CO ２ The cells were cultured under stirring for 3 days while blowing oxygen into the atmosphere so that the DO did not fall below 70%. After 3 days, the membrane area in contact with the liquid was 200 cm ２ Filtration was carried out using a module of porous hollow fiber membranes (manufactured by Asahi Kasei Medical, BioOptimal MF-SL, blocking pore size 0.4 μm) with the number and effective length adjusted so that the filtration rate was 100%. Continuous cultivation was then started, involving the addition of the culture medium to the culture tank.

[0144] The culture tank and a first opening in the hollow portion of the porous hollow fiber membrane, which is the primary side, were connected by a first flow path, and a second opening in the hollow portion of the porous hollow fiber membrane was connected to the culture tank by a second flow path. The culture solution filtered through the porous hollow fiber membrane was not returned to the culture tank. The culture tank was also provided with an outlet for sampling the culture medium inside and a supply port for supplying fresh culture medium.

[0145] The same amount of fresh medium as the amount of culture solution extracted from the culture tank by filtration was aseptically transferred from a 20 L bag (Thermo Fisher, Productainer BioProcess Container (BPC), 20 L) to the culture tank, and the amount of culture solution in the culture tank was controlled to be constant. The culture medium exchange rate was 1 vvd －１ (vessel volumes per day), and when the glucose concentration became 1 g / L or less or the glutamine concentration became 1 mmol / L or less, the medium exchange rate of the culture solution was changed to 0.375 vvd －１ minutes.

[0146] The temperature of the culture solution in the culture tank was controlled at 37 ° C, and the culture solution was stirred in the culture tank. The culture solution was sent from the culture tank to the porous hollow fiber membrane via the first flow path using a magnetic levitation centrifugal pump (Levitronix PuraLev 200MU), and tangential flow filtration was performed. The culture solution that passed through the hollow part of the porous hollow fiber membrane without being filtered by the porous hollow fiber membrane was returned to the culture tank via the second flow path. When the DO of the culture solution fell below 70%, oxygen was introduced into the culture solution in the culture tank using a sparger. In addition, air containing carbon dioxide at a concentration of 5% was constantly introduced into the culture tank.

[0147] Single-use pressure gauges (PREPS-N-038, manufactured by PendoTECH) were installed on the primary and secondary sides of the porous hollow fiber membrane, and the transmembrane pressure (TMP) was measured over time. The amount of culture medium sent from the culture tank to the porous hollow fiber membrane was adjusted so that the shear stress on the inner surface of the porous hollow fiber membrane was 2.10 N / m ２ The filtration flow rate through the porous hollow fiber membrane was increased stepwise by a pump to 1.0 LMH, 1.5 LMH, 2.0 LMH, 2.5 LMH, and 3.0 LMH, so that the amount of fresh medium introduced was increased to adjust the glucose and glutamine concentrations as the cells increased. In addition, when the cell density in the culture medium in the culture tank reached 7.5 x 10 ７ Furthermore, a liquid level sensor was placed in the culture vessel, and fresh medium was introduced into the culture vessel to keep the liquid level constant.

[0148] The culture medium in the culture tank was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As a result, as shown in Figure 9, the cell density in the culture medium continued to increase until bleeding began. As shown in Figure 10, the cell viability remained approximately constant. As shown in Figure 11, the antibody concentration tended to increase.

[0149] (Comparative Example 2) The membrane area of ​​the liquid-contacting portion is 200 cm ２The same method as in Comparative Example 1 was carried out, except that a porous hollow fiber membrane having a blocking pore diameter of 0.2 μm, manufactured by Asahi Kasei Corporation, Microza UMP, was used as the porous hollow fiber membrane module, the number and effective length of which were adjusted so as to satisfy the following.

[0150] The culture medium in the culture tank was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As a result, as shown in Figure 9, the cell density in the culture medium continued to increase until bleeding began. As shown in Figure 10, the cell viability remained approximately constant. As shown in Figure 11, the antibody concentration tended to increase.

[0151] (Reference Examples 1 to 3) 1.5 × 10 ７ The culture medium containing cells at a density of 1000 cells / mL was aseptically collected, and 600 mL of the culture medium was transferred into a spinner flask as a culture vessel that had been pre-sterilized by autoclaving.

[0152] The ambient temperature of the spinner flask was set to 4°C, and the culture medium was stirred in the spinner flask. The spinner flask was not connected to a hollow fiber membrane, and cells were cultured solely in the spinner flask. The culture medium in the spinner flask was sampled once or twice daily, and the cell density, cell viability, and antibody concentration in the culture medium were measured. Experiments were conducted under the same conditions except that the ambient temperature was set to room temperature (RT), and experiments were also conducted under the same conditions except that the ambient temperature was set to 37°C. As shown in Figure 12, the cell density tended to increase at room temperature. As shown in Figure 13, the cell viability tended to decrease at room temperature and 37°C. As shown in Figure 14, the antibody concentration tended to increase at room temperature and 37°C.

[0153] (Reference Examples 4 to 6) 1.5 × 10 ７ The culture medium containing cells at a density of 1000 cells / mL was aseptically collected, and 600 mL of the culture medium was transferred into a spinner flask as a culture vessel that had been pre-sterilized by autoclaving.

[0154] The ambient temperature of the spinner flask was set to 8°C, and the culture medium was stirred in the spinner flask. The hollow fiber membrane was not connected to the spinner flask, and cells were cultured only in the spinner flask. The culture medium in the spinner flask was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. Experiments were conducted under the same conditions except that the ambient temperature was set to 12°C, and under the same conditions except that the ambient temperature was set to 37°C. As shown in Figure 15, cell density tended to decrease at 37°C. As shown in Figure 16, cell viability tended to decrease at 37°C.

[0155] (Reference Examples 7 and 8) 1.5 × 10 ７ The culture medium containing cells at a density of 1000 cells / mL was aseptically collected, and 600 mL of the culture medium was transferred into a spinner flask as a culture vessel that had been pre-sterilized by autoclaving.

[0156] The ambient temperature of the spinner flask was set to 4°C, and the culture medium was stirred in the spinner flask. The spinner flask was not connected to a hollow fiber membrane, and cells were cultured solely in the spinner flask. The culture medium in the spinner flask was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. Experiments were also conducted under the same conditions, except that the culture medium was circulated using a pump. A magnetically levitated centrifugal pump (Levitronix PuraLevi i30SU) delivered the culture medium from the spinner flask to the pump via the first flow path, and the culture medium that passed through the pump was returned to the spinner flask via the second flow path. The pump delivered the culture medium at 50 mL / min. The first flow path was designed to allow sampling of the culture medium inside. The culture medium in the spinner flask was sampled from the first flow path once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As a result, as shown in Figures 17 to 19, the change in the culture solution over time was suppressed whether or not the culture solution was circulated by a pump.

[0157] (Analysis Method) The analysis methods used in the above-mentioned Examples, Comparative Examples, and Reference Examples are described below. The density and viability of cells contained in the culture medium were measured using a viable cell autoanalyzer (Vi-CELL XR, manufactured by Beckman Coulter). Samples were diluted with PBS(-) (Fujifilm Wako) and 600 μL was used for analysis. The image analysis method used was "CHO" in the Vi-CELL XR, and the settings for Minimum diameter (μm), Cell brightness (%), and Viable cell spot brightness (%) were changed appropriately to suit the actual situation.

[0158] HPLC measurement of antibody concentration was performed using the following method. (1) Detector: UV spectrophotometer (measurement wavelength: 280 nm) (2) Column: POROS G 20 μm Column, 4.6 × 50 mm, 0.8 mL (ThermoFisher) (3) Column temperature: Room temperature (4) Mobile phase. Mobile phase A: 7.098 g of disodium hydrogen phosphate (anhydrous) and 8.766 g of sodium chloride were dissolved in 800 mL of water, and 1 mol / L hydrochloric acid was added to adjust the pH to 7.0, followed by water to make a total volume of 1000 mL. Mobile phase B: 12 ​​mL of 1 mol / L hydrochloric acid and 8.766 g of sodium chloride were dissolved in water to make a total volume of 1000 mL. (5) Mobile phase delivery: Mobile phase A and mobile phase B were delivered at a flow rate of 2 mL / min, with the ratios of mobile phase A and mobile phase B varied as shown in Table 4 below.

[0159] The cell culture was centrifuged at 300 × g for 2 minutes, and the supernatant was sampled. Nine serial dilutions of commercially available human immunoglobulin G (Japan Blood Products Organization, Donated Blood Venoglobulin IH 5% Intravenous Injection 2.5 g / 50 mL) and the cell culture supernatant were then transferred using the same procedure. A standard curve was created using nine peak areas of human immunoglobulin G, and the antibody concentration in each solution was calculated from the standard curve and the peak areas of the samples.

[0160] The shear stress SS due to the culture medium on the liquid-contacting surface of the hollow fiber membrane is given by the product of the viscosity VC (Pa·s) of the culture medium flowing through the hollow portion of the hollow fiber membrane and the shear rate SV ( / s), as shown in the following formula: SS = VC × SV

[0161] The shear rate can be calculated from the linear velocity and the flow path diameter, and the linear velocity can be obtained from the pump output. Therefore, the shear stress was calculated based on the viscosity of the culture medium and the shear rate calculated from the linear velocity.

[0162] 11, 111... culture tank, 12, 112... hollow fiber membrane, 13, 14, 15, 113, 114, 115, 116, 117, 118, 119... flow path, 16... stirring device, 23, 25, 123, 125, 126... pump, 33, 34, 35, 133, 134, 135... pressure gauge, 50... temperature control tank, 201... container, 216... culture medium tank, 217... container, 218... container

Claims

1. Under conditions in which cell proliferation is suppressed, a culture solution containing cells is sent from a culture tank containing the culture solution to a porous membrane, and the culture solution that passes through the porous membrane without being filtered is returned to the culture tank, thereby circulating the culture solution between the culture tank and the porous membrane; evaluating one or more filtration conditions in the porous membrane; Including, Methods for evaluating filtration conditions.

2. 2. The method for evaluating filtration conditions according to claim 1, wherein the culture solution that has passed through the porous membrane without being filtered and the culture solution that has been filtered through the porous membrane are returned to the culture tank in returning the culture solution that has passed through the porous membrane without being filtered to the culture tank.

3. further comprising evaluating the filtration performance of the porous membrane under the one or more filtration conditions; The method for evaluating filtration conditions according to claim 1 , further comprising evaluating the one or more filtration conditions for the porous membrane based on the filtration performance of the porous membrane.

4. The method for evaluating filtration conditions according to claim 1 , wherein the conditions under which cell proliferation is inhibited are conditions under which progression of the cell cycle is inhibited.

5. The method for evaluating filtration conditions according to claim 1 , wherein the condition under which cell proliferation is inhibited is at least one of an atmospheric temperature and a culture solution temperature of 15° C. or lower.

6. The method for evaluating filtration conditions according to claim 5 , wherein at least one of the atmospheric temperature and the culture solution temperature of 15° C. or less is at least one of the atmospheric temperature and the culture solution temperature of 10° C. or less or 5° C. or less.

7. The method for evaluating filtration conditions according to claim 1 , wherein the conditions under which cell proliferation is inhibited are conditions under which an enzyme activity of the cells is inhibited.

8. The method for evaluating filtration conditions according to claim 7 , wherein the enzyme is a cyclin-dependent kinase.

9. The method for evaluating filtration conditions according to claim 1 , wherein the condition under which cell proliferation is suppressed is the presence of a cell cycle inhibitor in the culture medium.

10. The method for evaluating filtration conditions according to claim 1 , further comprising evaluating one or more filtration conditions in the porous membrane based on the permeability of the cell product through the porous membrane.

11. The method for evaluating filtration conditions according to claim 1 , further comprising evaluating one or more filtration conditions in the porous membrane based on a permeation flux in the porous membrane.

12. The method for evaluating filtration conditions according to claim 1 , further comprising evaluating one or more filtration conditions in the porous membrane based on a transmembrane pressure difference in the porous membrane.

13. The method for evaluating filtration conditions according to claim 1 , further comprising evaluating one or more filtration conditions in the porous membrane based on the turbidity of the filtrate.

14. The method for evaluating filtration conditions according to claim 1 , wherein the one or more filtration conditions are one or more conditions of the structure, material, or physical properties of the porous membrane.

15. The method for evaluating filtration conditions according to claim 1 , wherein the one or more filtration conditions are one or more conditions for the culture solution.

16. The method for evaluating filtration conditions according to claim 1 , wherein the one or more filtration conditions are one or more conditions regarding the density of the cells in the culture medium.

17. The method for evaluating filtration conditions according to claim 1 , wherein the one or more filtration conditions are one or more conditions of a flow rate of the culture solution fed to the porous membrane.

18. The method for evaluating filtration conditions according to claim 1 , wherein the one or more filtration conditions are one or more conditions regarding a flow rate of the culture solution filtered through the porous membrane.

19. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions of shear stress at the liquid-contacting surface of the porous membrane due to a flow of culture solution sent to the porous membrane.

20. The method for evaluating filtration conditions according to claim 1 , wherein a path through which the culture solution circulates, including the culture tank and the porous membrane, is disposed in a temperature controlled tank.

21. The method for evaluating filtration conditions according to claim 1, wherein the culture solution is circulated between the culture tank and the porous membrane without any active operation for maintaining the composition of the culture solution.

22. 2. The method for evaluating filtration conditions according to claim 1, wherein the culture medium is not added from the outside to a path through which the culture medium circulates, the path including the culture tank and the porous membrane, in circulating the culture medium between the culture tank and the porous membrane.

23. The method for evaluating filtration conditions according to claim 1, wherein at least one of dissolved oxygen and pH of the culture solution is not controlled when the culture solution is circulated between the culture tank and the porous membrane.

24. 2. The method for evaluating filtration conditions according to claim 1, wherein the culture solution is circulated between the culture tank and the porous membrane, and the cells are not bled from a path through which the culture solution circulates, including the culture tank and the porous membrane.

25. 2. The method for evaluating filtration conditions according to claim 1, further comprising collecting the culture solution for sampling from a path through which the culture solution circulates, the path including the culture tank and the porous membrane, in circulating the culture solution between the culture tank and the porous membrane.

26. The method for evaluating filtration conditions according to claim 25, further comprising measuring the density of the cells in the sampled culture medium.

27. The method for evaluating filtration conditions according to claim 25, further comprising measuring the viability of the cells in the sampled culture medium.

28. The method for evaluating filtration conditions according to claim 25, further comprising measuring a concentration of a product of the cells in the sampled culture medium.

29. The method for evaluating filtration conditions according to claim 25, further comprising measuring the turbidity of the culture medium sampled.

30. The method for evaluating filtration conditions according to claim 1 , wherein the porous membrane is a hollow fiber membrane.

31. The method for evaluating filtration conditions according to claim 1 , wherein the porous membrane is a microfiltration membrane.

32. The method includes: under conditions for cell growth, using filtration conditions, sending a culture solution containing cells from a culture tank containing the culture solution to a porous membrane; returning the culture solution that has passed through the porous membrane without being filtered back to the culture tank; recovering the culture solution containing the product of the cells that has been filtered through the porous membrane; and circulating at least a portion of the culture solution between the culture tank and the porous membrane; The filtration conditions are obtained by feeding the culture solution containing the cells from the culture tank containing the culture solution to the porous membrane under conditions in which cell growth is suppressed, returning the culture solution that has passed through the porous membrane without being filtered to the culture tank, and circulating the culture solution between the culture tank and the porous membrane, and evaluating one or more filtration conditions for the porous membrane. Methods for producing cell products.

33. 33. The method for producing a cell product according to claim 32, wherein when evaluating one or more filtration conditions for the porous membrane, the culture solution that passes through the porous membrane without being filtered is returned to the culture tank, and the culture solution that passes through the porous membrane without being filtered and the culture solution that is filtered through the porous membrane are returned to the culture tank.

34. The method for producing a cell product according to claim 32, wherein the conditions for cell proliferation are conditions for cell cycle progression.

35. The method for producing a cell product according to claim 32, wherein the conditions for the cells to grow are at least one of an atmospheric temperature and a culture solution temperature of 15°C or higher.

36. The method for producing a cell product described in claim 35, wherein at least one of the atmospheric temperature and culture solution temperature of 15°C or higher is at least one of the atmospheric temperature and culture solution temperature higher than 20°C, 25°C, 30°C, or 35°C.

37. The method for producing a cell product according to claim 32, wherein the conditions under which the cells grow are conditions under which enzymes in the cells are activated.

38. 38. The method for producing a cell product of claim 37, wherein the enzyme is a cyclin-dependent kinase.

39. 33. The method of claim 32, wherein the conditions under which the cells grow are the absence of cell cycle inhibitors in the culture medium.

40. The method for producing a cell product according to claim 32, wherein the conditions under which cell proliferation is inhibited are conditions under which progression of the cell cycle is inhibited.

41. The method for producing a cell product according to claim 32, wherein the condition for inhibiting cell proliferation is at least one of an atmospheric temperature and a culture solution temperature of 15°C or lower.

42. The method for producing a cell product described in claim 41, wherein at least one of the atmospheric temperature and culture solution temperature of 15°C or less is at least one of the atmospheric temperature and culture solution temperature of 10°C or less or 5°C or less.

43. The method for producing a cell product according to claim 32, wherein the conditions under which the proliferation of the cells is inhibited are conditions under which the enzymatic activity of the cells is inhibited.

44. 44. The method of claim 43, wherein the enzyme is a cyclin-dependent kinase.

45. The method for producing a cell product according to claim 32, wherein the condition under which cell proliferation is inhibited is the presence of a cell cycle inhibitor in the culture medium.

46. 33. The method of claim 32, wherein one or more filtration conditions for the porous membrane are evaluated based on the permeability of the product of the cells through the porous membrane.

47. The method of claim 32, wherein one or more filtration conditions for the porous membrane are evaluated based on the permeation flux through the porous membrane.

48. The method of claim 32, wherein one or more filtration conditions for the porous membrane are evaluated based on the transmembrane pressure across the porous membrane.

49. The method of claim 32, wherein one or more filtration conditions for the porous membrane are evaluated based on filtrate turbidity.

50. The method for producing a cell product described in claim 32, wherein the one or more filtration conditions are one or more conditions of the structure, material, or physical properties of the porous membrane.

51. 33. The method of claim 32, wherein the one or more filtration conditions are one or more conditions of the culture medium.

52. The method for producing a cell product of claim 32, wherein the one or more filtration conditions are one or more conditions of density of the cells in the culture medium.

53. The method for producing a cell product described in claim 32, wherein the one or more filtration conditions are one or more conditions of the flow rate of the culture medium fed to the porous membrane.

54. The method for producing a cell product described in claim 32, wherein the one or more filtration conditions are one or more conditions of flow rate of the culture solution filtered through the porous membrane.

55. The method for producing a cell product described in claim 32, wherein the one or more filtration conditions are one or more conditions of shear stress at the liquid-contacting surface of the porous membrane due to the flow of the culture medium sent to the porous membrane.

56. The method for producing a cell product described in claim 32, wherein, when evaluating one or more filtration conditions for the porous membrane, the culture tank and the path through which the culture solution containing the porous membrane circulates are arranged in a temperature-controlled tank.

57. The method for producing a cell product according to claim 32, wherein when evaluating one or more filtration conditions for the porous membrane, no active operation is performed to maintain the composition of the culture solution when circulating the culture solution between the culture tank and the porous membrane.

58. The method for producing a cell product according to claim 32, wherein when evaluating one or more filtration conditions for the porous membrane, the culture medium is circulated between the culture tank and the porous membrane without adding a medium from the outside to a path through which the culture medium circulates.

59. 33. The method for producing a cell product according to claim 32, wherein when evaluating one or more filtration conditions for the porous membrane, at least one of dissolved oxygen and pH of the culture solution is not controlled when circulating the culture solution between the culture tank and the porous membrane.

60. 33. The method for producing a cell product according to claim 32, wherein when one or more filtration conditions for the porous membrane are evaluated, the culture solution is circulated between the culture tank and the porous membrane, and the cells are not bled from a path through which the culture solution circulates, including the culture tank and the porous membrane.

61. 33. The method for producing a cell product according to claim 32, wherein when evaluating one or more filtration conditions in the porous membrane, the culture solution is circulated between the culture tank and the porous membrane, and the culture solution is collected for sampling from a path through which the culture solution circulates, the path including the culture tank and the porous membrane.

62. The method for producing a cell product of claim 61, further comprising measuring the density of the cells in the sampled culture medium.

63. The method for producing a cell product of claim 61, further comprising measuring the viability of the cells in the sampled culture medium.

64. 62. The method of claim 61, further comprising measuring the concentration of a product of the cells in the sampled culture medium.

65. The method for producing a cell product of claim 61, further comprising measuring the turbidity of the culture medium in the sampled culture medium.

66. The method for producing a cell product according to claim 32, wherein the porous membrane is a hollow fiber membrane.

67. The method for producing a cell product of claim 32, wherein the porous membrane is a microfiltration membrane.

68. 33. The method of claim 32, wherein the cells are in perfusion culture.