Method for evaluating filtration conditions and method for producing cell product

Abstract

The present invention pertains to a method for evaluating filtration conditions, the method comprising the steps of: feeding a culture solution to a porous membrane (12) from a culture tank (11) containing the culture solution containing cells under conditions that inhibit cell proliferation, returning the culture solution, which has passed through without being filtered by the porous membrane (12), to the culture tank (11), and circulating the culture solution between the culture tank (11) and the porous membrane (12); and evaluating one or more filtration conditions of the porous membrane (12).

Description

Technical Field

[0001] The present invention relates to a filtration technique, an evaluation method for filtration conditions, and a method for manufacturing cell products. Background Art

[0002] Cell culture technology is an essential technology for manufacturing various biopharmaceuticals such as antibodies, growth hormones, and insulin, and has made a great contribution to recent medical progress. Among biopharmaceuticals, antibody drugs have attracted particular attention. Efficiently and stably producing monoclonal antibodies by culturing antibody-producing cells is an important industrial issue.

[0003] Industrial cell culture methods for producing useful cell products such as antibodies are roughly divided into two types: adherent culture method and suspension culture (floating culture) method. In the adherent culture method, cells adhere to the inner surface of the culture tank. In suspension culture, cells float in the culture medium. Among these, the suspension culture method has become the mainstream because of its ease of large-scale production and easy control at a large scale.

[0004] In the suspension culture method, in order to culture cells in large quantities and at high density and continuously produce cell products efficiently, a method of culturing cells has been proposed in which fresh culture medium is supplied to the culture tank at a certain rate while filtering the old culture medium containing cell products and discharging it out of the culture tank at a certain rate. This type of culture is generally referred to as continuous culture or perfusion culture (for example, refer to Patent Documents 1 to 5). In continuous culture, control can be carried out in such a way that the amount of culture medium supplied to the culture tank is the same as the amount of culture medium discharged from the culture tank. In continuous culture, it is important to effectively separate cells in the culture medium from the old culture medium and cell products for a long time, take out the old culture medium and cell products outside the culture tank, and continuously maintain the cell growth environment in the culture tank under optimal conditions for a long time.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2018-76291

[0008] Patent Document 2: Japanese Unexamined Patent Application Publication No. 2009-45019

[0009] Patent Document 3: Japanese Unexamined Patent Application Publication No. 2021-48776

[0010] Patent Document 4: Japanese Patent No. 5696479

[0011] Patent Document 5: Japanese Patent Application Laid-Open No. 2022-516516 Summary of the Invention

[0012] Problems to be Solved by the Invention

[0013] In continuous cultivation, a porous membrane is used to separate cells in the culture solution of a culture tank from the old culture solution and the products of the cells. A method for appropriately evaluating the filtration conditions of the porous membrane is required. Therefore, one of the subjects of the present invention is to provide an evaluation method for filtration conditions capable of appropriately evaluating the filtration conditions of a cell culture solution and a method for producing cell products.

[0014] Means for Solving the Problem

[0015] [1] According to an aspect of the present invention, there is provided an evaluation method for filtration conditions, comprising the following steps: under conditions that suppress cell proliferation, a culture solution is transported from a culture tank containing the culture solution containing cells to a porous membrane, and the culture solution that has passed through without being filtered by the porous membrane is returned to the culture tank to circulate the culture solution between the culture tank and the porous membrane; and, one or more filtration conditions of the porous membrane are evaluated.

[0016] [2] In the evaluation method for filtration conditions of [1] above, in the process of returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, the culture solution that has passed through without being filtered by the porous membrane and the culture solution that has been filtered by the porous membrane can be returned to the culture tank.

[0017] [3] The evaluation method for filtration conditions of [1] or [2] above may be: further comprising a step of evaluating the filtration performance of the porous membrane under one or more filtration conditions, and evaluating one or more filtration conditions of the porous membrane based on the filtration performance of the porous membrane.

[0018] [4] In the evaluation method for filtration conditions according to any one of [1] to [3] above, the conditions for suppressing cell proliferation may be conditions for suppressing the progression of the cell cycle.

[0019] [5] In the evaluation method for filtration conditions according to any one of [1] to [4] above, the conditions for suppressing cell proliferation may be at least any one of an atmosphere temperature of 15°C or lower and a culture solution temperature.

[0020] [6] In the evaluation method for filtration conditions according to any one of [1] to [5] above, at least any one of an atmosphere temperature of 15°C or lower and a culture solution temperature may be at least any one of an atmosphere temperature of 10°C or lower and a culture solution temperature of 5°C or lower.

[0021] [7] In the evaluation method for filtration conditions according to any one of [1] to [6] above, the conditions for suppressing cell proliferation may be conditions for suppressing the enzyme activity of cells.

[0022] [8] In the evaluation method for filtration conditions of [7] above, the enzyme may be a cyclin-dependent kinase.

[0023] [9] In the method for evaluating the filtration conditions described in any one of [1] to [8] above, the condition for inhibiting cell proliferation may be the presence of a cell cycle inhibitor in the culture medium.

[0024]

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

[0025]

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

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

[0026]

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

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

[0027]

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

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

[0028]

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

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

[0029]

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

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

[0030]

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

[15] above, one or more filtration conditions may be one or more conditions of the density of the cells in the culture medium.

[0031]

[17] In the method for evaluating the filtration conditions described in any one of [1] to

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

[0032]

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

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

[0033]

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

[18] above, one or more filtration conditions may be one or more conditions of the shear stress on the liquid contact surface of the porous membrane generated by the flow of the culture medium delivered to the porous membrane.

[0034]

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

[19] above, the path of the culture medium circulation including the culture tank and the porous membrane can be arranged in the temperature control tank.

[0035]

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

[20] above, in the step of circulating the culture medium between the culture tank and the porous membrane, an active operation for maintaining the composition of the culture medium may not be performed.

[0036]

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

[21] above, in the step of circulating the culture medium between the culture tank and the porous membrane, a culture medium may not be added from the outside to the path of the culture medium circulation including the culture tank and the porous membrane.

[0037]

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

[22] above, in the step of circulating the culture medium between the culture tank and the porous membrane, at least either the dissolved oxygen or the pH of the culture medium may not be controlled.

[0038]

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

[23] above, in the step of circulating the culture medium between the culture tank and the porous membrane, cells may not be discharged (bled) from the path of the culture medium circulation including the culture tank and the porous membrane.

[0039]

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

[24] above, in the step of circulating the culture medium between the culture tank and the porous membrane, the culture medium may be collected for sampling from the path of the culture medium circulation including the culture tank and the porous membrane.

[0040]

[26] The method for evaluating the filtration conditions of

[25] above may further include the following step: measuring the density of cells in the sampled culture medium.

[0041]

[27] The method for evaluating the filtration conditions of

[25] or

[26] above may further include the following step: measuring the survival rate of cells in the sampled culture medium.

[0042]

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

[25] to

[27] above may further include the following step: measuring the concentration of the product of cells in the sampled culture medium.

[0043]

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

[25] to

[28] above may further include the following step: measuring the turbidity of the sampled culture medium.

[0044]

[30] In the method for evaluating the filtration conditions described in any one of [1] to

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

[0045]

[31] In the method for evaluating the filtration conditions described in any one of [1] to

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

[0046]

[32] According to the method of the present invention, there is provided a method for producing a cell product, which includes the following steps: under the conditions for cell proliferation, using the filtration conditions to transfer the culture solution from the culture tank containing the culture solution with cells to the porous membrane, returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, recovering the culture solution containing the cell product that has been filtered by the porous membrane, and circulating at least a part of the culture solution between the culture tank and the porous membrane, wherein the filtration conditions are the filtration conditions obtained as follows: under the conditions for inhibiting cell proliferation, transferring the culture solution from the culture tank containing the culture solution with cells to the porous membrane, returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, circulating the culture solution between the culture tank and the porous membrane, and evaluating one or more filtration conditions of the porous membrane to obtain the filtration conditions.

[0047]

[33] In the method for producing a cell product described in

[32] above, when evaluating one or more filtration conditions of the porous membrane, in the step of returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, the culture solution that has passed through without being filtered by the porous membrane and the culture solution that has been filtered by the porous membrane may be returned to the culture tank.

[0048]

[34] In the method for producing a cell product described in

[32] or

[33] above, the conditions for cell proliferation may be the conditions for promoting the cell cycle.

[0049]

[35] In the method for producing a cell product described in any one of

[32] to

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

[0050]

[36] In the method for producing a cell product described in

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

[0051]

[37] In the method for producing a cell product described in any one of

[32] to

[36] above, the conditions for cell proliferation may be the conditions under which the enzymes of the cells have been activated.

[0052]

[38] In the method for producing a cell product described in

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

[0053]

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

[32] to

[38] above, the condition for cell proliferation can be that there is no cell cycle inhibitor in the above culture solution.

[0054]

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

[32] to

[39] above, the condition for inhibiting cell proliferation can be the condition for inhibiting the progression of the cell cycle.

[0055]

[41] In the method for manufacturing the cell product according to any one of

[32] to

[40] above, the condition for inhibiting cell proliferation can be at least any one of an atmosphere temperature of 15°C or lower and a culture solution temperature of 15°C or lower.

[0056]

[42] In the method for manufacturing the cell product according to

[41] above, at least any one of an atmosphere temperature of 15°C or lower and a culture solution temperature of 15°C or lower can be at least any one of an atmosphere temperature of 10°C or lower and a culture solution temperature of 5°C or lower.

[0057]

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

[32] to

[42] above, the condition for inhibiting cell proliferation can be the condition for inhibiting the enzyme activity of the above cells.

[0058]

[44] In the method for manufacturing the cell product according to

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

[0059]

[45] In the method for manufacturing the cell product according to any one of

[32] to

[44] above, the condition for inhibiting cell proliferation can be that there is a cell cycle inhibitor in the culture solution.

[0060]

[46] In the method for manufacturing the cell product according to any one of

[32] to

[45] above, one or more filtration conditions of the porous membrane can be evaluated based on the permeation rate of the cell product through the porous membrane.

[0061]

[47] In the method for manufacturing the cell product according to any one of

[32] to

[46] above, one or more filtration conditions of the porous membrane can be evaluated based on the permeation flux of the porous membrane.

[0062]

[48] In the method for manufacturing the cell product according to any one of

[32] to

[47] above, one or more filtration conditions of the porous membrane can be evaluated based on the transmembrane pressure difference of the porous membrane.

[0063]

[49] In the method for manufacturing the cell product according to any one of

[32] to

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

[0064]

[50] In the method for manufacturing a cell product according to any one of

[32] to

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

[0065]

[51] In the method for manufacturing a cell product according to any one of

[32] to

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

[0066]

[52] In the method for manufacturing a cell product according to any one of

[32] to

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

[0067]

[53] In the method for manufacturing a cell product according to any one of

[32] to

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

[0068]

[54] In the method for manufacturing a cell product according to any one of

[32] to

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

[0069]

[55] In the method for manufacturing a cell product according to any one of

[32] to

[54] above, one or more filtration conditions may be one or more conditions of the shear stress on the liquid contact surface of the porous membrane generated by the flow of the culture medium delivered to the porous membrane.

[0070]

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

[32] to

[55] above, when evaluating one or more filtration conditions of the porous membrane, the path of the circulating culture medium, including the culture tank and the porous membrane, may be arranged in a temperature control tank.

[0071]

[57] In the method for manufacturing a cell product according to any one of

[32] to

[56] above, when evaluating multiple filtration conditions of the porous membrane, in the step of circulating the culture medium between the culture tank and the porous membrane, active operations for maintaining the composition of the culture medium may not be performed.

[0072]

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

[32] to

[57] above, when evaluating multiple filtration conditions of the porous membrane, in the step of circulating the culture medium between the culture tank and the porous membrane, culture medium may not be added from the outside to the path of the circulating culture medium, including the culture tank and the porous membrane.

[0073]

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

[32] to

[58] above, when evaluating a plurality of filtration conditions of the porous membrane, in the step of circulating the culture solution between the culture tank and the porous membrane, at least either the dissolved oxygen or the pH of the culture solution may not be controlled.

[0074]

[60] In the method for manufacturing a cell product according to any one of

[32] to

[59] above, when evaluating a plurality of filtration conditions of the porous membrane, in the step of circulating the culture solution between the culture tank and the porous membrane, cells may not be discharged from the path of the circulating culture solution including the culture tank and the porous membrane.

[0075]

[61] In the method for manufacturing a cell product according to any one of

[32] to

[60] above, when evaluating one or more filtration conditions of the porous membrane, in the step of circulating the culture solution between the culture tank and the porous membrane, the culture solution may be collected for sampling from the path of the circulating culture solution including the culture tank and the porous membrane.

[0076]

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

[61] above may further include the following step: measuring the density of cells in the sampled culture solution.

[0077]

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

[61] or

[62] above may further include the following step: measuring the viability of cells in the sampled culture solution.

[0078]

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

[61] to

[63] above may further include the following step: measuring the concentration of the product of cells in the sampled culture solution.

[0079]

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

[61] to

[64] above may further include the following step: measuring the turbidity of the sampled culture solution.

[0080]

[66] In the method for manufacturing a cell product according to any one of

[32] to

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

[0081]

[67] In the method for manufacturing a cell product according to any one of

[32] to

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

[0082]

[68] In the method for manufacturing a cell product according to any one of

[32] to

[67] above, perfusion culture of cells is performed.

[0083] Effects of the Invention

[0084] According to the present invention, an evaluation method for filtration conditions capable of appropriately evaluating the filtration conditions of a cell culture solution and a method for producing a cell product can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 It is a schematic diagram of an evaluation system for the filtration conditions of a hollow fiber membrane showing an embodiment.

[0086] Figure 2 It is a schematic diagram of a production system for a cell product showing an embodiment.

[0087] Figure 3 It is a graph showing the time change of the cell density in the culture solution circulated between the culture tank and the hollow fiber membrane in an example.

[0088] Figure 4 It is a graph showing the time change of the cell density in the culture solution circulated between the culture tank and the hollow fiber membrane in an example.

[0089] Figure 5 It is a graph showing the time change of the cell viability in the culture solution circulated between the culture tank and the hollow fiber membrane in an example.

[0090] Figure 6 It is a graph showing the time change of the cell viability in the culture solution circulated between the culture tank and the hollow fiber membrane in an example.

[0091] Figure 7 It is a graph showing the time change of the antibody concentration in the culture solution circulated between the culture tank and the hollow fiber membrane in an example.

[0092] Figure 8 It is a graph showing the time change of the antibody concentration in the culture solution circulated between the culture tank and the hollow fiber membrane in an example.

[0093] Figure 9 It is a graph showing the time change of the cell density in the culture solution circulated between the culture tank and the hollow fiber membrane in a comparative example.

[0094] Figure 10 It is a graph showing the time change of the cell viability in the culture solution circulated between the culture tank and the hollow fiber membrane in a comparative example.

[0095] Figure 11 It is a graph showing the time change of the antibody concentration in the culture solution circulated between the culture tank and the hollow fiber membrane in a comparative example.

[0096] Figure 12 It is a graph showing the time change of the cell density in the culture solution in the culture tank in a reference example.

[0097] Figure 13 A graph showing the time change of the survival rate of cells in the culture solution in the culture tank of the reference example.

[0098] Figure 14 A graph showing the time change of the antibody concentration in the culture solution in the culture tank of the reference example.

[0099] Figure 15 A graph showing the time change of the cell density in the culture solution in the culture tank of the reference example.

[0100] Figure 16 A graph showing the time change of the survival rate of cells in the culture solution in the culture tank of the reference example.

[0101] Figure 17 A graph showing the time change of the antibody concentration in the culture solution in the culture tank of the reference example.

[0102] Figure 18 A graph showing the time change of the cell density in the culture solution in the culture tank of the reference example.

[0103] Figure 19 A graph showing the time change of the survival rate of cells in the culture solution in the culture tank of the reference example.

[0104] Figure 20 A graph showing the time change of the antibody concentration in the culture solution in the culture tank of the reference example. Detailed implementation mode

[0105] The following is a detailed description of the mode for implementing the present invention (hereinafter referred to as "the present embodiment"). It should be noted that the present embodiment is for the purpose of making the present invention easy to understand and is not used for a restrictive interpretation of the present invention. The present invention is not limited by the present embodiment and can be implemented with various modifications within its gist.

[0106] Refer to Figure 1, the evaluation method of the filtration conditions of the present embodiment includes the following steps: Under conditions that inhibit cell proliferation, the culture solution containing cells is transported from the culture tank 11 accommodating the culture solution to the porous membrane 12, and the culture solution that has passed through without being filtered by the porous membrane 12 is returned to the culture tank 11 to circulate the culture solution between the culture tank 11 and the porous membrane 12; evaluate the filtration performance of the porous membrane 12 under one or more filtration conditions; and evaluate one or more filtration conditions of the porous membrane 12 based on the filtration performance of the porous membrane 12. Regarding returning the culture solution that has passed through without being filtered by the porous membrane 12 to the culture tank 11, the culture solution that has passed through without being filtered by the porous membrane 12 and the culture solution that has been filtered by the porous membrane 12 can be returned to the culture tank 11. Among them, as long as a culture medium equal to the amount of the recovered culture solution is supplemented into the culture solution in the culture tank 11, it does not prevent the recovery of the culture solution without returning the total amount of the culture solution to the culture tank 11. The recovery of the culture solution can be carried out without returning all or part of the culture solution that has been filtered by the porous membrane 12 to the culture tank 11. The amount of the recovered culture solution is not limited, and the change in the density of the cells and the product concentration contained in the culture solution in the culture tank 11 can be inhibited by supplementing an equal amount of the culture medium into the system. It should be noted that this recovery is different from the sampling described later for the purpose of analyzing the culture solution.

[0107] The culture tank 11 is, for example, a container having an internal space isolated from the outside gas. Among them, the culture tank 11 may be provided with a ventilation port and a filter for keeping the internal pressure constant. The ventilation port and the filter preferably have a pore diameter of 0.2 μm or less to prevent external bacteria from entering the inside of the culture tank 11. The culture tank 11 may be provided with a stirring device 16 for stirring the culture solution in the culture tank 11. Suspension culture of cells is carried out in the culture solution in the culture tank 11.

[0108] The cells cultured in the culture tank 11 are not particularly limited. The cells can be derived from animals including humans and can be derived from microorganisms. The cells can be eukaryotic cells or prokaryotic cells. Examples of animals include mammals, reptiles, birds, amphibians, fish, and insects. The cells can be genetically recombinant 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.

[0109] The cells produce products that can be used as drugs, for example, and release them into the culture solution. Examples of products that can be used as drugs include peptides, proteins, and viruses (including virus-like particles). Examples of proteins include antibodies, hormones, cytokines, growth factors, enzymes, and plasma proteins. The proteins can be recombinant proteins.

[0110] The antibody can be a monoclonal antibody or a polyclonal antibody. The antibody can be a human antibody or an antibody protein derived from mammals such as cows and mice other than humans. Alternatively, the antibody can be a chimeric antibody protein and a humanized antibody with human IgG. A chimeric antibody with human IgG is an antibody in which the variable region is derived from organisms other than humans such as mice and the remaining constant region is replaced with an immunoglobulin derived from humans. In addition, a humanized antibody refers to an antibody in which the complementarity-determining region (CDR) in the variable region is derived from organisms other than humans and the remaining framework region (FR) is derived from humans. Humanization further reduces immunogenicity compared to chimeric antibodies.

[0111] The shape of the porous membrane 12 is not particularly limited. Examples of the porous membrane include a hollow fiber membrane, a flat membrane, and a tubular membrane. Hereinafter, an example in which the porous membrane 12 is a hollow fiber membrane will be described. The surface of the porous membrane 12 to which the culture solution is supplied is referred to as the primary side of the hollow fiber membrane. In addition, the surface through which the permeate that has passed through 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 peripheral surface, the inner peripheral surface of the hollow fiber membrane becomes the primary side, and the outer peripheral surface of the hollow fiber membrane becomes the secondary side. In an embodiment in which the culture solution to be filtered is supplied to the outer peripheral surface, the outer peripheral surface of the hollow fiber membrane becomes the primary side, and the inner peripheral surface of the hollow fiber membrane becomes the secondary side.

[0112] The type of the porous membrane 12 is not particularly limited. Examples of the porous membrane include a coarse filter membrane, a microfiltration membrane, an ultrafiltration membrane, a dialysis membrane, a nanofiltration membrane, a reverse osmosis membrane, and a forward osmosis membrane.

[0113] The filtration method of the porous membrane 12 can be a tangential flow filtration (TFF) method. The tangential flow filtration method is a filtration method in which the culture solution flows along a direction parallel to the surface of the primary side of the hollow fiber membrane on the surface of the primary side of the hollow fiber membrane. The tangential flow filtration method includes an alternating tangential flow filtration (ATF) method. In the present embodiment, when simply referred to as the tangential flow filtration (TFF) method, it sometimes refers to a filtration method in which the culture solution flows unidirectionally on the surface of the primary side of the hollow fiber membrane. The alternating tangential flow filtration (ATF) method is a filtration method in which the culture solution flows back and forth on the surface of the primary side of the hollow fiber membrane.

[0114] Figure 1In the example shown, a flow path 13 for delivering 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 an optional flow path 15 for returning the culture solution that has been filtered by the porous membrane 12 to the culture tank 11 are arranged between the culture tank 11 and the porous membrane 12. The culture solution flowing in the flow path 13 may contain cells and cell products. The culture solution flowing in the flow path 14 that has passed through the porous membrane 12 without being filtered may contain cells and cell products. The culture solution flowing in the flow path 15 that has been filtered by the porous membrane 12 may contain cell products.

[0115] The flow path 13 is provided with, for example, a pump 23 for delivering the culture solution in the culture tank 11 to the porous membrane 12. Examples of the pump include a diaphragm pump, a tube pump, a centrifugal pump, and a rotary pump, but are not limited to these. 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 velocity and the 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 section for sampling the culture solution flowing in the flow path 13. The sampling section is closed except during sampling.

[0116] The flow path 14 may be provided with a pump for delivering the culture solution that has not passed through the pore portion of the porous membrane 12 but has passed through the hollow portion and has not been filtered by the porous membrane 12 to the culture tank 11. It should be noted that the pump may be provided in both the flow path 13 and the flow path 14, or may be provided in either one of them. The flow path 14 may be provided with a pressure gauge 34 for measuring 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 for measuring at least one of the flow velocity and the flow rate of the culture solution flowing in the flow path 14. A thermometer for measuring the temperature of the culture solution flowing in the flow path 14 may be provided in the flow path 14. The flow path 14 may be provided with a sampling section for sampling the culture solution flowing in the flow path 14. The sampling section is closed except during sampling.

[0117] The flow path 15 is provided with, for example, a pump 25 for delivering the culture solution that has passed through the pore portion of the porous membrane 12 and has been filtered by the porous membrane 12 to the culture tank 11. The flow path 15 is provided with a pressure gauge 35 for measuring 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 for measuring at least one of the flow velocity and the flow rate of the culture solution flowing in the flow path 15. The flow path 15 may be provided with a sampling section for sampling the culture solution flowing in the flow path 15. The sampling section is closed except during sampling.

[0118] The culture tank 11 and the porous membrane 12 constitute at least a part of the path for circulating the culture solution. In addition, the flow paths 13, 14, and 15 constitute at least a part of the path for circulating the culture solution. The path for circulating the culture solution can be closed from the outside, or as described above, an air vent and a filter for keeping the internal pressure constant can be provided in the culture tank 11.

[0119] The culture tank 11 and the porous membrane 12 can be arranged in a temperature management tank 50 that manages the internal temperature. The flow paths 13, 14, and 15 can be arranged in the temperature management tank 50. The temperature management tank 50 manages the ambient temperature of the culture tank 11, the porous membrane 12, and the flow paths 13, 14, and 15. The temperature management tank 50 can be a constant temperature tank, a refrigerator, or a booth, room, or building that maintains a constant temperature.

[0120] The conditions for inhibiting cell proliferation are not particularly limited. For example, they are conditions for inhibiting the progression of the cell cycle. Conditions for inhibiting the progression of the cell cycle are, for example, a low ambient temperature of 15°C or lower in the culture tank 11 and the porous membrane 12 or a temperature of 15°C or lower in the culture solution. The ambient temperature or the temperature of the culture solution is preferably 10°C or lower or 5°C or lower. It should be noted that from the viewpoint of inhibiting cell death, the ambient temperature of the culture tank 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. For the temperature of the culture solution, the temperature of the culture solution can be directly measured, or it can be inferred and measured from the ambient temperature of the culture tank 11 and the porous membrane 12. The temperature of the culture solution can be directly controlled by a temperature control device based on the measured temperature of the culture solution, or the ambient temperature of the culture tank 11 and the porous membrane 12 can be controlled by a temperature control device. Conditions for inhibiting the progression of the cell cycle can be, for example, conditions for inhibiting the enzyme activity of cyclin-dependent kinases in cells. Conditions for inhibiting the progression of the cell cycle can be the presence of cell cycle inhibitors in the culture solution. Under the conditions for inhibiting cell proliferation, the activity of the cells decreases, the progression of the cell cycle is inhibited, cell division is inhibited, and product production is inhibited.

[0121] In the present embodiment, both the culture solution that has passed through without being filtered by the porous membrane 12 and the culture solution that has been filtered by the porous membrane 12 can be returned to the culture tank 11. In this case, the total amount of cells and the total amount of products in the system are maintained even under the conditions for inhibiting cell proliferation, so that fluctuations in the density of cells and the concentration of products contained in the culture solution circulating between the culture tank 11 and the porous membrane 12 can be suppressed.

[0122] Under conditions of inhibiting cell proliferation, the activity of cells decreases, and the amount of culture medium components required by the cells also decreases. Therefore, it is not necessary to add medium to the circulation path of the culture medium from the outside. In addition, by not adding medium, fluctuations in the density of cells and the concentration of products contained in the culture medium can be suppressed. Furthermore, under conditions of inhibiting cell proliferation, the activity of cells decreases, and fluctuations in the dissolved oxygen concentration (DO) and pH in the culture medium are suppressed. Therefore, it is not necessary to add gases such as oxygen and carbon dioxide to the circulation path of the culture medium from the outside to control the dissolved oxygen concentration and pH. In addition, furthermore, under conditions of inhibiting cell proliferation, the density of cells in the culture medium is almost constant. Therefore, it is not necessary to discharge cells from the circulation path of the culture medium to the outside. Therefore, according to the evaluation method of the filtration conditions of the present embodiment, the operations for cell culture can be simplified. Among them, in order to monitor, for example, the density of cells, the survival rate of cells, and the concentration of cell products, a part of the culture medium can also be sampled. Sampling is not for adjusting the density of cells, so it is different from discharging.

[0123] One or more filtration conditions of the porous membrane 12 can be evaluated based on the filtration performance of the porous membrane 12. The filtration performance of the porous membrane 12 can be represented, for example, by at least any one of the following: the level of the permeation rate of cell products through the porous membrane 12; the level of the interception rate of inclusions by the porous membrane 12; the level of the transmembrane pressure difference of the porous membrane 12; the amount of liquid that can be filtered until the transmembrane pressure difference of the porous membrane 12 rises; the amount of liquid that can be filtered until the permeation flux of the porous membrane 12 starts to decrease; the level of the pressure loss of the porous membrane 12; the magnitude of the flow rate of the porous membrane 12; the speed of the flow velocity of the porous membrane 12; and the speed of the permeation flux of the porous membrane 12.

[0124] One or more filtration conditions of the porous membrane 12 can be evaluated according to whether one or more filtration conditions based on the porous membrane 12 make the filtration performance of the porous membrane 12 meet a specified standard or are relatively excellent. Alternatively, at least a part of the filtration conditions can be extracted based on whether the filtration performance of the porous membrane 12 meets a specified standard or is relatively excellent among the multiple filtration conditions of the porous membrane 12.

[0125] If the density of cells and the concentration of products contained in the culture solution supplied to the porous membrane 12 vary, it may be difficult to accurately evaluate the filtration performance of the porous membrane 12. In contrast, as described above, this embodiment can suppress the proliferation of cells and suppress the variation in the density of cells and the concentration of products contained in the culture solution. If the culture solution filtered by the porous membrane 12 is returned to the culture tank 11, the variation in the density of cells and the concentration of products contained in the culture solution can be further suppressed. Therefore, according to this embodiment, it is possible to evaluate the filtration performance of the porous membrane 12 while keeping the density of cells and the concentration of products contained in the culture solution substantially constant. In addition, if the culture solution filtered by the porous membrane 12 is returned to the culture tank 11, the step of supplementing the culture medium to the culture tank 11 can be omitted.

[0126] One or more filtration conditions are, for example, one or more conditions of the structure of the porous membrane 12. Conditions of the structure of the porous membrane 12 are, for example, film thickness, inner diameter, outer diameter, aperture ratio of the membrane surface, porosity, pore diameter, average pore diameter, pore diameter deviation, rejection pore diameter, ratio of pore diameter to fiber diameter, anisotropy of the membrane surface, anisotropy of the membrane pores, membrane surface roughness, and change in pore diameter from the primary side to the secondary side. For example, a plurality of porous membranes 12 having various structures are prepared, and the filtration performance of each porous membrane 12 is evaluated. Further, it is evaluated whether one or more conditions of the structure of the porous membrane 12 can provide filtration performance that meets a specified standard or is relatively excellent.

[0127] One or more filtration conditions are, for example, one or more conditions of the material of the porous membrane 12. For example, a plurality of porous membranes 12 formed of various materials are prepared, and the filtration performance of each porous membrane 12 is evaluated. Further, it is evaluated whether one or more conditions of the material of the porous membrane 12 can provide filtration performance that meets a specified standard or is relatively excellent.

[0128] One or more filtration conditions are, for example, one or more conditions of the physical properties of the porous membrane 12. Conditions of the physical properties of the porous membrane 12 refer to, 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. Further, it is evaluated whether one or more conditions of the physical properties of the porous membrane 12 can provide filtration performance that meets a specified standard or is relatively excellent.

[0129] One or more filtration conditions are, for example, one or more conditions of the culture medium. Conditions of the culture medium refer to, for example, the components of the culture medium, the concentration of each component in the culture medium, the viscosity of the culture medium, the pH of the culture medium, the conductivity of the culture medium, and the turbidity of the culture medium. Examples of the components of the culture medium include the composition of the culture medium, an antifoaming agent, salts, nucleic acids such as DNA and RNA, host cell-derived proteins (HCP), lipids, and polysaccharides. For example, a plurality of culture media with different conditions are prepared, and the filtration performance of the porous membrane 12 when using each culture medium is evaluated. Further, it is evaluated whether one or more conditions of the culture medium can provide filtration performance that meets a specified standard or is relatively excellent.

[0130] One or more filtration conditions are, for example, a plurality of conditions of the cell density in the culture medium. For example, a plurality of culture media with different cell densities are prepared, and the filtration performance of the porous membrane 12 when using each culture medium is evaluated. Further, it is evaluated whether one or more conditions of the cell density can provide filtration performance that meets a specified standard or is relatively excellent.

[0131] One or more filtration conditions are, for example, one or more conditions of the flow rate of the culture medium delivered to the porous membrane 12. For example, the culture medium is delivered to the porous membrane 12 at different flow rates, and the filtration performance of the porous membrane 12 when using each flow rate is evaluated. Further, it is evaluated whether one or more conditions of the flow rate of the culture medium delivered to the porous membrane 12 can provide filtration performance that meets a specified standard or is relatively excellent. The flow rate can be expressed as a volumetric flow rate, a mass flow rate, a linear velocity, or a shear velocity on the surface of the porous membrane 12.

[0132] One or more filtration conditions are, for example, one or more conditions of the flow rate of the culture medium filtered by the porous membrane 12. For example, the culture medium filtered by the porous membrane 12 is fed at different flow rates, and the filtration performance of the porous membrane 12 when using each flow rate is evaluated. Further, it is evaluated whether one or more conditions of the flow rate of the culture medium filtered by the porous membrane 12 can provide filtration performance that meets a specified standard or is relatively excellent.

[0133] One or more filtration conditions are, for example, one or more conditions of the shear stress on the liquid contact surface of the porous membrane 12. The liquid contact surface refers to the surface of the porous membrane 12 that comes into contact with the liquid being filtered. When the porous membrane 12 is a hollow fiber membrane, it is the surface on the primary side. For example, the shear stress on the liquid contact surface of the porous membrane 12 is changed, and the filtration performance of the porous membrane 12 when using each shear stress is evaluated. Further, it is evaluated whether one or more conditions of the shear stress on the liquid contact surface of the porous membrane 12 can provide filtration performance that meets a specified standard or is relatively excellent.

[0134] The evaluation method of the filtration conditions according to the present embodiment can suppress fluctuations in the density of cells and the concentration of products contained in the culture solution circulated between the culture tank 11 and the porous membrane 12, and select one or more filtration conditions that enable the porous membrane 12 to exhibit filtration performance satisfying a specified standard.

[0135] Next, referring to Figure 2 , the method for producing a cell product according to the present embodiment includes the following steps: Under the conditions for cell proliferation, using the filtration conditions obtained by the above-described evaluation method of filtration conditions, the culture solution is transported from the culture tank 111 containing the culture solution containing cells to the porous membrane 112, the culture solution that has passed through without being filtered by the porous membrane 112 is returned to the culture tank 111, the culture solution containing the cell product that has been filtered by the porous membrane 112 is recovered, and at least a part of the culture solution is circulated between the culture tank 111 and the porous membrane 112.

[0136] The conditions for cell proliferation are not particularly limited as long as they are conditions under which cells produce products and continuously supply products to the culture solution, for example, conditions for promoting the cell cycle. Conditions for promoting the cell cycle are, for example, an atmosphere temperature of 15°C or higher in the culture tank 111 and the porous membrane 112 or a temperature of 15°C or higher of the culture solution. The atmosphere temperature or the temperature of the culture solution is preferably a temperature higher than 20°C, higher than 25°C, higher than 30°C, or higher than 35°C. In addition, from the viewpoint of suppressing cell death, the atmosphere temperature of the culture tank 111 and the porous membrane 112 or the temperature of the culture solution is preferably 60°C or lower, 50°C or lower, or 40°C or lower. For the temperature of the culture solution, the temperature of the culture solution can be directly measured, or it can be inferred and measured from the atmosphere temperature of the culture tank 111 and the porous membrane 112. Based on the measured temperature of the culture solution, the temperature of the culture solution can be directly controlled by a temperature control device, or the atmosphere temperature of the culture tank 111 and the porous membrane 112 can be controlled by a temperature control device. Conditions for promoting the cell cycle can be, for example, conditions under which enzymes such as cyclin-dependent kinases in cells have been activated. The conditions for promoting the cell cycle can be that there is no cell cycle inhibitor in the culture solution.

[0137] The cells cultured in the culture tank 111 are preferably the same as the cells used in the evaluation method of the filtration conditions, but they can also be different from the cells used in the evaluation method of the filtration conditions. In addition, the cell product filtered by the porous membrane 112 is preferably the same as the cell product filtered by the evaluation method of the filtration conditions, but it can also be different from the cell product filtered by the evaluation method of the filtration conditions.

[0138] A flow path 113 for delivering the culture solution in the culture tank 111 to the porous membrane 112 is disposed between the culture tank 111 and 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 in the flow path 113 may contain cells and cell products. The culture solution that has not been filtered by the porous membrane 112 and has passed through the hollow portion rather than through the pore portion of the porous membrane 112 and flows in the flow path 114 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 filtered by the porous membrane 112 and flowing in the flow path 115, which has passed through the pore portion of the porous membrane 112, may contain cell products. The culture solution filtered by the porous membrane 112 is, for example, recovered into the container 201. The container 201 may be aseptically connected to the flow path 115. The flow path 115 through which the culture solution filtered by the porous membrane 112 flows may be directly connected to a column for the following purification process, for example. The culture solution filtered by the porous membrane 112 does not return to the culture tank 111.

[0139] The flow path 113 is provided with a pump 123 for delivering the culture solution in the culture tank 111 to the porous membrane 112, for example. 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 any one of the flow rate and the flow volume 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 section for sampling the culture solution flowing in the flow path 113. The sampling section is closed except during sampling.

[0140] The flow path 114 may be provided with a pump for delivering the culture solution that has not passed through the pore portion of the porous membrane 112 but has passed through the hollow portion and has not been filtered by the porous membrane 112 to the culture tank 111. It should be noted that the pump may be provided in both the flow path 113 and the flow path 114, or may be provided in either one of them. The flow path 114 may be provided with a pressure gauge 134 for measuring 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 for measuring at least any one of the flow rate and the flow volume of the culture solution flowing in the flow path 114. The flow path 114 may be provided with a thermometer for measuring the temperature of the culture solution flowing in the flow path 114. The flow path 114 may be provided with a sampling section for sampling the culture solution flowing in the flow path 114. The sampling section is closed except during sampling.

[0141] The flow path 115 is provided with, for example, a pump 125 for transporting the culture solution filtered by 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 by the porous membrane 112. The flow path 115 may be provided with a flow meter for measuring at least either the flow velocity or the flow rate of the culture solution flowing in the flow path 115. The flow path 115 may be provided with a sampling section for sampling the culture solution flowing in the flow path 115. The sampling section is closed except during sampling.

[0142] The culture tank 111 and the porous membrane 112 constitute at least a part of the path for circulating the culture solution. In addition, the flow paths 113 and 114 constitute at least a part of the path for circulating the culture solution.

[0143] The culture tank 111 may be connected to a flow path 116 for supplying a culture solution to the culture tank 111. The flow path 116 is connected, for example, to a culture solution tank 216 that houses the culture solution. The flow path 116 is provided with, for example, a pump 126 for transporting the culture solution to the culture tank 111. For example, the pumps 125 and 126 are controlled such that the amount of the culture solution filtered by the porous membrane 112 and not returned to the culture tank 111 is the same as the amount of the culture solution supplied to the culture tank 111. This control can be performed as follows: observing the liquid level height using a liquid level sensor (level sensor) provided in the culture tank 111 and keeping the liquid level height fixed; or measuring the total weight of the culture tank 111 including the culture solution and keeping the weight fixed.

[0144] The culture tank 111 may be connected to a flow path 117 for supplying air containing carbon dioxide to the culture tank 111. The flow path 117 may be connected, for example, to a container 217 that houses air containing carbon dioxide. In addition, the culture tank 111 may be connected to a flow path 118 for supplying oxygen to the culture tank 111. The flow path 118 may be connected, for example, to a container 218 that houses oxygen.

[0145] The culture tank 111 may be connected to a flow path 119 for discharging at least a part of the cells in the culture tank 111. For example, by discharging at least a part of the cells in the culture tank 111 using the flow path 119, the density of the cells in the culture solution in the culture tank 111 can be kept constant. Thereby, an increase in the cell density can be suppressed, and a shortage of oxygen and culture solution components, or an increase in the impurity concentration in the culture solution can be prevented. The step of discharging at least a part of the cells in the culture tank 111 is called discharging.

[0146] 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.

[0147] In the method for producing a cell product according to this embodiment, since the cell product is filtered using the previously obtained filtration conditions, the cell product can be obtained efficiently.

[0148] (Example 1)

[0149] After thawing a Chinese hamster ovary (CHO) cell (ATCC CRL-12445) strain that produces monoclonal antibodies and has been acclimatized and suspended in a serum-free medium and then cryopreserved and screened, it was added to a 125 mL conical flask pre-dispensed with 10 mL of a serum-free medium having the composition shown in Table 1, and the cells and the medium were mixed in the conical flask. The cell count was confirmed using a viable cell / dead cell automated analyzer (Vi-CELL XR, Beckman), and the cells were diluted with the medium so that the cell density became 3.5×10 5 cells / mL. Thereafter, the cells were cultured with shaking for 4 days in an incubator at 37°C in a 5% CO 2 atmosphere.

[0150] [Table 1]

[0151]

[0152] On the 4th day after cell thawing, 50 mL of a culture solution containing cells at a density of 3.5×10 5 cells / mL was added to two new 125 mL conical flasks, and the cells were cultured with shaking for 3 days in an incubator at 37°C in a 5% CO 2 atmosphere. After 3 days, 120 to 130 mL of a culture solution containing cells at a density of 3.5×10 5 cells / mL was added to two new 250 mL conical flasks, and the cells were cultured with shaking for 3 days in an incubator at 37°C in a 5% CO 2 atmosphere. After 3 days, 500 mL of a culture solution containing cells at a density of 3.5×10 5 cells / mL was added to three new 1 L conical flasks, and the cells were cultured with shaking for 3 days in an incubator at 37°C in a 5% CO 2 atmosphere.

[0153] To a 12 L-capacity culture tank that had been autoclaved, 6 L of a culture solution containing cells at a density of 5.5×10 5 cells / mL was added by aseptic operation. While blowing in oxygen so that the DO was not less than 70%, the cells were cultured with stirring for 3 days at 37°C in a 5% CO 2 atmosphere. After 3 days, the operation of adding a serum-free medium having the composition shown in Table 2 to the culture tank was continuously performed at 0.28 mL / min for 3 days.

[0154] [Table 2]

[0155] Add raw materials for culture medium composition Manufacturer Amount contained in 1 L of culture medium <![CDATA[GIBCO TM CHO CD EfficientFeed TM A chemically Defined Feed Supplement]]> Thermo Fisher Scientific 450 mL <![CDATA[GIBCO TM CHO CD EfficientFeed TM B chemically Defined Feed Supplement]]> Thermo Fisher Scientific 450 mL <![CDATA[GIBCO TM L-Glutamine 200mM]]> Thermo Fisher Scientific 100 mL

[0156] Then, the culture solution containing cells at a density of 1.5×10 7 cells / mL was recovered by aseptic operation, and 600 mL of the culture solution was transferred to a rotating bottle pre-sterilized by autoclave and used as a culture tank. A mini-module of a porous hollow fiber membrane (manufactured by Asahi Kasei Medical Co., Ltd., BioOptimal MF-SL, exclusion pore size 0.4 μm) with an effective length adjusted so that the membrane area of the liquid contact part was 3 cm 2 was prepared. The rotating bottle and the first opening of the hollow part on the primary side of the porous hollow fiber membrane were connected by a first flow path, the second opening of the hollow part of the porous hollow fiber membrane and the rotating bottle were connected by a second flow path, and the secondary side of the porous hollow fiber membrane and the rotating bottle were connected by a third flow path.

[0157] The atmospheric temperature of the rotating bottle and the porous hollow fiber membrane was set at 4°C, and the culture solution in the rotating bottle was stirred. The culture solution was pumped from the rotating bottle to the porous hollow fiber membrane via the first flow path by a magnetic levitation centrifugal pump (PuraLev i30SU manufactured by Levitronix) for tangential flow filtration. 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 rotating bottle via the second flow path. The culture solution filtered by the porous hollow fiber membrane was returned to the rotating bottle via the third flow path. The first and third flow paths had structures capable of sampling the culture solution inside.

[0158] A disposable pressure gauge (PREPS-N-000 or PREPS-N-012 manufactured by PendoTECH) was provided on the primary side and the secondary side of the porous hollow fiber membrane, and the transmembrane pressure difference (TMP) was measured over time. The viscosity of the culture solution was measured with an EMS viscometer (EMS-1000S), and the feed rate of the culture solution from the rotating bottle to the porous hollow fiber membrane was set so that the shear stress on the inner surface of the porous hollow fiber membrane was 4.58 N / m 2 . The filtration flow rate of the porous hollow fiber membrane was set to 5 μL / min with a pump to achieve a constant value (1 LMH).

[0159] The culture solution circulating between the rotating bottle and the porous hollow fiber membrane was sampled once or twice a day, and the density of cells, the survival rate of cells, and the antibody concentration in the culture solution were measured. The results are as shown in Figure 3 , Figure 5 and Figure 7 . The density of cells, the survival rate of cells, and the antibody concentration in the culture solution were each approximately constant. The culture solution was filtered at 300 L / m with BioOptimal MF-SL 2The transmittance of the monoclonal antibody of BioOptimal MF-SL during filtration was 78.8%, and the transmembrane pressure difference was 43.4 kPa.

[0160] (Example 2)

[0161] As a mini-module of a porous hollow fiber membrane with an adjusted effective length to make the membrane area of the liquid contact part 3 cm 2 of, a Microza UMP manufactured by Asahi Kasei was used, and a porous hollow fiber membrane with an exclusion pore size of 0.2 μm was used. Otherwise, the same method as in Example 1 was carried out. As a result, as Figure 3 、 Figure 5 and Figure 7 shown, the density of cells in the culture medium, the survival rate of cells, and the antibody concentration were respectively approximately constant. When filtering the culture medium at 300 L / m with MICROZA UMP 2 the transmittance of the monoclonal antibody of MICROZAUMP was 80.2%, and the transmembrane pressure difference was 81.0 kPa.

[0162] (Example 3)

[0163] As a mini-module of a porous hollow fiber membrane with an adjusted effective length to make the membrane area of the liquid contact part 3 cm 2 of, a Microza UJP manufactured by Asahi Kasei was used, and a porous hollow fiber membrane with an exclusion pore size of 0.65 μm was used. Otherwise, the same method as in Example 1 was carried out. As a result, as Figure 3 、 Figure 5 and Figure 7 shown, the density of cells in the culture medium, the survival rate of cells, and the antibody concentration were respectively approximately constant. When filtering the culture medium at 300 L / m with MICROZA UJP 2 the transmittance of the monoclonal antibody of MICROZAUJP was 99.2%, and the transmembrane pressure difference was 1.3 kPa.

[0164] (Example 4)

[0165] As a mini-module of a porous hollow fiber membrane with an adjusted effective length to make the membrane area of the liquid contact part 3 cm 2 of, a product manufactured by Repligen was used, and a porous hollow fiber membrane with an exclusion pore size of 0.2 μm was used. Otherwise, the same method as in Example 1 was carried out. As a result, as Figure 3 、 Figure 5 and Figure 7 shown, the density of cells in the culture medium, the survival rate of cells, and the antibody concentration were respectively approximately constant. When filtering the culture medium at 300 L / m with the hollow fiber manufactured by Repligen 2The transmittance of monoclonal antibody through the hollow fiber made by Repligen during filtration was 86.4%, and the transmembrane pressure difference was 1.0 kPa.

[0166] (Example 5)

[0167] As a mini-module of a porous hollow fiber membrane with an adjusted effective length to make the membrane area of the liquid contact part 3 cm 2 of the porous hollow fiber membrane, a porous hollow fiber membrane made by Cytiva with an exclusion pore size of 0.45 μm was used, and the same method as in Example 1 was carried out except for this. As a result, as Figure 3 、 Figure 5 and Figure 7 shown, the density of cells, the survival rate of cells, and the antibody concentration in the culture medium were respectively approximately constant. When filtering the culture medium at 300 L / m 2 using the hollow fiber made by Cytiva, the transmittance of monoclonal antibody through the hollow fiber made by Cytiva was 80.6%, and the transmembrane pressure difference was 2.0 kPa.

[0168] (Example 6)

[0169] After thawing the Chinese hamster ovary (CHO) cell (ATCC CRL-12445) strain that produces monoclonal antibody and has been domesticated and suspended in a serum-free medium and then screened and frozen, it was added to a 125 mL conical flask pre-filled with 10 mL of a serum-free medium having the composition shown in Table 3, and the cells and the medium were mixed in the conical flask. The cell number was confirmed using a live / dead cell automatic analyzer (Vi-CELL XR, Beckman), and the cells were diluted with the medium so that the cell density became 3.5×10 5 cells / mL. After that, the cells were cultured with shaking for 4 days in an incubator at 37 °C and 5% CO 2 atmosphere.

[0170] [Table 3]

[0171]

[0172] On the 4th day after cell thawing, 50 mL of the culture medium containing cells at a density of 3.5×10 5 cells / mL was added to 2 new 125 mL conical flasks, and the cells were cultured with shaking for 3 days in an incubator at 37 °C and 5% CO 2 atmosphere. After 3 days, 120 to 130 mL of the culture medium containing cells at a density of 3.5×10 5 cells / mL was added to 2 new 250 mL conical flasks, and the cells were cultured with shaking for 3 days in an incubator at 37 °C and 5% CO 2The cells were cultured with shaking for 3 days under the atmosphere. After 3 days, 500 mL of the culture medium containing cells at a density of 3.5×10 5 cells / mL was added to 3 new 1-L conical flasks, and the cells were cultured with shaking for 3 days in an incubator at 37 °C under 5% CO 2 atmosphere.

[0173] To a 12-L capacity culture tank that had been autoclaved, 5 L of the culture medium containing cells at a density of 5.5×10 5 cells / mL was added aseptically. Under the atmosphere of 37 °C and 5% CO 2 , the cells were cultured with stirring for 3 days while blowing in oxygen in such a way that the DO was not less than 70%. After 3 days, continuous culture was started, accompanied by filtration using a porous hollow fiber membrane (manufactured by Asahi Kasei Medical Co., Ltd., BioOptimal MF-SL0190, exclusion pore size 0.4 μm) and addition of the culture medium to the culture tank.

[0174] An amount of fresh medium equal to the amount of the culture medium withdrawn from the culture tank by filtration was aseptically transferred from a 50-L bag (manufactured by ThermoFisher, Productainer BioProcess Container (BPC), 50 L) to the culture tank to control the amount of the culture medium in the culture tank to be constant. The medium exchange rate of the culture medium was set to 1 vvd -1 (vessel volumes per day), and when the glucose concentration reached below 1 g / L or the glutamine concentration reached below 1 mmol / L, the medium exchange rate of the culture medium was increased by 0.5 vvd -1 .

[0175] Seven days after the start of continuous culture, the culture medium containing cells at a density of 1×10 8 cells / mL was aseptically recovered, and 600 mL of the culture medium was transferred to a spinner flask that had been autoclaved and used as a culture tank. A mini-module of a porous hollow fiber membrane (manufactured by Asahi Kasei Medical Co., Ltd., BioOptimal MF-SL, exclusion pore size 0.4 μm) with the effective length adjusted so that the membrane area of the liquid contact part became 3 cm 2 was prepared. The spinner flask was connected to the first opening of the hollow part on the primary side of the porous hollow fiber membrane by the first flow path, the second opening of the hollow part of the porous hollow fiber membrane was connected to the spinner flask by the second flow path, and the secondary side of the porous hollow fiber membrane was connected to the spinner flask by the third flow path.

[0176] The atmosphere temperature of the rotating bottle and the porous hollow fiber membrane is set to 4°C, and the culture solution is stirred in the rotating bottle. The culture solution is transported from the rotating bottle to the porous hollow fiber membrane via the first flow path using a magnetic levitation centrifugal pump (PuraLev i30SU manufactured by Levitronix) for tangential flow filtration. The culture solution that passes through the hollow portion of the porous hollow fiber membrane without being filtered by the porous hollow fiber membrane is returned to the rotating bottle via the second flow path. The culture solution filtered by the porous hollow fiber membrane is returned to the rotating bottle via the third flow path. The first and third flow paths have a structure that can sample the internal culture solution.

[0177] Disposable pressure gauges (PendoTECH, PREPS-N-000 or PREPS-N-012) were installed on the primary and secondary sides of the porous hollow fiber membrane to measure the transmembrane pressure difference (TMP) over time. The viscosity of the culture solution was measured with an EMS viscometer (EMS-1000S) so that the shear stress on the inner surface of the porous hollow fiber membrane reached 1.69 N / m 2 The amount of culture solution delivered from the spinner bottle to the porous hollow fiber membrane was set in the manner of . The filtration flow rate of the porous hollow fiber membrane was set to 5 μL / min by a pump so as to be constant (1 LMH).

[0178] The culture medium circulating between the spinner flask and the porous hollow fiber membrane was sampled once or twice a day to measure the cell density, cell survival rate and antibody concentration in the culture medium. Figure 4 , Figure 6 and Figure 8 As shown, the cell density, cell survival rate, and antibody concentration in the culture medium were approximately constant.

[0179] (Example 7)

[0180] The effective length was adjusted so that the membrane area in contact with the liquid was 3 cm 2 The same method as in Example 6 was used except that the porous hollow fiber membrane mini module of Asahi Chemical Industry Co., Ltd. Microza UMP with a pore size of 0.2 μm was used. Figure 4 , Figure 6 and Figure 8 As shown, the cell density, cell survival rate, and antibody concentration in the culture medium were approximately constant.

[0181] (Example 8)

[0182] The effective length was adjusted so that the membrane area in contact with the liquid was 3 cm 2Miniature module of a porous hollow fiber membrane, using a porous hollow fiber membrane manufactured by Asahi Kasei, Microza UJP, with an exclusion pore size of 0.65 μm, and otherwise the same method as in Example 6 was carried out. As a result, as shown in Figure 4 , Figure 6 and Figure 8 shown, the density of cells, the survival rate of cells, and the antibody concentration in the culture medium were each approximately constant.

[0183] (Example 9)

[0184] As a miniature module of a porous hollow fiber membrane with an adjusted effective length to make the membrane area of the liquid contact part 3 cm 2 , using a porous hollow fiber membrane manufactured by Repligen, with an exclusion pore size of 0.2 μm, and otherwise the same method as in Example 6 was carried out. As a result, as shown in Figure 4 , Figure 6 and Figure 8 shown, the density of cells, the survival rate of cells, and the antibody concentration in the culture medium were each approximately constant.

[0185] (Example 10)

[0186] As a miniature module of a porous hollow fiber membrane with an adjusted effective length to make the membrane area of the liquid contact part 3 cm 2 , using a porous hollow fiber membrane manufactured by Cytiva, with an exclusion pore size of 0.45 μm, and otherwise the same method as in Example 6 was carried out. As a result, as shown in Figure 4 , Figure 6 and Figure 8 shown, the density of cells, the survival rate of cells, and the antibody concentration in the culture medium were each approximately constant.

[0187] (Comparative Example 1)

[0188] Similar to the example, 120 to 130 mL of a culture medium containing cells at a density of 3.5×10 5 cells / mL was added to 2 new 250 mL conical flasks, and the cells were cultured with shaking in an incubator at 37 °C and 5% CO 2 atmosphere for 3 days. Then, 1.28 L of a culture medium containing cells at a density of 5.5×10 5 cells / mL was added to a 3 L capacity culture tank that had been autoclaved in advance by aseptic operation. Under an atmosphere of 37 °C and 5% CO 2 , while blowing in oxygen so that the DO was not less than 70%, the cells were cultured with stirring for 3 days. After 3 days, start carrying out with the membrane area of the liquid contact part made to be 200 cm by adjusting the number of roots and the effective length 2Filtration of the module of the porous hollow fiber membrane (manufactured by Asahi Kasei Medical Co., Ltd., BioOptimal MF-SL, exclusion pore size 0.4 μm) and continuous culture with addition of the culture medium to the culture tank.

[0189] Connect the culture tank and the first opening of the hollow part on the primary side of the porous hollow fiber membrane with the first flow path, and connect the second opening of the hollow part of the porous hollow fiber membrane and the culture tank with the second flow path. Ensure that the culture medium filtered by the porous hollow fiber membrane does not return to the culture tank. In addition, the culture tank is provided with an outlet for sampling the internal culture medium and an inlet for supplying fresh culture medium.

[0190] Transfer an equal amount of fresh culture medium as the amount of the culture medium withdrawn from the culture tank by filtration from a 20 L bag (manufactured by Thermo Fisher, Productainer BioProcess Container (BPC), 20 L) to the culture tank by aseptic operation, and control it in such a way that the amount of the culture medium in the culture tank remains constant. Set the medium exchange rate of the culture medium to 1 vvd -1 (vessel volumes per day), and when the glucose concentration reaches 1 g / L or less or the glutamine concentration reaches 1 mmol / L or less, increase the medium exchange rate of the culture medium by 0.375 vvd -1 .

[0191] Control the temperature of the culture medium added to the culture tank at 37 °C, and stir the culture medium in the culture tank. Use a magnetic levitation centrifugal pump (PuraLev 200MU manufactured by Levitronix) to transfer the culture medium from the culture tank to the porous hollow fiber membrane via the first flow path for tangential flow filtration. The culture medium that passes through the hollow part of the porous hollow fiber membrane without being filtered by the porous hollow fiber membrane is returned to the culture tank via the second flow path. When the DO of the culture medium is lower than 70%, introduce oxygen into the culture medium in the culture tank using a sparger. In addition, continuously introduce air containing carbon dioxide at a concentration of 5% into the culture tank.

[0192] Disposable pressure gauges (PREPS-N-038 manufactured by PendoTECH) are provided on the primary side and secondary side of the porous hollow fiber membrane to measure the transmembrane pressure difference (TMP) over time. The shear stress on the inner surface of the porous hollow fiber membrane reaches 2.10 N / m 2The amount of culture medium transported from the culture tank to the porous hollow fiber membrane is set in the following manner. Using a pump, the filtration flow rate of the porous hollow fiber membrane is increased stepwise in the manner of 1.0 LMH, 1.5 LMH, 2.0 LMH, 2.5 LMH, and 3.0 LMH. Correspondingly with the increase in cells, the introduction amount of fresh culture medium is increased to adjust the glucose concentration and glutamine concentration. In addition, discharge is carried out so that the density of cells in the culture medium in the culture tank is 7.5x10 7 cells / mL. Furthermore, a liquid level sensor is arranged in the culture tank, and fresh culture medium is introduced into the culture tank in such a way that the liquid level remains unchanged.

[0193] The culture medium in the culture tank is sampled once or twice a day, and the density of cells, the survival rate of cells, and the antibody concentration in the culture medium are measured. As a result, as Figure 9 shown, the density of cells in the culture medium continuously rises until the start of discharge. As Figure 10 shown, the survival rate of cells is roughly constant. As Figure 11 shown, the antibody concentration has an upward tendency.

[0194] (Comparative Example 2)

[0195] As a module of a porous hollow fiber membrane with the membrane area of the liquid contact part being 200 cm 2 after adjusting the number of roots and the effective length, a porous hollow fiber membrane manufactured by Asahi Kasei, Microza UMP, with an exclusion pore size of 0.2 μm is used, and the same method as in Comparative Example 1 is implemented except for this.

[0196] The culture medium in the culture tank is sampled once or twice a day, and the density of cells, the survival rate of cells, and the antibody concentration in the culture medium are measured. As a result, as Figure 9 shown, the density of cells in the culture medium continuously rises until the start of discharge. As Figure 10 shown, the survival rate of cells is roughly constant. As Figure 11 shown, the antibody concentration has an upward tendency.

[0197] (Reference Examples 1 to 3)

[0198] By aseptic operation, the culture medium containing cells at a density of 1.5×10 7 cells / mL prepared in the same manner as in Example 1 is recovered, and 600 mL of the culture medium is transferred to a spinner flask, which has been autoclaved in advance and serves as a culture tank.

[0199] The atmospheric temperature of the rotating flask was set at 4°C, and the culture solution was stirred in the rotating flask. The rotating flask was not connected to the hollow fiber membrane, and cells were cultured only in the rotating flask. The culture solution in the rotating flask was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture solution were measured. Experiments were also conducted under the same conditions except that the atmospheric temperature was set at room temperature (RT) and under the same conditions except that the atmospheric temperature was set at 37°C. The results are as Figure 12 shown, and there is a tendency for the cell density to increase at room temperature. As Figure 13 shown, there is a tendency for the cell viability to decrease at room temperature and 37°C. As Figure 14 shown, there is a tendency for the antibody concentration to increase at room temperature and 37°C.

[0200] (Reference Examples 4 to 6)

[0201] The culture solution containing cells at a density of 1.5×10 7 cells / mL prepared in the same manner as in Example 1 was recovered by aseptic operation, and 600 mL of the culture solution was transferred to a rotating flask that had been autoclaved and served as a culture tank.

[0202] The atmospheric temperature of the rotating flask was set at 8°C, and the culture solution was stirred in the rotating flask. The rotating flask was not connected to the hollow fiber membrane, and cells were cultured only in the rotating flask. The culture solution in the rotating flask was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture solution were measured. Experiments were also conducted under the same conditions except that the atmospheric temperature was set at 12°C and under the same conditions except that the atmospheric temperature was set at 37°C. The results are as Figure 15 shown, and there is a tendency for the cell density to decrease at 37°C. As Figure 16 shown, there is a tendency for the cell viability to decrease at 37°C.

[0203] (Reference Examples 7, 8)

[0204] The culture solution containing cells at a density of 1.5×10 7 cells / mL prepared in the same manner as in Example 1 was recovered by aseptic operation, and 600 mL of the culture solution was transferred to a rotating flask that had been autoclaved and served as a culture tank.

[0205] The atmosphere temperature of the rotating flask was set at 4 °C, and the culture medium was stirred in the rotating flask. The rotating flask was not connected to the hollow fiber membrane, and cells were cultured only in the rotating flask. The culture medium in the rotating flask was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. The culture medium was circulated using a pump, and the experiment was conducted under the same conditions otherwise. The culture medium was transported from the rotating flask to the pump via the first flow path using a magnetic levitation centrifugal pump (PuraLev i30SU manufactured by Levitronix), and the culture medium that passed through the pump was returned to the rotating flask via the second flow path. The transport of the culture medium using the pump was carried out at 50 mL / min. The first flow path had a structure capable of sampling the culture medium inside, and the culture medium in the rotating 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. The results are as Figures 17 to 19 shown. Regardless of whether the culture medium was circulated using a pump, the time-dependent change of the culture medium was similarly suppressed.

[0206] (Analysis method)

[0207] The analysis methods used in the above-mentioned examples, comparative examples, and reference examples are described below. The cell density and viability contained in the culture medium were measured using a live / dead cell automatic analyzer (Vi-CELLXR manufactured by Beckman Coulter). The sample was diluted with PBS(-) (FUJIFILM Wako) and analyzed with 600 μL. The image analysis method used "CHO" in Vi-CELL XR, and the method of appropriately changing the set values of the minimum diameter (μm), cell brightness (%), and live cell spot brightness (%) according to the actual situation was used.

[0208] The HPLC determination of the antibody concentration was carried out using the following method.

[0209] (1) Detector: Ultraviolet absorption photometer (measurement wavelength: 280 nm)

[0210] (2) Column: POROS G 20 μm column, 4.6×50 mm, 0.8 mL (ThermoFisher)

[0211] (3) Column temperature: Room temperature

[0212] (4) Mobile phase

[0213] Mobile phase A: Dissolve 7.098 g of disodium hydrogen phosphate (anhydrous) and 8.766 g of sodium chloride in 800 mL of water, adjust the pH to 7.0 with 1 mol / L hydrochloric acid, and then add water to 1000 mL.

[0214] Mobile phase B: Dissolve 12 mL of 1 mol / L hydrochloric acid and 8.766 g of sodium chloride in water, and make up to 1000 mL.

[0215] (5) Delivery of the mobile phase

[0216] Deliver the liquid at a flow rate of 2 mL / min, and change the ratios of mobile phase A and mobile phase B as shown in Table 4 below.

[0217] [Table 4]

[0218] Time after sample injection (minutes) Mobile phase A (vol%) Mobile phase B (vol%) 0～4 100 0 4～11 0 100 11～16 100 0

[0219] Centrifuge the cell culture solution at 300×g for 2 minutes, and then sample the supernatant. Use the above method to deliver 9 series of serial dilution solutions of commercially available human immunoglobulin G (manufactured by the Japanese Blood Products Organization, donated Venoglobulin IH 5% intravenous injection 2.5 g / 50 mL) and the supernatant of the cell culture solution according to the above steps. After making a standard curve using the peak areas of 9 peaks of human immunoglobulin G, calculate the antibody concentration in each solution from the standard curve and the peak area of the sample.

[0220] As shown in the following formula, the shear stress SS generated by the culture solution at the liquid contact surface of the hollow fiber membrane can be provided by the product of the viscosity VC (Pa·s) of the culture solution flowing in the hollow part of the hollow fiber membrane and the shear rate SV ( / s).

[0221] SS = VC × SV

[0222] 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, calculate the shear stress based on the viscosity of the culture solution and the shear rate calculated from the linear velocity.

[0223] Explanation of reference numerals

[0224] 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 solution tank, 217... container, 218... container.

Claims

1. A method for evaluating filtration conditions, comprising the following steps: Under conditions that inhibit cell proliferation, the culture medium containing cells is transported from a culture tank accommodating the culture medium to a porous membrane, and the culture medium that passes through without being filtered by the porous membrane is returned to the culture tank, so that the culture medium circulates between the culture tank and the porous membrane; and Evaluating one or more filtration conditions of the porous membrane.

2. The method for evaluating filtration conditions according to claim 1, wherein, In the step of returning the culture medium that passes through without being filtered by the porous membrane to the culture tank, the culture medium that passes through without being filtered by the porous membrane and the culture medium that has been filtered by the porous membrane are returned to the culture tank.

3. The method for evaluating filtration conditions according to claim 1, wherein, It further includes a step of evaluating the filtration performance of the porous membrane under the one or more filtration conditions, Evaluating the one or more filtration conditions of 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 condition for inhibiting cell proliferation is a condition for inhibiting the progression of the cell cycle.

5. The method for evaluating filtration conditions according to claim 1, wherein, The condition for inhibiting cell proliferation is at least any one of an atmosphere temperature of 15°C or lower and a culture medium temperature of 15°C or lower.

6. The method for evaluating filtration conditions according to claim 5, wherein, At least any one of the atmosphere temperature of 15°C or lower and the culture medium temperature of 15°C or lower is at least any one of an atmosphere temperature of 10°C or lower and a culture medium temperature of 10°C or lower, or at least any one of an atmosphere temperature of 5°C or lower and a culture medium temperature of 5°C or lower.

7. The method for evaluating filtration conditions according to claim 1, wherein, The condition for inhibiting cell proliferation is a condition for inhibiting the enzyme activity of the cells.

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 for inhibiting cell proliferation is the presence of a cell cycle inhibitor in the culture medium.

10. The method for evaluating filtration conditions according to claim 1, wherein, Evaluating one or more filtration conditions of the porous membrane based on the transmittance of the product of the cells through the porous membrane.

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

12. The method for evaluating filtration conditions according to claim 1, wherein, Evaluating one or more filtration conditions of the porous membrane based on the transmembrane pressure difference of the porous membrane.

13. The method for evaluating filtration conditions according to claim 1, wherein, Evaluating one or more filtration conditions of 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 of the culture solution.

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

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

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

19. The method for evaluating a filtration condition according to claim 1, wherein, the one or more filtration conditions are one or more conditions of the shear stress on the liquid contact surface of the porous membrane generated by the flow of the culture solution delivered to the porous membrane.

20. The method for evaluating a filtration condition according to claim 1, wherein, The path of the circulation of the culture solution including the culture tank and the porous membrane is arranged in a temperature control tank.

21. The method for evaluating a filtration condition according to claim 1, wherein, In the step of circulating the culture solution between the culture tank and the porous membrane, no active operation for maintaining the composition of the culture solution is performed.

22. The method for evaluating a filtration condition according to claim 1, wherein, In the step of circulating the culture solution between the culture tank and the porous membrane, no medium is added from the outside to the path of the circulation of the culture solution including the culture tank and the porous membrane.

23. The method for evaluating a filtration condition according to claim 1, wherein, In the step of circulating the culture solution between the culture tank and the porous membrane, at least any one of the dissolved oxygen and pH of the culture solution is not controlled.

24. The method for evaluating a filtration condition according to claim 1, wherein, In the step of circulating the culture solution between the culture tank and the porous membrane, the cells are not discharged from the path of the circulation of the culture solution including the culture tank and the porous membrane.

25. The method for evaluating a filtration condition according to claim 1, wherein, The step of circulating the culture solution between the culture tank and the porous membrane further includes the following step: collecting the culture solution for sampling from the path of the circulation of the culture solution including the culture tank and the porous membrane.

26. The method for evaluating a filtration condition according to claim 25, wherein, It further includes a step of measuring the density of the cells in the culture solution obtained by the sampling.

27. The method for evaluating a filtration condition according to claim 25, wherein, It further includes a step of measuring the survival rate of the cells in the culture solution obtained by the sampling.

28. The method for evaluating a filtration condition according to claim 25, wherein, It further includes a step of measuring the concentration of the products of the cells in the culture solution obtained by the sampling.

29. The method for evaluating a filtration condition according to claim 25, Among them, it further includes the step of measuring the turbidity of the culture solution in the culture solution obtained by sampling.

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

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

32. A method for manufacturing a cell product, which includes the following steps: under the conditions for cell proliferation, using the filtration conditions to transport the culture solution from a culture tank containing the culture solution containing cells to a porous membrane, returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, recovering the culture solution containing the product containing the cells that has been filtered by the porous membrane, and circulating at least a part of the culture solution between the culture tank and the porous membrane. wherein, the filtration conditions are the filtration conditions obtained as follows: under the conditions for inhibiting cell proliferation, transporting the culture solution from the culture tank containing the culture solution containing the cells to the porous membrane, returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, circulating the culture solution between the culture tank and the porous membrane, and evaluating one or more filtration conditions of the porous membrane to obtain the filtration conditions.

33. The method for manufacturing a cell product according to claim 32, wherein, when evaluating one or more filtration conditions of the porous membrane, in the step of returning the culture solution that has passed through without being filtered by the porous membrane to the culture tank, the culture solution that has passed through without being filtered by the porous membrane and the culture solution that has been filtered by the porous membrane are returned to the culture tank.

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

35. The method for manufacturing a cell product according to claim 32, wherein, the conditions for cell proliferation are at least any one of an atmosphere temperature of 15°C or higher and a culture solution temperature.

36. The method for manufacturing a cell product according to claim 35, wherein, at least any one of the atmosphere temperature and the culture solution temperature higher than 15°C is at least any one of an atmosphere temperature and a culture solution temperature higher than 20°C, higher than 25°C, higher than 30°C, or higher than 35°C.

37. The method for manufacturing a cell product according to claim 32, wherein, the conditions for cell proliferation are the conditions under which the enzyme of the cell has been activated.

38. The method for manufacturing a cell product according to claim 37, wherein, the enzyme is a cyclin-dependent kinase.

39. The method for manufacturing a cell product according to claim 32, wherein, the conditions for cell proliferation are that there is no cell cycle inhibitor in the culture solution.

40. The method for manufacturing a cell product according to claim 32, wherein, the conditions for inhibiting cell proliferation are the conditions for inhibiting the progression of the cell cycle.

41. The method for manufacturing a cell product according to claim 32, wherein, The condition for inhibiting cell proliferation is at least any one of an atmosphere temperature and a culture solution temperature below 15°C.

42. The method for producing a cell product according to claim 41, wherein at least any one of the atmosphere temperature and the culture solution temperature below 15°C is at least any one of an atmosphere temperature and a culture solution temperature below 10°C or below 5°C.

43. The method for producing a cell product according to claim 32, wherein the condition for inhibiting cell proliferation is a condition for inhibiting the enzyme activity of the cells.

44. The method for producing a cell product according to 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 for inhibiting cell proliferation is the presence of a cell cycle inhibitor in the culture solution.

46. The method for producing a cell product according to claim 32, wherein one or more filtration conditions of the porous membrane are evaluated based on the permeation rate of the product based on the cells in the porous membrane.

47. The method for producing a cell product according to claim 32, wherein one or more filtration conditions of the porous membrane are evaluated based on the permeation flux in the porous membrane.

48. The method for producing a cell product according to claim 32, wherein one or more filtration conditions of the porous membrane are evaluated based on the transmembrane pressure difference in the porous membrane.

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

50. The method for producing a cell product according to 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. The method for producing a cell product according to claim 32, wherein the one or more filtration conditions are one or more conditions of the culture solution.

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

53. The method for producing a cell product according to claim 32, wherein the one or more filtration conditions are one or more conditions of the flow rate of the culture solution delivered to the porous membrane.

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

55. The method for producing a cell product according to claim 32, wherein the one or more filtration conditions are one or more conditions of the shear stress on the liquid contact surface of the porous membrane generated by the flow of the culture solution delivered to the porous membrane.

56. The method for producing a cell product according to claim 32, wherein when evaluating one or more filtration conditions of the porous membrane, the path of the circulation of the culture solution including the culture tank and the porous membrane is arranged in a temperature control tank.

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

58. The method for producing a cell product according to claim 32, wherein, when evaluating one or more filtration conditions of the porous membrane, in the step of circulating the culture solution between the culture tank and the porous membrane, no medium is added to the path of the circulating culture solution from the outside.

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

60. The method for producing a cell product according to claim 32, wherein, when evaluating one or more filtration conditions of the porous membrane, in the step of circulating the culture solution between the culture tank and the porous membrane, the cells are not discharged from the path of the circulating culture solution including the culture tank and the porous membrane.

61. The method for producing a cell product according to claim 32, wherein, when evaluating one or more filtration conditions of the porous membrane, the following step is included during the process of circulating the culture solution between the culture tank and the porous membrane: collecting the culture solution for sampling from the path of the circulating culture solution including the culture tank and the porous membrane.

62. The method for producing a cell product according to claim 61, wherein, it further includes a step of measuring the density of the cells in the culture solution obtained by sampling.

63. The method for producing a cell product according to claim 61, wherein, it further includes a step of measuring the viability of the cells in the culture solution obtained by sampling.

64. The method for producing a cell product according to claim 61, wherein, it further includes a step of measuring the concentration of the cell product in the culture solution obtained by sampling.

65. The method for producing a cell product according to claim 61, wherein, it further includes a step of measuring the turbidity of the culture solution in the culture solution obtained by sampling.

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 according to claim 32, wherein, the porous membrane is a microfiltration membrane.

68. The method for producing a cell product according to claim 32, wherein, the cells are subjected to perfusion culture.

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