Production method for product and cell culture device

Abstract

According to the present invention, a production method for a product involves using a hollow fiber membrane filter to separate a product. The hollow fiber membrane filter includes a primary channel that is supplied a culture liquid that has been extracted from a culture tank during cell culture and includes cells and a product of the cells and a secondary channel at which the product flows from the primary channel through a hollow fiber membrane. The production method also involves backflow cleansing for causing a liquid to flow backward from the secondary channel side to the primary channel side to cleanse the hollow fiber membrane filter. The backflow cleansing includes a period that satisfies the condition that a linear velocity (cm / min) that is the backflow flow rate (mL / min) per unit area (cm2) of the hollow fiber membrane is at least 0.003 (cm / min) but no more than 0.03 (cm / min).

Description

Method for producing products and cell culture apparatus

[0001] The technology disclosed herein relates to a method for producing a product and a cell culture apparatus.

[0002] Cell culture is a known technique for producing cell-derived antibodies and other substances used in biopharmaceuticals (see Japanese Patent Publication No. 2023-135069 and Japanese Patent Publication No. 2024-522745). As described in Japanese Patent Publication No. 2023-135069 and Japanese Patent Publication No. 2024-522745, the recovery of products from the culture medium is performed by separating the products in the culture medium from the cells using a hollow fiber membrane filter and then extracting the separated products. The hollow fiber membrane filter includes a primary channel through which a culture medium containing cells and products is supplied, and a secondary channel through which a culture medium containing products separated from the cells flows after passing through the hollow fiber membrane from the primary channel.

[0003] As hollow fiber membrane filters are used over time, some of the substances contained in the culture medium accumulate on the hollow fiber membrane, causing clogging. To resolve this clogging, Japanese Patent Publication No. 2023-135069 and Japanese Patent Publication No. 2024-522745 describe performing backflow washing, which involves flowing the liquid back from the secondary channel side to the primary channel side.

[0004] As described in Japanese Patent Publication No. 2024-522745, perfusion culture is a known method of cell culture. Perfusion culture is a culture method that includes a process of recovering a portion of the culture medium containing the cell products during cell culture and continuously supplying an equal amount of new culture medium. In perfusion culture, the recovery of products is carried out in parallel with cell culture, and the culture period is relatively long, ranging from several days to several tens of days, so the hollow fiber membrane filter is washed multiple times during cell culture.

[0005] When backwashing is performed during cell culture, the recovery of the product is temporarily stopped. To minimize the decrease in product productivity, the number of washes should be kept to a minimum. Therefore, when backwashing is performed during cell culture, it is especially important that the washing effect is good so that the number of washes is reduced.

[0006] In conventional backwashing methods, depending on the backwash flow rate per unit area of ​​the hollow fiber membrane, a satisfactory cleaning effect may not be achieved even with extended washing time. Poor cleaning effectiveness leads to an increase in the number of washing cycles, reducing productivity.

[0007] The technology disclosed herein provides a method for producing a product and a cell culture apparatus that can achieve a better cleaning effect than conventional methods when cleaning hollow fiber membrane filters.

[0008] A method for producing a product according to the technology disclosed herein is a method for producing a product that separates the product using a hollow fiber membrane filter, which includes a primary channel to which the culture medium containing cells and cell products is supplied, and a secondary channel through which the product flows from the primary channel through a hollow fiber membrane, wherein the hollow fiber membrane filter is washed by backflow washing in which liquid is backflowed from the secondary channel side to the primary channel side, and the membrane area of ​​the hollow fiber membrane [cm² ２ This includes performing backwashing for a period of time that satisfies the condition that the linear velocity [cm / min], which is the backwash flow rate [mL / min] per [unit], is 0.003 [cm / min] or more and 0.03 [cm / min] or less.

[0009] Backwashing may be started at a linear velocity that satisfies the conditions. Backwashing may satisfy the conditions at a linear velocity within 2 minutes from the start. In backwashing, the backwash flow rate may be gradually increased at 0.1 [mL / min / min] or more. In backwashing, the duration of one cycle may be 10 minutes or more. Backwashing may be repeated at a predetermined timing. Backwashing may be performed once a day or more. In backwashing, culture medium may be used as the liquid. In backwashing, the permeate that has permeated from the primary channel to the secondary channel may be used as the liquid. The hollow fiber membrane filter may be an ATF type where a reciprocating flow of culture medium occurs in the primary channel. In backwashing, the reciprocating flow in the primary channel may be stopped, and in that state, the liquid may be backflowed and the liquid may be collected before it reaches the culture vessel from the hollow fiber membrane filter. The viable cell density of the culture medium is 30 × 10⁶. ６ The concentration may be 1.0 g / L or higher. The potency of the product is 1.0 g / L or higher.

[0010] The cell culture apparatus according to the technology disclosed herein includes a separation apparatus for separating products using a hollow fiber membrane filter, which includes a primary channel to which a culture medium containing cells and cell products is supplied and which is removed from the culture tank during cell culture, and a secondary channel through which the products flow from the primary channel through a hollow fiber membrane, and a backflow washing method for washing the hollow fiber membrane filter by backflowing liquid from the secondary channel side to the primary channel side, wherein the membrane area of ​​the hollow fiber membrane [cm² ２ The system includes a processor that controls the execution of backwashing, which includes a period during which the linear velocity [cm / min], which is the backwash flow rate [mL / min] per unit, satisfies the condition that it is 0.003 [cm / min] or more and 0.03 [cm / min] or less.

[0011] According to the technology disclosed herein, a better cleaning effect can be obtained in cleaning hollow fiber membrane filters than in conventional methods.

[0012] This figure shows the overall configuration of the cell culture apparatus. This is an explanatory diagram of the hollow fiber membrane filter. This is a cross-sectional view taken along line III-III in Figure 2. This figure schematically shows the primary and secondary channels of the hollow fiber membrane filter. This figure schematically shows the state of membrane clogging. This is an example of a graph showing the change in channel pressure over time when backwashing is not performed. This figure shows the operation under normal conditions and during backwashing. This figure schematically shows the state inside the hollow fiber membrane filter under normal conditions and during backwashing. This is an example of a graph showing the change in channel pressure over time when backwashing is performed. This figure shows an example of the conditions for backwashing. This figure shows an example of the correlation between linear velocity and pressure drop. This figure schematically shows the state inside the hollow fiber membrane filter when the linear velocity is high. This figure schematically shows the state inside the hollow fiber membrane filter when the linear velocity is low. This is a flowchart showing the control processing procedure during cell culture. This is a graph showing an example of changing the linear velocity during a single backwash. This figure shows an example of using permeate as the washing solution. This figure shows an example of using culture medium as the washing solution and not recovering the washing solution as waste liquid. This figure shows a modified version of Figure 17. This figure shows an example where permeate is used as the cleaning solution and the cleaning solution is not collected as waste liquid.

[0013] The cell culture method relating to the technology of this disclosure will be described below. However, the embodiments relating to this disclosure are not limited to the embodiments described below and can be implemented with appropriate modifications.

[0014] In this disclosure, numerical ranges indicated using "~" mean a range that includes the numbers indicated before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the value shown in the example. In this disclosure, the term "process" includes not only independent processes but also processes that are indistinguishable from other processes as long as the intended purpose of the process is achieved. Components indicated by the same reference numeral in each drawing mean that they are the same component.

[0015] A method for producing a product according to the technology disclosed herein is a method for producing a product that separates the product using a hollow fiber membrane filter, which includes a primary channel to which the culture medium containing cells and cell products is supplied, and a secondary channel through which the product flows from the primary channel through a hollow fiber membrane, wherein the hollow fiber membrane filter is washed by backflow washing in which liquid is backflowed from the secondary channel side to the primary channel side, and the membrane area of ​​the hollow fiber membrane [cm² ２ The method includes performing backwashing for a period of time that satisfies the condition that the linear velocity [cm / min], which is the backwash flow rate [mL / min] per [unit], is 0.003 [cm / min] or more and 0.03 [cm / min] or less. The linear velocity [cm / min] is preferably 0.004 to 0.025 [cm / min], more preferably 0.004 to 0.02 [cm / min], and even more preferably 0.01 to 0.02 [cm / min].

[0016] The culture medium contains a culture medium, cells, and products produced by the cells. The cells, for example, have a diameter of 5 μm or more, and the volume fraction of cells in the culture medium is, for example, 1% or more.

[0017] The cells are not particularly limited, but examples include animal cells, plant cells, eukaryotic cells such as yeast, prokaryotic cells such as Bacillus subtilis, and Escherichia coli. Animal cells such as CHO (Chinese Hamster Ovary cells) cells, BHK (Baby Hamster Kidney)-21 cells, HEK (Human Embryonic Kidney) cells, C127 cells, NS0 cells, and SP2 / 0-Ag14 cells are preferred, with CHO cells or HEK cells being more preferred due to the numerous analyses that have been performed and the establishment of genetic engineering techniques. Even if the cells do not originally produce the desired product or produce it in small quantities, the desired product can be efficiently produced by introducing an expression vector, such as a plasmid encoding the necessary protein, into the cells.

[0018] The products relating to the technology disclosed herein are not particularly limited as long as they are substances produced by the cells in the culture medium, and include, for example, alcohol, enzymes, antibiotics, and proteins. Among these, proteins are preferred as products, and antibodies are more preferred.

[0019] If the product is, for example, an antibody, animal cells such as CHO cells may be made to produce the antibody. The antibodies to be produced by animal cells are not particularly limited, but examples include anti-IL-6 receptor antibodies, anti-IL-6 antibodies, anti-glypican-3 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-GPIIb / IIIa antibodies, anti-TNF antibodies, anti-CD25 antibodies, anti-EGFR antibodies, anti-Her2 / neu antibodies, anti-RSV antibodies, anti-CD33 antibodies, anti-CD52 antibodies, anti-IgE antibodies, anti-CD11a antibodies, anti-VEGF antibodies, and anti-VLA4 antibodies. Antibodies include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, and monkeys, but also artificially modified antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies.

[0020] By culturing cells selected according to the purpose, the desired product can be manufactured.

[0021] [First Embodiment] Below, an embodiment of the method for producing a product according to the technology of this disclosure and an embodiment of a cell culture apparatus will be described. The cell culture apparatus 10 shown as an example in Figure 1 is an example of a cell culture apparatus that realizes the method for producing a product according to the technology of this disclosure, and is also an example of a cell culture apparatus according to the technology of this disclosure. Figure 1 is a diagram showing the overall configuration of the cell culture apparatus 10.

[0022] The cell culture apparatus 10 is a device that implements perfusion culture as a culture method. Perfusion culture is a culture method that includes a process of recovering a portion of the culture medium 30 during culture and supplying a new medium 31 equal to the recovered amount. The medium 31 contains nutrients for the cells 32. Perfusion culture differs from fed-batch culture in that it recovers waste products, including used medium 31, by recovering a portion of the culture medium 30 while leaving the cells 32 intact. In this way, because the medium 31 is replaced during culture in perfusion culture, it is easier to keep the culture environment of the cells 32 fresh and in good condition compared to fed-batch culture and batch culture where no medium 31 is added. As a result, perfusion culture makes it easier to ensure high productivity and is also suitable when culturing large quantities of cells. For example, with perfusion culture, 100 × 10 ６ It is possible to achieve a high viable cell density exceeding cells / mL.

[0023] As shown in Figure 1, the cell culture apparatus 10 comprises a culture tank 11, a separation device 12, a culture medium supply tank 13, an oxygen supply tank 14, a recovery tank 15, and a processor 16. In addition, the cell culture apparatus 10 includes a plurality of conduits 51-54, 65, 67 for supplying the culture medium 30, and a pump 18.

[0024] The culture vessel 11 is a culture container that holds the culture medium 30 and cultures cells in the culture medium 30, and is also called a bioreactor. The culture vessel 11 has a capacity of, for example, 100 L or more. From the viewpoint of further improving productivity, the capacity of the culture vessel 11 is more preferably 400 L or more, and even more preferably 1000 L or more.

[0025] The culture tank 11 is equipped with a stirring device having a stirring blade 11A. By rotating the stirring blade 11A, the culture medium 30 contained in the culture tank 11 is stirred, and the homogeneity of the cells 32 and other components contained in the culture medium 30 is maintained within the culture tank 11.

[0026] Furthermore, a sparger 11B is provided inside the culture tank 11 to supply oxygen 26 into the culture medium 30. The oxygen 26 is dissolved in the culture medium 30 and consumed by the cells 32. The sparger 11B is made of a porous material and generates bubbles of oxygen 26. The sparger 11B is connected to an oxygen supply tank 14 by a conduit 53. A mass flow controller 17 is located on the conduit 53 to supply oxygen 26 from the oxygen supply tank 14 to the sparger 11B. The amount of oxygen 26 supplied is adjusted by adjusting the output of the mass flow controller 17 on the conduit 53. This controls the dissolved oxygen concentration in the culture medium 30.

[0027] Furthermore, the culture tank 11 is connected to the culture medium supply tank 13 by a conduit 54. The culture medium supply tank 13 contains fresh culture medium 31. Fresh culture medium 31 is supplied to the culture tank 11 from the culture medium supply tank 13 through the conduit 54. A pump 18 for supplying the culture medium 31 is provided in the conduit 54, and the pump 18 is controlled by the processor 16. As described above, in perfusion culture, a portion of the culture medium 30 is recovered during cultivation, and the recovered culture medium 30 contains used culture medium 31. An amount of fresh culture medium 31 equivalent to the recovered amount is supplied from the culture medium supply tank 13 to the culture tank 11. This replaces the culture medium 31, and the culture environment in the culture medium 30 in the culture tank 11 is kept fresh.

[0028] The separation device 12 is a device for recovering a portion of the culture medium 30 discharged from the culture tank 11 during cell culture. The recovered culture medium 30 contains antibodies 33, which are an example of products of cells 32, and waste products such as used culture medium 31. The separation device 12 separates the cells 32 and antibodies 33 in the culture medium 30. The separation device 12 is connected to the recovery tank 15 by a conduit 52. A pump 18 is provided in the conduit 52. The culture medium 30 containing the cells 32 and the separated antibodies 33 is recovered into the recovery tank 15 when the pump 18 is driven. Since the recovered culture medium 30 also contains waste products, a portion of the waste products is recovered along with the products. Meanwhile, the culture medium 30 containing the cells 32 is resupplied from the separation device 12 to the culture tank 11. Hereinafter, the culture medium 30 recovered into the recovery tank 15 will be referred to as permeate 35 because it is the liquid that has permeated through the hollow fiber membrane filter 20 of the separation device 12, which will be described later.

[0029] The separation device 12 includes a hollow fiber membrane filter 20, a circulation pump 57, and a conduit 51. The hollow fiber membrane filter 20 separates cells 32 in the culture medium 30 from antibodies 33, which are products of the cells 32, as will be described in detail later. The hollow fiber membrane filter 20 is connected to the culture tank 11 by a conduit 51. The hollow fiber membrane filter 20 is provided with a first opening 20a, a second opening 20b, an outlet 20c, and an inlet 20d. The conduit 51 is connected to the first opening 20a, and the circulation pump 57 is connected to the second opening 20a. The conduit 52 is connected to the outlet 20c, and the conduit 65 is connected to the inlet 20d. The inlet 20d is an opening into which washing solution 62 is introduced to the hollow fiber membrane filter 20, as will be described later.

[0030] The circulation pump 57 draws a portion of the culture medium 30 from the culture tank 11 into the hollow fiber membrane filter 20, and resupplies a portion of the culture medium 30 drawn into the hollow fiber membrane filter 20 back into the culture tank 11. This allows the culture medium 30 to be circulated between the culture tank 11 and the separation device 12. The circulation pump 57 is, for example, a diaphragm pump that generates suction pressure and discharge pressure through the periodic pulsation of a diaphragm 57A located inside the circulation pump 57. When the circulation pump 57 generates suction pressure, it draws the culture medium 30 inside, and conversely, when it generates discharge pressure, it discharges the culture medium 30 that has been drawn inside.

[0031] In the suction operation, the circulation pump 57 generates negative pressure inside due to the displacement of the diaphragm 57A, drawing in a portion of the culture medium 30 in the hollow fiber membrane filter 20. As a result, a portion of the culture medium 30 is drawn from the culture tank 11 and into the hollow fiber membrane filter 20. On the other hand, in the discharge operation, the circulation pump 57 generates positive pressure inside due to the displacement of the diaphragm 57A, discharging the culture medium 30 that has been drawn in towards the hollow fiber membrane filter 20. As a result, the culture medium 30 in the hollow fiber membrane filter 20 is resupplied towards the culture tank 11. Thus, the separation device 12 is an ATF (Alternating Tangential Flow filtration) system in which a reciprocating flow of culture medium 30 occurs within the hollow fiber membrane filter 20.

[0032] The circulation pump 57 is equipped with an actuator 57B that drives the diaphragm 57A by drawing in and exhausting air. The processor 16 controls the circulation pump 57 through the actuator 57B. By controlling the circulation pump 57, the processor 16 can control the flow rate per unit time (i.e., the flow velocity of the culture medium 30) and the cycle time of the culture medium 30 flowing through the hollow fiber membrane filter 20. The cycle time is the total time of one cycle, where one suction operation and one discharge operation are combined to form one cycle in the periodically repeated suction and discharge operations of the culture medium 30.

[0033] Further, the cleaning liquid tank 61, the waste liquid tank 63, the pipeline 65, and the pipeline 67 are configured to realize backwashing, which is a cleaning process of the hollow fiber membrane filter 20. The cleaning liquid tank 61 is a tank that stores the cleaning liquid 62. As an example, the culture medium 31 is used as the cleaning liquid 62. The culture medium 31 is the same as the new culture medium 31 stored in the culture medium supply tank 13. The cleaning liquid tank 61 is connected to the hollow fiber membrane filter 20 via the pipeline 65. The pipeline 65 is a pipeline for introducing the cleaning liquid 62 into the hollow fiber membrane filter 20, and is connected to the inlet 20d of the hollow fiber membrane filter 20 as described above. A pump 18 is arranged in the pipeline 65, and the flow rate of the cleaning liquid 62 and the like are controlled by driving the pump 18.

[0034] Further, the pipeline 67 is a pipeline for recovering the used cleaning liquid 62 introduced into the hollow fiber membrane filter 20 and used for backwashing as the waste liquid 64 into the waste liquid tank 63. The pump 18 arranged in the pipeline 67 is a pump 18 for recovering the used cleaning liquid 62. One end of the pipeline 67 is connected to the waste liquid tank 63, and the other end is connected to the pipeline 51 via the valve 66. The valve 66 is a three-way valve, and selectively switches between a first state in which a flow path for circulating the culture liquid 30 between the culture tank 11 and the hollow fiber membrane filter 20 is opened, and a second state in which a flow path for flowing the used cleaning liquid 62 from the hollow fiber membrane filter 20 to the pipeline 67 is opened. As will be described later, the first state is selected when circulating the culture liquid 30 between the culture tank 11 and the hollow fiber membrane filter 20, and the second state is selected when flowing the cleaning liquid 62 to the waste liquid tank 63 during backwashing of the hollow fiber membrane filter 20 (see FIG. 7). These pump 18 and valve 66 are controlled by the processor 16 in the same manner as the circulation pump 57 and the like.

[0035] The processor 16 controls the overall operation of the cell culture device 10. The processor 16 is constituted by, for example, a CPU (Central Processing Unit) and a memory 16A. The memory 16A includes a working RAM (Random Access Memory) into which the program executed by the CPU is loaded, and a data storage for storing various data. The data storage is constituted by an NVM (Non-volatile Memory), a hard disk drive, and the like. Various data used for controlling the cell culture device 10 are stored in the memory 16A.

[0036] FIG. 2 is a side view showing a schematic configuration of the hollow fiber membrane filter 20, and FIG. 3 is a cross-sectional view showing a cross-section taken along line III-III in FIG. 2.

[0037] As shown in FIG. 2, the hollow fiber membrane filter 20 includes a hollow fiber membrane 22 having a primary flow path 23 extending in the longitudinal direction therein, and a housing 21 that houses a plurality of hollow fiber membranes 22. The hollow fiber membrane 22 is a separation membrane formed by processing a porous membrane 22a having a large number of pores 22h (see also FIG. 4 described later) into a cylindrical shape. The hollow portion of the hollow fiber membrane 22 constitutes the primary flow path 23. In the hollow fiber membrane filter 20, the space outside each hollow fiber membrane 22 in the housing 21 is the secondary flow path 24 (see FIG. 3).

[0038] The housing 21 has a cylindrical outer shape, and has a first opening 20a and a second opening 20b for flowing the culture solution 30 into or out of the primary flow path 23 at both ends thereof. Further, the housing 21 is provided with a discharge port 20c on the outer periphery for discharging the permeate 35 (see also FIG. 4) to the outside of the hollow fiber membrane filter 20. The permeate 35 is a part of the culture solution 30.

[0039] Figure 4 is a schematic diagram showing the primary channel 23 and secondary channel 24 of the hollow fiber membrane filter 20. As shown in Figures 2 and 3, in reality, multiple hollow fiber membranes 22 are housed inside the housing 21, but in Figure 4, in order to clearly explain the function of a single hollow fiber membrane 22, a configuration in which one hollow fiber membrane 22 is housed in the housing 21 is shown. In the hollow fiber membrane filter 20, a culture medium 30 containing culture medium 31, cells 32, and antibodies 33 is delivered to the primary channel 23. The hollow fiber membrane 22 separates the cells 32 and antibodies 33 in the culture medium 30 by allowing a permeate 35 containing antibodies 33 and waste products such as used culture medium 31 to pass through to the secondary channel 24.

[0040] In the hollow fiber membrane 22, components of the culture medium 30 passing through the primary channel 23 that are smaller than the pores 22h of the porous membrane 22a (hereinafter referred to as the pores 22h of the hollow fiber membrane 22) pass through the pores 22h and are transported to the secondary channel 24. In other words, components smaller than the pores 22h of the hollow fiber membrane 22 permeate the hollow fiber membrane 22. On the other hand, components of the culture medium 30 passing through the primary channel 23 that are larger than the pores 22h remain in the primary channel 23. Here, the pores 22h are smaller than the cells 32 and larger than the antibodies 33. The antibodies 33 in the culture medium 30 being delivered through the primary channel 23 of the hollow fiber membrane 22 are smaller than the pores 22h of the hollow fiber membrane 22 and therefore permeate to the secondary channel 24, while the cells 32 are larger than the pores 22h and therefore do not permeate to the secondary channel 24 and remain in the primary channel 23. Using such a hollow fiber membrane 22, cells 32 and antibodies 33 are separated.

[0041] This filtration method, in which a hollow fiber membrane filter 20 is used, a culture medium 30 is flowed through a primary channel 23 along the membrane surface of the hollow fiber membrane 22, and a permeate 35 containing the antibody 33, which is the product, is passed through a secondary channel, is called the tangential filtration method.

[0042] As shown in Figure 5, in the hollow fiber membrane filter 20, membrane clogging of the hollow fiber membrane 22 progresses as the culture medium 30 circulates back and forth in the primary channel 23. Membrane clogging occurs when clogging material 36, which is too large to pass through the pores 22h of the hollow fiber membrane 22 from the substances contained in the culture medium 30, enters the pores 22h and clogs them. The clogging material 36 eventually accumulates on the membrane surface of the hollow fiber membrane 22 on the primary channel 23 side. Figure 5 schematically shows how the clogging material 36 accumulates on the membrane surface of the hollow fiber membrane 22 in the primary channel 23. Figure 5(A) shows a state where the pores 22h are not clogged with clogging material 36, and Figure 5(B) shows a state where the pores 22h are beginning to be clogged by the clogging material 36. Furthermore, as time passes, as shown in Figure 5(C), the clogging material 36 accumulates to cover the membrane surface of the hollow fiber membrane 22. The clogging substance 36 specifically consists of antibody aggregates and dead cell fragments, etc.

[0043] As membrane clogging progresses, the culture medium 30 becomes less able to permeate from the primary channel 23 to the secondary channel 24, resulting in a decrease in the antibody 33 separation performance of the hollow fiber membrane filter 20. This makes it impossible to continue perfusion culture, and therefore the hollow fiber membrane filter 20 with reduced separation performance needs to be replaced. As membrane clogging progresses, the flow pressure P of the primary channel 23 increases, so the timing for replacing the hollow fiber membrane filter 20 can be determined, for example, by measuring the flow pressure P of the primary channel 23.

[0044] Figure 6 is a graph showing an example of the change over time of the flow path pressure P [psi] of the primary flow path 23 during the perfusion culture period. In Figure 6, the perfusion culture period is the number of days elapsed since the start of perfusion culture. The perfusion culture period is the number of days elapsed since the start of circulation of the culture medium 30. Figure 6 is an example where backwashing of the hollow fiber membrane filter 20 is not performed during the perfusion culture period. The graph in Figure 6 shows three lines: a solid line, a dashed line, and a dotted line. The solid line graph shows that the flow path pressure P rises to 10 [psi] after 20 days. The dashed and dotted line graphs show even shorter elapsed times, with the flow path pressure P reaching 10 [psi] after 10 to 15 days. A shorter elapsed time means that membrane clogging progresses faster and the lifespan of the hollow fiber membrane filter 20 is shorter. The rate of membrane clogging progresses differs depending on the type of cells 32 contained in the culture medium 30 and the type of antibodies 33 produced by the cells 32. In the graph shown in Figure 6, the dashed line graph represents the fastest rate of membrane clogging, resulting in the shortest lifespan for the hollow fiber membrane filter 20. Backwashing of the hollow fiber membrane filter 20 is performed to suppress such membrane clogging and extend the lifespan of the hollow fiber membrane filter 20.

[0045] Figure 7 shows the operation during backwashing in comparison to the normal operation during the perfusion culture period. Normal operation refers to the period of perfusion culture other than backwashing. As shown in Figure 7(A), under normal conditions, the valve 66 is set to the first state. In the first state, the flow path between the culture tank 11 and the hollow fiber membrane filter 20 is open, and the flow path from the hollow fiber membrane filter 20 to the waste liquid tank 63 is blocked. In the first state, circulation between the culture tank 11 and the hollow fiber membrane filter 20 is possible. In this state, the circulation pump 57 and the pump 18 of the pipeline 52 operate (indicated as [ON] in Figure 7). The pumps 18 of the pipeline 65 and pipeline 67 are stopped (indicated as [OFF] in Figure 7). As a result, while the culture medium 30 is circulating, the antibody 33 permeates the hollow fiber membrane 22 in the hollow fiber membrane filter 20, and the antibody 33 is separated from the cells 32. The culture medium 30 containing the separated antibody 33 is collected in the recovery tank 15 as permeate 35.

[0046] In contrast, as shown in Figure 7(B), during backflow cleaning, the valve 66 is set to the second state, opening the flow path from the hollow fiber membrane filter 20 to the waste liquid tank 63, and blocking the flow path between the culture tank 11 and the hollow fiber membrane filter 20. In this state, the pumps 18 in pipelines 65 and 67 operate (indicated as [ON] in Figure 7). The circulation pump 57 and the pump 18 in pipeline 52 stop (indicated as [OFF] in Figure 7). As a result, the circulation of the culture medium 30 is temporarily interrupted, and in this state, the cleaning solution 62 is introduced into the hollow fiber membrane filter 20, performing backflow cleaning of the hollow fiber membrane filter 20. The cleaning solution 62 that has passed through the hollow fiber membrane filter 20 is collected as waste liquid 64 in the waste liquid tank 63 via pipeline 67.

[0047] Figure 8 schematically shows the state inside the hollow fiber membrane filter 20 during normal operation and during backwashing. In the normal operation shown in Figure 8(A), a portion of the culture medium 30 containing the antibody 33 flows from the primary channel 23 into the secondary channel 24 as permeate 35 within the hollow fiber membrane filter 20. Under normal conditions, over time, clogging of the membrane by clogging material 36 progresses, and the clogging material 36 clogs the pores 22h of the hollow fiber membrane 22 and eventually accumulates on the membrane surface on the primary channel 23 side. On the other hand, during backwashing shown in Figure 8(B), washing solution 62 is introduced into the hollow fiber membrane filter 20 from the inlet 20d. The washing solution 62 then flows out from the secondary channel 24 side through the hollow fiber membrane 22 to the primary channel 23 side. During this process, the cleaning solution 62, due to the pressure of its flow, detaches the clogging material 36 that has accumulated in the pores 22h of the hollow fiber membrane 22, as well as a portion of the clogging material 36 that has accumulated on the membrane surface on the primary channel 23 side, from the hollow fiber membrane 22. The clogging material 36 detached from the hollow fiber membrane 22 mixes with the cleaning solution 62 and is discharged outside the hollow fiber membrane filter 20 through the primary channel 23. The cleaning solution 62 containing the clogging material 36 is collected in the waste liquid tank 63 through the pipeline 67. In this way, during cleaning, the cleaning solution 62 flows from the secondary channel 24 towards the primary channel 23. This flow direction of the cleaning solution 62 is opposite to the normal flow direction of the culture medium 30, which flows from the primary channel 23 to the secondary channel 24, and is therefore called backflow cleaning.

[0048] Figure 9 is a graph showing the change in flow pressure P of the primary channel 23 over time when such backwashing is performed during the perfusion culture period. In the example shown in Figure 9, for example, backwashing is performed once a day from the beginning of the perfusion culture period (for example, on the second day of the perfusion culture period). The washing time for each backwash is 80 minutes. When backwashing is performed, some of the clogging material 36 is removed, causing a temporary decrease in flow pressure P. After that, when the circulation of the culture medium 30 is resumed, membrane clogging progresses again, and the flow pressure P rises. Therefore, if backwashing is repeated during the perfusion culture period, the decrease and increase in flow pressure P will be repeated daily, as shown in the graph in Figure 9. Of course, even if backwashing is performed, not all of the clogging material 36 is removed and the hollow fiber membrane filter 20 returns to its initial state, and some of the clogging material 36 remains deposited on the hollow fiber membrane 22. Therefore, even when backwashing is performed, membrane clogging is not completely resolved, and membrane clogging gradually progresses. Therefore, as shown in the graph in Figure 9, even with backflow cleaning, the flow path pressure P increases in the long term. However, compared to the graph in Figure 6, where backflow cleaning is not performed, the time required to reach the same flow path pressure P (for example, 10 [psi]) is longer in the graph in Figure 9. ΔX in Figure 9 represents the number of days that have been extended (hereinafter referred to as the extended number of days) compared to the solid line graph in Figure 6.

[0049] Performing backwashing in this manner suppresses clogging of the hollow fiber membrane filter 20, thereby extending its lifespan. However, the same cleaning effect is not obtained under all cleaning conditions, and if appropriate cleaning conditions are not selected, the expected cleaning effect may not be achieved.

[0050] As shown in Figure 10, the memory 16A of the processor 16 is set with appropriate cleaning conditions according to the technology of this disclosure as cleaning conditions for backflow cleaning. As an example of the cleaning conditions, frequency, cleaning time, and linear velocity LV are set. The frequency is, for example, once a day, and the cleaning time is 80 mins per cleaning. The linear velocity LV is set to be between 0.003 [cm / min] and 0.03 [cm / min]. The membrane area of ​​the hollow fiber membrane 22 is S [cm ２ If the flow rate per unit time of the washing liquid 62 flowing back from the secondary flow path 24 to the primary flow path 23 (called the backwash flow rate) is Q [mL / min], then LV [cm / min] = Q [mL / min] / S [cm ２ This is defined as follows. The processor 16 performs backflow cleaning control to satisfy these cleaning conditions. Here, the frequency and cleaning time of these cleaning conditions can be changed as appropriate. An essential requirement in the art of this disclosure is that backflow cleaning is performed including a period during which the linear velocity LV satisfies the above conditions. In this example, the linear velocity LV is constant for the entire duration of one backflow cleaning.

[0051] Figure 11 shows the relationship between linear velocity LV ([cm / min]) and pressure drop ΔP ([psi]) as revealed by the experiment. Pressure drop ΔP is a value that indicates the degree of decrease in flow path pressure P when backflow cleaning is performed once. As shown in Figure 11, the lower the linear velocity LV, the higher the pressure drop ΔP. In other words, when performing backflow cleaning, a higher cleaning effect can be obtained by flowing the cleaning fluid 62 relatively slowly than by flowing it forcefully. The reason for this is presumed to be as follows.

[0052] Figure 12 schematically shows the progress of backwashing when the linear velocity LV is high, and Figure 13 schematically shows the progress of backwashing when the linear velocity LV is low. Figure 12(A) shows the state in which membrane clogging of the hollow fiber membrane 22 is progressing and clogging material 36 is accumulating on the membrane surface, and backwashing with the cleaning liquid 62 is started in this state. If the linear velocity LV of the cleaning liquid 62 is high, as shown in Figure 12(B), the detachment of clogging material 36 accumulated on the membrane surface of the hollow fiber membrane 22 tends to proceed intensively in the parts where the flow velocity of the cleaning liquid 62 is relatively high. Then, as shown in Figure 12(C), it is thought that the subsequent cleaning liquid 62 will concentrate and flow into the parts in which the clogging material 36 has detached and the resistance has decreased. In that case, even if the cleaning solution 62 is continuously flowed thereafter, the cleaning solution 62 will not flow to the parts where the clogging material 36 has not detached and remains accumulated, and as a result, it is thought that the clearing of the film blockage will not progress.

[0053] In contrast, when the linear velocity LV is small, it is thought that backflow cleaning proceeds as shown in Figure 13. Figure 13(A) shows the same state as Figure 12(A), and in this state, backflow cleaning with the cleaning solution 62 is started. When the linear velocity LV of the cleaning solution 62 is small, the partial peeling of the clogging material 36 is suppressed, and as shown in Figure 13(B), the cleaning solution 62 can easily spread over a wide area of ​​the membrane surface of the hollow fiber membrane 22 in the secondary flow path 24. As a result, as shown in Figure 13(C), peeling of the clogging material 36 proceeds over a wide area of ​​the membrane surface of the hollow fiber membrane 22. As a result, as shown in Figure 13(D), the clogging material 36 is removed over a wide area of ​​the membrane surface of the hollow fiber membrane 22. Therefore, in the case of Figure 13, it is thought that a better cleaning effect can be obtained compared to the case of Figure 12, in which the peeling of the clogging material 36 is concentrated in a part of the area.

[0054] The reason why a lower linear velocity LV results in a higher cleaning effect is presumed to be as described above. Of course, if the linear velocity LV is too low, there will be insufficient energy to detach the clogging substance 36 from the surface of the hollow fiber membrane 22, and therefore no cleaning effect will be obtained. For this reason, in the technology disclosed herein, the linear velocity LV is set to an appropriate range of 0.003 [cm / min] or more and 0.03 [cm / min] or less.

[0055] Figure 14 is a flowchart showing the processing procedure executed by the processor 16 of the cell culture apparatus 10 during cell culture. When perfusion culture is instructed, in step S301, as shown in Figure 7(A), the processor 16 sets the valve 66 to the first state and operates the circulation pump 57 and the pump 18 of the pipeline 52 to start circulating the culture medium 30. Then, in step S302, when a preset timing arrives, the processor 16 moves to step S303. Then, in step S303, the circulation of the culture medium 30 is temporarily stopped, and in step S304, as shown in Figure 7(B), the valve 66 is set to the second state and the pumps 18 of the pipelines 65 and 67 are operated to start backwashing under the set conditions. The linear velocity LV is set in the range of 0.003 [cm / min] or more and 0.03 [cm / min] or less. In the example in Figure 10, the frequency of backwashing is set to once per day, so the pre-set timing is, for example, a pre-set time within a day. The above process is repeated in step S305 until it is determined that the perfusion culture is complete. The conditions for determining the end of the perfusion culture include, for example, input of a termination command by the operator, or the elapsed of a pre-set perfusion culture period.

[0056] By performing backwashing during cell culture, including a period that satisfies the above-mentioned linear velocity LV condition, a better cleaning effect than conventional methods can be obtained. As a result, the lifespan of the hollow fiber membrane filter 20 is extended compared to cases where the above conditions are not met, and an improvement in the productivity of cell culture can be expected.

[0057] Here, "during cell culture" refers to the perfusion culture period which continues for several days to several tens of days. As described above, in order to recover products such as antibodies 33 during perfusion culture, the culture medium 30 is circulated through a circulation pathway between the hollow fiber membrane filter 20 and the culture tank 11. As described above, during backwashing, the circulation of the culture medium 30 is temporarily stopped, but this backwashing period is also included in the cell culture period. In other words, the perfusion culture period refers to the period starting from day 1, when cells 32 are seeded in the culture tank 11, until the predetermined period of several days to several tens of days arrives. From day 1 onward, circulation through the circulation pathway between the hollow fiber membrane filter 20 and the culture tank 11 begins.

[0058] Furthermore, in the above embodiment, the duration of one backwash cycle is 80 minutes. To enhance the cleaning effect, a cycle duration of 10 minutes or more is preferable, 20 minutes or more is more preferable, and 40 minutes or more is even more preferable. Within the upper limit range, 40 to 100 minutes is preferable, and 60 to 100 minutes is even more preferable. A longer cleaning time can reliably improve membrane clogging, but if it is too long, productivity will decrease. The cleaning time is set taking these factors into consideration.

[0059] Furthermore, in the above embodiment, backwashing is repeated at a predetermined timing. Repeating backwashing multiple times during cell culture can suppress a significant decrease in the separation performance of the hollow fiber membrane filter 20. Moreover, in the above embodiment, the frequency of backwashing is set to once per day. To enhance the washing effect, the frequency of backwashing is preferably 1 to 10 times per day, more preferably 2 to 8 times per day, and even more preferably 3 to 6 times per day. This prevents the washing interval from becoming too long, and thus further suppresses a significant decrease in the separation performance of the hollow fiber membrane filter 20.

[0060] Furthermore, in the above embodiment, the culture medium 31 is used as the cleaning solution 62, which is an example of a liquid that flows from the secondary channel 24 to the primary channel 23. Since the culture medium 31 is a fresh culture medium and does not contain substances that will turn into clogging material 36, it has a high cleaning effect. In addition, there is less waste compared to the case where a liquid specifically for cleaning is prepared as the cleaning solution 62.

[0061] Also, in the above embodiment, the hollow fiber membrane filter 20 is of the ATF method in which a reciprocating flow of the culture solution 30 occurs in the primary flow path 23. The ATF method is said to be less likely to clog because a reciprocating flow occurs, as compared with the TFF method in which the culture solution 30 flows in one direction without a reciprocating flow. By performing backwashing by such an ATF method, further extended life of the hollow fiber membrane filter 20 can be expected.

[0062] Also, in the above embodiment, in the countercurrent washing, the reciprocating flow in the primary flow path 23 is stopped, and in this state, the cleaning liquid 62 (an example of a liquid) is made to flow countercurrently, and the cleaning liquid 62 is recovered before reaching the culture tank 11 from the hollow fiber membrane filter 20. Therefore, it is possible to suppress the liquid containing the clogging substance 36 from flowing into the culture tank 11. Since the clogging substance 36 once peeled off from the hollow fiber membrane 22 does not flow into the hollow fiber membrane filter 20 again, the progress of membrane clogging is suppressed, contributing to the extended life of the hollow fiber membrane filter 20.

[0063] Also, in the above embodiment, the viable cell density of the culture solution 30 is preferably 30×10 ６ cells / mL or more. The viable cell density is more preferably 50×10 ６ cells / mL or more, still more preferably 80×10 ６ cells / mL or more, still more preferably 120×10 ６ cells / mL or more, and even more preferably 150×10 ６ cells / mL or more. Also, the viable cell density is preferably 400×10 ６ cells / mL or less, and more preferably 250×10 <00S0014> cells / mL or less.

[0064] The viable cell density can be obtained by measuring the number of viable cells excluding dead cells by a conventional method and dividing the measured number of cells by the amount of the culture solution. Here, the viable cell density is, as an example, the viable cell density in the production period. This is because the incubator before the production period generally increases the viable cell density, making it difficult to keep the viable cell density constant.

[0065] The higher the live cell density, the more likely the hollow fiber membrane filter 20 is to become clogged. Therefore, the technology of this disclosure is particularly effective when the live cell density is above the above value. In addition, in perfusion culture, a higher live cell density is required than in fed-batch culture in order to improve productivity. In perfusion culture, 30 × 10 ６ Since the viable cell density often exceeds cells / mL, the technology described herein is particularly effective.

[0066] Furthermore, in the above embodiment, it is preferable that the titer of the product of cell 32 is 1.0 g / L or higher. More preferably 1.4 g / L or higher, and even more preferably 1.8 g / L or higher. When producing the product, the minimum target titer from the viewpoint of productivity should be 1.0 g / L or higher. The higher the titer of the product, the more likely the hollow fiber membrane filter 20 is to become clogged. The technology of this disclosure is particularly effective when the titer is equal to or higher than the above value.

[0067] The titer of the product is measured, for example, by removing coarse matter such as cells from the culture medium through a 0.2 μm syringe filter and then measuring it using a metabolic analyzer. The Cedexbio metabolic analyzer manufactured by Roche is used. Similar to the viable cell density, the titer is also measured, for example, as the titer during the production phase.

[0068] [Second Embodiment] The first embodiment was shown as an example in which the linear velocity LV was kept constant during one backwash cycle, but the linear velocity LV may be varied during one backwash cycle.

[0069] In Figure 15, graph G1 is an example of the first embodiment where the linear velocity LV is kept constant. Graphs G2 and G3 are examples of the second embodiment where the linear velocity LV is varied. Specifically, these are examples in which the linear velocity LV gradually increases from the initial stage during a single backwash cycle.

[0070] As shown in graphs G2 and G3, it is preferable that a single backwash cycle be started when the linear velocity LV is between 0.003 [cm / min] and 0.03 [cm / min]. This is because, as shown in Figure 13, it is estimated that a lower linear velocity LV at the beginning of the backwash cycle can be expected to result in a higher cleaning effect.

[0071] If the above conditions are met in the initial stages of backwashing, the linear velocity LV may not meet the above conditions afterward. In graphs G2 and G3, the linear velocity LV exceeds the upper limit of the above conditions approximately 10 minutes and 30 minutes after the start, respectively. Specifically, it is preferable that the linear velocity LV meets the above conditions for the first two minutes after the start. This results in a better cleaning effect.

[0072] Furthermore, as shown in graphs G2 and G3, it is preferable to gradually increase the linear velocity LV from the beginning during a single backwash cycle. Specifically, it is preferable to gradually increase the backwash flow rate Q by 0.1 [mL / min / min] or more. This allows for a higher cleaning effect. As shown in Figure 10, the linear velocity LV is the value obtained by dividing the backwash flow rate Q by the film area S, so the linear velocity LV increases with increasing backwash flow rate Q. Graph G2 shows an example where the backwash flow rate Q is increased by 0.2 [mL / min / min], and graph G3 shows an example where the backwash flow rate Q is increased by 0.4 [mL / min / min].

[0073] [Third Embodiment] In the first embodiment, the culture medium 31 was used as the washing solution 62, but as shown in the third embodiment in Figure 16, the permeate 35 collected in the recovery tank 15 may be used as the washing solution. In this case, a washing solution tank 61 separate from the recovery tank 15 is not required. In this configuration, under normal circumstances, as shown in Figure 16(A), the valve 66 is set to the first state and the circulation pump 57 and the pump 18 of the pipeline 52 are operated. The pump 18 of the pipeline 67 connected to the waste liquid tank 63 is stopped. Then, during backflow washing, as shown in Figure 16(B), the valve 66 is set to the second state, the rotation direction of the pump 18 of the pipeline 52 is reversed and the pump 18 of the pipeline 67 is operated. This allows the permeate 35 from the recovery tank 15 to be introduced into the hollow fiber membrane filter 20 as the washing solution, and the permeate 35 that has passed through the hollow fiber membrane filter 20 to be flowed into the waste liquid tank 63.

[0074] According to the third embodiment, since the permeate 35 is used as the liquid for cleaning, a tank dedicated to the cleaning liquid is not required, and the structure for achieving backflow cleaning can be simplified. Also, from the perspective of not using a liquid dedicated to cleaning, there is less waste.

[0075] [Other] In the first embodiment shown in Figure 7 and the third embodiment shown in Figure 16, the liquid introduced into the hollow fiber membrane filter 20 during backflow washing and used as washing solution 62 is collected in the waste liquid tank 63 instead of being discharged into the culture tank 11. However, as shown in Figures 17 to 19, the liquid used as washing solution 62 may be discharged into the culture tank 11 without being collected. According to these configurations, backflow washing can be performed without stopping the circulation of the culture solution 30.

[0076] Figure 17 shows an example where culture medium 31 is used as the washing solution 62. In the normal operation shown in Figure 17(A), the circulation pump 57 and the pump 18 in the conduit 52 are operating. The pump 18 in the conduit 65 is stopped. As a result, the permeate 35 containing the antibody 33 separated by the hollow fiber membrane filter 20 is collected in the recovery tank 15. On the other hand, in the backwash operation shown in Figure 17(B), the pump 18 in the conduit 52 is stopped, and the pump 18 in the conduit 65 is operating. The circulation pump 57 remains operating. As a result, the washing solution 62 introduced into the hollow fiber membrane filter 20 through the conduit 65 circulates back and forth within the hollow fiber membrane filter 20 and between it and the culture tank 11. In the configuration shown in Figure 17, since the washing solution 62 is the same as in the first embodiment, which is new culture medium 31, there is no problem in returning the washing solution 62 to the culture tank 11.

[0077] Figure 18 shows a modified version of the configuration shown in Figure 17, the difference being the connection point of the pipeline 65 that introduces the cleaning solution 62. In the configuration shown in Figure 17, the pipeline 65 was directly connected to the hollow fiber membrane filter 20, but in the configuration shown in Figure 18, the pipeline 65 is connected to pipeline 52, and the cleaning solution 62 is introduced to the hollow fiber membrane filter 20 via pipeline 52.

[0078] The configuration shown in Figure 19 is similar to the third embodiment in that permeate 35 is used as the washing solution, and the permeate 35 used for washing is recovered in the culture tank 11. In the configuration shown in Figure 19, the only difference between the normal operation and the operation during backflow washing is the rotation direction of the pump 18 in the pipeline 52. During backflow washing, the pump 18 is reversed to introduce the permeate 35 in the recovery tank 15 into the hollow fiber membrane filter 20. The permeate 35 used for washing is returned to the culture tank 11.

[0079] In this way, the liquid used for washing may be returned to the culture tank 11. This simplifies the configuration for achieving backflow washing, for example, by eliminating the need for a waste liquid tank 63. Furthermore, if it is permissible to return the liquid used for washing to the culture tank 11, it becomes possible to perform backflow washing while continuing the circulation of the culture medium 30.

[0080] Examples and comparative examples relating to the technology of this disclosure will be described below with reference to Tables 1 and 2.

[0081]

[0082] Table 1 shows the experimental results from actual experiments, and Table 2 shows the simulation results derived based on the experimental results in Table 1. The membrane area S is common to all backwashing conditions in experimental results 1-5 shown in Table 1. The membrane area S is 1300 [cm²]. ２ The starting time was 35 days or more after the start of the perfusion culture period, and the conditions of experimental results 1 to 5 were performed once per day. In addition, the washing solution 62 was the culture medium 31, and the used washing solution 62 was collected as waste liquid 64.

[0083] In Experiment 1-5, the backwash flow rate Q, linear velocity LV, and the washing time of a single backwash cycle were varied. Furthermore, in Experiment 1-4, the backwash flow rate Q was kept constant during the washing time, meaning the linear velocity LV was also kept constant. In contrast, in Experiment 5, the backwash flow rate Q was increased every minute. The rate of increase, shown as "acceleration" in Table 1, was 0.4 [mL / min / min]. Also, in Experiment 1-5, the backwash flow rate Q was varied from 9 to 32 [mL / min]. As a result, the linear velocity LV varied from 0.07 to 0.025 [mL / min]. The washing time also varied from 25 to 80 [min].

[0084] As shown in Table 1, comparing experimental results 1 to 3 for the same cleaning time among experimental results 1 to 4 when the linear velocity LV is constant, the pressure drop ΔP for experimental result 1 was 2.1 [psi], while the pressure drop ΔP for experimental result 3 was 2.6 [psi]. Experimental result 3, with a lower linear velocity LV, had a larger pressure drop ΔP, indicating a higher cleaning effect. As shown in Figure 11, the relationship between experimental results 1 to 4 shows that there is a correlation between linear velocity LV and pressure drop ΔP. Furthermore, the pressure drop ΔP for experimental result 5, in which the backwash flow rate Q was gradually increased to increase the speed, was 3.5 [psi], which was the largest among experimental results 1 to 5. Experimental result 5 showed that speed increase is effective.

[0085] Table 2 shows the simulation results of calculating the pressure drop ΔP for each of Examples 1 to 14 and Comparative Examples 1 to 3, based on the approximate curve representing the correlation between linear velocity LV and pressure drop ΔP derived from experimental results 1 to 5 shown in Table 1. Furthermore, based on the calculated pressure drop ΔP, the number of days until the flow path pressure P reaches 10 [psi] is shown in Figures 6 and 9.

[0086] Examples 1-14 and Comparative Examples 1-3 shown in Table 2 share the same membrane area S and the same starting timing for initiating backwashing during the perfusion culture period. The membrane area S is 1300 [cm²]. ２ The starting time is two days after the start of the perfusion culture period.

[0087] Examples 1 to 14 include a period in which the linear velocity LV during a single backwash cycle satisfies the condition of being between 0.003 [cm / min] and 0.03 [cm / min]. Of Examples 1 to 14, all but Examples 7 to 10 show a constant linear velocity LV during a single backwash cycle, while Examples 7 to 10 show an increasing velocity. The rate of increase in the backwash flow rate Q in Example 7 is 0.1 [mL / min / min], in Example 8 it is 0.2 [mL / min / min], in Example 9 it is 0.4 [mL / min / min], and in Example 10 it is 0.6 [mL / min / min]. As a result, the linear velocity LV also increases. Example 8 corresponds to G2 shown in Figure 15, and Example 9 corresponds to G3 shown in Figure 15. In both cases, the linear velocity LV satisfies the condition of being 0.003 [cm / min] or higher and 0.03 [cm / min] or lower for at least 10 minutes from the start of backwashing. In Examples 11 and 12, the frequency of backwashing is 2 times / day and 3 times / day, respectively. The frequency for all other examples and comparative examples is 1 time / day. The washing time for one backwash is 80 minutes, except for Example 5, which was 20 minutes, and Comparative Example 1, which did not perform backwashing.

[0088] In Comparative Example 1, where backflow cleaning is not performed, the number of days it takes for the flow path pressure P to reach 10 [psi] is 20 days. The evaluation was based on the 20 days in Comparative Example 1, with conditions where the extension number of days (corresponding to ΔX in Figure 9) was 2 days or more being judged as OK, and all other conditions being judged as NG. If the number of days it takes for the flow path pressure P to reach 10 [psi] is 22 days, then ΔX = 22 days - 20 days = 2. Based on these simulation results, the condition that the linear velocity LV is 0.003 [cm / min] or more and 0.03 [cm / min] or less was derived. The conditions that were judged as OK are Examples 1 to 14 related to the technology of this disclosure, and the conditions that were judged as NG are Comparative Examples 1 to 3. In Comparative Example 2, the linear velocity LV is 0.001 [cm / min], which is less than 0.003 [cm / min], and in Comparative Example 3, it is 0.04, which is greater than 0.03 [cm / min]. In both cases, the pressure drop ΔP was smaller compared to Examples 1 to 14, and the extension period ΔX was 1 day or less, and less than 2 days.

[0089] In Examples 1 to 14, among the examples where no speed increase was performed and the frequency was once a day, Example 2 had the largest ΔX (6.2 days = 26.2 days - 20 days). The linear velocity LV in Example 2 was 0.01 [cm / min]. Also, among the examples where the frequency was once a day, the ΔX for Examples 7 to 10, where speed increase was performed, was 6.0 days, 8.6 days, 15.6 days, and 27.1 days, respectively. The ΔX for Examples 7 to 10, where speed increase was performed, was generally larger than the ΔX for the example where no speed increase was performed. The linear velocity LV for Examples 7 to 10 was all 0.02 [cm / min]. According to these simulation results, assuming that the period includes a time when the linear velocity LV is between 0.003 [cm / min] and 0.03 [cm / min], it can be seen that performing speed increase or increasing the frequency contributes to improving the cleaning effect.

[0090] As described above, according to the above examples, it can be seen that including a period in the backwashing during cell culture where the linear velocity LV is 0.003 [cm / min] or higher and 0.03 [cm / min] or lower yields better results compared to the comparative example.

[0091] The above embodiments and their various modifications can be combined with each other, as long as no contradictions arise.

[0092] The above embodiments further disclose the following additional information: [Additional Information 1] A method for producing a product, comprising separating the product using a hollow fiber membrane filter, the filter having a primary channel to which a culture medium containing cells and cell products is supplied, and a secondary channel through which the product flows from the primary channel through a hollow fiber membrane, wherein the hollow fiber membrane filter is washed by backflow washing in which liquid is backflowed from the secondary channel to the primary channel, and the membrane area of ​​the hollow fiber membrane [cm² ２A method for producing a product, comprising performing a backwash wash for a period of time during which the linear velocity [cm / min], which is the backwash flow rate [mL / min] per unit, is 0.003 [cm / min] or more and 0.03 [cm / min] or less. [Note 2] The method for producing a product according to Note 1, wherein the backwash wash is started at a linear velocity that satisfies the conditions. [Note 3] The method for producing a product according to Note 2, wherein the linear velocity within 2 minutes from the start of the backwash wash satisfies the conditions. [Note 4] The method for producing a product according to Note 2 or Note 3, wherein the backwash flow rate is gradually increased to 0.1 [mL / min / min] or more during the backwash wash. [Note 5] The method for producing a product according to any one of Note 1 to Note 4, wherein the duration of one backwash wash is 10 minutes or more. [Note 6] The method for producing a product according to any one of Note 1 to Note 5, wherein the backwash wash is repeated at a predetermined timing. [Note 7] The method for producing the product according to Note 6, wherein backwashing is performed at least once per day. [Note 8] The method for producing the product according to any one of Notes 1 to 7, wherein in backwashing, the culture medium is used as the liquid. [Note 9] The method for producing the product according to any one of Notes 1 to 8, wherein in backwashing, the permeate that has permeated from the primary channel to the secondary channel is used as the liquid. [Note 10] The method for producing the product according to any one of Notes 1 to 10, wherein the hollow fiber membrane filter is an ATF type, where a reciprocating flow of culture medium occurs in the primary channel. [Note 11] The method for producing the product according to Note 10, wherein in backwashing, the reciprocating flow in the primary channel is stopped, and in that state, the liquid is backflowed, and the liquid is recovered before it reaches the culture tank from the hollow fiber membrane filter. [Note 12] The live cell density of the culture medium is 30 × 10 ６A method for producing the product according to any one of the appendix items 1 to 11, wherein the titer of the product is 1.0 g / L or more. [Appendix 13] A method for producing the product according to any one of the appendix items 1 to 12, wherein the titer of the product is 1.0 g / L or more. [Appendix 14] A separation device for separating the product using a hollow fiber membrane filter, which includes a primary channel to which the culture medium removed from the culture tank during cell culture is supplied, and a secondary channel through which the product flows permeate the hollow fiber membrane from the primary channel; and a backflow washing method for washing the hollow fiber membrane filter by backflowing liquid from the secondary channel to the primary channel, wherein the membrane area of ​​the hollow fiber membrane is [cm² ２ A cell culture apparatus comprising a processor that controls the execution of backwashing, which includes a period of time during which the linear velocity [cm / min], which is the backwashing flow rate [mL / min] per unit, satisfies the condition of being 0.003 [cm / min] or more and 0.03 [cm / min] or less.

[0093] In the above embodiment, the processes performed by the cell culture apparatus 10 are performed on any computer. Alternatively, any computer may perform these processes using a processor as hardware, a program as software, or a combination thereof. In this case, the processor is configured to work in cooperation with the program to perform the various processes in this embodiment, and can function as a unit or means in this embodiment. Furthermore, the execution order of the processes by the processor is not limited to the order described and may be changed as appropriate.

[0094] Any computer may be a general-purpose computer, a computer designed for a specific purpose, a workstation, or any other system capable of performing each process. A processor may consist of one or more pieces of hardware, and the type of hardware is not limited. For example, a processor may consist of programmable logic devices such as a CPU (Central Processing Unit), MPU (Micro Processing Unit), FPGA (Field Programmable Gate Array), dedicated circuits for performing specific processes such as an ASIC (Application Specific Integrated Circuit), GPU (Graphic Processing Unit), or NPU (Neural Processing Unit). Furthermore, the type of hardware may be a combination of different types of hardware. When multiple hardware components are configured to execute one or more processes of a processor, these components may reside in physically separate devices or in the same device. Furthermore, in any embodiment, the order of the processes performed by the processor is not limited to the order described above and may be changed as appropriate. The hardware components are composed of electrical circuits (circuits) and the like, which are combinations of circuit elements such as semiconductor elements.

[0095] Furthermore, the program may be firmware or software such as microcode. Alternatively, the program may be, for example, a set of program modules, each function of which may be implemented by a processor configured to perform its respective function. The program may be program code or multiple code segments stored on one or more non-temporary computer-readable media (e.g., storage media or other storage). The program may be divided and stored on multiple non-temporary computer-readable media located in physically separate devices. Program code or code segments may represent any combination of procedures, functions, subprograms, routines, subroutines, modules, software packages, classes, or instructions, data structures, or program statements. Program code or code segments may be connected to other code segments or hardware circuits by sending and receiving information, data, arguments, parameters, or memory contents.

[0096] The technology of this disclosure can be appropriately combined with the various embodiments and / or variations described above. Furthermore, it is understood that various configurations can be adopted without departing from the gist of the invention, and the invention is not limited to the embodiments described above. In addition, the technology of this disclosure extends to storage media for storing programs non-temporarily. The storage media are computer-readable non-temporarily storage media such as USB (Universal Serial Bus) memory, flexible disks, and CD-ROMs (Compact Disc Read Only Memory). Programs may also be provided online via a network such as the Internet. Furthermore, the technology of this disclosure extends to program products in addition to programs. Program products include all forms of products for providing programs. Like programs, program products may be stored and provided on computer-readable non-temporarily storage media, or they may be provided online.

[0097] The descriptions and illustrations presented above are detailed explanations of the technical aspects of this disclosure and are merely examples of the technical aspects. For example, the above descriptions of the structure, function, operation, and effect are examples of the structure, function, operation, and effect of the technical aspects of this disclosure. Therefore, it goes without saying that you may delete unnecessary parts, add new elements, or replace elements in the descriptions and illustrations presented above, as long as you do not deviate from the essence of the technical aspects of this disclosure. Furthermore, in order to avoid confusion and facilitate understanding of the technical aspects of this disclosure, explanations of common technical knowledge and the like that do not require special explanation to enable the implementation of the technical aspects of this disclosure have been omitted from the descriptions and illustrations presented above.

[0098] In this specification, "A and / or B" is synonymous with "at least one of A and B." That is, "A and / or B" means that it may be A alone, or B alone, or a combination of A and B. Furthermore, in this specification, the same concept as "A and / or B" applies when expressing three or more things linked by "and / or."

[0099] The disclosure of Japanese Patent Application No. 2025-006310, filed on 16 January 2025, is incorporated herein by reference in its entirety. Furthermore, all documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually indicated as being incorporated by reference.

Claims

1. A method for producing a product, comprising separating the product using a hollow fiber membrane filter, which includes a primary channel to which a culture medium containing cells and cell products is supplied, and a secondary channel through which the product flows from the primary channel through a hollow fiber membrane, wherein the hollow fiber membrane filter is washed by backflowing liquid from the secondary channel to the primary channel, and the membrane area of ​​the hollow fiber membrane [cm²] ２ A method for producing a product, comprising performing backwashing for a period of time that satisfies the condition that the linear velocity [cm / min] of the backwash flow rate [mL / min] per unit is 0.003 [cm / min] or more and 0.03 [cm / min] or less.

2. The method for producing the product according to claim 1, wherein the backwash is started at the linear velocity that satisfies the above conditions.

3. The method for producing the product according to claim 2, wherein the linear velocity within two minutes from the start of the backwash satisfies the above conditions.

4. The method for producing the product according to claim 2, wherein the backwash flow rate is gradually increased to 0.1 [mL / min / min] or more during the backwash.

5. The method for producing the product according to claim 1, wherein the time for one backwash is 10 minutes or more.

6. The method for producing the product according to claim 1, wherein the backwashing is repeated at a predetermined timing.

7. The method for producing the product according to claim 6, wherein the backwashing is performed once or more times per day.

8. The method for producing an agricultural product according to claim 1, wherein a culture medium is used as the liquid in the backwashing.

9. The method for producing an agricultural product according to claim 1, wherein, in the backwashing, the liquid used is the permeate that has permeated from the primary channel to the secondary channel.

10. The method for producing an organism according to claim 1, wherein the hollow fiber membrane filter is an ATF type in which a reciprocating flow of the culture medium occurs in the primary channel.

11. The method for producing an organism according to claim 10, wherein, in the backflow washing, the reciprocating flow in the primary channel is stopped, and in that state, the liquid is backflowed, and the liquid is recovered before it reaches the culture tank from the hollow fiber membrane filter.

12. The viable cell density of the culture medium is 30 × 10 ６ A method for producing the product according to claim 1, wherein the cells / mL is 1 or higher.

13. The method for producing the product according to claim 1, wherein the potency of the product is 1.0 g / L or more.

14. A separation device for separating the products using a hollow fiber membrane filter, which includes a primary channel to which a culture medium containing cells and cell products is supplied, and a secondary channel through which the products flow permeate a hollow fiber membrane from the primary channel; and a backflow washing method for washing the hollow fiber membrane filter by backflowing liquid from the secondary channel side to the primary channel side, wherein the membrane area of ​​the hollow fiber membrane [cm²] ２ A cell culture apparatus comprising a processor that controls the execution of backwashing, which includes a period of time during which the linear velocity [cm / min], which is the backwashing flow rate [mL / min] per unit, satisfies the condition of being 0.003 [cm / min] or more and 0.03 [cm / min] or less.

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