JP2021048776A - Culture method and culture device - Google Patents

Abstract

To provide a culture method and a culture device capable of suppressing a discharge amount of a bleeding liquid discharged to outside of a culture system, and capable of efficiently performing stable perfusion culture.SOLUTION: A culture method is configured to extract a culture fluid including cells from a culture tank in which cells are cultured, for performing cell separation processing, then return a cell concentrated solution generated in the cell separation processing to the culture tank for performing perfusion culture, and during the perfusion culture, discharge the cell concentrated solution to outside of a culture system for adjusting cell concentration of the culture fluid. A culture device 100 comprises: a culture tank 1 in which cells are cultured; a cell separation device 13 for performing cell separation processing of the culture fluid including cells and being extracted from the culture tank 1 for generating the cell concentrated solution; a circulation pipeline 11 for extracting the culture fluid from the culture tank 1 and sending the same to the cell separation device 13, and returning the cell concentrated solution from the cell separation device 13 to the culture tank 1 for circulating cells; and a discharge pipeline 17 for discharging the cell concentrated solution to outside of a circulation system.SELECTED DRAWING: Figure 1

Description

The present invention relates to a culture method and a culture device used for perfusion culture.

Biopharmaceuticals such as antibody drugs are produced by culturing cells in a culture medium and then purifying the substances produced by the cells. For the production of a substance, a cell line into which a gene for producing a target substance, including an antibody gene, has been introduced is used. As the cell line used for the production of the substance, a cell line that secretes the target substance with high efficiency is selected from the transgenic transformants into which the gene has been introduced.

As a method for culturing cells, there are batch culture, fed-batch culture (half batch culture), and continuous culture (perfusion culture). Batch culture is a culture method in which no medium is supplied during the culture. Fed-batch culture is a culture method in which a medium is supplied during culture but is not discharged until the culture is completed. Continuous culturing is a culturing method in which a medium is continuously supplied during culturing and the same amount of medium is continuously discharged.

Batch culture simplifies the structure and operation of the culture apparatus, thus reducing the risk of contamination. On the other hand, there is a demerit that cell proliferation is restricted because overnutrition in the early stage of culture, undernourishment in the late stage of culture, accumulation of waste products and the like occur. According to fed-batch culture, overnutrition and undernutrition are eliminated to some extent as compared with batch culture, so that long-term culture is possible, but the problem of accumulation of waste products remains.

On the other hand, according to the perfusion culture, the culture environment can be easily kept constant, and the production of the substance can be stably performed. In addition, perfusion culture allows cells to grow to high cell densities. Since it is possible to reduce the culture scale and simplify the expanded culture while ensuring the cell density required for the production of the substance, there is an advantage that the culture equipment can be downsized. It is expected as a culture method that can efficiently produce unmodified, high-purity, high-quality products while reducing equipment costs and manufacturing costs.

In general, perfusion culture is performed while discharging the cells in the culture tank together with the culture medium to a cell separation device, separating the cells from small molecules such as waste products by the cell separation device, and returning the cells to the culture tank. .. The culture tank is continuously supplied with an equal amount of fresh medium as the discharged culture medium. As the cell separation device, a device that performs filtration using a separation membrane is common.

The culture tank during perfusion culture is controlled so that the cell density of the culture solution is within a certain range. If the cell density of the culture solution in the culture tank becomes too high, oxygen and nutrients will be insufficient, and impurities will increase due to cell death. Therefore, during perfusion culture, some of the cells proliferated in the culture tank are discharged out of the culture system together with the culture solution. Such an operation of discharging a part of living cells together with a culture medium is called bleeding.

Patent Document 1 describes a technique for circulating a cell culture in a separation system as a technique that can be used for perfusion culture (see paragraph 0028 and the like). The circulation flow for the filter in the separation system includes dead-end flow, which is a flow that is substantially perpendicular to the filter surface, and cross flow (tangential flow), which is a flow that is substantially parallel to the filter surface. It is listed.

The Tangent Flow Filtration (TFF) method, which performs cross-flow filtration, is known to have a high effect of washing away foulants adhering to the separation membrane. In general, the separation membrane used for cell separation is prone to adherence and accumulation of cell-derived foulants, and therefore, when perfusion culture is continued, the membrane pores are blocked and the separation ability is reduced. It is known that fine particles and the like generated due to cell crushing, cell death and the like form gel-like substances and cause fouling even when they are smaller than the membrane pore size of the separation membrane.

According to the TFF method, the foulant adhering to the separation membrane is easily washed away, and the separation membrane is less likely to be clogged as compared with dead end flow filtration. Further, even if the foulant adheres and accumulates and the differential pressure between the membranes increases, the separation membrane can be easily backwashed, so that the service life of the separation membrane is extended. In addition, compared to dead-end flow filtration, shearing force is less likely to be applied, and cells tend to be less likely to be damaged. Therefore, as a cell separation method, a membrane separation treatment by the TFF method is widely used.

Patent Document 2 describes a closed filtration system by the Alternating Tangential Flow Filtration (ATF) method as a technique that can be used for perfusion culture (see claims, paragraphs 0009 to 0011, etc.). .. In the alternating tangential flow filtration method, filtration is performed while alternately reversing the direction of the cross flow.

According to the ATF method, since suction and discharge by the pump are repeated alternately, the flow along the film surface is repeatedly reversed, and a high effect of washing away the foulant can be obtained. Further, when suction and discharge by the pump are repeated, the flow passing through the separation membrane also repeats inversion, so that a backwashing action can be applied to the separation membrane. Therefore, according to the ATF method, as compared with the normal TFF method, the increase in the differential pressure between the membranes is less likely to proceed, and the clogging of the separation membrane is further reduced, so that the service life of the separation membrane is extended.

Patent Document 3 describes a technique for providing two outlets in a culture tank as a technique that can be used for perfusion culture. In this technique, a culture medium outlet from the cell separator and an outlet for a mixed solution of the culture medium and cells are separately provided for the culture tank. The bleeding solution (the culture solution discharged to the outside of the culture system for the purpose of bleeding) is extracted from the outlet of the mixed solution of the medium and the cells provided in the culture tank.

Japanese Unexamined Patent Publication No. 2014-217395 Japanese Patent No. 6164487 Japanese Unexamined Patent Publication No. 4-126072

In the conventional perfusion culture, bleeding for adjusting the cell density of the culture solution is performed directly on the culture tank as in Patent Document 3. Excess cells grown in the culture tank during perfusion culture are directly withdrawn from the culture tank together with a certain amount of culture medium. However, when the bleeding solution is directly extracted from the culture tank, there is a problem that the substances produced in the culture solution and the nutrients necessary for the culture are also extracted from the culture system.

When the bleeding solution is removed from the culture tank, at least a part of the substance produced by the cells is excreted from the culture system without being sent to the cell separator, so that the substance produced by the cells is properly separated.・ Recovery becomes difficult. The substance produced by the cells is not separated and recovered by the cell separator, but is recovered outside the system, so that there is a problem that the efficiency of substance production deteriorates. Substances produced by cells can be individually separated and recovered from the bleeding solution, but such separation and recovery requires extra cost.

Further, when the bleeding solution is withdrawn from the culture tank, at least a part of the nutrients in the culture tank is discharged to the outside of the culture system, which causes a problem that the medium cost is increased. It is conceivable to reuse the extracted bleeding liquid without discarding it, but if it is reused, a separate separation / recovery system is required, resulting in extra equipment costs and operating costs. In addition, since it is necessary to add a fresh medium with the excretion of nutrients, there is a possibility that the fluctuation of nutrient concentration becomes large.

As described in Patent Document 1 and Patent Document 2, the TFF method and ATF method membrane separation treatment uses cross flow, and the backwash of the separation membrane is easy, so that it is effective for cell separation. It can be said that there is. However, even if these techniques are simply applied to perfusion culture, there is room for improvement in the efficiency of perfusion culture because bleeding involves a large excretion of products and nutrients. Further, even if the separation membrane is backwashed, when the filtrate is used, the foulant is reintroduced and the foulant remains in the culture system, so that the increase in the intermembrane differential pressure is suppressed. There is room for improvement in terms of points.

On the other hand, as described in Patent Document 3, if a medium outlet from the cell separator and an outlet for a mixed solution of the medium and cells are provided, the amount of the culture solution in the culture tank can be kept constant. it can. At this time, if the flow rate ratios of each other are appropriately adjusted, the cell density of the culture solution in the culture tank can also be adjusted within a certain range. However, it is not clear how to reduce the amount of substances produced in the culture solution and the amount of nutrients required for culture.

Therefore, an object of the present invention is to provide a culture method and a culture apparatus capable of stably and efficiently performing perfusion culture by suppressing the discharge amount of the bleeding liquid discharged to the outside of the culture system.

As a result of diligent studies, the present inventors extracted the cell concentrate concentrated by the cell separation treatment to the outside of the culture system as a bleeding solution, and found that the cell density in the perfusion culture was controlled to a certain range outside the culture system. It was found that the amount of bleeding solution discharged per cell amount to be discharged was suppressed. When the cell concentrate with high cell density is discharged as a bleeding solution, the amount of cells to be extracted outside the culture system is relative to the amount of cells to be extracted outside the culture system, as compared with the case where excess cells proliferated during perfusion culture are directly extracted from the culture tank. Since the ratio of the amount of culture medium extracted to the cells is small, the amount of useful substances (products) produced by cells and the amount of nutrients discharged are also suppressed, and the cost is suppressed in a stable and efficient manner. We have found that various perfusion cultures are possible, and have completed the present invention.

That is, in order to solve the above-mentioned problems, in the culture method according to the present invention, a culture solution containing the cells is extracted from the culture tank in which the cells are cultured and subjected to cell separation treatment, and the cell concentrate produced by the cell separation treatment is performed. Is returned to the culture tank for perfusion culture, and during the perfusion culture, the cell concentrate is discharged out of the culture system to adjust the cell density of the culture solution.

Further, the culture apparatus according to the present invention includes a culture tank for culturing cells, a cell separation device for producing a cell concentrate by cell separation treatment of a culture solution containing the cells extracted from the culture tank. A circulation pipe for extracting the culture solution from the culture tank and sending it to the cell separation device, returning the cell concentration solution from the cell separation device to the culture tank to circulate the cells, and a circulation system for the cell concentration solution. It is equipped with a discharge pipe for discharging to the outside.

According to the present invention, it is possible to provide a culturing method and a culturing apparatus capable of stably and efficiently performing perfusion culturing by suppressing the amount of bleeding liquid discharged to the outside of the culturing system.

It is a schematic diagram which shows an example of the culture apparatus which concerns on 1st Embodiment of this invention. It is a schematic diagram which shows the membrane separation apparatus provided with the hollow fiber membrane filter. It is a schematic diagram which shows an example of the culture apparatus which concerns on 1st Embodiment of this invention.

Hereinafter, the culturing method and the culturing apparatus according to the embodiment of the present invention will be described with reference to the drawings. The same reference numerals are given to the configurations common to each of the following figures, and duplicate description will be omitted.

The culturing method according to the present embodiment relates to a culturing method for perfusing and culturing cells. In perfusion culture, the culture medium containing cells is extracted from the culture tank in which the cells are cultured, subjected to cell separation treatment, and the cell concentrate produced by this cell separation treatment is returned to the culture tank to continuously produce the target cells. Cultivate.

The cells to be cultured are not particularly limited. The cell to be cultured may be any of animal cells, insect cells, plant cells, microalgae, orchid algae, bacteria, yeast, fungi, algae, yeast and the like. As cells to be cultured, various antibodies such as human antibody, humanized antibody, chimeric antibody, mouse antibody, various physiologically active substances, and various useful substances useful as pharmaceutical raw materials, chemical raw materials, food raw materials, etc. are used. Producing cells are preferred, and antibody-producing cells are particularly preferred. Further, as a culture target, floating cells such as Chinese Hamster Ovary (CHO) cells are preferable.

During perfusion culture, bleeding is performed so that the cell density of the culture solution in the culture tank is within a certain range. However, in conventional perfusion culture, overgrown cells are extracted directly from the culture tank. When the bleeding solution is directly withdrawn from the culture tank, a large amount of useful substances secreted into the culture solution and nutrients required for culture are also withdrawn from the culture system.

In such a case, the extracted products and nutrients are wasted, and even if they are separated and recovered, another system and treatment are required, which is costly. In addition, since it is necessary to add a fresh medium, the fluctuation of the nutrient concentration in the culture tank becomes large, and the culture environment may become unstable.

Therefore, in the culture method according to the present embodiment, during the perfusion culture, the cell concentrate produced by the cell separation treatment is discharged to the outside of the culture system as a bleeding solution. The cell separation treatment is a treatment for separating the cells contained in the culture medium from small molecules smaller than the cells, for example, useful substances (products) produced by the cells, waste products, and the like.

By discharging the cell concentrate as a bleeding solution to the outside of the culture system during perfusion culture, the cell density of the culture solution in which the cells are cultured can be adjusted. Since the cell concentrate is concentrated in a state where the cell density is higher than that of the culture solution in the culture tank, the amount of useful substances produced by the cells and the amount of nutrients discharged are suppressed.

Hereinafter, the culture method according to the present embodiment will be specifically described together with the culture apparatus used for perfusion culture.

FIG. 1 is a schematic view showing an example of a culture apparatus according to an embodiment of the present invention.
As shown in FIG. 1, the culture apparatus 100 according to the present embodiment separates the culture tank 1, the medium supply pipe 6, the circulation pipe 11 (extraction pipe 11a, return pipe 11b), and the cell separation device 13. Liquid discharge pipe 14, bleeding liquid discharge pipe (drainage pipe) 17, backwash liquid supply pipe 20, extraction pipe valve 23, return pipe valve 24, discharge pipe valve 25, equipment associated therewith, etc. It has.

The culture device 100 is a device for perfusing and culturing cells. The culture device 100 extracts the cells cultured in the culture tank 1 together with the culture solution into the cell separation device 13, performs cell separation treatment in the cell separation device 13, and returns the cells separated by the cell separation treatment to the culture tank 1. It is composed.

The culture device 100 includes a membrane separation device that performs filtration (membrane separation treatment) using a separation membrane as the cell separation device 13 that performs the cell separation treatment. The cell separation device 13 shown in FIG. 1 is a tangential flow filtration (TFF) type membrane separation device.

The culture tank 1 is a tank for culturing cells in a culture solution. The culture tank 1 can be provided with various devices necessary for perfusion culture of cells. In FIG. 1, the culture tank 1 is provided with a spurger 2 for aeration, a stirrer 3 for mechanical stirring, and an insertion type cell densitometer 4 for measuring the cell density of the culture tank 1. However, the culture tank 1 may be provided with an appropriate device as a ventilation device, a stirring device, and a sensor for monitoring the culture environment and the cell mass.

As the stirring device, the culture solution can be flowed in each of the radial direction, the circumferential direction, and the height direction of the culture tank 1 from the viewpoint of promoting cell suspension, oxygen dissolution, homogenization of the culture solution, and the like. The device is preferably used. Examples of such a stirring device include a device for moving the stirring blade up and down or three-dimensionally, a device for moving the culture tank up and down or three-dimensionally, a device having a plurality of stirring axes multiaxial in a plurality of directions, and stirring. An example is a device equipped with a baffle that changes the direction of the flow.

The culture tank 1 can be formed of a metal such as stainless steel or an aluminum alloy, or an appropriate material such as plastic or glass. The stainless steel tank has excellent detergency and sterility and is suitable for repeated use. The resin tank is suitable for use as a disposable product, and cleaning and sterilization of the culture tank 1 can be omitted.

The culture tank 1 can also be provided as a flexible culture bag. The flexible culture bag can be used as a disposable product. The flexible culture bag can be formed using, for example, a multilayer film made of a resin such as ethylene vinyl acetate, ethylene vinyl alcohol, low density polyethylene, or polyamide.

As shown in FIG. 1, a medium supply tank 7 is connected to the culture tank 1 via a medium supply pipe 6. The culture medium supply pipe 6 is provided with a culture medium supply pump 8. The medium supply tank 7 is a container in which the fresh medium required for perfusion culture is prepared. The fresh medium prepared in the medium supply tank 7 is supplied to the culture tank 1 by the medium supply pump 8 during the perfusion culture.

Further, an oxygen gas supply device 10 is connected to the culture tank 1 via an oxygen gas supply pipe 9. The oxygen gas supply device 10 is a device that supplies oxygen gas necessary for culturing cells. During the perfusion culture, the oxygen gas is supplied from the oxygen gas supply device 10 to the spurger 2 of the culture tank 1 and is dispersed in the culture solution.

Further, a cell separation device 13 is connected to the culture tank 1 via a circulation pipe 11. The circulation pipe 11 extracts the culture solution from the culture tank 1 and sends it to the cell separation device 13, and returns the culture solution (cell concentrate) in which the cells are concentrated in the cell separation device 13 from the cell separation device 13 to the culture tank 1. It is a pipe that circulates cells in a closed ring circulation system.

The circulation pipe 11 is composed of an extraction pipe 11a for extracting the culture solution from the culture tank 1 and a return pipe 11b for returning the cell concentrate to the culture tank 1. The circulation pipe 11 and the cell separation device 13 form a circulation system for circulating the culture solution containing cells to the culture tank 1.

One end of the extraction pipe 11a is connected to the culture tank 1. The other end of the extraction pipe 11a is connected to the liquid inlet of the cell separation device 13. The extraction pipe 11a is provided with a culture solution extraction pump 12 and an extraction pipe valve 23 in this order from the upstream side.

One end of the return pipe 11b is connected to the liquid outlet on the primary side of the cell separation device 13. The other end of the return pipe 11b is connected to the culture tank 1. The return pipe 11b is provided with a return pipe valve 24. As the return piping valve 24, either a flow rate control valve that can adjust the flow rate or a switching valve that switches between fully open and fully closed can be used.

At the time of perfusion culture, the culture solution in which the cells are suspended is filled in the culture tank 1 so as to have a predetermined amount. The extraction pipe valve 23 and the return pipe valve 24 are normally controlled to be in an open state during perfusion culture. The culture solution in the culture tank 1 is withdrawn into the cell separation device 13 by the culture solution extraction pump 12.

The cell separation device 13 is provided for cell separation processing of the culture solution extracted from the culture tank 1 to separate cells contained in the culture solution and small molecules smaller than the cells from each other. There is. In FIG. 1, as the cell separation device 13, a TFF type membrane separation device is provided. As the separation membrane of the membrane separation device, an ultrafiltration membrane having a membrane pore size smaller than the cell size is used.

A separation liquid tank 15 is connected to the cell separation device 13 via a separation liquid discharge pipe 14. The separation liquid discharge pipe 14 is provided with a separation liquid pump 16. The separation liquid tank 15 is a container for collecting small molecules separated from cells in the cell separation device 13, for example, useful substances (products) produced by the cells, waste products, and the like. The culture solution that has flowed into the cell separation device 13 is sucked through the separation membrane by the separation solution pump 16 during perfusion culture. Negative pressure is applied to the secondary side of the separation membrane, and operating pressure is applied between the separation membranes.

FIG. 2 is a schematic view showing a membrane separation device including a hollow fiber membrane filter.
As shown in FIG. 2, as the cell separation device 13, for example, an internal pressure filtration type membrane separation device 130 including a hollow fiber membrane filter can be used. The membrane separation device 130 includes a casing 131 and a plurality of hollow fiber membrane filters 132.

The casing 131 is provided, for example, in a tubular shape, and an inlet side header 133 is attached to one end side and an outlet side header 134 is attached to the other end side. The inlet side header 133 is provided with a liquid inlet on the primary side of the hollow fiber membrane filter 132, and one end side of each hollow fiber membrane filter 132 is connected. Further, the outlet side header 134 is provided with a liquid outlet on the primary side of the hollow fiber membrane filter 132, and the other end side of each hollow fiber membrane filter 132 is connected.

The inner space of the hollow fiber membrane filter 132 is provided so as to communicate with the primary side liquid inlet provided in the inlet side header 133 and the primary side liquid outlet provided in the outlet side header 134, respectively. An extraction pipe 11a can be connected to the inlet side header 133. Further, the return pipe 11b can be connected to the outlet side header 134. A liquid outlet 135 on the secondary side of the hollow fiber membrane filter 132 is provided in the space inside the other end side of the casing 131. A separation liquid discharge pipe 14 can be connected to the liquid outlet 135.

In the TFF type membrane separation device, the liquid to be treated flows into the inner space of the hollow fiber membrane filter 132 and flows along the length direction. When a predetermined operating pressure is applied between the membranes by the negative pressure on the secondary side, substances smaller than the pore size of the hollow fiber membrane filter 132 permeate from the primary side to the secondary side and flow out from the space inside the casing 131. To do. On the other hand, a substance larger than the pore size of the hollow fiber membrane filter 132 does not permeate from the primary side to the secondary side, flows through the inner space of the hollow fiber membrane filter 132 along the length direction, and flows out from the outlet side header 134. ..

According to the internal pressure filtration type membrane separation device 130 provided with the hollow fiber membrane filter 132, cells in the culture solution flowing in as the liquid to be treated flow along the inner space of the hollow fiber membrane filter 132 along the length direction, so that friction occurs. , Cells are less likely to be damaged by shearing force due to collision or the like. Further, since the membrane separation treatment can be performed by increasing the flow rate of the liquid to be treated, the speed efficiency of cell separation can be increased.

According to such a cell separation device 13, the culture solution extracted from the culture tank 1 is subjected to cell separation treatment, and the culture solution (cell concentrate) in which the target cells are concentrated and the target cells are substantially separated. It is separated into a culture solution (separation solution) that does not contain but contains small molecules such as useful substances and waste products. The separation liquid that has permeated to the secondary side of the separation membrane flows out to the separation liquid discharge pipe 14 and is collected in the separation liquid tank 15. On the other hand, the cell concentrate that does not permeate the separation membrane and is concentrated on the primary side flows out to the return tube 11b.

As shown in FIG. 1, a backwash liquid supply tank 21 is connected to the cell separation device 13 via a backwash liquid supply pipe 20. The backwash liquid supply pipe 20 is a pipe for supplying the backwash liquid to the secondary side of the separation membrane. The backwash liquid supply pipe 20 can be connected to, for example, a wash liquid inlet 136 (see FIG. 2) provided on the secondary side of the separation membrane of the cell separation device 13. The backwash liquid supply pipe 20 is provided with a backwash liquid supply pump 22. The backwash liquid supply tank 21 is a container in which a backwash liquid for backwashing the separation membrane is prepared. The backwash liquid prepared in the backwash liquid supply tank 21 is pressure-fed to the secondary side of the separation membrane of the cell separation device 13 by the backwash liquid supply pump 22 at a predetermined time.

In the culture apparatus 100, the bleeding liquid discharge pipe 17 is connected to the return pipe 11b constituting the circulatory system. The bleeding liquid discharge pipe 17 branches from the return pipe 11b and is connected to the bleeding liquid tank 18. The bleeding liquid discharge pipe 17 is provided with a discharge pipe valve 25 and a bleeding liquid pump 19 in this order from the upstream side. As the discharge pipe valve 25, either a flow rate control valve that can adjust the flow rate or a switching valve that switches between fully open and fully closed can be used. The bleeding liquid discharge pipe 17 is a bleeding pipe for discharging the cell concentrate generated by the cell separation device 13 to the outside of the circulatory system.

In a circulatory system in which a culture solution containing cells is circulated to a culture tank 1, if a bleeding solution discharge pipe 17 is provided after the cell separation device 13, the cell concentrate returned to the culture tank 1 is circulated as a bleeding solution. It can be discharged to the outside of the system. That is, in order to control the cell density in the perfusion culture within a certain range, the cell concentrate in which the cells are concentrated is discharged out of the circulatory system, and the cell density of the culture solution in the culture tank 1 is adjusted to the decreasing side. Can be done.

The cell concentrate is concentrated in a state of high cell density in which the amount of cells per unit amount is larger than that of the culture solution in the culture tank 1. Therefore, when the cell concentrate is used as the bleeding solution, the ratio of the amount of the culture solution extracted outside the culture system to the amount of cells to be extracted outside the culture system is higher than that in the case of directly extracting the culture solution in the culture tank 1. It becomes smaller. Since the amount of useful substances produced by cells and the amount of nutrients excreted are suppressed, stable and efficient perfusion culture with reduced cost can be performed.

In the culture apparatus 100, the cell density of the culture solution in the culture tank 1 can be adjusted by adjusting the discharge amount of the bleeding solution (cell concentrate) discharged to the outside of the circulatory system through the bleeding solution discharge pipe 17. For example, if the cell density of the culture solution in the culture tank 1 becomes too high, cell death or unnecessary differentiation occurs. Therefore, the amount of bleeding solution discharged is increased to increase the cell density of the culture solution in the culture tank 1. Can be reduced.

In the culture apparatus 100, the following mathematical formulas (1) and (2) are established with respect to the balance of the amount of the culture solution and the amount of cells.
F ₃ = F _0- F _1- F ₂ ... (1)
_{V · (N-N 0)} = F 2 · Δt · N · F 0 / (F 0 -F 1) ··· (2)

However, each symbol in the formula is as follows. It is assumed that Δt is a time sufficiently shorter than the doubling time of cells.
V: Amount of culture solution charged into culture tank 1 [m ³ ]
N: Cell density of culture tank 1 [cells / m ³ ]
N ₀ : Control target value of cell density in culture tank 1 [cells / m ³ ]
F ₀ : Flow rate of extraction pipe 11a [m ³ / s]
F ₁ : Flow rate of the separation liquid discharge pipe 14 [m ³ / s]
F ₂ : Flow rate of bleeding liquid discharge pipe 17 [m ³ / s]
F ₃ : Flow rate of medium supply pipe 6 [m ³ / s]
Δt: Operating time of the bleeding liquid pump 19 [s]

Since the concentration rate in the cell separator 13 is _{represented by F 0} / (F ₀ −F ₁ ), the increase in cell density due to concentration is represented by NF ₀ / (F _{0 −F 1).} As expressed by the mathematical formula (2), when the increment of the cell density is discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17, the cell density of the culture solution in the culture tank 1 does not increase.

F ₃ is represented by _{F 3} = CSPR · N ₀ · V using the medium consumption per cell (Cell Specific Perfusion Rate: CSPR). _{Therefore, the discharge flow rate F 2} and the operation time Δt required for the operation of the bleeding liquid pump 19 are expressed by the following mathematical formulas (3) to (4).
F ₂ = F _0- F _1- CSPR ・ N ₀・ V ・ ・ ・ (3)
Δt = V · (N−N ₀ ) · (F ₀ −F ₁ )
/ {(F _0- F _1- CSPR / N ₀ / V) / N / F ₀ } ... (4)

Also, emissions F ₂ · Delta] t bleeding liquid to be discharged out of the circulatory system through the bleeding liquid discharge tube 17 is expressed by the following equation (5).
F ₂ · Δt = V · (N−N ₀ ) · (F ₀ −F ₁ ) / ( _{NF 0} ) ··· (5)

On the other hand, when the bleeding solution is directly withdrawn from the culture tank 1 as in the conventional case, the following mathematical formula (6) is established if the _{withdrawal amount is V 1} [m ^3].
_{V · (N-N 0)} = V 1 · N ··· (6)

Comparing the discharge amount F ₂ · Δt of the formula (5) with the withdrawal amount V ₁ of the formula (6), in order to control the cell density in the perfusion culture within a certain range, the bleeding liquid discharge pipe 17 is used outside the circulatory system. It can be seen that the discharge amount F _{2 · Δt of the bleeding liquid to be discharged to is smaller by (F 0} −F ₁ ) / F ₀ times as compared with the case where the bleeding liquid is directly withdrawn from the culture tank 1.

The magnification of (F ₀ −F ₁ ) / F ₀ represents the reciprocal of the enrichment rate in the cell separator 13. This ratio, when discharged bleeding fluid through bleeding liquid discharge tube 17, as compared with the case where withdrawn directly from the culture tank 1, which means that the flow amount corresponding to the discharged flow rate F ₁ is reduced. When the bleeding liquid discharge pipe 17 is used, it means that the flow rate of the separation liquid discharged through the separation liquid discharge pipe 14 is kept in the circulation system.

Further, since the discharge amount F ₂ · Δt of _{the bleeding liquid becomes smaller by (F 0} −F ₁ ) / F ₀ times, F ₁ is fixed to make F ₀ smaller, or F ₀ is fixed to make F ₁ It can be seen that if the amount is increased, the amount of emissions will be even smaller (however, F ₁ <F ₀ ). By reducing F _{0 or increasing F 1} , the cell density of the culture medium can be reduced with less bleeding solution, so that the amount of useful substances and nutrients discharged can be further suppressed. Become.

In the culture apparatus 100, the control apparatus 5 includes the measurement result (N) of the cell density of the culture tank 1 measured by the cell densitometer 4, the current flow rate (F ₀ ) of the extraction pipe 11a, and the current discharge of the separated liquid. Based on the flow rate of the pipe 14 (F ₁ ), the flow rate of the bleeding liquid discharge pipe 17 (F ₂ ), the operating time of the bleeding liquid pump 19 (Δt), and the discharge amount of the bleeding liquid (F ₂ · Δt) are calculated. It can be equipped with a function.

Further, the control device 5 displays calculation results such as the flow rate of the bleeding liquid discharge pipe 17 (F ₂ ), the operating time of the bleeding liquid pump 19 (Δt), and the discharge amount of the bleeding liquid (F ₂ · Δt) on the display device. Can be displayed. Examples of the display device include a liquid crystal display, a plasma display, an organic EL display, a cathode ray tube, and the like. With such a display, the user of the incubator 100 can manually operate the bleeding and monitor the bleeding.

Further, the control device 5 includes the output of the medium supply pump 8, the culture solution extraction pump 12, the separation solution pump 16 and the bleeding solution pump 19, and the opening degree of the extraction piping valve 23, the return piping valve 24, and the discharge piping valve 25. And other functions to control the liquid feeding system and ventilation system can be provided. The output of bleeding pump 19, opening of the return line valve 24 and the exhaust pipe valve 25, F _2, Delta] t, may be controlled according to the result of such F 2 _· Δt.

In the culture device 100, the cell concentrate that has flowed out of the cell separation device 13 during perfusion culture can be intermittently discharged to the outside of the circulatory system, or can be continuously discharged to the outside of the circulatory system. That is, the return pipe valve 24 and the discharge pipe valve 25 can be opened and closed alternately, and both the return pipe valve 24 and the discharge pipe valve 25 can be maintained at an intermediate opening degree.

When intermittent bleeding is performed, the return pipe valve 24 is controlled to be fully closed, and the discharge pipe valve 25 is controlled to be fully open. The return pipe valve 24 and the discharge pipe valve 25 have a duty ratio of opening and closing, and the total opening time when the bleeding liquid discharge pipe 17 side is opened under _{a predetermined flow rate (F 2) of the bleeding liquid discharge pipe 17 is bleeding.} It can be set so as to be substantially equal to the operating time (Δt) of the liquid pump 19.

Intermittent bleeding can be performed at appropriate time intervals during perfusion culture. When bleeding is performed, substantially all of the cell concentrate flowing out of the cell separation device 13 is flowed from the return pipe 11b to the bleeding liquid discharge pipe 17 and discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17. During this period, the cell separation process is continued, but the return to the culture tank 1 is interrupted. According to the intermittent bleeding, the return pipe valve 24 and the discharge pipe valve 25 can be easily controlled.

On the other hand, when continuous bleeding is performed, the return pipe valve 24 and the discharge pipe valve 25 are controlled to have an intermediate opening degree. The operating time (Δt) of the bleeding liquid pump 19 can be set within the range of the cell doubling time, and the flow rate (F ₂ ) of the bleeding liquid discharge pipe 17 can be set below that. The opening degree of each of the return pipe valve 24 and the discharge pipe valve 25 can be set, and the output of the bleeding liquid pump 19 can be selected.

When performing continuous bleeding, a part of the cell concentrate flowing out of the cell separation device 13 is diverted from the return pipe 11b to the bleeding liquid discharge pipe 17 and discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17. .. During this period, the cell separation process and the return to the culture tank 1 are continued. According to continuous bleeding, fluctuations in cell density of the culture medium and fluctuations in the culture environment in the culture tank 1 can be reduced.

Regarding the balance of the circulation system, the culture apparatus 100 has an average discharge amount of the bleeding liquid discharged through the bleeding liquid discharge pipe 17 per unit time and a cell concentrate returned from the cell separation device 13 to the culture tank 1 per unit time. The total of the average return amount and the average amount of the separated solution extracted from the cell separation device 13 to the separation solution discharge pipe 14 per unit time is the unit of the culture solution extracted from the culture tank 1 to the cell separation device 13. It is operated so as to be approximately equal to the average withdrawal amount per hour.

Therefore, in the medium supply pump 8 of the culture device 100, the average supply amount of fresh medium supplied through the medium supply pipe 6 per unit time is the average per unit time of the culture solution extracted from the culture tank 1 to the cell separation device 13. It can be controlled so as to be substantially equal to the difference between the withdrawal amount and the average return amount of the cell concentrate returned from the cell separation device 13 to the culture tank 1 per unit time. When intermittent bleeding is performed, the flow rate of fresh medium supplied by the medium supply pump 8 can be variably controlled.

In the culture device 100, the concentration rate in the cell separation device 13 may be controlled to be constant or variably. The amount of bleeding liquid discharged F ₂ · Δt means the reciprocal of the concentration rate (F ₀ −F ₁ ) / F ₀ times, so it becomes smaller when F ₀ is made smaller or F ₁ is made larger. The cell density of the culture solution in the culture tank 1 can be reduced with a small amount of bleeding solution.

From the viewpoint of reducing the amount of such bleeding solution discharged, when the bleeding solution (cell concentrate) is discharged out of the culture system, an operation of increasing the cell concentration rate in the cell separation process is performed and the cells are discharged out of the culture system. It is also possible to increase the cell density of the cell concentrate. For example, in the case of intermittent bleeding, an operation of increasing the cell concentration rate in the cell separation process can be performed so as to synchronize with the return pipe valve 24 and the discharge pipe valve 25.

Examples of the operation of increasing the cell concentration rate include an operation of reducing the flow rate of the culture solution extracted from the culture tank 1, an operation of increasing the intermembrane differential pressure of the separation membrane used for the cell separation treatment, and a plurality of lines. Examples thereof include an operation of limiting the line of use of the cell separation device 13. These operations may be used alone or in combination of two or more.

Examples of operations for reducing the flow rate of the culture solution extracted from the culture tank 1 include an operation of adjusting the flow rate of the culture solution extraction pump 12 in the decreasing direction and an operation of controlling the opening degree of the extraction piping valve 23 in the closing direction. Can be used. Further, as an operation of increasing the intermembrane differential pressure of the separation membrane used for the cell separation treatment, for example, an operation of adjusting the flow rate of the separation liquid pump 16 in the increasing direction can be used.

Further, in the culture apparatus 100, the separation membrane of the cell separation apparatus 13 can be backwashed by using the system of the backwash liquid supply pipe 20. The backwash of the separation membrane can be performed before the start of the perfusion culture, after the end of the perfusion culture, or intermittently during the perfusion culture. When performed during perfusion culture, the culture solution extraction pump 12, the separation solution pump 16, and the backwash solution supply pump 22 are operated alternately to perform backwash intermittently.

Generally, in a cell separation treatment using a separation membrane, a foulant such as a host cell protein (HCP) may adhere to the separation membrane and deposit on the membrane surface as the treatment is continued. Since the separation membrane is covered with a gel-like substance due to foulant, the differential pressure between the membranes gradually increases, eventually leading to rupture and loss of separation ability.

On the other hand, when the system of the backwash liquid supply pipe 20 is provided, the separation membrane used for the cell separation treatment is intermittently backwashed with the backwash liquid supplied from outside the culture system, and the foulant is contained in the cell concentrate. Can be floated in. When the backwash solution is pumped to the secondary side of the separation membrane of the cell separation device 13, the backwash solution flows from the secondary side to the primary side of the separation membrane, and the foulant adhering to the separation membrane moves to the cell concentrate side. It will peel off. Therefore, the foulant can be discharged from the cell separation device 13 together with the cell concentrate on the primary side.

As the backwash solution, for example, a fresh medium, a buffer solution, or the like can be used. The fresh medium and buffer solution are liquids that do not contain cells or fine particles such as HCP and have a small effect on the culture environment. Therefore, when a fresh medium, a buffer solution, or the like is used, new foulant is rarely introduced into the circulatory system, and an increase in the intermembrane differential pressure due to backwashing of the separation membrane is suppressed. In addition, fluctuations in the culture environment due to backwashing of the separation membrane are less likely to occur.

The backwash of the separation membrane of the cell separation device 13 can be performed at the same time as the intermittent bleeding, at a different time from the intermittent bleeding, or intermittently during the continuous bleeding. You can also do it. When the separation membrane is backwashed at the same time as intermittent bleeding or during continuous bleeding, the foulant suspended in the cell concentrate is discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17. Therefore, the differential pressure between the membranes is less likely to increase, and the service life of the separation membrane is extended.

The backwashing of the separation membrane of the cell separation device 13 can be performed, for example, when the intermembrane differential pressure of the separation membrane exceeds a preset threshold value. As the fouling of the separation membrane progresses, many of the membrane pores of the separation membrane are blocked, and the differential pressure between the membranes does not decrease even if backwashing is performed. In such a case, it is necessary to replace the separation membrane, but replacement involving opening work may cause contamination. Therefore, from the viewpoint of reducing the frequency of replacement of the separation membrane, it is preferable to set a small intermembrane differential pressure value close to the normal pressure range, such as the maximum value of the normal intermembrane differential pressure, as the threshold value.

The intermembrane differential pressure of the separation membrane can be measured, for example, by setting a differential pressure gauge 26 at the outlet of the cell separation device 13. The intermembrane differential pressure of the separation membrane is based on, for example, an apparent value such as a pressure difference between the inlet and outlet of the cell separation device 13, a flow rate of a culture solution, a flow rate of a cell concentrate, a flow rate of a separation solution, a hydrostatic pressure, and the like. It can also be obtained as an effective value to be analyzed.

In the culture apparatus 100, the cell concentrate discharged to the outside of the culture system through the bleeding liquid discharge pipe 17 is subjected to an appropriate separation / purification treatment, and the substance produced by the cells can be recovered. As the separation / purification treatment, a semipermeable membrane separation treatment or a centrifugation treatment is preferably used. As the semipermeable membrane separation treatment, total filtration by dead end flow filtration is particularly preferable. By using these separation / purification treatments, high-purity useful substances with few impurities can be efficiently recovered from the cell concentrate in the state where the cells are concentrated.

According to the above-mentioned culture apparatus 100 and the culture method using the above, during perfusion culture, the cell concentrate produced by the cell separation treatment is discharged out of the culture system to adjust the cell density of the culture solution in the culture tank. can do. Therefore, it is possible to suppress the discharge amount of the bleeding solution per cell discharged to the outside of the culture system during perfusion culture, as compared with the case where the excess cells that have proliferated are directly extracted from the culture tank. By suppressing the excretion of the culture solution, the excretion of useful substances produced by the cells and the excretion of nutrients are also suppressed, so that it is necessary to spend a great deal of cost on separation and recovery, and a large amount of fresh medium is used. No need to add. For example, it is possible to reduce the recovery load of the product and the waste of the medium without separately providing the separation / recovery equipment for the bleeding liquid. Therefore, it is possible to simplify such separation / recovery equipment, and if the lost value of the product or medium is lower than the operating cost of the separation / recovery equipment, the separation / recovery equipment can be omitted. become. That is, since the culture environment and substance productivity can be stabilized while suppressing the cost and the supply of the fresh medium, the perfusion culture can be performed stably and efficiently.

Further, according to the above-mentioned culture apparatus 100 and the culture method using the above, the foulant in the circulatory system is discharged to the outside of the system together with bleeding, so that the service life of the separation membrane used for the cell separation treatment can be extended. The action of discharging such foulants is further enhanced by supplying a backwash solution to the secondary side of the separation membrane used for the cell separation treatment. Therefore, it is possible to reduce the replacement cost of the separation membrane and the risk of contamination associated with the replacement work. Since the cell separation device 13 is a TFF type membrane separation device, the cell separation process and the recovery of useful substances can be continuously performed with less pulsation.

FIG. 3 is a schematic view showing an example of the culture apparatus according to the embodiment of the present invention.
As shown in FIG. 3, the culture device 200 according to the present embodiment includes the culture tank 1, the medium supply pipe 6, the cell separation device 13, the separation liquid discharge pipe 14, and the separation liquid discharge pipe 14, similarly to the culture device 100 described above. It is provided with a bleeding liquid discharge pipe (discharge pipe) 17, a backwash liquid supply pipe 20, a discharge pipe valve 25, and equipment associated therewith.

The difference between the culture device 200 according to the present embodiment and the culture device 100 is that the method of the cell separation device 13 is changed. In the culture apparatus 200, the reciprocating pipe 29, the diaphragm pump 30, and the reciprocating pipe are replaced with the circulation pipe 11 (extraction pipe 11a, return pipe 11b), culture solution extraction pump 12, extraction pipe valve 23, and return pipe valve 24. A piping valve 31 is provided.

The culture device 200 includes a membrane separation device that performs filtration (membrane separation treatment) using a separation membrane as the cell separation device 13 that performs the cell separation treatment. The cell separation device 13 shown in FIG. 3 is an alternating tangential flow filtration (ATF) type membrane separation device.

In the culture device 200, the cell separation device 13 is connected to the culture tank 1 via a reciprocating pipe 29. The reciprocating pipe 29 extracts the culture solution from the culture tank 1 and sends it to the cell separation device 13, and returns the culture solution (cell concentrate) in which the cells are concentrated in the cell separation device 13 from the cell separation device 13 to the culture tank 1. It is a pipe that circulates cells by reciprocating in a linear flow path. The reciprocating pipe 29, together with the cell separation device 13, forms a circulatory system that reciprocally circulates the culture solution containing cells to the culture tank 1.

One end of the reciprocating pipe 29 is connected to the culture tank 1. The other end of the reciprocating pipe 29 is connected to the liquid inlet / outlet of the cell separation device 13. The reciprocating pipe 29 is connected to, for example, the inlet side header 133 of the membrane separation device 130. The reciprocating pipe 29 is provided with a reciprocating pipe valve 31. As the reciprocating piping valve 31, either a flow rate control valve that can adjust the flow rate or a switching valve that switches between fully open and fully closed can be used.

Similar to the culture device 100, the cell separation device 13 of the culture device 200 is connected to the separation liquid tank 15 via the separation liquid discharge pipe 14. Further, the backwash liquid supply tank 21 is connected to the cell separation device 13 of the culture device 200 via the backwash liquid supply pipe 20 as in the culture device 100.

In the cell separation device 13 of the culture device 200, similarly to the culture device 100, the culture solution extracted from the culture tank 1 is subjected to cell separation treatment, and the cells contained in the culture solution, small molecules smaller than the cells, etc. And are provided to separate from each other. In FIG. 3, as the cell separation device 13, an ATF type membrane separation device is provided. As the separation membrane of the membrane separation device, an ultrafiltration membrane having a membrane pore size smaller than the cell size is used.

An air-driven diaphragm pump 30 is connected to the cell separation device 13 of the culture device 200 via a pipe. The diaphragm pump 30 includes a chamber 30a for driving a liquid feed and a compressor 30b for supplying compressed air to the chamber 30a.

The chamber 30a has a hollow structure, and the inside thereof is divided into a liquid to be treated chamber and a driving fluid chamber by a flexible diaphragm. The liquid chamber to be treated communicates with the primary side of the separation membrane of the cell separation device 13. The liquid chamber to be treated is connected to, for example, the outlet side header 134 of the membrane separation device 130 via a pipe or the like. The drive fluid chamber communicates with the fluid outlet of the compressor 30b.

In the diaphragm pump 30, when the compressed air in the driving fluid chamber is exhausted, the diaphragm is deformed, the volume of the driving fluid chamber is reduced, and the volume of the liquid chamber to be treated is expanded. By expanding the volume of the liquid to be treated chamber, the liquid to be treated is sucked. On the other hand, when compressed air is introduced into the driving fluid chamber from the compressor 30b, the diaphragm is deformed, the volume of the driving fluid chamber is expanded, and the volume of the liquid chamber to be treated is reduced. By reducing the volume of the liquid chamber to be treated, the liquid to be treated is discharged.

At the time of perfusion culture, the culture solution in which the cells are suspended is filled in the culture tank 1 so as to have a predetermined amount. The reciprocating piping valve 31 is normally controlled to be in an open state during perfusion culture. The culture solution in the culture tank 1 is extracted into the cell separation device 13 by the suction operation of the diaphragm pump 30. The culture solution that has flowed into the cell separation device 13 is sucked through the separation membrane by the separation solution pump 16 during perfusion culture. Negative pressure is applied to the secondary side of the separation membrane, and operating pressure is applied between the separation membranes.

The culture solution extracted from the culture tank 1 is separated into a cell concentrate and a separation solution by applying an operating pressure. The separation liquid that has permeated to the secondary side of the separation membrane flows out to the separation liquid discharge pipe 14 and is collected in the separation liquid tank 15. On the other hand, the cell concentrate that does not permeate the separation membrane and is concentrated on the primary side flows out to the reciprocating pipe 29 by the discharge operation of the diaphragm pump 30. During the discharge operation, a part of the separation liquid that has permeated to the secondary side of the separation membrane moves to the primary side, and a backwashing action is applied to the separation membrane. The cell concentrate flowing out to the reciprocating pipe 29 is returned to the culture tank 1 at normal times during perfusion culture.

In the culture apparatus 200, the bleeding liquid discharge pipe 17 is connected to the reciprocating pipe 29 that constitutes the circulatory system. The bleeding liquid discharge pipe 17 branches from the reciprocating pipe 29 and is connected to the bleeding liquid tank 18. Similar to the culture apparatus 100, the bleeding liquid discharge pipe 17 is provided with a discharge pipe valve 25 and a bleeding liquid pump 19 in order from the upstream side. As the discharge pipe valve 25 of the incubator 200, a switching valve for switching between fully open and fully closed is preferably used.

When the bleeding liquid discharge pipe 17 is connected to the reciprocating pipe 29 as in the culture device 200, the cell concentrate returned to the culture tank 1 can be discharged to the outside of the circulatory system as the bleeding liquid. That is, in order to control the cell density in the perfusion culture within a certain range, the cell concentrate in which the cells are concentrated is discharged out of the circulatory system, and the cell density of the culture solution in the culture tank 1 is adjusted to the decreasing side. Can be done.

In the culture device 200, similarly to the culture device 100, the amount of the bleeding liquid (cell concentrate) discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17 is adjusted to adjust the cell density of the culture liquid in the culture tank 1. Can be adjusted. Further, the control device 5 uses the average flow rate per unit time in the reciprocating pipe 29 instead _{of the flow rate (F 0 ) of the extraction pipe 11a, and uses the flow rate (F 2} ) of the bleeding liquid discharge pipe 17 and the bleeding liquid pump. 19 operation time of (Delta] t), can be calculated emissions bleeding fluid _{(F 2} · Δt).

The control device 5 of the culture device 200 includes the output of the medium supply pump 8, the separation liquid pump 16 and the bleeding liquid pump 19, the output and operation of the diaphragm pump 30, the opening degree of the discharge pipe valve 25 and the reciprocating pipe valve 31, and the opening degree of the reciprocating pipe valve 31. It can be equipped with a function to control other liquid feeding systems and ventilation systems. The output of bleeding pump 19, opening of the exhaust pipe valve 25 and the reciprocating line valve 31, F _2, Delta] t, may be controlled according to the result of such F 2 _· Δt.

In the culture device 200, the cell concentrate that has flowed out of the cell separation device 13 during perfusion culture can be intermittently discharged to the outside of the circulatory system. That is, the reciprocating piping valve 31 and the discharging piping valve 25 can be opened and closed alternately. The discharge pipe valve 25 is opened during the period when the diaphragm pump 30 is performing the discharge operation, and is closed during the period when the diaphragm pump 30 is performing the suction operation.

When intermittent bleeding is performed, the reciprocating pipe valve 31 is controlled to be fully closed, and the discharge pipe valve 25 is controlled to be fully open. The reciprocating pipe valve 31 and the discharge pipe valve 25 have a duty ratio of opening and closing, and the total opening time when the bleeding liquid discharge pipe 17 side is opened under _{a predetermined flow rate (F 2) of the bleeding liquid discharge pipe 17 is bleeding.} It can be set so as to be substantially equal to the operating time (Δt) of the liquid pump 19.

Intermittent bleeding can be performed at appropriate time intervals during perfusion culture. When bleeding is performed, substantially all of the cell concentrate flowing out of the cell separation device 13 is flowed from the reciprocating pipe 29 to the bleeding liquid discharge pipe 17 and discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17. During this period, the cell separation process is continued, but the return to the culture tank 1 is interrupted.

Similar to the culture device 100, the culture device 200 returns the average discharge amount of the bleeding liquid discharged through the bleeding liquid discharge pipe 17 per unit time and the cell separation device 13 to the culture tank 1 with respect to the balance of the circulation system. The total of the average return amount of the cell concentrate per unit time and the average amount of the separation solution extracted from the cell separation device 13 to the separation solution discharge pipe 14 per unit time is transferred from the culture tank 1 to the cell separation device 13. The operation is performed so as to be substantially equal to the average withdrawal amount of the withdrawn culture solution per unit time.

Therefore, in the medium supply pump 8 of the culture device 200, the average supply amount of fresh medium supplied through the medium supply pipe 6 per unit time is extracted from the culture tank 1 to the cell separation device 13 in the same manner as the culture device 100. The difference between the average withdrawal amount of the culture medium per unit time and the average return amount of the cell concentrate returned from the cell separation device 13 to the culture tank 1 per unit time is controlled to be substantially equal. The flow rate of fresh medium supplied by the medium supply pump 8 can also be variably controlled.

Further, in the culture device 200, similarly to the culture device 100, the concentration rate in the cell separation device 13 may be controlled to be constant or variably. Similar to the culture apparatus 100, when the bleeding solution (cell concentrate) is discharged out of the culture system, an operation is performed to increase the cell concentration rate in the cell separation process, and the cells of the cell concentrate discharged out of the culture system are performed. The density can also be increased.

Similar to the culture apparatus 100, the operations for increasing the cell concentration rate include an operation of reducing the flow rate of the culture solution extracted from the culture tank 1, an operation of increasing the intermembrane differential pressure of the separation membrane used for the cell separation treatment, and a plurality of operations. An operation of limiting the line to be used for the cell separation device 13 provided with the above line can be mentioned.

Examples of the operation of reducing the flow rate of the culture solution extracted from the culture tank 1 include an operation of adjusting the average suction flow rate per unit time of the diaphragm pump 30 in a decreasing direction and a reciprocating piping valve 31 during the suction operation of the diaphragm pump 30. An operation of controlling the opening degree of is in the closing direction can be used.

Further, in the culture device 200, the separation membrane of the cell separation device 13 can be backwashed by using the system of the backwash liquid supply pipe 20 as in the culture device 100. The backwash of the separation membrane can be performed before the start of the perfusion culture, after the end of the perfusion culture, or intermittently during the perfusion culture. When performed during perfusion culture, the diaphragm pump 30, the separation liquid pump 16, and the backwash liquid supply pump 22 are operated alternately to perform backwash intermittently.

The backwashing of the separation membrane of the cell separation device 13 can be performed at the same time as the intermittent bleeding, or can be performed at a different time from the intermittent bleeding. When the separation membrane is backwashed at the same time as the intermittent bleeding, the foulant suspended in the cell concentrate is discharged to the outside of the circulatory system through the bleeding liquid discharge pipe 17, so that the differential pressure between the membranes increases. It becomes difficult to do so, and the service life of the separation membrane is extended.

In the culturing device 200, similarly to the culturing device 100, the cell concentrate discharged to the outside of the culture system through the bleeding liquid discharge pipe 17 is subjected to an appropriate separation / purification treatment to recover the substance produced by the cells. Can be done. As the separation / purification treatment, a semipermeable membrane separation treatment or a centrifugation treatment is preferably used. As the semipermeable membrane separation treatment, total filtration by dead end flow filtration is particularly preferable.

According to the above-mentioned culturing apparatus 200 and the culturing method using the same, the cell concentrate produced by the cell separation treatment is discharged to the outside of the culturing system during the perfusion culture, as in the case of the culturing apparatus 100 and the culturing method. , The cell density of the culture solution in the culture tank can be adjusted. Therefore, it is possible to suppress the discharge amount of the bleeding solution per cell discharged to the outside of the culture system during perfusion culture, as compared with the case where the excess cells that have proliferated are directly extracted from the culture tank. Since the culture environment and substance productivity can be stabilized while suppressing the cost and the supply of fresh medium, perfusion culture can be performed stably and efficiently.

Further, according to the above-mentioned culture apparatus 200 and the culture method using the above, the foulant in the circulatory system is discharged to the outside of the system together with the bleeding, so that the service life of the separation membrane used for the cell separation treatment can be extended. The action of discharging such foulants is further enhanced by supplying a backwash solution to the secondary side of the separation membrane used for the cell separation treatment. Therefore, it is possible to reduce the replacement cost of the separation membrane and the risk of contamination associated with the replacement work. Since the cell separation device 13 is an ATF type membrane separation device, the action of washing away the foulant adhering to the circulatory system is higher than that of the TFF method.

Although the present invention has been described above, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to those having all the configurations included in the above-described embodiment. Replacing part of the configuration of one embodiment with another, adding part of the configuration of one embodiment to another, or omitting part of the configuration of one embodiment. Can be done.

For example, the culture devices 100 and 200 described above do not deviate from the gist of the invention, such as the shape and model of the culture tank, cell separation device, etc., the model and arrangement of devices such as pumps and valves, and the connection of pipes. It can be changed to an appropriate form. For example, the bleeding liquid discharge pipe 17 can be directly connected to the cell separation device 13. Further, the backwash liquid supply pipe 20 can be connected to an appropriate position on the secondary side of the separation membrane of the cell separation device 13. The culture devices 100 and 200 include a membrane separation device as the cell separation device 13, but the cell separation process may be performed by a device based on other principles such as gravity sedimentation separation, centrifugation, and acoustic separation. it can.

Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited thereto.

<Comparative example 1>
During the perfusion culture, the culture solution was cultured by directly extracting the culture solution containing the cells from the culture tank.

As the culturing device, a culturing device provided with a circulatory system as shown in FIG. 1 was used. CHO cells were used as the cells to be cultured. The amount of the culture solution charged into the culture tank was 2 L. Control target value of the cell density of the culture tank, for viable cells, was 1.0 × ¹⁰ 7 cells / mL. The extraction flow rate from the culture tank to the cell separator and the circulation flow rate in the circulatory system were set to 40 L / d (20 times the amount charged in the culture tank per day).

Perfusion culture was continued for several days, bleeding by removing the culture broth directly from the culture tank. The medium consumption (CSPR) per cell of the cultured CHO cells is 1.0 × 10 ^-10 L / cells · d. The perfusion rate was 1.0 v / v · d. Since the doubling time of CHO cells was about 2 days, the culture solution was directly withdrawn from the culture tank and discharged out of the culture system every 2 days to maintain the amount of viable cells in the culture tank. .. The amount of the bleeding solution discharged to the outside of the culture system during this period was 1 L.

<Example 1>
During the perfusion culture, the cells were cultured by a method of discharging the cell concentrate from which the cells were separated and concentrated to the outside of the culture system.

As the culturing device, a culturing device provided with a circulatory system as shown in FIG. 1 was used. CHO cells were used as the cells to be cultured. The amount of the culture solution charged into the culture tank was 2 L. Control target value of the cell density of the culture tank, for viable cells, was 1.0 × ¹⁰ 7 cells / mL. The extraction flow rate from the culture tank to the cell separator and the circulation flow rate in the circulatory system were set to 40 L / d (20 times the amount charged in the culture tank per day).

Perfusion culture was continued for several days, bleeding by draining the cell concentrate out of the culture system. The medium consumption (CSPR) per cell of the cultured CHO cells is 1.0 × 10 ^-10 L / cells · d. The perfusion rate was 1.0 v / v · d. Since the doubling time of CHO cells was about 2 days, bleeding was performed every time about 2 days passed, with the flow rate of the cell concentrate discharged to the outside of the circulatory system set to 37 L / d and the discharge time set to 0.6 h. The amount of viable cells in the culture tank was maintained. The amount of the bleeding solution discharged to the outside of the culture system during this period was 950 mL.

As described above, the amount of the bleeding liquid of Example 1 was reduced by 5% as compared with Comparative Example 1.

100 Culture device 1 Culture tank 2 Spager 3 Stirrer 4 Cell densitometer 5 Control device 6 Medium supply pipe 7 Medium supply tank 8 Medium supply pump 9 Oxygen gas supply pipe 10 Oxygen gas supply pipe 11 Oxygen gas supply equipment 11 Circulation pipe 11a Extraction pipe 11b Return pipe 12 Culture medium extraction pump 13 Cell separator 14 Separation liquid discharge pipe 15 Separation liquid tank 16 Separation liquid pump 17 Bleeding liquid discharge pipe (discharge pipe)
18 Bleeding liquid tank 19 Bleeding liquid pump 20 Backwashing liquid supply pipe 21 Backwashing liquid supply tank 22 Backwashing liquid supply pump 23 Extraction piping valve 24 Return piping valve 25 Discharge piping valve 29 Reciprocating piping 30 Diaphragm pump 30a Chamber 30b Compressor 31 Reciprocating piping valve 130 Film separator 131 Casing 132 Hollow thread film filter 133 Inlet side header 134 Outlet side header 135 Liquid outlet 136 Cleaning liquid inlet

Claims (10)

The culture solution containing the cells is extracted from the culture tank in which the cells are cultured, subjected to cell separation treatment, and the cell concentrate produced by the cell separation treatment is returned to the culture tank to perform perfusion culture, and the perfusion culture is performed. A culture method in which the cell concentrate is discharged out of the culture system to adjust the cell density of the culture solution. The culture method according to claim 1.
A culture method in which the cell concentrate is intermittently discharged out of the culture system during the perfusion culture, or a part of the cell concentrate is continuously discharged out of the culture system. The culture method according to claim 2.
During the perfusion culture, the cell concentrate is intermittently discharged out of the culture system.
When the cell concentrate is discharged to the outside of the culture system, an operation of increasing the concentration rate of the cells in the cell separation treatment is performed to increase the cell density of the cell concentrate discharged to the outside of the culture system. Method. The culture method according to claim 3.
The operation of increasing the concentration rate of the cells is an operation of reducing the flow rate of the culture solution extracted from the culture tank, or an operation of increasing the intermembrane differential pressure of the separation membrane used for the cell separation treatment. The culture method according to claim 1.
A culture method in which the cell concentrate discharged to the outside of the culture system is subjected to a semipermeable membrane separation treatment or a centrifugation treatment to recover the substance produced by the cells. The culture method according to claim 1.
During the perfusion culture, the separation membrane used for the cell separation treatment is intermittently backwashed with a fresh medium or a buffer solution supplied from outside the culture system, and the foulant of the separation membrane is put into the cell concentrate. Floating culture method. A culture tank for culturing cells and
A cell separation device that produces a cell concentrate by performing cell separation treatment on a culture solution containing the cells extracted from the culture tank.
A circulation pipe that extracts the culture solution from the culture tank and sends it to the cell separation device, returns the cell concentrate from the cell separation device to the culture tank, and circulates the cells.
A culture device including a discharge pipe for discharging the cell concentrate to the outside of the circulatory system. The culture apparatus according to claim 7.
The circulation pipe is an extraction pipe connecting the culture tank and the liquid inlet of the cell separation device, and a return pipe connecting the liquid outlet of the cell separation device and the culture tank. ,
The cell separation device is a device that performs a membrane separation process using a separation membrane.
The membrane separation treatment is a culture apparatus using a tangential flow filtration method. The culture apparatus according to claim 7.
The circulation pipe is a reciprocating pipe that connects the culture tank and the liquid inlet / outlet of the cell separation device.
The cell separation device is a device that performs a membrane separation process using a separation membrane.
The membrane separation treatment is a culture apparatus using an alternating tangential flow filtration method. The culture apparatus according to claim 7.
The cell separation device is a device that performs a membrane separation process using a separation membrane, and a supply pipe for supplying a fresh medium or a buffer solution is connected to the secondary side of the separation membrane.
A culture apparatus in which the separation membrane is intermittently backwashed with the fresh medium or the buffer solution during perfusion culture of the cells, and the foulant adhering to the separation membrane is suspended in the cell concentrate.

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