JP5217407B2 - Biological cell separation device, culture device, and biological cell separation method - Google Patents

Description

  The present invention relates to a separation device that separates cells from a culture solution of biological cells, a culture device having the separation device, and a separation method that separates cells from the culture solution of biological cells.

  In the culture of biological cells, it is desired to culture biological cells at high density in order to improve the productivity of the target product, and there is a continuous culture method as a culture technique for achieving this. In continuous culture of animal cells, it is essential to separate cells in the culture medium, extract only the liquid components from the culture tank, and supply a new medium. Various cell separation means have been developed so far. .

  Non-Patent Document 1 and Patent Document 1 have a description of a continuous culture apparatus using a gravity sedimentation method. Patent Documents 2 and 3 describe a technique for sedimentation and separation of cells by centrifugation. Patent Document 4 describes a culture device characterized in that a culture region surrounded by a filter medium that prevents passage of cells is provided in a culture tank, and the filtrate that has passed outside the culture region is drawn out. Patent Document 5 describes that a porous hollow fiber membrane is used as a filtration membrane and backwashing is performed using a culture medium. Patent Document 6 describes that two pairs of filtration membranes are installed, one is used as an aeration means, and the other is used as a filtration means, and filtration and backwashing are repeatedly performed by alternately switching them. Patent Document 7 describes a culture apparatus provided with a rotating filtration membrane in a culture tank. Patent Literature 8 describes a culture apparatus in which a rotating filtration membrane is installed outside the culture tank.

  However, each of these conventional methods has significant problems. That is, the gravity sedimentation method requires a large stationary region because of the low sedimentation rate of cells, and is extremely difficult to apply to a large culture apparatus. The centrifugal separator has a complicated apparatus configuration and is difficult to increase in size. Although the filtration separation device has a simple device configuration and is easy to scale up, clogging of the filtration membrane is substantially inevitable, and it is difficult to carry out the separation operation for a long period of time.

Tissue culture Vol.15, No.8, pp.283-287 (issued in 1989) JP 6-209761 A Japanese Unexamined Patent Publication No. 5-192607 JP-A-6-90737 JP-A-5-95778 Japanese Unexamined Patent Publication No. 1-281072 JP-A-6-98758 Japanese National Patent Publication No. 03-505041 Japanese Patent Laid-Open No. 06-237754

  Therefore, the present invention provides a separation apparatus that separates cells from a culture solution of living cells using a cylindrical rotary filter, and that can effectively clean the clogging of the filter, and the separation apparatus. It is an object of the present invention to provide a culture apparatus and a separation method capable of effectively cleaning clogged filters.

The present invention that has achieved the above-described object includes the following.
That is, the separation device according to the present invention is a separation device that separates biological cells from a culture solution of biological cells, and includes a rotary filter provided with a filter that prevents the passage of cells on the outer peripheral surface of a cylindrical rotating body, and a tank A pressure detection means for detecting an internal pressure, a backwash liquid storage tank filled with a backwash liquid to be supplied to the inside of the rotary filter, and the backwashing measured by the pressure detection means of the backwash liquid storage tank And a control device for controlling the backwash liquid supplied from the backwash liquid storage tank to the inside of the rotary filter according to the pressure value inside the liquid storage tank. In the separation apparatus according to the present invention, the control means includes a backwash liquid supplied from the washing liquid storage tank with an air pressure that is greater than the pressure outside the rotary filter and smaller than the bubble point pressure of the filter. Is discharged from the inside of the rotary filter to the outside of the rotary filter through the filter.

  The separation apparatus according to the present invention includes a second pressure detecting means for detecting the pressure inside and outside the rotary filter, and a pressure value inside and outside the rotary filter measured by the second pressure inspection means. Depending on the above, a second control means for controlling the pressure inside and outside the rotary filter may be further provided. In this case, the second control means in the separation device can separate the filtrate containing no living cells from the living cell culture solution by making the pressure inside the rotary filter lower than the outside pressure.

  In particular, in the separation apparatus according to the present invention, it is preferable to rotate the rotary filter when supplying the backwash liquid from the backwash liquid storage tank to the rotary filter. At this time, the rotational speed of the rotary filter is more preferably higher than the rotational speed at the time of separating the filtrate not containing biological cells from the culture solution of biological cells.

  On the other hand, the culture apparatus according to the present invention includes the separation apparatus according to the present invention described above and a culture tank filled with a culture solution of living cells. In the culture apparatus according to the present invention, the separation means separates a filtrate that does not contain biological cells from the culture solution of biological cells.

  In the culture apparatus according to the present invention, the outside of the rotary filter in the separation apparatus and the culture tank are connected, and a culture solution containing biological cells filled in the culture tank is outside the rotary filter. To be supplied. Furthermore, it is preferable that the culture apparatus according to the present invention further includes a conduit for communicating between the gas phase part of the backwashing liquid storage tank and the gas phase part of the culture tank in the separation apparatus.

  On the other hand, the biological cell separation method according to the present invention allows a culture solution to pass from the outside of the filter to the filter using a rotary filter provided with a filter that prevents the passage of cells on the outer peripheral surface of the cylindrical rotating body. From the filtration step for separating the filtrate not containing biological cells from the culture solution, and the backwash solution storage tank filled with the backwash solution, the bubble point pressure of the filter is greater than the pressure outside the rotary filter. The backwashing step of alternately discharging the backwashing liquid from the inside of the rotary filter through the filter to the outside of the rotary filter with a lower air pressure is performed.

  In particular, in the biological cell separation method according to the present invention, in the filtration step, the filtrate containing no biological cells is separated from the culture solution of the biological cells by making the pressure inside the rotary filter lower than the external pressure. Is preferred. Furthermore, in the biological cell separation method according to the present invention, in the backwashing step, it is preferable to rotate the rotary filter when supplying the backwash liquid from the backwash liquid storage tank to the rotary filter. Furthermore, in the biological cell separation method according to the present invention, it is preferable that the rotational speed of the rotary filter in the backwashing step is larger than the rotational speed of the rotary filter in the filtration step.

  According to the separation device and the separation method of the present invention, clogging of a filter can be effectively washed when cells are separated from a culture solution of living cells using a cylindrical rotary filter. In addition, according to the culture device of the present invention, it is possible to effectively remove the clogging of the filter when separating the cells from the culture solution of the living cells by using the cylindrical rotary filter by using the separation device. This makes it possible to continue cell culture efficiently over a long period of time.

  Hereinafter, a biological cell separation apparatus, separation method, and culture apparatus according to the present invention will be described in detail with reference to the drawings. The biological cell separation apparatus, separation method, and culture apparatus according to the present invention can be applied when culturing cells that produce substances that are main raw materials such as pharmaceuticals. In the present invention, the substance to be produced is not limited in any way, and examples thereof include proteins such as antibodies and enzymes, physiologically active substances such as low molecular compounds and high molecular compounds. Further, the cells to be cultured are not limited in any way, and examples include animal cells, plant cells, insect cells, bacteria, yeasts, fungi, and algae. In particular, the biological cell separation apparatus, separation method, and culture apparatus according to the present invention preferably target animal cells that produce proteins such as antibodies and enzymes.

  An example of a biological cell separation apparatus to which the present invention is applied is schematically shown in FIG. Moreover, although mentioned later for details, an example of the culture apparatus to which this invention is applied is typically shown in FIG. First, the separation apparatus according to the present invention will be described. As shown in FIG. 1, the cell separation device 32 includes a rotary filter 1 provided with a filter 2 that prevents passage of cells on the outer peripheral surface, a filter storage container 4 that stores the rotary filter 1, and a filter for rotating the filter. A filter drive motor 5, a pressure gauge 6 for detecting the pressure inside the rotary filter 1, a pressure gauge 7 for detecting the pressure outside the rotary filter 1 (a second pressure detecting means in combination with the pressure gauges 6 and 7), rotation A backwash liquid storage tank 8 communicating with the inside of the filter 1, a pressure gauge 9 for detecting the pressure inside the backwash liquid storage tank 8, and a pressure adjusting valve 10 for adjusting the pressure inside the backwash liquid storage tank 8 are provided. Note that the separation device includes the pressure value inside the rotary filter 1 measured by the pressure gauge 6, the pressure value outside the rotary filter measured by the pressure gauge 7, and the pressure value inside the backwash liquid storage tank 8 measured by the pressure gauge 9. It has a control device 23 that inputs and controls the pressure regulating valve 10 in accordance with these input values and preset conditions.

  Culture medium circulation pipes 20 and 21 are provided between the filter container 4 and the culture tank, and the culture medium 22 is circulated by liquid feeding means such as a pump. The culture tank and liquid feeding means are not shown in FIG. As a result, the culture solution inside the filter container 4 is constantly renewed, and the culture is caused by partial densification of the cells due to extraction of the filtrate by filtration and deterioration of the environment due to prolonged residence. The adverse effect of can be suppressed.

  The filter container 4 has a function of preventing the flow of the culture medium inside and preventing the entry of germs from the outside. Specifically, the filter container 4 is provided with a shaft seal 15 using a mechanical seal. The mechanical seal prevents leakage of gas and liquid by causing the sliding member fixed to the rotating shaft 13 to rotate and the sliding member fixed to the shaft seal 15 to slide closely. In this example, two mechanical seals of a mechanical seal 16a for preventing the culture medium from flowing out of the filter container 4 and a mechanical seal 16b for preventing entry from the outside are used. A seal chamber 17 is formed between the mechanical seal 16a and the mechanical seal 16b and serves as a flow path that connects the inside and the outside of the rotary filter 1. In the present invention, the mechanical seal is not particularly limited, and any mechanical seal such as a mechanical seal used in a normal culture apparatus or a dry type mechanical seal can be used. Further, the usage method and arrangement of the mechanical seal are not limited to this example.

  The rotating shaft 13 has a hollow structure sealed at both ends, and has one end opened to the seal chamber 17 and the other end opened to communicate with a filtrate channel 12 provided in a cylinder 3 to be described later. Thus, the axial flow path 14 is formed. The shaft seal 15 is provided with a communication nozzle 18 that connects the seal chamber 17 and the outside, and is used for taking in and out filtrate and backwash liquid.

  The rotary filter 1 has a structure in which the cylinder 3 and the filter 2 are coaxially attached to the rotary shaft 13 and the upper end and the lower end are sealed with a sealing member, and the pressure inside the filter 2 is made lower than the outside. Filtration is performed, and a filtrate that has passed through the pores of the filter is obtained. In the filtration, when the rotary filter 1 is rotated inside the filter container 4, a flow of the culture solution parallel to the surface of the filter 2 is generated, and a so-called cross-flow filtration state is obtained. If the space between the inner wall surface of the filter container 4 and the surface of the filter 2 is reduced to make the flow turbulent, the cells and finer densified layers formed by extracting the filtrate are diffused. be able to. Thereby, clogging of the filter can be suppressed, and a larger amount of filtrate can be obtained at a higher speed. The rotation speed of the rotary filter is determined by the resistance of the cells of the living body to physical external force and the shape of the separation device.

  The filtrate that has passed through the filter 2 passes through a cylindrical gap 11 formed between the cylinder 3 and the filter 2, passes through a filtrate channel 12 provided in the cylinder 3, and an axial channel 14 provided in the rotary shaft 13. Then, it is guided to the seal chamber 17 of the shaft seal 15 and transferred to the backwash liquid storage tank 8 via the valves 40 and 41 by the communication flow path connected to the communication nozzle 18.

  The material of the filter 2 used in the present invention is not particularly limited as long as it can prevent the passage of cells, such as a commonly used filter cloth and membrane filter. In particular, it is preferable to select one having mechanical strength, heat resistance, and corrosion resistance that can withstand sterilization by blowing steam into the rotary filter 1 and injection of cleaning liquid during cleaning. In addition, since there are a large number of fine cell fragments produced by the decay of dead cells in the culture solution, a filter having filtration characteristics that prevents the passage of healthy cells and allows the passage of fine cell fragments is particularly preferable. . In this example, a metal filter is used in which a thin stainless steel wire is wound into a cylindrical shape with a predetermined interval to form a slit-like opening. In a normal filter, there are many pores smaller than the particle diameter to be blocked, and clogging is caused by blocking even fine cell fragments. The filter 2 is substantially free of pores other than the slits. Thereby, in this filter, only a cell larger than the slit width can be prevented from passing, and fine particles such as cell fragments smaller than the slit width can be passed. The slit width is determined by the size of the cells to be cultured, and is usually 5 to 30 μm.

  The backwash liquid storage tank 8 includes a liquid level meter 19 for measuring the position of the liquid level, a pressure gauge 9 for detecting the internal pressure, and a pressure adjusting valve 10 for adjusting the pressure inside the backwash liquid storage tank 8. It has. The backwash liquid storage tank 8 is connected to the communication nozzle 18 by opening the valves 40 and 41, the harvest tank by opening the valves 41 and 44, and the culture tank by opening the valve 42, respectively. The liquid can be moved. In addition, the description in FIG. 1 of the harvest tank and the culture tank was abbreviate | omitted.

  The pressure inside the backwash liquid storage tank 8 is adjusted by adjusting the amount of air blown by opening and closing the valve 43 and by adjusting the exhaust amount by opening and closing the pressure adjusting valve 10. In the present invention, it is preferable that the internal pressure of the backwash liquid storage tank 8 is always kept higher than the atmospheric pressure. As the air used for adjusting the pressure, it is preferable to use aseptic air from which fine particles such as bacteria have been previously removed.

  Although it does not specifically limit as the liquid level meter 19, it has mechanical strength, heat resistance, and corrosion resistance that can withstand sterilization by blowing steam into the backwash liquid storage tank 8 and injection of cleaning liquid at the time of washing. It is preferable to select what has. In addition, there are many cells and fine cell fragments produced by cell collapse in the culture solution, and there is a risk that bubbles may accumulate on the liquid surface. It is preferable to select. In this example, a capacitance type level sensor is used.

  Hereinafter, using the separation apparatus configured as described above, various steps including a filtration step for filtering a culture solution containing biological cells and a backwashing step for washing the filter 2 will be described in detail.

(1) Filtration step First, the culture solution is circulated in the space formed by the inner wall of the filter container 4 and the outer periphery of the rotary filter via the culture solution circulation pipes 20 and 21 by a liquid feeding means such as a pump. However, the rotary filter 1 is rotated by the filter drive motor 5. The pressure gauge 7 measures the pressure inside the filter container 4 (synonymous with the pressure outside the rotary filter 1), and the pressure gauge 9 measures the pressure inside the backwash liquid storage tank 8. The pressure inside the filter container 4 is the same as the pressure in the culture tank, and is usually pressurized to 0.01 to 0.05 MPa. The control device 23 adjusts the pressure inside the backwash liquid storage tank 8 with the pressure adjustment valve 10 so that the pressure inside the backwash liquid storage tank 8 becomes slightly lower than the pressure inside the filter container 4. Subsequently, when the valve 41 and the valve 40 are sequentially opened, the culture solution in the filter container 4 is filtered by the filter 2 and the shaft seal 15 is passed through the filtrate channel 12 and the shaft channel 14 provided in the rotary shaft 13. The filtrate is guided to the seal chamber 17 and transferred to the backwash liquid storage tank 8 through the valves 40 and 41 by the communication flow path connected to the communication nozzle 18.

  The liquid level position of the filtrate is measured by the liquid level meter 19, and when it reaches a predetermined position, the valve 40 and the valve 41 are sequentially closed, and the filtration process is completed. In the filtration operation, it is not preferable to rapidly apply a large filtration differential pressure because clogging is promoted. In this example, the pressure difference between the filter container 4 and the backwash liquid storage tank 8, that is, the filtration differential pressure is set to 0.04 MPa or less. This value can be input to the control device 23 in advance. The filtration speed is adjusted by adjusting the pressure difference between the filter container 4 and the backwash liquid storage tank 8. When it is necessary to keep it lower than the filtration rate, the valve 40 may be opened and closed intermittently and the length of the opening time may be adjusted.

  Further, the opening / closing timing of the valves 40 and 41 may be controlled by a control device using the signal output from the liquid level meter 19 as an input value, but may be controlled by the control device 23 described above. Alternatively, the opening / closing timing of the valves 40 and 41 may be performed by an operator.

(2) Filtrate discharging step The filtrate discharging step is a step of transferring the filtrate transferred to the backwash liquid storage tank 8 to a harvest tank (not shown). First, the valve 43 is opened and the internal pressure of the backwash liquid storage tank 8 is set higher than the internal pressure of the harvest tank. Thereafter, the valve 41 and the valve 44 are sequentially opened to transfer the filtrate in the backwash liquid storage tank 8 to the harvest tank. The liquid level position of the filtrate is measured by the liquid level gauge 19, and when it reaches the predetermined position, the valve 44 and the valve 41 are closed sequentially, and the filtrate discharging step is completed.

  In the filtrate discharging step, when the liquid level position of the harvest tank is lower than the liquid level position of the backwash liquid storage tank 8, the valve 44 and the valve 41 are sequentially opened to filter the inside of the backwash liquid storage tank 8. The liquid may be transferred into the harvest tank. However, in this case, it is preferable to inject air by opening the valve 43 so that the internal pressure of the backwash liquid storage tank 8 does not become lower than the atmospheric pressure due to the outflow of the filtrate from the backwash liquid storage tank 8.

(3) Medium transfer process The medium transfer process is a process of supplying the culture solution used in the subsequent backwashing process from the medium tank (not shown) to the backwashing liquid storage tank 8. First, the internal pressure of the backwash liquid storage tank 8 is adjusted to be lower than the internal pressure of the medium tank by the pressure adjusting valve 10 according to the control of the control device 23. Thereafter, the culture medium flows from the culture medium tank into the backwash liquid storage tank 8 by opening the valve 42. The liquid level of the medium is measured by the liquid level meter 19, and when it reaches a predetermined position, the valve 42 is closed and the supply of the medium is terminated.

  In setting the pressure, it is desirable to consider the liquid level height of the medium tank and the backwash liquid storage tank 8. That is, when the liquid level position of the medium tank is higher than the liquid level position of the backwash liquid storage tank 8, it is preferable to reduce the pressure difference so that the transfer speed of the medium does not become too high. Moreover, when the liquid level position of a culture tank is lower than the liquid level position of the backwashing liquid storage tank 8, it is preferable to enlarge a pressure difference so that a culture medium may be transferred. When it is necessary to adjust the transfer rate of the culture medium, the valve 42 may be opened and closed intermittently to adjust the length of the opening time.

  Further, when the liquid level position of the medium tank is higher than the liquid level position of the backwash liquid storage tank 8, the pressure difference between the backwash liquid storage tank 8 and the medium tank inside is increased by sequentially opening the valve 42 and the valve 43. You may transfer the culture medium inside a culture tank into the backwashing liquid storage tank 8 inside, without setting.

(4) Backwashing step The backwashing step is to eliminate clogging by washing the filter 2 (which may be clogged) clogged by living cells through the above-mentioned (1) filtration step. It is a process. First, the valve 43 is opened and the internal pressure of the backwash liquid storage tank 8 is set higher than the internal pressure of the filter container 4. When the internal pressure of the filter container 4 is increased to 0.01 to 0.05 MPa, the internal pressure of the backwash liquid storage tank 8 is controlled by the pressure adjusting valve 10 under the control of the control device 23. Adjust to be higher than the internal pressure. Thereafter, by sequentially opening the valve 41 and the valve 40, the medium inside the backwashing liquid storage tank 8 is provided in the seal chamber 17 of the shaft seal 15 and the rotary shaft 13 through the communication flow path via the valves 40 and 41. It is sequentially guided to the axial flow path 14 and the filtrate flow path 12, passes through the cylindrical gap 11, passes through the pores of the filter 2 in the direction opposite to the filtration step, and flows into the filter container 4. Thereby, the clogged filter 2 (which may be clogged) can be washed to eliminate clogging. Thereafter, the medium is supplied to the culture tank through the culture solution circulation line 21. That is, in this example, when the culture medium stored in the backwash liquid storage tank 8 is supplied to the culture tank, it plays a role as a backwash liquid for the filter 2.

  In particular, at this time, it is preferable that the rotational speed of the rotary filter 1 be larger than the rotational speed in the filtration step. By performing the backwash while rotating the rotary filter 1 at a high speed, the backwash liquid can be uniformly distributed over the entire inner surface of the filter 2. Moreover, a larger backwashing effect can be obtained by adding a cleaning action by centrifugal force generated by rotation.

  In this step, the pressure regulating valve 10 is set so that the internal pressure of the backwash liquid storage tank 8 is higher than the internal pressure of the filter container 4 and smaller than the pressure obtained by adding the bubble point pressure of the filter 2 to the internal pressure of the filter container 4. It is preferable to adjust by. In particular, this pressure adjustment is preferably performed at the end of the backwash process when the liquid level of the medium is measured by the liquid level meter 19 and reaches the predetermined position. By adjusting the internal pressure of the backwash liquid storage tank 8 to be higher than the internal pressure of the filter storage container 4 and lower than the pressure obtained by adding the bubble point pressure of the filter 2 to the internal pressure of the filter storage container 4, All of the medium in the filtrate channel 12, the shaft channel 14, and the communication channel can be discharged to the filter container 4. That is, by setting in this way, it becomes possible to use the entire culture medium without wasting it. Moreover, by setting in this way, it can prevent that a bubble arises from the filter 2, and clogging can be eliminated reliably. The bubble point pressure of the filter can be obtained by submerging the rotary filter 1 in the medium, injecting air into the medium, and measuring the pressure at the time when bubbles started to be released from the filter surface.

  Next, after a preset time has elapsed, the valve 44 and the valve 41 are sequentially closed, the rotational speed of the rotary filter 1 is returned to the rotational speed in the filtration step, and the backwashing step is completed.

  In this example, the biological cell separation operation can be continued by repeating the operations (1) to (4). Moreover, the separation apparatus detailed above can be applied to a culture apparatus as shown in FIG. That is, the culture apparatus to which the present invention is applied includes a culture tank 31, a cell separation apparatus 32, a medium tank 33, and a harvest tank 34 as shown in FIG. As the cell separation device 32, the biological cell separation device to which the present invention shown in FIG. 1 is applied is used. Moreover, although not shown in FIG. 2, it has gas supply facilities, such as air, oxygen, nitrogen, and a carbon dioxide gas, a hot / cold water supply facility, a steam supply facility, and a water supply / drainage facility. Further, the culture tank 31 includes a measuring means 38 for measuring the properties of the culture solution 22, and the measured values 39 of the dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, temperature, ammonia concentration, lactic acid concentration and glutamine concentration are obtained. obtain. As for the measuring means 38, one detecting means is used for each detection item or each control item in the actual apparatus, but only one is shown in FIG. 2 for the sake of simplicity.

  The culture tank 31 is represented by a cross section. The culture solution 22 stuck in the culture tank 31 is stirred by a stirrer 36 driven by a driving motor 35 and mixed uniformly. Oxygen necessary for the culture is supplied by two methods: a submerged aeration method in which oxygen-containing gas is supplied into the liquid from the aeration means 37 disposed at the bottom of the tank, and a top aeration method in which the gas is supplied to the upper gas phase of the tank. The

  The ventilation system to the liquid and to the upper surface includes individual operation means 50a and 50b for controlling the respective supply gases. The individual operation means 50a and 50b have a flow rate control function and a supply amount measurement function for each of air, oxygen, and carbon dioxide. The ventilation to the upper surface is controlled by the individual operation means 50a for controlling the supply gas, and the composition and the amount of ventilation. In this embodiment, air is aerated at a constant amount, and carbon dioxide gas is mixed corresponding to the pH of the culture solution. The carbon dioxide gas concentration was controlled by normal proportional control using pH as the control amount and carbon dioxide flow rate as the operating factor. The aeration from the aeration means 37 into the culture solution is controlled by the individual operation means 50b for controlling the supply gas.

  The culture tank 31 is held at a constant pressure by a pressure adjustment valve 52 based on the measurement result of the pressure gauge 51. Usually, the pressure is 0.01 to 0.05 MPa in order to prevent the entry of bacteria and the like from the outside. In addition, the pressure gauge 51 in FIG. 2 is synonymous with the pressure gauge 7 in FIG. Culture medium circulation lines 20 and 21 are provided between the culture tank 31 and the cell separation device 32, and the culture medium 22 is circulated by a pump 48. The culture fluid circulation lines 20 and 21 are provided with a valve 45, a valve 46 and a valve 47, which are opened and closed as necessary when stopping the culture fluid circulation or withdrawing the culture fluid from the cell separation device 32. The

  It is preferable that a communication conduit 53 and a valve 54 are provided between the culture tank 31 and the backwash liquid storage tank 8 so that the respective gas phase portions communicate with each other. When the communication pipe 53 and the valve 54 are provided, the pressure inside the culture tank 31 and the backwash liquid storage tank 8 can be easily made the same by opening the valve 54. This is particularly effective when the internal pressure of the backwash liquid storage tank 8 is adjusted slightly lower than the internal pressure of the culture tank 31 when starting the filtration process.

  The medium tank 33 is a tank for storing a medium to be supplied to the culture tank 31, and includes a stirrer 55, a stirrer driving motor 56, a pressure gauge 57 for detecting internal pressure, a pressure adjusting valve 58, and an air supply valve 59. It has. The culture medium tank 33 is connected to the backwash liquid storage tank 8 by a valve 42, a valve 60, and a communication pipe line, so that the culture medium can be transferred to the backwash liquid storage tank 8 (Description of the above (3) Medium Transfer Process) reference). The medium tank 33 is held at a constant pressure by the pressure adjustment valve 58 based on the measurement result of the pressure gauge 57. Usually, the pressure is 0.01 to 0.05 MPa in order to prevent the entry of bacteria and the like from the outside. Moreover, in order to prevent the quality change during storage of a culture medium, it cools to 5 to 10 degreeC.

  The harvest tank 34 is a tank for storing a filtrate in which a substance intended for production separated by the cell separation device 32 is dissolved, and includes a stirrer 61, a stirrer driving motor 62, and a pressure gauge for detecting an internal pressure. 63, a pressure regulating valve 64 and an air supply valve 65 are provided. The harvest tank 34 is connected to the backwashing liquid storage tank 8 by a valve 41, a valve 44 and a communication pipe line, and can receive the filtrate in the backwashing liquid storage tank 8 ((2) in the filtrate discharging step). See description). The harvest tank 34 is held at a constant pressure by the pressure adjustment valve 64 based on the measurement result of the pressure gauge 63. Usually, the pressure is 0.01 to 0.05 MPa in order to prevent the entry of bacteria and the like from the outside. Moreover, in order to prevent a target substance from changing in quality during storage, it cools to 5 to 10 degreeC.

  The continuous culture in the main culture apparatus is the above-described biological cell separation method, (1) filtration step, (2) filtrate discharge step, (3) medium transfer step, and (4) backwash step. This is done by iteratively repeating.

  In particular, in the main culture apparatus, the separation device 32 is configured so that the internal pressure of the backwashing liquid storage tank 8 is higher than the internal pressure of the filter container 4 and lower than the pressure obtained by adding the bubble point pressure of the filter 2 to the internal pressure of the filter container 4. The backwashing process is performed by adjusting to the above. Accordingly, in the main culture apparatus, the cylindrical gap 11, the filtrate flow path 12, the axial flow path 14, and the medium in the communication flow path can all be discharged to the filter container 4 in the backwashing step. Thereby, the main culture apparatus can use all of the expensive medium used in the backwashing process for the culture without wasting it. Moreover, in this culture apparatus, it can prevent that a bubble arises from the filter 2 in a backwashing process, and can eliminate clogging reliably. Thereby, in this culture apparatus, it becomes possible to continue continuous culture for a long time, and the improvement of productivity can be achieved.

  In the separation apparatus and the culture apparatus described above, the culture medium is used as the backwashing liquid used in the backwashing process, but the technical scope of the present invention is not limited to this. The backwashing solution used in the backwashing step may be a solution containing at least one of the medium components, a solution dedicated to backwashing, sterilized water, or the like. In consideration of adding the backwash solution to the culture tank, it is preferable to use a solution containing at least one of the medium and the medium components as the backwash solution. In other words, as long as the backwash solution is not added to the culture tank, the backwash solution is not particularly limited, and a solution having any composition may be used.

  Moreover, although the separation apparatus and culture apparatus mentioned above were provided with the culture tank which supplies backwash liquid to the backwash liquid storage tank 8, the technical scope of this invention is not limited to this. That is, the separation apparatus and the culture apparatus to which the present invention is applied include a plurality of medium component tanks stored for each of various medium components, and a solution prepared by mixing so as to obtain a desired composition ratio is the backwash liquid storage tank 8. Can also be supplied.

It is a schematic block diagram which shows typically an example of the biological cell separation apparatus to which this invention is applied. It is a schematic block diagram which shows typically an example of the culture apparatus to which this invention is applied.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Rotary filter, 2 ... Filter, 4 ... Filter storage container, 8 ... Backwash liquid storage tank, 31 ... Culture tank, 32 ... Cell separation apparatus, 33 ... Medium tank, 34 ... Harvest tank.

Claims (11)

A separation device for separating biological cells from a culture solution of biological cells,
A rotary filter provided with a filter for preventing the passage of cells on the outer peripheral surface of the cylindrical rotating body;
A pressure detection means for detecting the pressure inside the tank, and a backwash liquid storage tank filled with a backwash liquid to be supplied to the inside of the rotary filter;
Control means for controlling the backwash liquid supplied from the backwash liquid storage tank to the inside of the rotary filter according to the pressure value inside the backwash liquid storage tank measured by the pressure detection means of the backwash liquid storage tank,
The control means filters backwash liquid supplied from the cleaning liquid storage tank from the inside of the rotary filter with an air pressure that is larger than the pressure outside the rotary filter and lower than the bubble point pressure of the filter. A separation device for discharging to the outside of the rotary filter through Second pressure detecting means for detecting the pressure inside and outside the rotary filter, and the inside and outside of the rotary filter according to the pressure values inside and outside the rotary filter measured by the second pressure detecting means. Second control means for controlling the external pressure;
The said 2nd control means isolate | separates the filtrate which does not contain a living cell from the culture solution of the said living cell by making the pressure inside the said rotary filter lower than an external pressure. Separation device.   2. The separation apparatus according to claim 1, wherein when the backwash liquid is supplied from the backwash liquid storage tank to the rotary filter, the rotary filter is rotated.   The rotational speed of the rotary filter when supplying the backwash liquid from the backwash liquid storage tank to the rotary filter is higher than the rotational speed when separating the filtrate not containing biological cells from the culture solution of the biological cells. The separation device according to claim 3, wherein: A separation device according to any one of claims 1 to 4,
A culture tank filled with a culture solution of biological cells,
The separation apparatus separates a filtrate containing no biological cells from a culture solution of biological cells.   The outside of the rotary filter and the culture tank in the separation device are connected to each other, and a culture solution containing biological cells filled in the culture tank is supplied to the outside of the rotary filter. Item 6. The culture apparatus according to Item 5.   6. The culture apparatus according to claim 5, further comprising a conduit for communicating between the gas phase part of the backwashing liquid storage tank and the gas phase part of the culture tank in the separation device. A method for separating a living cell from a living cell culture,
Using a rotary filter with a filter that prevents the passage of cells on the outer peripheral surface of the cylindrical rotating body, the culture solution is separated from the culture solution by allowing the culture solution to pass through the filter from the outside of the filter. Filtration step to
From the backwash liquid storage tank filled with backwash liquid, the backwash liquid is filtered from the inside of the rotary filter with an air pressure that is greater than the pressure outside the rotary filter and smaller than the bubble point pressure of the filter. And a backwashing step for discharging the rotating filter to the outside of the rotating filter alternately.   9. The biological cell according to claim 8, wherein, in the filtration step, a filtrate not containing biological cells is separated from the culture solution of the biological cells by making a pressure inside the rotary filter lower than an external pressure. Separation method.   9. The method of separating biological cells according to claim 8, wherein in the backwashing step, the rotary filter is rotated when backwashing liquid is supplied from the backwash liquid storage tank to the rotary filter.   The method for separating biological cells according to claim 10, wherein the rotational speed of the rotary filter in the backwashing step is larger than the rotational speed of the rotary filter in the filtration step.

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