Method for producing active pharmaceutical ingredient of biopharmaceutical, system for producing active pharmaceutical ingredient of biopharmaceutical, and active pharmaceutical ingredient of biopharmaceutical

Abstract

A culturing unit for performing a culture step for culturing cells in culture broth retained in a culture tank and removing the cells from the culture broth to thereby obtain a culture supernatant containing a product of the cells, and a purification unit for performing a purification step for purifying the product from the culture supernatant to thereby obtain an active pharmaceutical ingredient of a biopharmaceutical, are connected by a connecting line. The culture supernatant is allowed to flow through the connecting line from the culturing unit to the purification unit. A flexible single-use tank having a capacity of 0.2 to 10 times the volume of the culture broth in the culture tank is joined to a branch line provided between the culturing unit and the purification unit. The culture supernatant is temporarily retained in the tank.

Description

Manufacturing method for biopharmaceutical drug substances, manufacturing system for biopharmaceutical drug substances, and biopharmaceutical drug substances

[0001] The technology of the present disclosure relates to a method for producing a drug substance for a biopharmaceutical, a system for producing a drug substance for a biopharmaceutical, and a drug substance for a biopharmaceutical.

[0002] It is widely practiced to obtain drug substances for biopharmaceuticals by culturing cells such as antibody-producing cells in which antibody genes have been introduced into Chinese hamster ovary cells (CHO cells) to obtain cell products such as antibodies, and then purifying the products. In the manufacture of drug substances for such biopharmaceuticals, batch production, in which the cell culture process and the product purification process are carried out separately, has traditionally been used. However, in recent years, continuous production has become mainstream, replacing batch production. Continuous production is a method in which a culture section, which performs the culture process including a culture tank or the like, and a purification section, which performs the purification process including a chromatography device or the like, are physically connected, thereby consistently and continuously carrying out the culture process and the purification process. Continuous production can significantly improve the quality and productivity of drug substances compared to batch production.

[0003] JP 2017-515501 A describes a continuous production system for manufacturing active pharmaceutical ingredients for biopharmaceuticals. In the manufacturing system described in JP 2017-515501 A, in addition to a connecting line connecting the culture unit and the purification unit, a branch line is provided between the culture unit and the purification unit, and a tank (container m in Figure 5 of JP 2017-515501 A) is connected to the branch line.

[0004] As can be seen from Figure 5, the tank described in JP 2017-515501 A is made of glass and is intended for repeated use. This raises concerns about contamination of the culture supernatant. Furthermore, when considering the need to replace a tank with a new one after it has been filled with culture supernatant, glass is heavy and requires time and effort for sterilization, making it difficult to handle. These problems of contamination and difficulty in handling become more pronounced as the tank's capacity increases. The culture supernatant is obtained by removing cells from the culture solution and contains the cell product. The culture supernatant flows from the culture section to the purification section through a connecting line.

[0005] One embodiment of the technology of the present disclosure provides a method for producing a drug substance for a biopharmaceutical, a system for producing a drug substance for a biopharmaceutical, and a drug substance for a biopharmaceutical that can reduce concerns about contamination and are easy to handle.

[0006] The method for producing a drug substance for a biopharmaceutical according to the present disclosure comprises connecting a culture unit, which performs a culture step in which cells are cultured in a culture medium stored in a culture tank and a culture supernatant containing a cell product is obtained by removing the cells from the culture medium, and a purification unit, which performs a purification step in which a drug substance for the biopharmaceutical is obtained by purifying the product from the culture supernatant, via a connecting line; allowing the culture supernatant to flow from the culture unit to the purification unit via the connecting line; connecting a flexible, single-use tank having a capacity that is 0.2 to 10 times the volume of the culture medium in the culture tank to a branch line provided between the culture unit and the purification unit; and temporarily storing the culture supernatant in the tank.

[0007] It is preferable that all of the flow paths for the culture supernatant from the culture section to the tank be connected in a sterile manner.

[0008] It is preferable that the connection portion of the branch line to the tank and the connection portion of the tank to the branch line are both made of sterile connectors, and that the branch line and the tank are connected aseptically via the sterile connectors.

[0009] It is preferable to aseptically connect the tubes constituting the branch line to the inlet tube connected to the inlet for the culture supernatant of the tank by heat welding.

[0010] It is preferable to filter the culture supernatant flowing into the purification section through a sterile filter provided in the connecting line downstream of the connection with the branch line.

[0011] The amount of culture medium in the culture tank is preferably 50 L or more and 5000 L or less.

[0012] It is preferable to discharge the temporarily stored culture supernatant from a discharge port provided in the tank.

[0013] It is preferable to allow the temporarily stored culture supernatant to flow into the purification section through an outlet provided in the tank.

[0014] It is preferable to use a plurality of branch lines between the culture section and the purification section.

[0015] When a tank connected to one of the branch lines is filled with culture supernatant, it is preferable to switch use to a tank connected to another branch line.

[0016] If a problem occurs in the purification section, it is preferable to switch the flow path of the culture supernatant from the connecting line to the branch line.

[0017] It is preferable to switch the flow path of the culture supernatant using a non-wetted valve that can switch the flow path without coming into contact with the culture supernatant.

[0018] It is preferable to switch the flow path of the culture supernatant using a non-wetted valve provided in the branch line and a non-wetted valve provided in the connecting line downstream of the junction with the branch line.

[0019] The non-wetted valve is preferably a type in which a tube is clamped from the outside.

[0020] When a signal indicating that a malfunction has occurred is output from the purification section, it is preferable to perform control to switch the flow path of the culture supernatant from the connecting line to the branch line.

[0021] It is preferable to measure the flow rate of the culture supernatant with a flow meter provided in the connecting line upstream of the connection with the branch line, and to control the flow rate of the culture supernatant based on the measurement results.

[0022] The purification section preferably continuously purifies the culture supernatant.

[0023] Preferably the product is an antibody.

[0024] The antibody concentration in the culture supernatant is preferably 1.0 g / L or more.

[0025] The cell concentration in the culture medium was 8.0 × 10 ７ It is preferably 1000 cells / mL or more.

[0026] The manufacturing system for a drug substance for a biopharmaceutical according to the present disclosure comprises: a culture unit that performs a culture step in which cells are cultured in a culture medium stored in a culture tank and a culture supernatant containing a cell product is obtained by removing the cells from the culture medium; a purification unit that performs a purification step in which a drug substance for the biopharmaceutical is obtained by purifying the product from the culture supernatant; a connecting line that connects the culture unit and the purification unit and allows the culture supernatant to flow from the culture unit to the purification unit; a branch line provided between the culture unit and the purification unit; and a flexible single-use tank connected to the branch line, which has a capacity of 0.2 to 10 times the volume of the culture medium in the culture tank and temporarily stores the culture supernatant.

[0027] The biopharmaceutical drug substance of the present disclosure is produced by the method for producing a biopharmaceutical drug substance described above.

[0028] According to the technology of the present disclosure, it is possible to provide a method for producing a drug substance for a biopharmaceutical, a system for producing a drug substance for a biopharmaceutical, and a drug substance for a biopharmaceutical that can reduce concerns about contamination and are easy to handle.

[0029] 1 is a diagram showing a manufacturing system for active pharmaceutical ingredients for biopharmaceuticals. FIG. 2 is a diagram showing the structure of a pinch valve. FIG. 3 is a block diagram of a computer constituting a control device. FIG. 4 is a block diagram of the CPU of the computer constituting the control device. FIG. 5 is a diagram showing the processing of the intermediate section control unit when a malfunction occurs in the purification section. FIG. 6 is a diagram showing how, when one tank is filled with culture supernatant, use is switched to another tank. FIG. 7 is a flowchart for explaining the operation of the technology of the present disclosure. FIG. 8 is a diagram showing an embodiment in which tubes constituting a branch line and an inlet tube connected to the inlet for culture supernatant of the tank are aseptically connected by heat welding. FIG. 9 is a diagram showing details of heat welding. FIG. 10 is a diagram showing a screw-type tube clamp. FIG. 11 is a diagram showing an embodiment in which culture supernatant temporarily stored in a tank is allowed to flow into the purification section from an outlet provided in the tank. FIG. 12 is a diagram showing an embodiment in which culture supernatant temporarily stored in a tank is allowed to flow into the waste liquid or the purification section from an outlet provided in the tank. FIG. 13 is a table showing examples and comparative examples.

[0030] As shown in FIG. 1 as an example, a biopharmaceutical drug substance manufacturing system 2 that implements the method for manufacturing a biopharmaceutical drug substance according to the technology of the present disclosure includes a culture unit 10 that performs a culture step, an intermediate unit 11, a purification unit 12 that performs a purification step, and a control device 13. The culture unit 10 has a culture tank 14 and a filter 15. A culture solution (culture medium) 16 is stored in the culture tank 14. The amount of the culture solution 16 in the culture tank 14 is 50 L (liters) or more and 5000 L or less. Antibody-producing cells 17 are seeded in the culture tank 14, and the antibody-producing cells 17 are cultured in the culture solution 16. The concentration of the antibody-producing cells 17 in the culture solution 16 is 8.0 x 10 ７ The concentration is 1000 cells / mL or more. Note that "cells" refers to the number of cells.

[0031] The antibody-producing cells 17 are cells established by incorporating an antibody gene into cells such as CHO cells. The antibody-producing cells 17 produce immunoglobulins, i.e., antibodies 18, as a product during the culture process. Therefore, not only the antibody-producing cells 17 but also the antibodies 18 are present in the culture medium. The antibodies 18 are, for example, monoclonal antibodies, and are the active ingredients of biopharmaceuticals.

[0032] The culture tank 14 is provided with a culture solution supply channel 19, a gas supply channel 20, an exhaust channel 21, a culture solution delivery / recovery channel 22, a sparger 23, a gas supply channel 24, and an agitator blade 25. The culture solution supply channel 19 is a flow channel for continuously supplying fresh culture solution 16 into the culture tank 14, as indicated by arrow A. In other words, the culture unit 10 performs perfusion culture. The gas supply channel 20 is a flow channel for supplying gas containing air and carbon dioxide from above, as indicated by arrow B. The exhaust channel 21 is a flow channel for exhausting gas supplied from the gas supply channel 20 to the outside of the culture tank 14, as indicated by arrow C. An exhaust filter 26 is provided in the exhaust channel 21.

[0033] The culture solution delivery / recovery path 22 is connected to an inlet / outlet 27 of the filter 15. The culture solution delivery / recovery path 22 is a flow path for delivering the culture solution 16 in the culture tank 14 to the filter 15, as shown by arrow D. The culture solution delivery / recovery path 22 is also a flow path for returning the culture solution 16 (concentrated solution) from the filter 15 into the culture tank 14, as shown by arrow E. Although not shown, the culture solution supply path 19 is provided with a pump for supplying the culture solution 16 into the culture tank 14.

[0034] The sparger 23 is disposed at the bottom of the culture tank 14. As indicated by arrow F, the sparger 23 releases oxygen-containing gas supplied from a gas supply channel 24 into the culture tank 14. The oxygen released from the sparger 23 dissolves in the culture solution 16 and assists the antibody-producing cells 17 in producing antibodies 18. The agitator blades 25 are rotated at a predetermined rotation speed by a motor or the like to agitate the culture solution 16 in the culture tank 14. This maintains the homogeneity of the culture solution 16 in the culture tank 14. In addition to the above, the culture tank 14 is also provided with a flow path and the like for a cell bleed process in which a portion of the culture solution 16 is intentionally extracted.

[0035] The filter 15 has a filter membrane 28 therein. The filter membrane 28 captures the antibody-producing cells 17 and allows the antibodies 18 to pass through. The filter 15 removes the antibody-producing cells 17 from the culture solution 16 using the filter membrane 28, for example, by tangential flow filtration (TFF), thereby obtaining a culture supernatant 29.

[0036] More specifically, the filter 15 has a diaphragm pump 31 having an elastic membrane 30 therein, and a degassing / air supply path 32. While air below the elastic membrane 30 is being degassed through the degassing / air supply path 32, the elastic membrane 30 is elastically deformed so as to adhere to the lower end of the diaphragm pump 31, whereby the culture solution 16 in the culture tank 14 flows into the filter 15 via the culture solution delivery / recovery path 22. Furthermore, while air is being supplied to the lower side of the elastic membrane 30 from the degassing / air supply path 32, the elastic membrane 30 is elastically deformed so as to adhere to the upper side of the diaphragm pump 31, whereby the culture solution 16 (concentrated solution) that did not pass through the filter membrane 28 is returned to the culture tank 14 via the culture solution delivery / recovery path 22.

[0037] The culture supernatant 29 flows out from the outlet 33 of the filter 15 as indicated by arrow G. The culture supernatant 29 mainly contains the antibody 18. In addition to the antibody 18, the culture supernatant 29 also contains impurities such as cell-derived proteins, cell-derived DNA (deoxyribonucleic acid), and aggregates of the antibody 18, as well as viruses. The concentration of the antibody 18 in the culture supernatant 29 is 1.0 g / L or more.

[0038] A connecting line 34 is connected to the outlet 33 of the filter 15. The connecting line 34 is provided with a delivery pump 35 and a flow meter 36. The delivery pump 35 sends the culture supernatant 29 flowing out from the outlet 33 downstream toward the intermediate section 11 and the purification section 12. The flow meter 36 measures the flow rate of the culture supernatant 29 flowing through the connecting line 34.

[0039] In the intermediate section 11, the connecting path 34 is aseptically connected to a connecting path 41 via a three-way joint 40. The connecting path 41 is connected to the purification section 12. In other words, the connecting paths 34 and 41 connect the culture section 10 and the purification section 12. The connecting paths 34 and 41 are an example of a "connecting line" according to the technology of the present disclosure.

[0040] The connecting path 34 is also aseptically connected to a branch path 42 via a three-way joint 40. The branch path 42 is aseptically connected to branch paths 44 and 45 via a three-way joint 43. Furthermore, the branch path 44 is aseptically connected to branch paths 47 and 48 via a three-way joint 46. Although not shown, a plurality of branch paths are further aseptically connected beyond the branch path 47. These branch paths 42, 44, 45, 47, and 48 are examples of "branch lines" according to the technology of the present disclosure. In other words, a plurality of branch lines are provided between the culture section 10 and the purification section 12. Furthermore, the three-way joint 40 is an example of a "connection portion" in a "connecting line downstream of the connection portion with the branch line" according to the technology of the present disclosure.

[0041] The connecting path 41 is provided with a pinch valve 49 and a sterile filter 50. The pinch valve 49 and the sterile filter 50 are provided in the connecting path 41 downstream of the three-way joint 40, which is the connection point with the branch path 42. The pinch valve 49 is a valve for switching the flow path of the culture supernatant 29 from the connecting path 41 to the branch path 42. The pinch valve 49 is an example of a "non-wetted valve" according to the technology of the present disclosure. The sterile filter 50 filters the culture supernatant 29 flowing into the purification unit 12. The sterile filter 50 incorporates a filter membrane with a pore size of, for example, 0.2 μm.

[0042] A tank 51 is connected to the branch paths 45 and 48. The tank 51 temporarily stores the culture supernatant 29 from the culture unit 10 that flows in from the branch path 42 and the like. The tank 51 is made of a resin film such as polyolefin (PO) or ethylene-vinyl acetate (EVA) copolymer, molded into a rectangular parallelepiped shape, and is flexible. The tank 51 is a single-use product that can be used only once.

[0043] The tank 51 has a capacity that is 0.2 to 10 times the volume of the culture solution 16 in the culture tank 14. Therefore, for example, when the volume of the culture solution 16 is 50 L, which is the lower limit, the capacity of the tank 51 is 10 L to 500 L. Furthermore, when the volume of the culture solution 16 is 5000 L, which is the upper limit, the capacity of the tank 51 is 1000 L to 50000 L. 0.2 to 10 times means 0.2 to 10 times. Note that the tank 51 does not necessarily have to be connected to the branch line, as in the case of the branch paths 45 and 48. The capacity of the tank 51 is preferably 1 to 5 times, and more preferably 1.5 to 5 times, the volume of the culture solution 16 in the culture tank 14.

[0044] A sterile connector 52 is provided at the tip of the branch path 45. A sterile connector 55 is also provided at the tip of an inflow tube 54 connected to an inlet 53 for the culture supernatant 29 of the tank 51. The branch path 45, the inflow tube 54, and ultimately the tank 51 are aseptically connected via these sterile connectors 52 and 55. Similarly, a sterile connector 56 is provided at the tip of the branch path 48, and the branch path 48 is aseptically connected to the tank 51 via the sterile connectors 55 and 56. The entire flow path of the culture supernatant 29 from the culture unit 10 to the tank 51 is aseptically connected via these sterile connectors 52, 55, and 56 and the three-way joints 40, 43, and 46. The tips of the branch path 45 and the branch path 48 are examples of "connections between the branch line and the tank" according to the technology disclosed herein. The tip of the inflow tube 54 is also an example of "connections between the tank and the branch line" according to the technology disclosed herein. The sterile connector 52 may be, for example, a ReadyMate manufactured by Global Life Science Technologies Japan Co., Ltd. ＴＭ A DAC (Disposable Aseptic Connector) can be used.

[0045] A pinch valve 57 is provided in the branch path 45. Furthermore, a pinch valve 58 is provided in the branch path 48. The pinch valve 57 is a valve for allowing the culture supernatant 29 to flow into the tank 51 via the branch path 45. The pinch valve 58 is a valve for allowing the culture supernatant 29 to flow into the tank 51 via the branch path 48. Like the pinch valve 49, the pinch valves 57 and 58 are examples of the "non-liquid-contacting valve" according to the technology of the present disclosure.

[0046] A liquid level detection sensor (not shown) is provided in the tank 51. When the liquid level detection sensor detects that the culture supernatant 29 has reached a predetermined level, a signal indicating that the tank 51 is filled with the culture supernatant 29 is transmitted to the control device 13.

[0047] A drain port 59 is provided at the bottom of tank 51. A drain tube 60 is connected to drain port 59. A sterile connector 61 is provided at the tip of drain tube 60. A waste liquid path 62 is connected to drain tube 60. A sterile connector 63 is provided at the tip of waste liquid path 62. Discharge tube 60, and therefore tank 51, are aseptically connected to waste liquid path 62 via these sterile connectors 61 and 63.

[0048] The waste liquid path 62 is provided with a manual pinch valve 64. When the pinch valve 64 is opened, the culture supernatant 29 temporarily stored in the tank 51 is discharged into a waste liquid tank (not shown) via the outlet 59, the discharge tube 60, and the waste liquid path 62. The culture supernatant 29 from the culture unit 10 is not directly discharged into the waste liquid path 62, but is first stored in the tank 51 and then discharged into the waste liquid path 62 in order to prevent contamination from the waste liquid path 62. Although not shown, a sterile filter may be installed between the waste liquid path 62 and the waste liquid tank.

[0049] The purification unit 12 has an immunoaffinity chromatography device 70, a cation chromatography device 71, and an anion chromatography device 72, and the culture supernatant 29 is continuously purified by these chromatography devices 70 to 72. The culture supernatant 29 flows into the immunoaffinity chromatography device 70 via a connecting path 41. The immunoaffinity chromatography device 70 extracts the antibody 18 from the culture supernatant 29 using a column on which a ligand such as protein A or protein G, which has affinity for the antibody 18, is immobilized on a carrier, thereby producing a first purified solution 73. The first purified solution 73 is subjected to a virus inactivation treatment 74 (hereinafter referred to as a virus inactivation treatment).

[0050] A first purified solution 73 that has been subjected to a virus inactivation treatment 74 flows into the cation chromatography device 71. The cation chromatography device 71 extracts antibodies 18 from the first purified solution 73 using a column with a cation exchanger as the stationary phase, thereby producing a second purified solution 75. The second purified solution 75 flows into the anion chromatography device 72. The anion chromatography device 72 extracts antibodies 18 from the second purified solution 75 using a column with an anion exchanger as the stationary phase, thereby producing a third purified solution 76. The third purified solution 76 is passed through a filter 77 to remove viruses. The third purified solution 76 is then subjected to concentration and filtration treatments using ultrafiltration (UF) and diafiltration (DF) using a filter 78. This results in a drug substance 79 for a biopharmaceutical. By sequentially performing component separation processes using these multiple types of chromatography devices 70 to 72, contaminants and viruses are gradually removed from the culture supernatant 29, and the purity of the antibody 18 is gradually increased. A single-pass TFF filter may be provided upstream of the immunoaffinity chromatography device 70.

[0051] As an example, as shown in Figure 2, the pinch valve 49 has a main body 80 and a holder 81. A cylindrical coil 82, a movable iron core 83, a fixed iron core 84, and a shaft 85 are provided within the main body 80. The movable iron core 83 is housed in the lower part of the cylindrical space of the coil 82. The fixed iron core 84 is fixed to the upper end of the cylindrical space of the coil 82. The lower end of the shaft 85 is attached to the movable iron core 83. A rectangular parallelepiped valve element 86, which is placed in the holder 81, is attached to the upper end of the shaft 85.

[0052] The holder 81 is a generally U-shaped member attached to the upper end surface 87 of the main body 80. A tube 88 that constitutes the connecting passage 41 is inserted into the holder 81. The tube 88 is held in a sandwiched state between the valve body 86 and an operating surface 89 of the holder 81 that faces the upper end surface 87 of the main body 80.

[0053] When no current is flowing through the coil 82, as shown in (A), the movable iron core 83 is located at the lower end of the main body 80, and thus the valve body 86 is located on the side of the upper end surface 87 of the main body 80. In this case, the tube 88 is not crushed by the valve body 86, and the connecting path 41 is open. Therefore, in the case of (A), the culture supernatant 29 flows from the culture unit 10 to the purification unit 12 via the connecting path 41. On the other hand, when a current is flowing through the coil 82, as shown in (B), the magnetic field generated in the coil 82 attracts the movable iron core 83 to the fixed iron core 84, and thus the valve body 86 moves toward the operating surface 89. The tube 88 is crushed by the valve body 86 and the operating surface 89, thereby blocking the connecting path 41. Therefore, in the case of (B), the flow of the culture supernatant 29 from the culture unit 10 to the purification unit 12 via the connecting path 41 is blocked. The fixed core 84 also serves as a stopper that restricts the upward movement of the movable core 83 and, in turn, the valve body 86 .

[0054] In this way, pinch valve 49 pinches tube 88 from the outside, and is capable of switching the flow path without coming into contact with culture supernatant 29. The other pinch valves 57 and 58 have the same configuration and function as pinch valve 49, except that the tube inserted into holder 81 is different, so a detailed description thereof will be omitted. Furthermore, pinch valve 64 has the same configuration and function as pinch valve 49, except that the tube inserted into holder 81 is different and that it is manually operated, so a detailed description thereof will be omitted.

[0055] 3 , the computer constituting the control device 13 includes a storage 95, a memory 96, a CPU (Central Processing Unit) 97, a communication unit 98, a display 99, and an input device 100. These are interconnected via a bus line 101.

[0056] The storage 95 is a hard disk drive built into the computer constituting the control device 13 or connected via a cable or network. Alternatively, the storage 95 is a disk array consisting of multiple hard disk drives. The storage 95 stores control programs such as an operating system, various application programs, and various data associated with these programs. Note that a solid state drive may be used instead of a hard disk drive.

[0057] The memory 96 is a work memory for the CPU 97 to execute processing. The CPU 97 loads programs stored in the storage 95 into the memory 96 and executes processing in accordance with the programs. In this way, the CPU 97 comprehensively controls each part of the computer. The memory 96 may be built into the CPU 97.

[0058] The communication unit 98 controls the transmission of various information to and from external devices. The display 99 displays various screens. Each screen is provided with an operation function using a GUI (Graphical User Interface). The computer constituting the control device 13 accepts input of operation instructions from an input device 100 via each screen. The input device 100 is a keyboard, a mouse, a touch panel, a microphone for voice input, etc.

[0059] 4, a control program 105 is stored in the storage 95 of the control device 13. The control program 105 is an application program for causing a computer to function as the control device 13. In addition to this, the storage 95 also stores data for various screens to be displayed on the display 99.

[0060] When the control program 105 is started, the CPU 97 of the computer constituting the control device 13 cooperates with the memory 96 and the like to function as a culture section control section 110, an intermediate section control section 111, and a purification section control section 112. The culture section control section 110 controls the operation of the culture section 10, the intermediate section control section 111 controls the operation of the intermediate section 11, and the purification section control section 112 controls the operation of the purification section 12.

[0061] The culture unit control unit 110 receives the measurement result of the flow rate of the culture supernatant 29 from the flow meter 36. The culture unit control unit 110 controls the flow rate of the culture supernatant 29 based on the measurement result. More specifically, the culture unit control unit 110 transmits a control signal to the delivery pump 35 to set the flow rate of the culture supernatant 29 to a preset value. In addition to this, the culture unit control unit 110 also controls the supply amount of the culture solution 16 through the culture solution supply path 19, the supply amount of the oxygen-containing gas through the sparger 23 and the gas supply path 24, the rotation speed of the agitator blades 25, etc.

[0062] The intermediate unit control unit 111 transmits control signals to the pinch valves 49, 57, and 58. The control signals are signals that transmit either an instruction to not pass current through the coil 82 of the pinch valve 49 or the like, or an instruction to pass current through the coil 82. When a control signal transmitting an instruction not to pass current through the coil 82 is received, the pinch valve 49 or the like opens the flow path as shown in FIG. 2A. On the other hand, when a control signal transmitting an instruction to pass current through the coil 82 is received, the pinch valve 49 or the like closes the flow path as shown in FIG. 2B. Hereinafter, a control signal transmitting an instruction not to pass current through the coil 82 will be referred to as a control signal (open). Furthermore, a control signal transmitting an instruction to pass current through the coil 82 will be referred to as a control signal (close).

[0063] The purification unit control unit 112 transmits control signals to the chromatography devices 70 to 72. The chromatography devices 70 to 72 operate their internal pumps, valves, and the like in response to the control signals. The purification unit control unit 112 also receives error signals from the chromatography devices 70 to 72. The error signals are issued by the chromatography devices 70 to 72 when a malfunction occurs in the chromatography devices 70 to 72. Examples of such malfunctions include a clogged column in the chromatography devices 70 to 72 or a malfunction of a pump in the chromatography devices 70 to 72. The purification unit control unit 112 transmits the error signal to the intermediate unit control unit 111. The error signal is an example of a "signal indicating that a malfunction has occurred" according to the technology of the present disclosure. Although not shown in the drawings, when the malfunction is resolved, the chromatography devices 70 to 72 transmit a notification signal indicating this to the purification unit control unit 112.

[0064] 5 , when an error signal is received from the purification unit control unit 112, the intermediate unit control unit 111 sends a control signal (close) to the pinch valve 49 and then sends a control signal (open) to the pinch valve 57. This switches the flow path of the culture supernatant 29 from the connecting channel 41 to the branch channels 42 and 45. In other words, when an error signal indicating a malfunction is output from the purification unit 12, the intermediate unit control unit 111 controls the switching of the flow path of the culture supernatant 29 from the connecting channel 41 to the branch channels 42 and 45. The culture supernatant 29 flows into the tank 51 through the branch channels 42 and 45, the inflow tube 54, and the inflow port 53. Although not shown in the figure, when the malfunction in the purification section 12 is resolved and an alarm signal indicating this is received from the chromatography devices 70 to 72, the intermediate section control section 111 sends a control signal (close) to the pinch valve 57, and then sends a control signal (open) to the pinch valve 49, thereby controlling the flow path of the culture supernatant 29 to return from the branch paths 42 and 45 to the connecting path 41.

[0065] 6, (A) shows a state in which the intermediate control unit 111 transmits a control signal (open) to the pinch valve 57 of the branch path 45 and a control signal (close) to the pinch valve 58 of the branch path 48, and the culture supernatant 29 is temporarily stored in the tank 51 connected to the branch path 45. In the state shown in (A), when the intermediate control unit 111 receives a signal from the liquid level detection sensor of the tank 51 connected to the branch path 45 indicating that the tank 51 is filled with the culture supernatant 29, as shown in (B), the intermediate control unit 111 transmits a control signal (close) to the pinch valve 57 of the branch path 45 and then transmits a control signal (open) to the pinch valve 58 of the branch path 48. As a result, the flow path of the culture supernatant 29 switches from the branch path 45 to the branch paths 44 and 48, and the culture supernatant 29 flows into the tank 51 connected to the branch path 48. That is, when the tank 51 connected to the branch path 45, which is one of the branch lines, is filled with the culture supernatant 29, the intermediate section control unit 111 switches use to the tank 51 connected to another branch line, the branch path 48. Although not shown, when the tank 51 connected to the branch path 48 is filled with the culture supernatant 29, the intermediate section control unit 111 switches use to the tank 51 connected to the branch path 45 or another branch path beyond the branch path 47.

[0066] The tank 51 connected to the branch path 45 filled with the culture supernatant 29 is separated from the branch path 45 by manually opening the pinch valve 64 to discharge the culture supernatant 29, and then removing the sterile connector 55 from the sterile connector 52. The sterile connector 55 of a new empty tank 51 is attached to the sterile connector 52. In this way, the used tank 51 is replaced with the new empty tank 51.

[0067] Next, the operation of the above-described configuration will be described with reference to the flowchart shown in Figure 7. First, antibody-producing cells 17 are seeded in culture tank 14 of culture unit 10 and cultured in culture solution 16. As a result, antibodies 18 are produced from antibody-producing cells 17, and antibodies 18 are dispersed in culture solution 16.

[0068] The culture medium 16 is sent to the filter 15 via the culture medium delivery / recovery path 22 and the inlet / outlet 27. The antibody-producing cells 17 are removed from the culture medium 16 by the filter membrane 28 of the filter 15, and the culture medium 16 is turned into a culture supernatant 29. The culture supernatant 29 flows out of the outlet 33 into the connecting path 34 and is sent downstream by the delivery pump 35. At this time, the culture unit control unit 110 sends a control signal to the delivery pump 35 in accordance with the measurement result of the flow meter 36, and the flow rate of the culture supernatant 29 is controlled based on the measurement result of the flow meter 36.

[0069] If no malfunction has occurred in the purification unit 12 (NO in step ST100), the intermediate unit controller 111 sends a control signal (open) to pinch valve 49 and a control signal (close) to pinch valves 57 and 58. This opens the connecting channel 41 and closes the branch channel 45 and other channels. In this way, the culture supernatant 29 flows from the connecting channel 34 into the connecting channel 41. The culture supernatant 29 that has flowed into the connecting channel 41 is filtered by the sterile filter 50 and then flows into the purification unit 12 (step ST110). In the purification unit 12, the antibody 18 in the culture supernatant 29 is continuously purified by the chromatography devices 70 to 72, and ultimately a drug substance 79 for the biopharmaceutical is obtained.

[0070] On the other hand, if a malfunction occurs in the purification unit 12 (YES in step ST100), an error signal is issued from the chromatography devices 70-72 to the purification unit control unit 112. The error signal is transmitted from the purification unit control unit 112 to the intermediate unit control unit 111. In this case, as shown in FIG. 5 , the intermediate unit control unit 111 transmits a control signal (close) to the pinch valve 49, and then transmits a control signal (open) to the pinch valve 57. This closes the connecting channel 41 and opens the branch channel 45. In this way, the culture supernatant 29 flows into the tank 51 via the branch channels 42 and 45, the inflow tube 54, and the inflow port 53 (step ST120). This state in which the culture supernatant 29 flows into the tank 51 and is temporarily stored therein continues until the malfunction in the purification unit 12 is resolved (NO in step ST130).

[0071] 6 , when the tank 51 connected to the branch path 45 is filled with the culture supernatant 29, the intermediate control unit 111 sends a control signal (close) to the pinch valve 57 of the branch path 45, and then sends a control signal (open) to the pinch valve 58 of the branch path 48. This switches the flow path of the culture supernatant 29 from the branch path 45 to the branch paths 44 and 48, and the culture supernatant 29 flows into the tank 51 connected to the branch path 48.

[0072] When the malfunction in the purification unit 12 is resolved (YES in step ST130), the intermediate unit control unit 111 sends a control signal (close) to the pinch valve 57 or 58, and then sends a control signal (open) to the pinch valve 49. This reopens the connecting channel 41, closes the branch channel 45 or 48, and allows the culture supernatant 29 to flow from the connecting channel 34 into the connecting channel 41.

[0073] As described above, the method for producing a drug substance for a biopharmaceutical according to the technology of the present disclosure connects a culture unit 10, which performs a culture step of culturing antibody-producing cells 17 in a culture solution 16 stored in a culture vessel 14 and removing the antibody-producing cells 17 from the culture solution 16 to obtain a culture supernatant 29 containing an antibody 18, to a purification unit 12, which performs a purification step of purifying the antibody 18 from the culture supernatant 29 to obtain a drug substance 79 for a biopharmaceutical, via connecting paths 34 and 41, and allows the culture supernatant 29 to flow from the culture unit 10 to the purification unit 12 through the connecting paths 34 and 41. A flexible, single-use tank 51, which has a capacity of 0.2 to 10 times the volume of the culture solution 16 in the culture vessel 14, is connected to branch paths 45 and 48 provided between the culture unit 10 and the purification unit 12, and the culture supernatant 29 is temporarily stored in the tank 51.

[0074] Because the tank 51 is flexible, it can be folded compactly when not in use, making it easier to handle than the glass tank described in JP-A-2017-515501. Furthermore, the tank 51 is single-use and is not reused like the glass tank described in JP-A-2017-515501, so there is less concern about contamination of the culture supernatant 29. Furthermore, because the tank 51 is single-use, it does not require the same amount of sterilization work as the glass tank described in JP-A-2017-515501. Furthermore, it is easier to dispose of than the glass tank described in JP-A-2017-515501.

[0075] The tank 51 has a capacity of 0.2 to 10 times the volume of the culture medium 16 in the culture tank 14. If the tank 51 had a capacity less than 0.2 times the volume of the culture medium 16 in the culture tank 14, the tank 51 would quickly fill with the culture supernatant 29, necessitating frequent drainage. Alternatively, if the tank 51 does not have a drain outlet 59, the tank 51 would need to be replaced frequently, resulting in poor work efficiency. Conversely, if the tank 51 had a capacity greater than 10 times the volume of the culture medium 16 in the culture tank 14, the tank 51 would be too large and difficult to handle. Therefore, a tank 51 with a capacity of 0.2 to 10 times the volume of the culture medium 16 in the culture tank 14 is an appropriate size for temporarily storing the culture supernatant 29. Therefore, the method for producing a drug substance for a biopharmaceutical according to the technology of the present disclosure can reduce concerns about contamination and is easy to handle. Furthermore, the biopharmaceutical drug substance 79 produced by the method for producing a biopharmaceutical drug substance according to the technology of the present disclosure is less susceptible to contamination and is easy to produce. As mentioned above, the capacity of the tank 51 is preferably 1 to 5 times, and more preferably 1.5 to 5 times, the volume of the culture solution 16 in the culture tank 14.

[0076] The entire flow path of the culture supernatant 29 from the culture section 10 to the tank 51 is connected in a sterile manner, which further reduces the risk of contamination of the culture supernatant 29.

[0077] The connections of the branch paths 45 and 48 with the tank 51 and the connections of the tank 51 with the branch paths 45 and 48 are both made of sterile connectors 52, 55, and 56, and the branch paths 45 and 48 are aseptically connected to the tank 51 via the sterile connectors 52, 55, and 56. This makes it possible to further reduce the risk of contamination of the culture supernatant 29.

[0078] The culture supernatant 29 flowing into the purification section 12 is filtered by a sterile filter 50 provided in the connecting path 41 downstream of the three-way joint 40, which is the connection point with the branch path 42. This prevents bacteria and other contaminants from entering the purification section 12 into the intermediate section 11 and ultimately the culture section 10, and maintains a sterile state upstream of the sterile filter 50, including the branch line 42 and the tank 51. This further reduces concerns about contamination of the culture supernatant 29. The sterile filter 50 is not particularly limited as long as it does not contain live bacteria or other microorganisms and prevents their permeation upstream. However, the pore size of the filter membrane is preferably 0.001 μm to 0.5 μm, more preferably 0.02 μm to 0.3 μm, and particularly preferably 0.1 μm to 0.3 μm.

[0079] The amount of culture solution 16 in the culture tank 14 is 50 L or more and 5000 L or less. When the amount of culture solution 16 in the culture tank 14 is within this range, as described above, the capacity of the tank 51 is 10 L to 500 L, or 1000 L to 50,000 L. Manufacturing a tank 51 of this size out of glass would be quite costly and would also require a lot of effort for sterilization. Therefore, if the amount of culture solution 16 in the culture tank 14 is 50 L or more and 5000 L or less, it is possible to reduce concerns about contamination and the effect of ease of handling can be more effectively exhibited. The amount of culture solution 16 in the culture tank 14 is more preferably 200 L or more and 5000 L or less, and particularly preferably 200 L or more and 3000 L or less.

[0080] The quality of the antibodies 18 in the culture supernatant 29 temporarily stored in the tank 51 deteriorates over time. For this reason, the temporarily stored culture supernatant 29 is discharged from a discharge port 59 provided in the tank 51. This eliminates the risk of culture supernatant 29 with deteriorated quality of the antibodies 18 being mistakenly flowed into the purification unit 12. It is also possible to reliably prevent the culture supernatant 29 from overflowing from the tank 51. Furthermore, this method is more efficient than a case in which the tank 51 is not provided with a discharge port 59 and the tank 51 must always be replaced with a new empty tank 51 when it becomes filled with the culture supernatant 29.

[0081] As in the branch lines 45 and 48, a plurality of branch lines are provided between the culturing unit 10 and the purifying unit 12. Therefore, if a tank 51 is connected to each of the plurality of branch lines, the frequency of replacement of the tank 51 can be reduced compared to, for example, a case in which one tank 51 is connected to one branch line, making handling easier. The number of branch lines is more preferably two or more, and particularly preferably two to ten.

[0082] When the tank 51 connected to one of the branches 45 and 48 is filled with the culture supernatant 29, the tank 51 connected to the other branch is switched over to use, thereby reliably preventing the culture supernatant 29 from overflowing from the tank 51.

[0083] If a malfunction occurs in the purification unit 12, the flow path of the culture supernatant 29 is switched from the connecting path 41 to the branch path 42, etc. This makes it possible to avoid situations in which the operation of the culture unit 10 must be stopped due to a malfunction in the purification unit 12, which would significantly reduce production efficiency.

[0084] The flow path of the culture supernatant is switched using pinch valves 49, 57, and 58, which are non-wetted valves that can switch the flow path without coming into contact with the culture supernatant 29. This makes it possible to further reduce the risk of contamination of the culture supernatant 29.

[0085] The flow path of the culture supernatant 29 is switched using pinch valves 57 and 58 provided in the branch paths 45 and 48, and pinch valve 49 provided in the connecting path 41 downstream of three-way joint 40, which is the connection part with the branch path 42. Therefore, the flow path of the culture supernatant 29 can be reliably switched between the connecting path 41 and the branch path 42, etc.

[0086] The pinch valves 49, 57, and 58 are of a type that pinch the tube 88 or the like that constitutes the connecting path 41 from the outside. Therefore, it is possible to reduce the risk of contamination of the culture supernatant 29 with a simple configuration.

[0087] When an error signal indicating that a malfunction has occurred is output from the purification unit 12, the intermediate unit control unit 111 performs control to switch the flow path of the culture supernatant 29 from the connecting path 41 to the branch path 42, etc. This eliminates the need to manually switch the flow path of the culture supernatant 29 from the connecting path 41 to the branch path 42, etc.

[0088] The flow rate of the culture supernatant 29 is measured by a flow meter 36 provided in the connecting path 34 upstream of a three-way joint 40, which is the connection point of the branch path 42, and the flow rate of the culture supernatant 29 is controlled based on the measurement results. Therefore, the flow rate of the culture supernatant 29 flowing through the connecting path 41 etc. can be kept constant.

[0089] The purification unit 12 continuously purifies the culture supernatant 29. This makes it possible to obtain the drug substance 79 of the biopharmaceutical more efficiently.

[0090] Biopharmaceuticals that contain antibody 18 as an active ingredient are called antibody drugs and are widely used to treat chronic diseases such as cancer, diabetes, and rheumatoid arthritis, as well as rare diseases such as hemophilia and Crohn's disease. Therefore, this example, in which antibody 18 is used as the product, can contribute to the development of antibody drugs that are widely used to treat various diseases.

[0091] The concentration of antibody 18 in culture supernatant 29 is 1.0 g / L or more. This allows for more efficient production of biopharmaceutical drug substance 79. The concentration of antibody 18 in culture supernatant 29 is more preferably 1.2 g / L or more and 15.0 g / L or less, even more preferably 1.3 g / L or more and 13.0 g / L or less, and particularly preferably 1.4 g / L or more and 10.0 g / L or less.

[0092] The concentration of antibody-producing cells 17 in the culture medium 16 was 8.0 × 10 ７ The concentration of antibody-producing cells 17 in the culture medium 16 is 8.0 × 10 ７ If the concentration is 8.0×10 cells / mL or more, the antibody-producing cells 17 are more likely to die when the operation of the culture unit 10 is stopped. Therefore, it is possible to more effectively avoid situations that would significantly reduce production efficiency, such as when the operation of the culture unit 10 has to be stopped due to a malfunction in the purification unit 12. The concentration of the antibody-producing cells 17 in the culture solution 16 is preferably 8.0×10 ７ cells / mL or more 5.0×10 ８ cells / mL or less, more preferably 1.0 x 10 ８ cells / mL or more 4.0×10 ８ cells / mL or less, and particularly preferably 1.0 x 10 ８ cells / mL or more 3.0×10 ８ cells / mL or less.

[0093] Although the sterile connectors 52, 55, and 56 are used as examples of means for aseptically connecting the branch line and the tank 51, the present invention is not limited to this. For example, as shown in Figure 8, the tube 120 constituting the branch line 45 and the inlet tube 54 may be aseptically connected by heat welding.

[0094] For example, a biowelder 121 manufactured by Sartorius Stedim Japan (Internet URL (Uniform Resource Locator): https: / / premium.ipros.jp / sartorius-stedim / product / detail / 2000608451 / ?hub=163&categoryId=52772, see FIG. 9 ) is used for the thermal welding. When replacing a tank 51 filled with culture supernatant 29 with a new empty tank 51, as shown in FIG. 9A for example, a tube 120 constituting a branch path 45 connected to the tank 51 filled with culture supernatant 29 (referred to as a "used tank" in FIG. 9 ) and an inlet tube 54 of a new empty tank 51 (referred to as a "new tank" in FIG. 9 ) are set in the biowelder 121, and thermal welding is instructed. Then, as shown in (B), the connecting line side of the tube 120 constituting the branch path 45 and the tank 51 side of the inflow tube 54 of the new empty tank 51 are aseptically connected by heat welding using the bio-welder 121. At the same time, the tank 51 side filled with culture supernatant 29 of the tube 120 constituting the branch path 45 and the open end side of the inflow tube 54 of the new empty tank 51 are aseptically connected by heat welding using the bio-welder 121.

[0095] In this way, the tube 120 constituting the branch path 45 and the inflow tube 54 connected to the inflow port 53 for the culture supernatant 29 of the tank 51 may be aseptically connected by heat welding. This embodiment also makes it possible to further reduce concerns about contamination of the culture supernatant 29. Note that aseptic connection using the aseptic connector 52 or the like and aseptic connection by heat welding may be combined.

[0096] Although the pinch valves 49, 57, and 58 are shown as examples of non-wetted valves, the present invention is not limited to these. Instead of the pinch valve 49, a screw-type tube clamp 125 shown in FIG. 10 may be used as an example.

[0097] Figure 10 illustrates an example in which a screw-type tube clamp 125 is used instead of the pinch valve 49. In Figure 10, the screw-type tube clamp 125 has a substantially U-shaped holder 126 and a screw 127. The head of the screw 127 forms a knob 128 that the user can grip with their fingers. A rectangular parallelepiped valve body 129 is attached to the tip of the screw 127. The tube 88 that forms the connecting path 41 is set in the holder 126. The tube 88 is held in a sandwiched state between the valve body 129 and an operating surface 130 of the holder 126.

[0098] In the state shown in (A) where the screw 127 is loosened all the way to the top, the tube 88 is not crushed by the valve body 129, and the connecting path 41 is open. Therefore, in the state shown in (A), the culture supernatant 29 flows from the culture unit 10 to the purification unit 12 via the connecting path 41. On the other hand, in the state shown in (B) where the screw 127 is tightened all the way to the bottom, the tube 88 is crushed by the valve body 129 and the operating surface 130, thereby blocking the connecting path 41. Therefore, in the state shown in (B), the flow of the culture supernatant 29 from the culture unit 10 to the purification unit 12 via the connecting path 41 is blocked.

[0099] In this way, the non-wetted valve may be a non-electric type such as a screw-type tube clamp 125. Note that, in addition to the screw type, alligator type, roller type, ratchet type, or the like may also be used as the tube clamp.

[0100] When a non-electrical, non-wetted valve such as the screw tube clamp 125 is used, an indicator such as a warning light may be provided in the purification unit 12 to notify the user of a malfunction in the purification unit 12. In this case, it is not necessary to issue an error signal from the chromatography devices 70 to 72 to the purification unit control unit 112.

[0101] [Second Embodiment] In the first embodiment, the culture supernatant 29 stored in the tank 51 is discarded, but this is not limiting. For example, as in a second embodiment shown in Figures 11 and 12, the culture supernatant 29 stored in the tank 51 may be allowed to flow into the purification unit 12.

[0102] 11 , a return flow path 140 is aseptically connected to a discharge tube 60 connected to a discharge port 59 of a tank 51 via a sterile connector 141, instead of the waste liquid path 62 of the first embodiment. The return flow path 140 is connected to connecting paths 41A and 41B via a three-way joint 142. The three-way joint 142 is provided between the pinch valve 49 and the sterile filter 50. Therefore, the connecting path 41 is divided into an upstream connecting path 41A having the pinch valve 49 and a downstream connecting path 41B having the sterile filter 50.

[0103] The return flow path 140 is provided with a pinch valve 143 and a delivery pump 144. The pinch valve 143 has the same configuration as the pinch valve 49, etc. A control signal is sent to the pinch valve 143 from the intermediate control unit 111. The pinch valve 143 opens and closes the tube that constitutes the return flow path 140 in response to the control signal. The delivery pump 144 delivers the culture supernatant 29, which is temporarily stored in the tank 51, toward the connecting path 41B and, ultimately, the purification unit 12.

[0104] The intermediate section control unit 111 sends a control signal (close) to the pinch valve 49, and then sends a control signal (open) to the pinch valve 143. The intermediate section control unit 111 also sends a control signal to the delivery pump 144 to operate the delivery pump 144. As a result, the culture supernatant 29 stored in the tank 51 flows into the purification unit 12 through the outlet 59, the discharge tube 60, the return flow path 140, and the connecting path 41B.

[0105] 12 shows an example in which a wetted three-way valve 150 is provided in the return flow path 140 instead of the pinch valve 143, and the waste flow path 62 is connected to the wetted three-way valve 150. In this case, the intermediate unit control unit 111 sends a control signal (close) to the pinch valve 49, and then sends a control signal to the wetted three-way valve 150 to open the return flow path 140 side. The intermediate unit control unit 111 also sends a control signal to the delivery pump 144 to operate the delivery pump 144. This causes the culture supernatant 29 stored in the tank 51 to flow into the purification unit 12. When the culture supernatant 29 stored in the tank 51 is to be discharged, the intermediate unit control unit 111 sends a control signal to the wetted three-way valve 150 to open the waste flow path 62 side.

[0106] In this way, in the second embodiment, the culture supernatant 29 temporarily stored is allowed to flow into the purification unit 12 through the outlet 59 provided in the tank 51. Therefore, the culture supernatant 29 temporarily stored in the tank 51 can be effectively utilized with as little waste as possible.

[0107] In order to prevent culture supernatant 29 with deteriorated quality of antibody 18 from flowing into purification unit 12, it is preferable to time the storage time of culture supernatant 29 in tank 51, and to discard culture supernatant 29 whose storage time exceeds a set time without flowing it into purification unit 12. Furthermore, in cases where there is little advantage to flowing culture supernatant 29 into purification unit 12 and reusing it, such as when the amount of culture supernatant 29 stored in tank 51 is extremely small, culture supernatant 29 may be discarded without flowing it into purification unit 12.

[0108] Whether or not to allow the culture supernatant 29 to flow into the purification unit 12 may be determined depending on the amount of culture supernatant 29 stored in the tank 51 and the purification capacity of the purification unit 12. Specifically, if the amount of culture supernatant 29 stored in the tank 51 is an amount that can be processed within a predetermined time period using the purification capacity of the purification unit 12, the culture supernatant 29 is allowed to flow into the purification unit 12. On the other hand, if the amount of culture supernatant 29 stored in the tank 51 is an amount that cannot be processed within a predetermined time period using the purification capacity of the purification unit 12, the culture supernatant 29 is not allowed to flow into the purification unit 12 but is discarded.

[0109] [Examples] Examples of the techniques of the present disclosure will be described below.

[0110] In this example, antibody-producing cells 17 derived from CHO cells producing antibody 18 were cultured in a culture vessel 14 at a density of 12.0 × 10 ７ cells / mL (>8.0×10 ７ The cells were cultured until the concentration reached 12.0 × 10 ７Once the concentration of 10 ...

[0111] The results are shown in Table 155 of Figure 13. The amount of culture medium 16 in the culture tank 14 was 1 L (Examples 1 to 9 and Comparative Examples 1 to 4) or 500 L (Example 10).

[0112] The specific experimental method when the amount of the culture solution 16 was 1 L was as follows: 1) 1 L of the culture solution 16 was poured into a fermenter 14 with a capacity of 2 L manufactured by Biott ABLE. 2) 0.05 × 10 ７ CHO cell-derived antibody-producing cells 17 were seeded to a concentration of 180 cells / mL. 3) The agitator blade 25 was rotated at 180 rpm (rotations per minute), and air was supplied at 19 mL / min and carbon dioxide at 1 mL / min from the gas supply channel 20 at the top of the culture tank 14. Furthermore, oxygen-containing gas was supplied from the sparger 23 at the bottom of the culture tank 14 so that the oxygen concentration in the culture solution 16 was 80%. 4) The antibody-producing cells 17 were cultured while continuously supplying the culture solution 16 to the culture tank 14 from the culture solution supply channel 19 at a perfusion ratio of 1.0 vvd (vessel volume per day, also referred to as the average daily perfusion rate). Then, two days after seeding, the culture solution 16 was continuously filtered using a filter 15 manufactured by Repligen, ATF2, to obtain a culture supernatant 29. 5) Furthermore, on the fifth day after seeding, the perfusion ratio was changed to 2.0 vvd. 6) The concentration of antibody-producing cells 17 in the culture medium 16 was increased to 12.0 × 10 ７When the concentration reached 1000 cells / mL, a cell bleed process was performed in which a portion of the culture medium 16 was withdrawn while the culture medium 16 was being supplied to maintain the concentration. The cell bleed process was performed by feedback-controlling the amount of culture medium 16 withdrawn while monitoring the capacitance of the culture medium 16 with an Aber sensor. 7) Simultaneously with the cell bleed process in 6) above, the connecting paths 34 and 41 connected to the outlet 33 of the Repligen ATF2 were connected to the purification unit 12, and the culture supernatant 29 was continuously purified by each of the chromatography devices 70-72 in the purification unit 12. 8) After maintaining the condition in 7) above for five days, assuming a malfunction in the purification unit 12, the culture supernatant 29 was allowed to flow into the tank 51 for two days while continuing to operate the delivery pump 35. The concentration of antibody 18 in culture supernatant 29 at this time was measured using CedexBio manufactured by Roche Diagnostics K.K. and was approximately 1.5 g / L to 2.0 g / L (>1.0 g / L or more) throughout the five days. In Comparative Example 1, tank 51 was not installed, and therefore operation of delivery pump 35 was stopped as a measure to be taken in case a malfunction occurred in purification unit 12.

[0113] The specific experimental method when the amount of culture solution 16 was 500 L was the same as when the amount of culture solution 16 was 1 L, except that the culture tank 14 was a Thermo Fisher Hypeforma 500, 500 L of culture solution 16 was added, the rotation speed of the stirring blades 25 was 150 rpm, air was supplied at 4.75 L / min and carbon dioxide was supplied at 0.25 L / min from the gas supply path 20 above the culture tank 14, and ATF10 manufactured by Repligen was used as the filter 15.

[0114] The items evaluated were the effect on the culture unit 10, work efficiency, installation space, and contamination inside the tank 51.

[0115] The impact on the culture unit 10 is the most important item. Specifically, the impact on the culture unit 10 was evaluated based on the cell viability of the antibody-producing cells 17 in the culture solution 16 in the culture tank 14 after the culture supernatant 29 was allowed to flow into the tank 51 for two days in 8) above. A cell viability of 85% or more was rated "good," and a cell viability of less than 85% was rated "unacceptable."

[0116] The cell viability was measured by extracting the culture medium 16 from the culture tank 14 after allowing the culture supernatant 29 to flow into the tank 51 for two days, and then introducing the extracted culture medium 16 into a Cell Viability Analyzer Vi-cell XR manufactured by Beckman Coulter. Vi-cell XR2.04 was used as the Vi-cell software, and the parameters used during measurement were set as follows: Min diameter: 6 μm Max diameter: 50 μm Dilution: The concentration of antibody-producing cells 17 in the culture medium 16 was 1.0 × 10 ７ If the concentration of antibody-producing cells 17 in the culture medium 16 is less than 1.0 × 10 cells / mL, the sample is not diluted and the measurement is performed at a dilution of 1. ７ If the concentration is more than 100 cells / mL, dilute the sample 10 times and measure at a dilution of 10. Cell brightness: 85% Cell sharpness: 100 Viable cell spot brightness: 75% Viable cell spot area: 5% Minimum circularity: 0 Decluster degree: Medium

[0117] Work efficiency is the second most important item after the impact on the culture unit 10. Work efficiency was evaluated based on the time required for switching the pinch valve 49 and the like after a malfunction occurred in the purification unit 12, connecting, switching, and replacing the tank 51, and discharging the waste liquid from the outlet 59. If the work time was less than 3 hours out of 48 hours over two days, it was rated as "good," if it was 3 hours or more but less than 6 hours, it was rated as "fair," and if it was 6 hours or more, it was rated as "poor."

[0118] Regarding the installation space, if the installation area of ​​the tank 51 is less than 1.5 times the installation area of ​​the culture tank 14, it is rated as "good", if it is 1.5 times or more but less than 2.5 times, it is rated as "acceptable", and if it is 2.5 times or more, it is rated as "unacceptable".

[0119] Contamination inside the tank 51 is not so important when the culture supernatant 29 temporarily stored in the tank 51 is discarded, but it becomes important when the culture supernatant 29 temporarily stored in the tank 51 is reused by flowing it into the purification unit 12, as in the second embodiment. Contamination inside the tank 51 was rated as "good" if there was no contamination, "fair" if there was contamination, and "poor" if the contamination was particularly severe.

[0120] Example 1 is an example in which the amount of culture solution 16 in the culture tank 14 is 1 L, the number of branch lines such as the branch channel 45 connected to the tank 51 is two, the capacity of the tank 51 is 2 L, and the magnification of the capacity of the tank 51 relative to the amount of culture solution 16 is 2 times. Furthermore, Example 1 is an example in which an outlet 59 is provided in the tank 51, and the means for connecting the branch line such as the branch channel 45 to the tank 51 is a sterile connector 52 or the like. Furthermore, Example 1 is an example in which both the switching valve provided in the branch line such as the branch channel 45 (referred to as "switching valve (branch line - tank)" in Table 155) and the switching valve provided in the connecting line such as the connecting channel 41 (referred to as "switching valve (branch line - purification section)" in Table 155) are tube clamps such as the screw-type tube clamp 125 shown in FIG. 10, and the switching method of the switching valve is manual. Furthermore, Example 1 is an example in which a sterile filter 50 is provided, and the culture supernatant 29 stored in the tank 51 is used as waste liquid. The evaluation of Example 1 was "good" in all aspects: the effect on the culture unit 10, the work efficiency, the installation space, and the contamination inside the tank 51.

[0121] Example 2 is the same as Example 1, except that the capacity of the tank 51 is 0.5 L and the ratio of the capacity of the tank 51 to the amount of culture solution 16 is 0.5. Example 2 was evaluated as "fair" only for work efficiency, with the others being "good." The reason for the "fair" work efficiency is that the capacity of the tank 51 is smaller than that of Example 1, which made the switching, replacement, and wastewater operations of the tank 51 somewhat more time-consuming. Note that when the capacity of the tank 51 was set to 0.2 L and the ratio of the capacity of the tank 51 to the amount of culture solution 16 was set to 0.2, evaluation results similar to those of Example 2 were obtained.

[0122] Example 3 is the same as Example 1, except that the capacity of the tank 51 is 5 L and the ratio of the capacity of the tank 51 to the amount of culture solution 16 is 5 times. The evaluation of Example 3 was "acceptable" only for the installation space, with the others being "good." The reason the installation space was "acceptable" is that the capacity of the tank 51 was 2.5 times that of Example 1 (10 times that of Example 2), which simply increased the installation area of ​​the tank 51. Regarding work efficiency, the work time in Example 1 was 2.5 hours, whereas in Example 3, this was improved to 2 hours due to the increased capacity of the tank 51. Note that when the capacity of the tank 51 was set to 10 L and the ratio of the capacity of the tank 51 to the amount of culture solution 16 was set to 10 times, evaluation results similar to those of Example 3 were obtained.

[0123] Example 4 is the same as Example 1, except that the means for connecting the branch line and tank 51 is a joint rather than a sterile connector 52 or the like, and no sterile filter 50 is provided. Example 4 was evaluated as "fair" only for contamination inside tank 51, and the others were evaluated as "good." The contamination inside tank 51 was rated "fair" because the branch line and tank 51 were not connected in a sterile manner, and the sterile filter 50 was not used to prevent the intrusion of bacteria and the like from the purification section 12.

[0124] Example 5 is the same as Example 1, except that the tank 51 is not provided with an outlet 59, and the means for connecting the branch line to the tank 51 is heat welding as shown in Figures 8 and 9 rather than a sterile connector 52 or the like. Example 5 was evaluated as "fair" only in terms of work efficiency, with the others being "good." The reason why work efficiency was evaluated as "fair" is that there was no way to dispose of the culture supernatant 29 stored in the tank 51, and so each time the tank 51 was filled with the culture supernatant 29, it had to be replaced with a new empty tank 51 using the method shown in Figure 9.

[0125] Example 6 is the same as Example 1, except that the number of branch lines connected to tank 51 is one instead of two, and the means for connecting the branch line to tank 51 is heat welding as shown in Figures 8 and 9 rather than sterile connector 52 or the like. Example 6 was evaluated as "fair" only for work efficiency, and the others were evaluated as "good." The reason why work efficiency was evaluated as "fair" is that there was only one branch line connected to tank 51, and only one tank 51 could be installed, so it was not possible to switch to another tank 51, and the culture supernatant 29 had to be frequently discarded.

[0126] Example 7 is the same as Example 1, except that the culture supernatant 29 stored in the tank 51 is used for purification rather than as waste liquid. As with Example 1, Example 7 was evaluated as "good" in terms of the impact on the culture unit 10, work efficiency, installation space, and contamination inside the tank 51.

[0127] Example 8 is the same as Example 1, except that a wetted three-way valve is used as both the switching valve provided in the branch line and the switching valve provided in the connecting line. The evaluation of Example 8 is that only the contamination inside tank 51 is "fair", and the others are "good". The reason why the contamination inside tank 51 is "fair" is because a wetted valve is used instead of a tube clamp (a non-wetted valve).

[0128] Example 9 is the same as Example 1, except that pinch valves 49, 57, and 58 shown in Figure 2 are used as the switching valves provided in the branch line and the connecting line, and the switching method of the switching valves is automatic. As with Example 1, Example 9 was evaluated as "good" in terms of the impact on the culture unit 10, work efficiency, installation space, and contamination inside the tank 51. Looking at work efficiency, the work time in Example 1 was 2.5 hours, whereas in Example 9, the work time was improved to 2 hours due to the automation.

[0129] Example 10 is an example in which the amount of culture solution 16 in the culture tank 14 is 500 L and the capacity of the tank 51 is 1000 L. Other than this, it is the same as Example 1. As with Example 1, the evaluation of Example 10 was "good" in terms of the impact on the culture unit 10, the work efficiency, the installation space, and the contamination inside the tank 51.

[0130] As described above, Comparative Example 1 is an example in which the tank 51 is not installed, and the operation of the delivery pump 35 is stopped as a measure to be taken when a malfunction occurs in the purification unit 12. The evaluation of Comparative Example 1 is "unacceptable" in terms of the impact on the culture unit 10. The reason why the impact on the culture unit 10 is "unacceptable" is that stopping the operation of the delivery pump 35 forced the operation of the culture unit 10 to be stopped, causing a large amount of cell death.

[0131] Comparative Example 2 is an example in which the capacity of the tank 51 is 0.1 L, the ratio of the capacity of the tank 51 to the volume of the culture medium 16 is 0.1, the connection means between the branch line and the tank 51 is a joint, the switching valve provided in the branch line and the switching valve provided in the connecting line are both wetted three-way valves, and further, no sterile filter 50 is provided. Other than these, it is the same as Example 1. The evaluation of Comparative Example 2 is "good" as in Example 1 in terms of the impact on the culture unit 10 and the installation space, but "poor" in terms of work efficiency and contamination inside the tank 51. The reason for the "poor" rating for work efficiency is that the capacity of the tank 51 is extremely small, 1 / 20 of that of Example 1, which made it very time-consuming to switch, replace, and waste the tank 51. The contamination inside tank 51 was judged to be "unacceptable" because the branch line and tank 51 were not connected in a sterile manner, the sterile filter 50 was not used to prevent the entry of bacteria and the like from the purification section 12, and a liquid-contacting valve was used instead of a tube clamp (a non-liquid-contacting valve).

[0132] Comparative Example 3 is the same as Comparative Example 2, except that the capacity of the tank 51 is 20 L, and the ratio of the capacity of the tank 51 to the volume of the culture solution 16 is 20 times. The evaluation of Comparative Example 3 is "good", the same as Example 1, for the impact on the culture unit 10 and the work efficiency, but "unacceptable" for the installation space and contamination inside the tank 51. The reason why the installation space is "unacceptable" is that, as in Example 3, the capacity of the tank 51 is 10 times that of Example 1, so the installation area of ​​the tank 51 simply increases. The reason why the contamination inside the tank 51 is "unacceptable" is the same as in Comparative Example 2.

[0133] Comparative Example 4 is the same as Example 1, except that the tank 51 is made of glass, as in JP-A-2017-515501, rather than a single-use tank. The evaluation of Comparative Example 4 is "good," as in Example 1, for work efficiency and installation space, but "unacceptable" for the impact on the culture unit 10 and the contamination inside the tank 51. The reason why the impact on the culture unit 10 and the contamination inside the tank 51 were "unacceptable" is that, because the tank 51 is made of glass, the culture supernatant 29 inside the tank 51 is first contaminated, and this contamination inside the tank 51 then affects the culture unit 10.

[0134] From Examples 1 to 10 and Comparative Examples 1 to 4, it was confirmed that the capacity of the tank 51 should be 0.2 to 10 times the volume of the culture medium 16 in the culture tank 14. It was also confirmed that having multiple branch lines is more efficient than having one, and that providing the tank 51 with an outlet 59 is more efficient. It was also confirmed that contamination inside the tank 51 can be effectively prevented by using a sterile connector 52 or heat welding as the connection means between the branch line and the tank 51 and providing a sterile filter 50. It was also confirmed that contamination inside the tank 51 can be effectively prevented by using a non-wetted valve such as a tube clamp or pinch valve as the switching valve that switches the flow path of the culture supernatant 29 from the connecting line to the branch line. It was also confirmed that contamination inside the culture unit 10 and the tank 51 can be effectively prevented by using a single-use tank 51.

[0135] The product is not limited to the antibody 18. It may be any product that can be used as a drug substance 79 for a biopharmaceutical, and may be a protein, a peptide, a nucleic acid (DNA, RNA (Ribonucleic Acid)), a lipid, a virus, a virus subunit, a virus-like particle, or the like.

[0136] In each of the above embodiments, the following various processors can be used as the hardware structure of processing units that perform various processes, such as the culture unit control unit 110, the intermediate unit control unit 111, and the purification unit control unit 112. As described above, the various processors include the CPU 97, which is a general-purpose processor that executes software (control program 105) to function as various processing units, as well as dedicated electrical circuits that are processors having a circuit configuration designed specifically to perform specific processes, such as a programmable logic device (PLD) that is a processor whose circuit configuration can be changed after manufacture, such as an FPGA (Field Programmable Gate Array), and an ASIC (Application Specific Integrated Circuit).

[0137] A single processing unit may be configured with one of these various processors, or may be configured with a combination of two or more processors of the same or different types (e.g., a combination of multiple FPGAs and / or a combination of a CPU and an FPGA).Furthermore, multiple processing units may be configured with a single processor.

[0138] Examples of configuring multiple processing units with a single processor include, first, a form in which one processor is configured with a combination of one or more CPUs and software, as typified by computers such as client and server, and this processor functions as multiple processing units. Second, a form in which a processor is used to realize the functions of the entire system including multiple processing units with a single IC (Integrated Circuit) chip, as typified by systems on chips (SoCs). In this way, various processing units are configured using one or more of the above-mentioned various processors as a hardware structure.

[0139] Furthermore, more specifically, the hardware structure of these various processors can be an electric circuit (circuitry) that combines circuit elements such as semiconductor elements.

[0140] The technology of the present disclosure can be appropriately combined with the various embodiments and / or various modified examples described above. Furthermore, it is not limited to the above-described embodiments, and various configurations can be adopted without departing from the spirit of the present disclosure. Furthermore, the technology of the present disclosure extends not only to programs but also to storage media that non-temporarily store programs.

[0141] The above-described description and illustrations are a detailed explanation of the parts related to the technology of the present disclosure and are merely an example of the technology of the present disclosure. For example, the above description of the configuration, functions, actions, and effects is an explanation of an example of the configuration, functions, actions, and effects of the parts related to the technology of the present disclosure. Therefore, it goes without saying that unnecessary parts may be deleted, new elements may be added, or replacements may be made to the above-described description and illustrations within the scope of the gist of the technology of the present disclosure. Furthermore, to avoid confusion and facilitate understanding of the parts related to the technology of the present disclosure, the above-described description and illustrations omit explanations of common technical knowledge that do not require particular explanation to enable the implementation of the technology of the present disclosure.

[0142] In this specification, "A and / or B" is synonymous with "at least one of A and B." In other words, "A and / or B" means that it may be only A, only B, or a combination of A and B. Furthermore, in this specification, the same concept as "A and / or B" is also applied when three or more things are expressed by connecting them with "and / or."

[0143] All publications, patent applications, and technical standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A culture unit that performs a culture step of culturing cells in a culture solution stored in a culture tank and removing the cells from the culture solution to obtain a culture supernatant containing a product of the cells, and a purification unit that performs a purification step of purifying the product from the culture supernatant to obtain a drug substance for a biopharmaceutical are connected by a connecting line, and the culture supernatant is made to flow from the culture unit to the purification unit through the connecting line, A flexible single-use tank having a capacity of 0.2 to 10 times the volume of the culture solution in the culture tank is connected to a branch line provided between the culture unit and the purification unit, The culture supernatant is temporarily stored in the tank. Manufacturing method for active pharmaceutical ingredients for biopharmaceuticals.

2. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein all of the flow paths for the culture supernatant from the culture section to the tank are connected in a sterile manner.

3. A connection portion of the branch line to the tank and a connection portion of the tank to the branch line are both sterile connectors; The method for producing an active pharmaceutical ingredient of a biopharmaceutical according to claim 2, wherein the branch line and the tank are aseptically connected via the sterile connector.

4. 3. The method for producing a drug substance for a biopharmaceutical according to claim 2, wherein a tube constituting the branch line and an inlet tube connected to an inlet of the tank for the culture supernatant are aseptically connected by heat welding.

5. 2. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the culture supernatant flowing into the purification section is filtered by a sterile filter provided in the connecting line downstream of a connection part with the branch line.

6. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the amount of the culture medium in the culture tank is 50 L or more and 5,000 L or less.

7. 2. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the temporarily stored culture supernatant is discharged from a discharge outlet provided in the tank.

8. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the temporarily stored culture supernatant is made to flow into the purification section through a discharge outlet provided in the tank.

9. The method for producing a drug substance for a biopharmaceutical according to claim 1 , wherein a plurality of branch lines are provided between the culture section and the purification section.

10. The method for producing an active pharmaceutical ingredient of a biopharmaceutical according to claim 9, wherein, when the tank connected to one of the plurality of branch lines is filled with the culture supernatant, use is switched to the tank connected to another branch line.

11. 2. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein, when a malfunction occurs in the purification section, the flow path of the culture supernatant is switched from the connecting line to the branch line.

12. The method for producing a drug substance for a biopharmaceutical according to claim 11, wherein the flow path of the culture supernatant is switched using a non-wetted valve capable of switching the flow path without coming into contact with the culture supernatant.

13. 13. The method for producing a drug substance for a biopharmaceutical according to claim 12, wherein a flow path for the culture supernatant is switched using the non-wetted valve provided in the branch line and the non-wetted valve provided in the connecting line downstream of a connection portion with the branch line.

14. The method for producing a drug substance for a biopharmaceutical according to claim 12, wherein the non-wetted valve is a type that clamps a tube from the outside.

15. The method for producing a drug substance for a biopharmaceutical according to claim 11, further comprising controlling to switch the flow path of the culture supernatant from the connecting line to the branch line when a signal indicating the occurrence of a malfunction is output from the purification unit.

16. measuring the flow rate of the culture supernatant using a flow meter provided in the connecting line upstream of a connection portion with the branch line; The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the flow rate of the culture supernatant is controlled based on the measurement results.

17. The method for producing a drug substance for a biopharmaceutical according to claim 1 , wherein the purification unit continuously purifies the culture supernatant.

18. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the product is an antibody.

19. The method for producing a drug substance for a biopharmaceutical according to claim 18, wherein the concentration of the antibody in the culture supernatant is 1.0 g / L or more.

20. 2. The method for producing a drug substance for a biopharmaceutical according to claim 1, wherein the concentration of the cells in the culture medium is 8.0 x 107 cells / mL or more.

21. A culture unit that performs a culture step of culturing cells in a culture solution stored in a culture tank and removing the cells from the culture solution to obtain a culture supernatant containing a product of the cells; a purification unit that performs a purification step of purifying the product from the culture supernatant to obtain a drug substance for a biopharmaceutical; a connection line connecting the culture unit and the purification unit and allowing the culture supernatant to flow from the culture unit to the purification unit; A branch line provided between the culture section and the purification section; A flexible single-use tank connected to the branch line, the tank having a capacity of 0.2 to 10 times the volume of the culture solution in the culture tank and temporarily storing the culture supernatant; A manufacturing system for active pharmaceutical ingredients for biopharmaceuticals.

22. Measuring the retention time of the culture supernatant in the tank, The method for producing a drug substance for a biopharmaceutical according to claim 8, wherein, when the retention time exceeds a set time, the culture supernatant in the tank is not allowed to flow into the purification section but is instead discharged.

23. A method for producing an active pharmaceutical ingredient of a biopharmaceutical as described in claim 8, wherein, when the amount of the culture supernatant stored in the tank is less than a set amount, the culture supernatant in the tank is discharged without being allowed to flow into the purification section.

24. When the amount of the culture supernatant in the tank is an amount that can be processed in the purification section within a predetermined time, the culture supernatant in the tank is allowed to flow into the purification section, 9. The method for producing a drug substance for a biopharmaceutical according to claim 8, wherein, when the amount of the culture supernatant stored in the tank is an amount that cannot be processed in the purification section within the predetermined time, the culture supernatant in the tank is not allowed to flow into the purification section but is instead discharged.