Cell culture perfusion feeding device

Abstract

The invention relates to a cell culture perfusion discharging device which comprises a material pipe used for discharging, a membrane material arranged on the periphery of the material pipe in a sleeving mode and a hollow magnetic driving device connected to the membrane material. The magnetic drive comprises a hollow motor used for adjusting rotation of the membrane material and a magnetic coupling assembly used for sterile isolation, and the magnetic coupling assembly is placed between the hollow motor and the membrane material. The invention aims to solve the problems of high risk and high cost caused by a complex system of perfusion liquid change of a hollow fiber column in cell culture.

Description

Technical Field

[0001] The present application relates to the technical field of biopharmaceuticals, and more particularly to a cell culture perfusion discharge device. Background Art

[0002] In the field of biopharmaceuticals, the target product is produced through large-scale culture through the expression system of animal cells, such as CHO and SF9; the expressed product is secreted by the cells in the culture medium of the culture system. Since the cells are in the process of continuous expression, it is necessary to continuously add nutrients to the culture system. At the same time, cells and metabolites are continuously separated while reducing cell damage and loss. This process is a continuous perfusion harvesting process. At present, the mainstream method is to separate by hollow fiber columns placed outside the reactor. As consumables in the process of use, hollow fiber columns have high pharmaceutical costs due to the complexity of the molding process. Secondly, since the hollow fiber columns are placed outside the reactor to reciprocate and extract and return the culture medium containing cells, the aseptic control requirements of the system are extremely high. Once a pipeline or a link is contaminated, the culture system of the reactor will be contaminated, resulting in the failure of the reactor culture. Furthermore, the hollow fiber system has a certain non-circulating volume, that is, dead volume, and it is necessary to conduct flow matching research in the control process. Otherwise, due to the mismatch of the circulation volume, the cell concentration in the hollow fiber column system increases, thereby blocking the hollow fiber column and making it impossible to continuously harvest the expression product. In view of the above key technologies, the present invention intends to solve the defects in the hollow fiber column perfusion system: 1: the external system is complex and bloated, with high risk of contamination and cumbersome operation; 2: there is blockage caused by process matching during the perfusion process; 3: high cost caused by consumables and complex systems. Summary of the invention

[0003] In order to solve the problems of complex and bloated external systems, high risk of contamination, and cumbersome operation, the present application provides a cell culture perfusion discharge device.

[0004] The present application provides a cell culture perfusion discharge device using the following technical solution:

[0005] A cell culture perfusion discharge device includes a hollow shaft motor for providing power and penetrating a discharge pipe, and a coupling connected between the hollow shaft motor and an outer rotor for transmission; through magnetic coupling, the outer rotor transmits the power of the motor to the inner rotor after penetrating an isolation sleeve, and the inner rotor drives the filter membrane material at the bottom to rotate with the assistance of upper and lower bearings; the hollow shaft motor, hollow shaft outer rotor, hollow shaft inner rotor, and the hollow circle inside are all designed for the discharge pipe to penetrate the entire structure, avoiding the rotating component to extract the cell culture fluid that has passed through the diaphragm, thereby continuously harvesting the product.

[0006] The present application replaces the existing hollow fiber column separation system that is placed outside the reactor with a rotating diaphragm system that is built into the reactor, so that the direction of material flow is a single flow toward the outside of the tank; magnetic coupling is introduced as a rotation drive for the filter membrane, and the transmission mechanism inside and outside the reaction is isolated by the use of an isolation sleeve, so that the risk of leakage pollution caused by the transmission mechanism is reduced to 0; the membrane material is rotated, and the centrifugal force on the periphery of the membrane material is used to reduce the accumulation of cells on the periphery of the membrane, thereby reducing the risk of the membrane being blocked by cells.

[0007] Preferably, the magnetic coupling drive adopts internal and external high-temperature magnet coupling drive, and the internal and external magnets are coated with biocompatible materials, such as PTFE, PEEK, PP, and other engineering plastics, so that the magnets can avoid contact with biological process materials and contamination of the stock solution during application.

[0008] Through the above scheme, the reactor avoids the risk of seal failure caused by mechanical seals, oil seals and other moving seals during the sterile control process, thereby avoiding the situation where the reactor seal failure causes the inside and outside of the reaction tank to be connected and the cell culture environment in the tank is polluted.

[0009] Preferably, the filter membrane is made of a high molecular polymer with a smooth surface and uniform pore size, such as PTFE, polyethersulfone, PET and other base materials; and during the perfusion process, it is connected to the inner rotor and rotates at a uniform speed.

[0010] Through the above scheme, cells and cell fragments in the perfusion discharge process cannot form agglomerations on the smooth membrane surface. While the perfusion discharge pipeline extracts the supernatant, the motor is started to drive the filter membrane to rotate, generating centrifugal force in the liquid. The centrifugal force throws the cells and other particles with higher density away from the circular motion, thereby reducing the probability of membrane clogging and increasing the perfusion discharge speed.

[0011] Preferably, the inner and outer magnetic drives are both pre-embedded with high temperature resistant magnets.

[0012] By adopting high-temperature resistant magnets, the magnetic field strength of the magnetic drive is guaranteed not to weaken during the high-temperature steam sterilization process of the reactor, ensuring that the strength of the magnet coupling is consistent during long-term use of the device, and stably controlling the consistency of internal and external rotation, so that the inner rotor will not rotate out of control, causing the membrane material rotation failure, resulting in the inability to generate centrifugal force, causing the membrane pores to be blocked.

[0013] Preferably, a concentric fixing seat is installed above the hollow shaft motor.

[0014] The discharge pipe is fixed at the center of the hollow shaft motor through a concentric fixing seat. When the external connecting hose is pulled to perfuse the fluid, the discharge pipe will not interfere with the inner wall of the hollow shaft due to the side force, causing the hollow shaft motor to rub the discharge pipe during rotation, resulting in the discharge pipe being worn out and the motor being damaged.

[0015] In summary, the present application includes at least one of the following beneficial technical effects:

[0016] The present application replaces the hollow fiber column filtration discharge system placed outside the reactor container. When continuously harvesting cell expression products, disinfection and sterilization are carried out together with the reactor. By rotating the membrane material that intercepts the cells, centrifugal force is generated to prevent the cells from clogging the filter membrane. Through the magnetic coupling of the internal and external magnetic drives, power transmission inside and outside the reactor is achieved, completely isolating the internal and external environments of the tank.

[0017] The transmission component adopts a hollow shaft with internal and external magnetic drives, and cooperates with a hollow shaft motor to simplify the complex perfusion discharge structure into an integrated hollow shaft structure, thereby avoiding the middle discharge pipe when rotating the membrane material for perfusion discharge, and no longer requiring other mechanical structures such as shaft seals, oil seals and other vulnerable parts.

[0018] The rotating membrane material adopts a single-layer polymer membrane material. During the rotating perfusion discharge process, its periphery is subjected to centrifugal force, smooth surface material, and reasonable filter pore size design to prevent cells and debris from clogging the filter membrane during perfusion discharge. At the same time, it avoids the shearing of cells caused by the high-speed fluid in the hollow fiber column when it reciprocates to extract the liquid and pushes back the liquid. Furthermore, during the operation of the membrane material, there are no living cells inside the rotating membrane material to cause challenges to mass transfer and oxygen transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Diagram of the perfusion discharge system of the existing hollow fiber column in cell culture;

[0020] Figure 2 is a system diagram of the present application in an example of a cell culture reactor;

[0021] Figure 3 This is a cross-sectional view of the hollow motor structure of the present application;

[0022] Figure 4 This is a cross-sectional view of the magnetic drive membrane isolation sleeve structure of the present application;

[0023] Figure 5 is a cross-sectional view of the magnetic drive structure;

[0024] Figure 6 It is a cross-sectional view of the fixing structure of the hollow shaft motor and the discharge pipe.

[0025] Explanation of the reference numerals: 01, bioreactor, 02, cell culture fermentation broth, 03, hollow fiber column, 04, diaphragm pump, 05, perfusion discharge peristaltic pump, 06, supernatant harvesting container, 07, harvesting supernatant, 08, feeding container for fresh culture medium, 09, feeding fresh culture medium, 10, perfusion feed peristaltic pump; 201, perfusion discharge pipe, 202, bearing, 203, protective cover, 204, outer rotor, 205, isolation sleeve, 206, inner rotor, 207, sealing ring, 208, bearing, 209, bearing chamber, 210, bearing, 211, filter membrane material, 312, concentric fixing seat, 313, motor, 314, motor fixing seat. Specific implementation cases

[0026] The following combination Figures 1 to 6 For further details, refer to Figure 1 , Figure 1 Disclosed is an existing method for harvesting supernatant; 01 is a bioreactor, 02 is a fermentation liquid in the bioreactor, 03 is a hollow fiber column composed of a plurality of fine hollow fiber filaments, 04 is a diaphragm pump, 05 is a supernatant harvesting peristaltic pump, 06 is a container for harvesting supernatant, 07 is harvested supernatant, 08 is a feeding container for fresh culture medium, and 09 is a feeding fresh culture medium; the existing Figure 1 The operation mode is that in the expression stage of cell culture, the feed speed and the discharge speed are basically consistent through the speed coordination of the feed pump 09 and the harvest pump 05, and the products expressed by the cells are continuously harvested from the reactor. The role of the hollow fiber column in the system is to extract the fermentation liquid with cells from the reactor through a diaphragm pump, and pass through multiple fiber columns by tangential flow filtration. On the other side of the hollow fiber column, the peristaltic pump extracts the supernatant in the fermentation liquid, and the cells are isolated by the hollow fiber membrane and then pushed back into the reaction tank by the diaphragm pump. The diaphragm pump periodically extracts the fermentation liquid and returns the fermentation liquid. The peristaltic pumps for feeding and discharging need to cooperate with the processing capacity of the hollow fiber column to avoid excessive concentration of cells in the dead volume in the hollow fiber column, which leads to excessive cell concentration in the hollow fiber column and blockage of the column.

[0027] Embodiment 1:

[0028] This application example discloses a perfusion discharge device, referring to Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6, install the perfusion discharge device on the top of the reactor, connect the top discharge pipe and the supernatant harvesting bottle, sterilize the reactor and the perfusion discharge device with high-pressure steam, and then carry out cell culture. When the cell density is cultured to a certain density, the cells express the target protein. At this time, the feeding and discharging operations are carried out. The outer rotor is driven by the rotation of the motor, and the outer rotor drives the inner rotor through magnetic coupling. The inner rotor drives the filter membrane material to rotate, and the perfusion discharge peristaltic pump continuously harvests the supernatant fermentation liquid containing the target product from the reactor.

[0029] Reference Figure 3 The hollow shaft motor is installed at the top of the device, with the discharge pipe concentrically fixed above and the base connected to the external magnetic drive below. The running speed of the motor is adjustable, and the corresponding speed is matched according to the speed of the perfusion discharge.

[0030] Reference Figure 4 The outermost layer of the device is a protective cover (203) to prevent foreign objects from being drawn into the system during the rotation of the device. At the same time, a bearing (202) is installed on the top to provide fixed and concentric support for the outer rotor (204). High-temperature resistant magnets are embedded in the outer rotor. After the magnetic field penetrates the isolation sleeve (205), it is coupled with the inner rotor (206), thereby transmitting the power of the motor to the rotating membrane material (211) at the bottom.

[0031] Reference Figure 5 The bearing chamber (209) of the device connecting with the reactor is the foundation of the whole device, providing a connection flange with the reactor, a rotating bearing installation of the inner rotor, and a support shield (203). The upper and lower installation positions are designed concentrically to ensure the concentricity between the various rotating parts.

[0032] Reference Figure 6 The gap protection between the discharge pipe of the device and the hollow shaft of the motor, the hollow shaft of the outer rotor, and the hollow shaft of the inner rotor is fixed at the top of the motor to ensure that the outer wall of the discharge pipe is not worn during each circular motion process.

[0033] The above is the preferred solution provided by this application, and it does not limit the protection scope of this application. Therefore, all equivalent changes based on the appearance, structure and principle of this application are included in the protection scope of this application.

Claims

1. A cell culture perfusion discharge device, characterized in that: The invention comprises a filter membrane (211) for intercepting reactor cells, which is connected to a rotating inner rotor (206). The filter membrane (211) is driven to rotate after magnetic coupling with an outer rotor (204) outside an isolation sleeve (205), and the outer rotor (204) is freely rotated through a bearing (202) fixed to a protective cover (203).

2. A cell culture perfusion effluent device according to claim 1, characterized in that: The support bearing of the inner rotor (206) is installed on the bearing chamber (209), and the center is designed as a hollow pipeline so as to avoid the discharge pipe (201) in the middle during rotation.

3. A cell culture perfusion discharge device according to claim 2, characterized in that: The discharge pipe (201) is fixed at the center of the isolation sleeve (205) and penetrates the center of the outer rotor (204). The outer rotor is designed as a hollow shaft. The rotational power is provided by a motor (313).

4. A cell culture perfusion discharge device according to claim 3, characterized in that: The outer rotor (204) is connected to the motor (313), and the motor (313) is designed as a hollow shaft.

5. A cell culture perfusion discharge device according to claim 1, characterized in that: The inner rotor (206) and the outer rotor (204) transmit power through magnetic coupling, and the built-in magnets are high-temperature resistant magnets.

6. A cell culture perfusion discharge device according to claim 1, characterized in that: The filter membrane (211) can isolate the cells in the fermentation liquid so that the cells are not drawn out of the reactor. The pore size of the filter membrane is smaller than the pore size of the cells.

7. A cell culture perfusion discharge device according to claim 4, characterized in that: The top end of the motor (313) is connected to the concentric fixing seat (312).

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