Waterfall flow type stackable fixed bed bioreactor

Abstract

The utility model discloses a waterfall flow type stackable fixed bed bioreactor which comprises a tank body, a baffle plate is arranged on the inner wall of the tank body, and a cover plate is arranged at the upper end of the tank body in a sealing manner; a shaft seal is rotatably arranged in the middle of the cover plate, a hollow stirring shaft is fixedly arranged at the lower end of the shaft seal, a lower stirrer is fixedly arranged at the tail of the other end of the stirring shaft, and an upper stirrer is arranged in the middle of the stirring shaft; the upper stirrer and the lower stirrer rotate to generate waterfall flow; the fixed bed is used for culturing and fixing the cells. A packaged fiber membrane is arranged in the middle of the fixed bed. The fixed bed bioreactor solves the problems of non-uniform distribution, non-synchronous growth and expression and the like of cells in the traditional fixed bed bioreactor; due to the stirring design, the mass transfer performance is improved, and better growth conditions are given to cells; the waterfall flow design improves the uniformity when the cells are inoculated to the fixed bed, provides possibility for subsequent consistent growth and expression, facilitates cell digestion operation, is milder in reaction, and saves pancreatin, serum and the like.

Description

Technical Field

[0001] The utility model belongs to the technical field of bioreactors, and particularly relates to a cascade-flow type stackable fixed-bed bioreactor. Background Art

[0002] Cell culture refers to a culture technique in which cells are taken from tissues in vivo, and are cultured under aseptic conditions, at an appropriate temperature, pH value, and certain nutrient conditions, simulating the in vivo survival environment, so that they grow and reproduce and maintain their structure and function. In the field of biopharmaceuticals, cell culture is widely used in the production of products such as recombinant protein drugs, monoclonal antibodies, vaccines, cell gene therapy, and other biological agents, bringing unprecedented changes to human health and well-being. The main cell culture methods include three types: adherent culture, suspension culture, and immobilized culture. Animal cells are relatively fragile and are easily affected by shear forces, etc. The cell immobilization technology can better protect cells from mechanical forces and environmental changes, improve the tolerance of cells, thereby achieving high-density culture and increasing the yield of target products.

[0003] At present, the fixed-bed bioreactors widely used in the biomedical field usually use non-woven polyester fiber membranes as fixed carriers, which is a relatively mild fixation method. The advantages of this method are its low shear force, strong anti-pollution performance, the ability to achieve high-density cell growth, and at the same time making the collection, separation, and purification of products more convenient. However, in the existing fiber membrane-based fixed-bed bioreactors, their structure usually consists of two mesh baffles and a tank body jointly forming a fixed hanging basket, and sheet carriers are placed in the fixed hanging basket. Cells and culture media flow through the fixed hanging basket through the bioreactor stirring system. Cells adsorb on the sheet carriers in the fixed hanging basket due to their adherent ability. The culture media flow through the sheet carriers through the bioreactor stirring system and provide nutrients for the cells to maintain the growth and expression of the cells.

[0004] However, due to the limited arrangement method of the sheet carriers in the fixed hanging basket in the prior art, they can only be stacked in multiple layers in an irregular manner, and the sheet carriers are tightly squeezed together, resulting in a large resistance when the cells to be cultured and the culture media flow through the sheet carriers, a short flow path, and it is difficult to distribute evenly inside and outside. At the same time, since the adherent cells adsorb on the tightly stacked sheet carriers, it is difficult to separate the cells from the carriers during digestion. Moreover, the existing fixed-bed bioreactors also have problems such as inconvenient loading of carriers and difficulty in re-amplifying cells. The common disadvantages of spherical carriers and sheet carriers are high cost, poor repeated use effect, and complex digestion and amplification operation procedures.

[0005] Therefore, aiming at the above problems, it is urgent to design a new type of cascade-flow type and multi-layer stackable fixed bioreactor. Summary of the Utility Model

[0006] The purpose of the present utility model is to provide a cascade - type stackable fixed - bed bioreactor to solve the problems raised in the above - mentioned background technology. The following technical solutions are provided: A cascade - type stackable fixed - bed bioreactor, comprising:

[0007] A tank body, with baffles provided on the inner wall of the tank body, and a cover plate hermetically provided at the upper end of the tank body;

[0008] A shaft seal is rotatably provided in the middle of the cover plate. A stirring shaft is fixedly provided at the lower end of the shaft seal. The stirring shaft is hollow inside. At the other end of the stirring shaft, a lower stirrer is fixedly provided, and an upper stirrer is provided in the middle of the stirring shaft. The upper stirrer and the lower stirrer rotate to generate a cascade flow;

[0009] A stackable fixed - bed is provided inside the tank body, which is used for the immobilized culture of cells. An insertion port is provided at the upper end of the fixed - bed, a groove is provided at the lower end of the fixed - bed, and a wound fiber membrane is provided in the middle of the fixed - bed.

[0010] A harvesting pipeline is also provided at the bottom end wall of the tank body, and an annular gas distributor is also provided at the bottom end wall of the tank body. An electrode socket, a temperature electrode socket, and a tail - gas condensation device are fixedly provided on the cover plate, and handles are provided on both sides of the cover plate.

[0011] In this technical solution, the fixed - bed is a fixed - bed prepared from a wound fiber membrane. Compared with the fixed - bed filled with sheet - like carriers, its structure is more uniform, and uniform gaps can be left, which is beneficial to the uniformity of cell immobilization and growth, and facilitates the penetration of the cascade flow in this patent.

[0012] The wound fiber membrane is prepared into a stackable modular component, which is convenient for installation and scale - up, and the stacking number of the modular components can be flexibly adjusted according to process requirements, simplifying the linear scale - up operation.

[0013] After draining the conditioned medium and rinsing the cells with PBS solution containing EDTA, an appropriate amount of digestive solution containing trypsin is added. With a suitable rotation speed (or periodically variable rotation speed), a cascade flow is formed above the fixed - bed, so that the digestive solution spills from above into the side gaps of the wound carrier of the fixed - bed and further uniformly penetrates into the interior of the fixed - bed to complete cell digestion (this method can save the consumption of digestive solution and the digestion effect is milder). After digestion, the digestive solution is drained through the collection pipeline, and an appropriate amount of fresh medium containing serum is added to terminate trypsin. With a suitable rotation speed (or periodically variable rotation speed), a cascade flow is formed above the fixed - bed, so that the medium spills from above into the side gaps of the wound carrier of the fixed - bed to wash the cells out of the fixed - bed (appropriate oscillation can be combined to accelerate cell detachment). After the cells are collected, they can be used for sub - culture and further scale - up production.

[0014] Cells can be fixed, grow and reproduce in the fiber voids of the wound fiber membrane. Multiple fixed beds can be stacked vertically. Operators can stack them according to the required cell attachment area under the premise that the volume of the bioreactor allows, so as to achieve a linear amplification of the cell culture scale.

[0015] In any of the above technical solutions, further, a single feeding port and a four-way feeding port are fixedly installed on the cover plate for feeding the inside of the tank body.

[0016] In this technical solution, the innovative immobilized inoculation method is adopted. During inoculation, the low liquid level is controlled (such as higher than the lowermost fixed bed but not higher than the uppermost fixed bed), and combined with a suitable rotation speed (or periodically variable rotation speed), a waterfall flow is formed above the fixed bed, so that the cell seed liquid containing is evenly sprinkled into the side gaps of the fixed bed roll carrier, helping the cells to be evenly fixed into the fixed bed. After inoculation is completed, the rotation speed is increased to provide a high-efficiency mass transfer driving force and enter the culture mode.

[0017] In any of the above technical solutions, further, a fixed bed support is fixedly provided in the middle of the tank body, and the fixed beds are fixedly stacked on the fixed bed support. The upper stirring includes a first upper stirring, a second upper stirring and a raised cross bar. The space between the first upper stirring and the second upper stirring is hollow and the raised cross bar is fixed on the first upper stirring and the second upper stirring.

[0018] In this technical solution, the cell digestion mode is innovated. An upper and lower double stirring is installed in the reactor. The lower stirring is a propeller type or an inclined blade type, providing downward and horizontal radial thrusts. Considering that the cells are protected by immobilization, the shear force sensitivity is reduced. The downward pressing stirring can extend the residence time of bubbles in the liquid and improve the oxygen transfer efficiency; the upper stirring is a flying saucer type. In addition to providing radial thrust, its interior is hollow and forms a through cavity with the hollow stirring shaft. Under suitable rotation speed conditions, a radial centrifugal force is generated in the hollow cavity of the flying saucer type stirring, extracting liquid from the lower opening of the stirring shaft and forming a downward or obliquely downward waterfall flow from the upper stirring opening.

[0019] The raised cross bar is used to fix the first upper stirring and the second upper stirring to form an A void, and at the same time plays a stirring role to solve the problem of insufficient stirring efficiency caused by the fixed bed blocking the lower stirring.

[0020] The first upper stirring, the second upper stirring and the raised cross bar form a flying saucer type stirring structure. The space between the first upper stirring and the second upper stirring is hollow, and the inside of the stirring shaft is hollow, and the hollow parts of the two are connected. Under suitable stirring speed conditions, a radial centrifugal force is generated in the hollow cavity of the flying saucer type stirring structure, extracting liquid from the lower opening of the stirring shaft and forming a downward or obliquely downward waterfall flow from the upper stirring opening.

[0021] In this device, the structure of the biological bed is designed so that the fixed beds are stacked in a spatial position. After stacking, a radial space remains in the middle of the fixed bed, into which a stirring shaft can be inserted.

[0022] Stirring blades are respectively arranged at the upper and lower ends of the stirring shaft, so that the reagent solution in the reactor can flow down in a waterfall manner from the lower end through the middle of the stirring shaft to the upper stirring part, thereby solving the problem of uneven distribution of nutrients and oxygen in the vertical direction.

[0023] Moreover, during the cultivation process, the cell digestion is less difficult, the operation procedure is complex, and it can ensure the uniform transfer of nutrients, oxygen, etc., enabling the cells in the inner layer to obtain sufficient growth support and promoting the normal growth and expression of the cells.

[0024] The beneficial effects of the present utility model are as follows: The fixed bed solves the problems of uneven distribution of cells, asynchronous growth and expression in traditional fixed bed bioreactors; and adopts modular stackable fixed beds, which are convenient for installation and scale-up, and the stacking quantity of modular components can be flexibly adjusted according to process requirements, simplifying the linear scale-up operation; the stirring design improves the mass transfer performance and provides better growth conditions for cells; the waterfall flow design improves the uniformity when cells are inoculated into the fixed bed, provides the possibility for subsequent consistent growth and expression, and at the same time facilitates the operation of cell digestion, the reaction is milder, and the use of trypsin, serum, etc. is saved. Description of the Drawings

[0025] Figure 1 is the external view schematic diagram of the present utility model;

[0026] Figure 2 is the top view of the upper stirring of the present utility model;

[0027] Figure 3 is the front view of the upper stirring and the lower stirring of the present utility model;

[0028] Figure 4 is the schematic diagram when the fixed beds of the present utility model are stacked for use.

[0029] Figure 5 is the schematic diagram of the internal waterfall flow of the present utility model.

[0030] The reference numerals in the figure are: 101, shaft seal; 102, stirring shaft; 103, upper stirring; 104, lower stirring; 105, tank body; 106, cover plate; 107, baffle; 108, fixed bed support; 109, bottom ventilation path; 110, annular gas distributor; 111, collection pipeline; 112, handle; 113, single feeding port; 114, four-way feeding port; 115, electrode socket; 116, temperature electrode socket; 117, tail gas condensation device; 118, first upper stirring; 119, second upper stirring; 120, raised cross bar; 121, lower stirring blade; 201, fixed bed; 202, socket; 203, wound fiber membrane; 204, groove. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present application will be clearly described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art belong to the scope protected by the present application.

[0032] In the description of the present application, it should be noted that the terms used here are only for describing specific implementation manners, and are not intended to limit the exemplary embodiments according to the present application. For the convenience of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. Technologies, methods, and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technologies, methods, and devices should be regarded as part of the authorization specification. In all the examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0033] Embodiment 1:

[0034] As Figures 1-5 shown, this embodiment provides a waterfall-flow type stackable fixed-bed bioreactor, including:

[0035] A tank body 105, a baffle 107 is provided at the inner wall of the tank body 105, and a cover plate 106 is hermetically provided at the upper end of the tank body 105;

[0036] In the middle of the cover plate 106, a shaft seal 101 is rotatably provided. At the lower end of the shaft seal 101, a stirring shaft 102 is fixedly provided. The inside of the stirring shaft 102 is hollow. At the other end of the stirring shaft 102, a lower stirrer 104 is fixedly provided. In the middle of the stirring shaft 102, an upper stirrer 103 is provided; the upper stirrer 103 and the lower stirrer 104 rotate to generate a waterfall flow;

[0037] Inside the tank body 105, a fixed bed 201 is stacked, and the fixed bed 201 is used for the culture and fixation of cells. At the upper end of the fixed bed 201, a socket 202 is provided. At the lower end of the fixed bed 201, a groove 204 is provided. And in the middle of the fixed bed 201, a wound fiber membrane 203 is provided.

[0038] At the bottom end wall of the tank body 105, a collection pipeline 111 is further provided. At the bottom end wall of the tank body 105, an annular gas distributor 110 is further provided. On the cover plate 106, an electrode socket 115, a temperature electrode socket 116 and a tail gas condensation device 117 are fixedly provided. And on one side of the cover plate 106, a handle 112 is provided.

[0039] In this technical solution, the tank body 101 is made of glass material, and the fixed bed 201 is a fixed bed prepared by a wound fiber membrane. Compared with the fixed bed filled with sheet carriers, its structure is more uniform, and uniform gaps can be left, which is beneficial to the uniformity of cell fixation and growth, and facilitates the penetration of the waterfall flow in this patent.

[0040] The wound fiber membrane is prepared into stackable modular components, which is convenient for installation and amplification, and the stacking quantity of the modular components can be flexibly adjusted according to process requirements, simplifying the linear amplification operation.

[0041] After emptying the conditioned medium and rinsing the cells with PBS solution containing EDTA, an appropriate amount of digestive solution containing trypsin is added. With a suitable rotation speed (or periodically variable rotation speed), a waterfall flow is formed above the fixed bed, so that the digestive solution spills from above into the side gaps of the wound carrier of the fixed bed, and further uniformly penetrates into the inside of the fixed bed to complete cell digestion (this method can save the consumption of digestive solution, and the digestion effect is more gentle). After digestion, the digestive solution is emptied through the collection pipeline 111, and an appropriate amount of fresh medium containing serum is added to terminate trypsin. With a suitable rotation speed (or periodically variable rotation speed), a waterfall flow is formed above the fixed bed, so that the medium spills from above into the side gaps of the wound carrier of the fixed bed to wash the cells out of the fixed bed (appropriate oscillation can be combined to accelerate cell detachment). After the cells are collected, they can be used for transfer and further scale-up production.

[0042] Cells can be fixed, grown and propagated in the fiber voids of the wound fiber membrane 203. Multiple fixed beds 201 can be stacked up and down. Operators can stack them according to the membrane area requirement under the premise that the volume of the bioreactor permits, so as to realize the linear amplification of the cell adherent area.

[0043] In a preferred embodiment of the present utility model:

[0044] As Figure 1 shown, specifically, a single feeding port 113 and a four-way feeding port 114 are fixedly installed on the cover plate 106 for feeding the inside of the tank body 105.

[0045] In this technical solution, the inoculation method of innovation immobilization is adopted. During inoculation, the low liquid level is controlled (such as higher than the lowest fixed bed but not higher than the uppermost fixed bed), and a suitable rotation speed (or periodically variable rotation speed) is coordinated to form a waterfall flow above the fixed bed, so that the cell seed liquid is evenly sprinkled into the side gap of the fixed bed roll carrier, helping the cells to be evenly fixed into the fixed bed. After inoculation: the rotation speed is increased to provide a high-efficiency mass transfer driving force and enter the culture mode.

[0046] In a preferred embodiment of the present utility model:

[0047] As Figures 2-5 shown, specifically, a fixed bed support 108 is fixedly arranged in the middle of the tank body 105, and the fixed beds 201 are fixedly stacked on the fixed bed support 108. The upper stirrer 103 includes a first upper stirrer 118, a second upper stirrer 119 and a convex cross bar 120. The space between the first upper stirrer 118 and the second upper stirrer 119 is hollow, and the convex cross bar 120 is fixed on the first upper stirrer 118 and the second upper stirrer 119.

[0048] In this technical solution, the cell digestion mode is innovated. Upper and lower double stirrers are installed in the reactor. The lower stirrer 104 is of a propeller type or an inclined blade type, providing downward and horizontal radial thrusts. Considering that the cells are protected by immobilization and the shear sensitivity is reduced, the downward pressing stirrer can extend the residence time of bubbles in the liquid and improve the oxygen transfer efficiency; the upper stirrer 103 is of a flying saucer type. In addition to providing radial thrust, its interior is hollow and forms a through cavity with the hollow stirring shaft 102. Under suitable rotation speed conditions, a radial centrifugal force is generated in the hollow cavity of the flying saucer type stirrer, sucking the liquid from the lower opening of the stirring shaft and forming a downward or obliquely downward waterfall flow from the upper opening of the upper stirrer.

[0049] The convex cross bar 120 is used to fix the first upper stirrer and the second upper stirrer to form an A gap, and at the same time plays a stirring role to solve the problem of insufficient stirring efficiency of the lower stirrer blocked by the fixed bed.

[0050] The first upper stirrer 118, the second upper stirrer 119 and the raised cross bar 120 form a flying saucer type stirring structure. The space between the first upper stirrer 118 and the second upper stirrer 119 is hollow, and the inside of the stirring shaft 102 is hollow, and the hollow parts of the two are connected. At an appropriate stirring speed, a radial centrifugal force is generated in the hollow cavity of the flying saucer type stirring structure, which extracts liquid from the lower opening of the stirring shaft and forms a downward or obliquely downward waterfall flow from the upper stirring opening.

[0051] In this device, the structure of the biological bed is designed so that the fixed beds are stacked in space. After stacking, a radial space remains in the middle of the fixed bed, into which the stirring shaft can be inserted.

[0052] Stirring blades are respectively arranged at the upper and lower ends of the stirring shaft, so that the reagent solution in the reactor can flow down in a waterfall manner from the lower end through the middle of the stirring shaft to the upper stirring part, thereby solving the problem of uneven distribution of nutrients and oxygen in the vertical direction.

[0053] Moreover, during the cultivation process, the cell digestion is less difficult, the operation procedure is simplified, and the transfer of nutrients, oxygen, etc. can be ensured to be uniform, enabling the cells in the inner layer to obtain sufficient growth support and promoting the normal growth and expression of the cells.

[0054] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Without conflict, the embodiments and the features in the embodiments in the present application can be combined with each other. The present application is not limited to the above specific embodiments. The above specific embodiments are merely illustrative rather than restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all of them fall within the protection scope of the present application.

Claims

1. A cascade-type stackable fixed-bed bioreactor, characterized in that: include: A tank body (105), wherein a baffle (107) is provided on the inner wall of the tank body (105), and a cover plate (106) is sealed at the upper end of the tank body (105); A shaft seal (101) is rotatably provided in the middle of the cover plate (106); a stirring shaft (102) is fixedly provided at the lower end of the shaft seal (101); the interior of the stirring shaft (102) is hollow; a lower stirring shaft (104) is fixedly provided at the other end of the stirring shaft (102); an upper stirring shaft (103) is provided in the middle of the stirring shaft (102); the upper stirring shaft (103) and the lower stirring shaft (104) rotate to generate a waterfall flow; A fixed bed (201) is stacked inside the tank body (105), and the fixed bed (201) is used for culturing and fixing cells.

2. A cascade-flow stackable fixed-bed bioreactor according to claim 1, characterized in that: A single feeding port (113) and a four-way feeding port (114) are also fixedly mounted on the cover plate (106) for feeding the inside of the tank body (105).

3. The cascade-flow stackable fixed-bed bioreactor according to claim 1, characterized in that: An electrode socket (115), a temperature electrode socket (116) and an exhaust gas condensation device (117) are also fixedly provided on the cover plate (106), and a handle (112) is also provided on one side of the cover plate (106).

4. The cascade-flow stackable fixed-bed bioreactor according to claim 1, characterized in that: A collecting pipeline (111) is also provided at the bottom end wall of the tank body (105), and an annular gas distributor (110) is also provided at the bottom end wall of the tank body (105).

5. The cascade-flow stackable fixed-bed bioreactor according to claim 1, characterized in that: A fixed bed support (108) is fixedly provided in the middle of the tank body (105), and the fixed bed (201) is fixedly stacked on the fixed bed support (108).

6. The cascade-flow stackable fixed-bed bioreactor according to claim 5, characterized in that: The upper end of the fixed bed (201) is provided with a socket (202), the lower end of the fixed bed (201) is provided with a groove (204), and the middle part of the fixed bed (201) is provided with a rolled fiber membrane (203).

7. The cascade-flow stackable fixed-bed bioreactor according to claim 5, characterized in that: The upper agitator (103) includes a first upper agitator (118), a second upper agitator (119) and a raised horizontal bar (120), wherein the first upper agitator (118) and the second upper agitator (119) are hollow and the raised horizontal bar (120) is fixed on the first upper agitator (118) and the second upper agitator (119).

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