CN206814791U - Disposable intelligent digester systems for animal cell culture - Google Patents

Abstract

The utility model discloses a disposable intelligent digester system for animal cell culture, comprising a disposable digester unit and an intelligent control unit, the disposable digester unit includes at least a closed chamber that can be filled with fluid, the A retention component and a stirring component are provided in the closed chamber, and the retention component is connected to the lower portion of the closed chamber, wherein the retention component is at least used to make the dispersed cells flow to the next-stage bioreactor and make the empty carrier and the carrier with cells are trapped in the closed chamber, and the agitation assembly is at least used to mix the fluids in the closed chamber. The disposable intelligent digester system of the utility model saves steps such as on-line disinfection and on-line cleaning, equipment installation and debugging, simplifies the operation process, saves production costs and labor costs, and avoids cross-contamination between batches; and By adopting an intelligent program to control the digestion process, the cell digestion loss rate can be as low as 2%.

Description

Disposable intelligent digester system for animal cell culture

technical field

The utility model relates to a technique for obtaining free cells by separating cells from microcarriers in the process of animal cell culture, and more particularly relates to a disposable intelligent digester system for animal cell culture.

Background technique

In the production of high value-added biological products such as vaccines, antibodies, and protein drugs, some mammalian cells used cannot be directly suspended in the culture medium for growth, and need to attach to the surface of a carrier with a small amount of positive charge to grow. The basic process is : Mammalian cells are mechanically dispersed or digested to make a cell suspension, filtered, centrifuged, purified, rinsed, and then inoculated into a reactor containing fresh medium for culture. The cells cultured by this method grow well and are easy to observe, but this method is only suitable for laboratory research. However, in the process of large-scale cell culture, inoculation and expansion, adherent cells have the characteristics of contact inhibition. Once the cells cover the surface, the growth will be inhibited, affecting the final cell yield. Therefore, it is necessary to digest and separate the cells from the carrier, and enter into a new carrier.

At present, the methods of cell digestion mainly include digestion inoculation, in situ digestion inoculation, direct inoculation and other methods.

However, regardless of the above digestion methods, the digesters used are still rotary bottles, Erlenmeyer flasks, mechanically stirred fermenters, etc. These digesters consist of a reaction vessel made of glass, steel or preferably stainless steel and a control unit. After each use, in order to ensure the aseptic cleaning effect of the digester, it must be disassembled, cleaned and sterilized repeatedly. The cleaning process itself is a time-consuming and laborious process, and the cleaning method used increases production. cost, and there is still a risk of contamination by residual organic matter.

In addition, the traditional digestion method simply separates the cells from the carrier or directly transfers the cells. The whole process only relies on technicians to manually operate the digester and empirically judge whether the digestion process is complete, which not only causes a large loss of cells during the digestion process, but also And the results are difficult to repeat from batch to batch. Therefore, the separation of cells and carriers to obtain free cells is still a major technical problem in the process of large-scale cell culture.

Utility model content

The main purpose of the utility model is to provide a disposable intelligent digester system for animal cell culture to overcome the deficiencies of the prior art.

In order to achieve the above object, the technical scheme of the utility model is as follows:

An embodiment of the utility model provides a disposable intelligent digester system for animal cell culture, including a disposable digester unit and an intelligent control unit, the disposable digester unit includes at least a fluid that can accommodate cultured cells and a sterile closed chamber for digestive fluid, at least one feed port, at least one gas outlet, at least one air inlet, at least one digestive fluid inlet, at least one cleaning solution inlet, at least one culture Base inlet, at least one outlet for cleaning liquid and digestive liquid, and at least one sampling port, and at least a retention component and a stirring component are provided in the closed chamber, and the retention component is at least used to remove the The carrier retains and passes the animal cells, and the agitation assembly is at least used to mix the substances in the closed chamber.

As one of the preferred embodiments, the disposable intelligent digester system for animal cell culture includes an airtight bag, the inner cavity of the airtight bag forms the closed chamber, and the airtight bag is provided with the inlet material inlet, gas outlet, air inlet, digestive fluid inlet, cleaning fluid inlet, medium inlet, and the lower part of the airtight bag is also provided with the cleaning fluid, digestive fluid outlet and sampling port;

Further, the intercepting component is arranged at the lower part of the inner cavity of the airtight bag, and is connected to the inner wall of the airtight bag in a surrounding and sealed connection; preferably, the airtight bag is also provided with at least one acid solution inlet and at least one alkali solution inlet ;

Further, the airtight bag is also provided with at least one cell transfer inlet and at least one cell transfer outlet;

Further, preferably, multiple ports are formed on the top and side of the airtight bag, and the multiple ports are respectively used as the feed inlet, gas outlet, air inlet, digestive liquid inlet, cleaning liquid inlet, cultivation Base inlet, cleaning solution and digestion solution outlet and sampling port;

Furthermore, the port is at least isolated from the external environment by any one of a cap, a Luer connector, a hose barb connector, a one-way valve, a sampling device, and a sterile filter.

Further, at least one air inlet pipe is inserted into the closed chamber through the air inlet, and the lower end of the air inlet pipe communicates with an air distributor, and the air distributor is located below the fluid level in the closed chamber;

Furthermore, the air distributor includes a ventilation ring, and a plurality of ventilation holes are evenly distributed on the ventilation ring, and the diameter of the ventilation holes is smaller than the diameter of the carrier;

Furthermore, a flow meter and a filter are also connected to the air intake pipe;

Furthermore, a pressure gauge or a pressure sensor and a pressure control valve are also connected to the outlet pipe;

Furthermore, the pressure in the closed chamber is maintained between 0.01-0.05Mpa;

Further, the material of the airtight bag includes rigid, semi-rigid or flexible plastic;

Further, the plastics include any one or two or more of polycarbonate, polyester, fluoropolymer, acrylic, PET, polyvinyl chloride, ABS, PC, PS, GAG, acrylic and polyolefin combination; still further, the plastic includes polyolefin.

As one of the preferred embodiments, the disposable digester unit includes a support structure, and the support structure is at least used to support the airtight bag;

Further, the support structure includes fixing buckles extending outward from the upper and lower parts of the airtight bag;

Further, the support structure includes a support frame arranged outside the airtight bag, the airtight bag is installed on the support frame or in the support frame, and the support frame is a fixed support frame or a movable support shelf;

Furthermore, the support structure includes a support frame arranged in the airtight bag;

Further, the support structure is at least selected from any one of metal barrels, plastic barrels, wooden barrels or glass barrels.

As one of the preferred implementations, the agitating assembly is in transmission connection with a driving mechanism, and the driving mechanism is connected with the support structure and arranged outside or inside the airtight bag;

Preferably, the drive mechanism includes a motor.

As one of the preferred embodiments, the stirring assembly includes a mechanical stirring mechanism and/or a magnetic stirring mechanism;

As one of the preferred embodiments, the stirring assembly includes a stirring shaft and a stirring paddle, the stirring paddle is fixedly connected to one end of the stirring shaft and arranged in the closed chamber, and the other end of the stirring shaft is located in the closed chamber. The outside of the chamber is in transmission connection with the drive mechanism, and the joint between the stirring shaft and the wall of the closed chamber is also sealed and connected by more than one sealing ring;

Further, the stirring blade is at least selected from any one of single-layer or multi-layer straight blade blades, arrow blade blades, curved blade blades, and propellers.

As one of the preferred embodiments, the retaining component includes at least one sieve or filter, and the pore size of the sieve or filter is larger than that of animal cells and smaller than that of microcarriers.

As one of the preferred implementations, the intelligent control unit includes any one or a combination of two or more of the following components:

a temperature control assembly for controlling at least the temperature within the enclosed chamber;

a pH control assembly for at least monitoring the pH of the fluid within the enclosed chamber;

a dissolved oxygen component, at least used to detect changes in dissolved oxygen in the closed chamber;

An in-line live cell sensor assembly for at least monitoring changes in the number of live cells that escape during digestion;

Weighing components, at least for on-line monitoring of fluid intake;

A central controller, at least used to control the operating status of each component;

The temperature control component, pH control component, dissolved oxygen component, online living cell sensor component and weighing component are all connected to the central controller and the closed chamber;

Further, the temperature control component, pH control component, dissolved oxygen component, online living cell sensor component and weighing component are all disposable components;

Preferably, the intelligent control unit further includes any one or a combination of two or more of the following components, all of which are connected to the central controller:

a carbon dioxide sensing component, at least used to detect changes in carbon dioxide in the closed chamber;

a mixing rate sensing component at least for detecting the mixing condition of the fluid in the closed chamber;

The photoelectric sensing component is at least used to detect the turbidity of the fluid in the closed chamber.

Further, the temperature control assembly includes a heater, a jacket circulating water system, and plug-in or non-plug-in temperature electrodes.

Further, the pH control component includes a pH value electrode.

Further, the dissolved oxygen component includes a dissolved oxygen electrode.

Further, the on-line living cell sensor assembly is installed on the side of the closed chamber in an inserting or non-inserting manner, and the on-line living cell sensor assembly is close to the retaining assembly.

Further, the weighing assembly includes more than one weighing module supporting the disposable digester unit and a junction box, and the weighing module is electrically connected to the junction box.

Furthermore, the weighing assembly includes 3-4 weighing modules installed at the feet of the disposable digester unit.

As one of the preferred embodiments, the intelligent control unit also includes a liquid pumping component, and the liquid pumping component includes a cleaning liquid pump, a digestive liquid pump, a culture medium pump, an acid pump, an alkali pump, and a cleaning liquid discharge pump , digestive fluid discharge pump and cell transfer pump, any one or a combination of two or more, the cleaning liquid pump, digestive juice pump, culture medium pump, acid pump, alkali pump, cleaning liquid discharge pump, digestive juice discharge pump And the cell transfer pumps are respectively connected with the central controller.

As one of the preferred implementations, the central controller further includes a wireless communication module, and the central controller is wirelessly connected to the remote terminal via the wireless communication module.

Compared with the prior art, the utility model has the advantages of at least:

1) The utility model saves steps such as SIP (on-line disinfection) and CIP (on-line cleaning), repeated installation and debugging of equipment, simplifies the operation process, and fundamentally reduces the daily maintenance work of instruments and equipment. The consumption of steam, water, and cleaning fluid saves the time for equipment installation and commissioning, thereby saving production costs and labor costs.

2) Disposable components are discarded after use and are not used for subsequent process operations, which avoids cross-contamination between batches and improves production stability and product quality.

3) The use of disposable components reduces the complexity of GMP verification, because there are almost no reusable components, which saves a lot of sterilization and cleaning verification processes, thus simplifying the GMP certification cycle in the R&D process and shortening the time to market.

4) The intelligent control unit designed by the utility model can automatically control the whole process of digestion reaction, greatly reducing the loss rate of cell digestion, and the loss rate can be as low as 2%.

5) The disposable digester unit designed by the utility model is consistent with the design principle of the classic multiple digester in terms of container design, stirring design, and ventilation strategy, and maintains geometric similarity on different reactor scales. Therefore, process transfer and Process scale-up does not increase additional risks, and it helps to transfer existing processes to existing industrial reaction equipment.

Description of drawings

In order to illustrate the structural features and technical points of the utility model more clearly, the utility model will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

Fig. 1 is the implementation flowchart of digestion reaction in comparative example 1;

Fig. 2 is a schematic structural view of the disposable digester unit in Example 1 of the utility model;

Fig. 3 is a front view of the closed chamber of the disposable digester unit in Embodiment 1 of the present utility model;

Fig. 4 is a rear view of the closed chamber of the disposable digester unit in Example 1 of the present utility model;

Fig. 5 is the implementation flowchart of digestion reaction in the utility model embodiment 1;

Fig. 6 is the implementation flow diagram of the digestion reaction in Example 2 of the present utility model.

Description of reference signs: 1-motor, 2-support structure, 3-weighing assembly, 4-acid solution inlet, 5-lye solution inlet, 6-cell transfer inlet, 7-cleaning solution inlet, 8-closed chamber , 9-stirring component, 10-temperature control component, 11-pH control component, 12-sampling port 2, 13-cleaning liquid/digestion liquid outlet, 14-feeding port, 15-digestion liquid inlet, 16-sampling port 1 , 17-dissolved oxygen component, 18-living cell sensor component, 19-cell transfer outlet, 20-outlet pipe, 21-intake pipe, 22-air distributor, 23-retention component.

detailed description

The technical solution in the embodiment will be specifically, clearly and completely described below in conjunction with the drawings in the embodiment.

Definition of Terms

The term "digestion" refers to the process of separating the cells from the old carrier and transferring them to the new carrier when the animal cells with the characteristics of adherent growth cover the surface of the carrier to avoid mutual inhibition between the cells.

"Disposable digester unit" mainly provides a disposable reaction place for the process of cell digestion reaction.

Referring to Figures 2-4, the embodiment of the present invention provides a disposable intelligent digester system for animal cell culture, including a disposable digester unit and an intelligent control unit, and the disposable digester unit includes at least A sterile closed chamber 8 for accommodating cell culture fluid and digestive juice. The closed chamber 8 is provided with a retaining component 23 and a stirring component 9. The retaining component 23 is connected with the inner wall of the closed chamber 8 in a surrounding and sealed manner, wherein the retaining The component 23 is at least used for retaining the carrier in the fluid in the closed chamber 8 and allowing the animal cells to pass through, and the stirring component 9 is at least used for mixing the substances in the closed chamber 8 .

Preferably, the intelligent control unit includes: a temperature control assembly 10, at least used to control the temperature in the closed chamber 8; a pH control assembly 11, at least used to detect the pH value of the fluid in the closed chamber 8; a dissolved oxygen assembly 17, It is at least used to detect the change of dissolved oxygen in the closed chamber 8; the online living cell sensor assembly 18 is at least used to monitor the change in the number of living cells flowing away during the digestion process; the weighing assembly 3 is at least used to monitor the amount of fluid entering online, The heavy component 3 can monitor the amount of fluid entering by monitoring the weight change of the digester; the central controller is at least used to control the operating status of each component; the temperature control component 10, the pH control component 11, the dissolved oxygen component 17, the online living cell The sensor assembly 18 and the weighing assembly 3 are connected to the central controller and the closed chamber 8 respectively.

More preferably, the intelligent control unit further includes any one or a combination of two or more of the following components: a carbon dioxide sensing component, at least used to detect changes in carbon dioxide in the closed chamber; a mixing rate sensing component, At least for detecting the mixing state of the fluid in the closed chamber; the photoelectric sensing component is at least for detecting the turbidity of the fluid in the closed chamber; the carbon dioxide sensing component, the mixing rate sensing component and the photoelectric sensing component Sensing components are all connected to the central controller:

More preferably, the temperature control component 10, the pH control component 11, the dissolved oxygen component 17, the online living cell sensor component 18 and the weighing component 3 are all disposable components. Moreover, each component can be fixed in the closed chamber 8 , or embedded or bonded on the surface of the closed chamber 8 .

Preferably, the closed chamber 8 is provided with ports for the entry and exit of materials and gases, and the ports include a feed port 14 communicated with the upper end of the closed chamber 8, a digestive liquid inlet 15, a cleaning liquid inlet 7, an acid liquid Inlet 4, lye inlet 5, cell transfer inlet 6 and each component interface, the port also includes a cleaning solution/digestion solution outlet 13, a sampling port 12 and a cell transfer outlet 19 connected to the bottom of the closed chamber 8, the port It also includes an air inlet and an air outlet communicating with the upper end of the closed chamber 8 .

Further, an air inlet pipe 21 and an air outlet pipe 20 are respectively inserted in the air inlet and the air outlet, and the air inlet pipe 21 and the air outlet pipe 20 extend into the closed chamber 8, and the lower end of the air inlet pipe 21 communicates with the air distributor, and the air distributor Located below the level of the fluid in the enclosed chamber.

Furthermore, the air distributor should choose a ventilation ring, which is evenly distributed with ventilation holes, the aperture of the ventilation holes is smaller than the diameter of the microcarrier, and gas is introduced into the air inlet pipe 21, so that the pressure in the closed chamber 8 is maintained at the set level. Fixed value, and make the airtight cavity rapid prototyping and shaping. Wherein, sterile air, oxygen or carbon dioxide are fed into the air inlet pipe 21, and the air, oxygen, and carbon dioxide inlet pipes 21 can be connected in parallel to provide bubbling of the contents of the airtight bag.

Furthermore, the air inlet pipe 21 is connected with a flowmeter and a filter, and the air outlet pipe 20 is connected with a pressure gauge or a pressure sensor and a pressure control valve. The pressure gauge or pressure sensor is used to detect the pressure in the closed chamber 8, so that the closed chamber 8 The internal pressure is kept between 0.01-0.05Mpa, so as to ensure the rapid molding of the closed chamber 8 and avoid the formation of wrinkles. The pressure control valve is used to discharge the waste gas in the closed chamber 8 .

Further, each of the above-mentioned ports is isolated from the outside by any means of caps, Luer connectors, hose barb connectors, one-way valves, sampling devices, and sterile filters.

Preferably, the disposable digester unit can adopt the simple form of an airtight bag, and the inner cavity of the airtight bag forms the closed chamber 8, wherein the airtight bag is provided with a feeding port 14, a digestive liquid inlet 15, a cleaning liquid Inlet 7, acid inlet 4, lye inlet 5, cell transfer inlet 6, cleaning solution/digestion solution outlet 13, sampling port 12, cell transfer outlet 19, air inlet and air outlet, at the inlet and outlet An air inlet pipe 21 and an air outlet pipe 20 are respectively inserted in the mouth.

Further, the airtight bag is made of rigid, semi-rigid or flexible plastic; more preferably, the plastic includes polycarbonate, polyester, fluoropolymer, acrylic, PET, polyvinyl chloride, ABS, PC, PS, Any one or a combination of two or more of GAG, acrylic and polyolefin; still further preferably, the plastic includes polyolefin.

Preferably, the disposable digester unit comprises at least one support structure 2 for supporting the closed chamber 8 .

Preferably, the upper part and the lower part of the closed chamber 8 extend outward to form fixing buckles for fixing and supporting the closed chamber 8 .

Further, the support structure 2 includes a support frame arranged outside the airtight bag, the airtight bag is installed on or in the support frame, and the support frame is a fixed support frame or a movable support frame; further, the The support structure includes a support frame arranged in the airtight bag.

Further, the supporting structure 2 adopts at least any one of metal barrel, plastic barrel, wooden barrel or glass barrel.

Preferably, at least the closed chamber 8 and each component are sterilized, and the sterilization treatment method includes any one or both of gamma ray irradiation, beta ray irradiation, ethylene oxide gas treatment and steam sterilization The above combinations can also be reused after being sterilized by gamma rays, beta rays, ethylene oxide, or steam.

Preferably, the retaining component 23 includes at least one layer of sieve plate or filter net made of plastic, and the pore size of the sieve plate or filter net is larger than the diameter of the cell and smaller than the diameter of the microcarrier.

Preferably, the stirring assembly 9 includes a mechanical stirring mechanism and/or a magnetic stirring mechanism; the stirring assembly 9 can be installed in a manner of central erection, central inclination, eccentric erection, and eccentric inclination.

Further, the stirring assembly 9 includes a stirring shaft and a stirring paddle, the stirring paddle is fixedly connected to one end of the stirring shaft and is arranged in the closed chamber 8, and the other end of the stirring shaft is located outside the closed chamber 8 and is connected to the motor 1 in transmission, and The joint between the stirring shaft and the chamber wall of the closed chamber 8 is also sealed and connected through more than one sealing ring.

Further, the agitating paddle adopts any one or a combination of two or more of single-layer or multi-layer straight blade paddles, arrow blade paddles, curved blade paddles, and propellers.

Furthermore, the material of the stirring shaft and/or the stirring paddle is high-density polyethylene sterilized by gamma rays, beta rays, ethylene oxide or steam.

Preferably, the temperature control assembly 10 includes a heater, a jacket circulating water system, and plug-in or non-plug-in temperature electrodes. The plug-in or non-plug-in temperature electrodes are used to adjust the opening and closing of the heater and the jacket circulating water system. Alternatively, the temperature control assembly 10 may be incorporated into the shell of the support structure 2 . The temperature control assembly 10 can also be applied to the condensation of the exhaust pipe, so that the exhausted gas condenses and prevents the filter from getting wet and affecting the gas filtering effect.

Preferably, the pH control component 11 includes a pH electrode to control the feeding of acid solution, lye solution and culture medium.

Preferably, the dissolved oxygen component 17 includes a dissolved oxygen electrode, and the dissolved oxygen is associated with the rotation speed, feeding and pressure.

Preferably, the online living cell sensor assembly 18 is installed on the side of the closed chamber 8 in an inserting or non-inserting manner, and the online living cell sensor assembly 18 is close to the retaining assembly 23 .

Preferably, the weighing assembly 3 includes 3-4 weighing modules and a junction box installed at the feet of the disposable digester unit, the junction box is electrically connected to each weighing module, and the junction box contacts the signals of each weighing module, And adjust the weight difference at each foot according to the loading conditions of each module, and then the junction box sends the collected signals to the central controller.

Preferably, the intelligent control unit further includes a liquid pumping component, and the liquid pumping component includes a cleaning liquid pump, a digestive liquid pump, a culture medium pump, an acid pump, an alkali pump, a cleaning liquid discharge pump, a digestive liquid discharge pump, and a cell transfer pump, The cleaning liquid pump, digestive liquid pump, medium pump, acid pump, alkali pump, cleaning liquid discharge pump, digestive liquid discharge pump and cell transfer pump are respectively connected to the central controller and used to transfer cleaning liquid, digestive liquid, culture Base, acid, lye, medium rich in free cells. The liquid pumping components are connected to the central controller by data lines, and each pump body can accept instructions from the central controller to realize automatic and manual control of pump operation.

Preferably, the central controller further includes a wireless communication module, and the central controller is wirelessly connected to the remote terminal via the wireless communication module.

The digestion process of the disposable intelligent digester system for animal cell culture is as follows:

(i) Installation and commissioning stage: the airtight bag is fixed on the outer wall of the digester support structure 2 through fixed buckles, and the gas and liquid pipelines on the airtight bag are aseptically connected through quick connectors, acid liquid, lye, digestive liquid, and cleaning liquid , culture medium, washing solution and digestion solution are discharged, and the cell transfer pipeline is installed on the corresponding peristaltic pump. Complete gas flow assembly 21, temperature control assembly 10, pH control assembly 11, dissolved oxygen control assembly 17, pressure control assembly (intake pipe, air outlet pipe, flowmeter, filter and pressure control valve form the described pressure control assembly), The weighing component 3, the online living cell sensor component 18, the stirring component 9 and the connection of the central controller. Introduce compressed air, oxygen or carbon dioxide into the airtight bag through the air source inlet, and maintain the pressure at 0.01-0.05Mpa to ensure the rapid formation of the airtight bag, and at the same time check the airtightness of the airtight bag and the connection of each pipeline. According to the digestion conditions, set the initial parameters, including ventilation, temperature, pH, dissolved oxygen, pressure, and rotation speed.

(ii) The initial stage of digestion: after the installation and commissioning are completed, start the digestive liquid entry step, and monitor the incoming amount of the digestive liquid online through the weighing component 3; at the same time, according to the preset pH, the system automatically starts the flow-feeding of acid or lye ;According to the preset temperature, the system automatically starts the heater for heating or starts the jacketed circulating water system for cooling; During the process, the number of free suspended living cells gradually increases, and after a certain period of time, the number of free suspended living cells no longer increases. The online live cell sensor 18 monitors the digestion process online and transmits the signal of the change in the number of free suspended living cells to the Central control system; when the number of free suspended living cells reaches the preset value, the disposable intelligent digester system automatically closes the pH control component 11 and the temperature control component 10, and starts the digestive juice discharge step, and the digestive juice is quickly discharged through the digestive juice pump. The carrier is trapped in the closed chamber 8 by the trapping assembly.

(iii) Digestion and cleaning stage: After the digestive juice is exhausted, start the cleaning step, reduce the speed, adjust the temperature to the cell culture temperature, set the amount of cleaning solution and the number of cleanings (usually 1-3 times), and the system will follow the preset According to the cleaning program, the cleaning solution pump in the cleaning solution channel is turned on, the cleaning solution enters the closed chamber 8, and the online living cell sensor 18 monitors the cell loss.

(iv) Digestion termination stage: after the cleaning is completed, the digestion termination step is started. Increase the stirring speed, adjust the temperature and pH to control the culture temperature and pH range, set the amount of medium added, associate dissolved oxygen with parameters such as ventilation, pressure, and rotational speed, and associate pH with feed, acid, and alkali. The system turns on the medium pump of the medium channel according to the preset digestion termination program, and the medium enters the closed chamber 8 . This process can last for a long time. When the number of free suspended cells remains stable, or the cell viability begins to decline, the digestion is considered to be terminated.

(v) Cell transfer stage: After the digestion termination procedure is completed, the cells can be transferred to the next-stage reactor in the following two ways: a) Increase the tank pressure of the digester to 0.03-0.05Mpa, reduce the pressure of the next-stage reactor The tank pressure is 0.01-0.02Mpa, and the culture hydraulic pressure is sent to the next-stage reactor by the differential pressure method; b) The peristaltic pump of the cell transfer channel is turned on, and the cell transfer pump rich in free suspended cells is sent to the next-stage reactor, And the empty carrier is trapped in the closed chamber 8 by the trapping component.

Below in conjunction with embodiment 1-2, comparative example 1 and accompanying drawing 1, 5 and 6, the technical solution of the utility model is further described.

Example 1

Referring to Figure 5, the 10L disposable bioreactor in this embodiment is used as a seed reactor to carry out microcarrier cell suspension culture; the 10L disposable digester is used as a place for the digestion reaction of the seed cells that have been cultivated to realize the PK-15 cell After separation from the microcarriers, the free cells are transferred to a 50L disposable bioreactor for further culture. The working volumes of 10L and 50L multiple bioreactors are 7L and 35L respectively. The height-to-diameter ratio of the reactor is 1.8:1.0. The structural design of the 10L disposable digester unit is shown in Figures 2, 3 and 4, and the working volume is 7L. The 10L disposable digester carrier medium used Cytodex-1 produced by GE Company, 3g/L, and the cell density was 2.5×106cells/mL. The implementation process of the digestion reaction is shown in Figure 2, and the specific implementation steps are as follows:

(i) Liquid preparation stage: Prepare 20L of cleaning solution, 1L of digestive solution, 5L of culture medium, 2L of acid solution (1N HCl), and 2L of lye solution (3N NaOH), wherein the culture medium contains 3% newborn bovine serum.

(ii) Installation and commissioning stage: the airtight bag is fixed on the outer wall of the digester bracket through a fixed buckle, and the gas pipeline and liquid pipeline (acid, lye, digestive solution, cleaning solution, culture medium) on the airtight bag are connected through a quick connector pipeline) and the corresponding peristaltic pump to achieve aseptic connection; complete the connection of temperature control components, pH control components, dissolved oxygen components, weighing components, online living cell sensor components, stirring components and the central controller. Introduce compressed air, oxygen or carbon dioxide into the inside of the airtight bag through the inlet pipe, and maintain the pressure at 0.01-0.05Mpa, so as to realize pressure control and ensure the rapid formation of the airtight bag, and at the same time check the airtightness of the airtight bag and the connection of each pipeline; According to the digestion conditions, the initial parameters were set, including ventilation volume 0.5vvm, temperature 37°C, pH 7.0, pressure 0.02Mpa, and rotation speed 25rpm.

(iii) Initial stage of digestion: After the installation and commissioning are completed, the culture medium cultivated in the 10L disposable reactor is directly transferred to the 10L disposable digester. Start the step of entering the digestive juice, monitor the amount of the digestive juice on-line through the weighing component, and pump 0.6L of the digestive juice into the closed bag of the digester through the digestive juice; set the temperature at 37°C, the speed at 25rpm, and the pH at 7.0, At the same time, according to the preset pH, the system automatically starts the feeding of acid or lye; according to the preset temperature, the system automatically starts the heater to heat or starts the jacket circulating water system to cool; according to the preset dissolved oxygen, the dissolved oxygen Associated control with ventilation volume, pressure, speed and other parameters. The online living cell sensor monitors the digestion process online and transmits the signal of the change in the number of free suspended living cells to the central control system; when the number of cells no longer increases, it is considered that the digestion process is complete; after reaching the preset value, the digester system Automatically close the pH control component and temperature control component, and open the digestive juice discharge step. The digestive juice is quickly discharged by the digestive juice pump, and the carrier is trapped in the airtight bag of the digester by the retention component 23 .

(iv) Digestion and cleaning stage: After the digestive juice is exhausted, start the cleaning step, increase the rotation speed to 50rpm, adjust the temperature to 37°C, set the amount of cleaning solution added to 5L, the number of cleaning times to 2 times, and the cleaning time to 10min. According to the preset cleaning program, the system turns on the cleaning liquid pump in the cleaning liquid channel, and the cleaning liquid enters the airtight bag of the digester; after cleaning, it automatically starts the cleaning liquid discharge pump to quickly drain the cleaning liquid, and the live cell sensor monitors the cells loss situation.

(v) Digestion termination stage: after the cleaning is completed, the digestion termination step is started. Increase the stirring speed, set the temperature at 37°C, the rotating speed at 100rpm, set the amount of medium added to 5L, and control the pH in relation to acid and alkali. According to the preset digestion termination program, the system turns on the medium pump of the medium channel, and the medium enters the airtight bag of the digester; after 5 minutes, the number of free suspended cells monitored by the online living cell sensor begins to increase, and then the number of free suspended cells begins to increase after a period of time Digestion is considered terminated when cell numbers remain stable.

(vi) Cell transfer stage: After the digestion termination procedure is completed, the cell transfer pump of the cell transfer channel is turned on, and the medium rich in free suspended cells is pumped to the 50L disposable bioreactor, while the carrier is trapped in 10L once by the retention component in the digestive tract. Data monitoring showed that the number of free cells was 0.49×10 ⁶ cells/mL. After 11 hours, the cells were attached to the new carrier, and the cell loss rate was only 2.01%.

Example 2

Referring to Figure 6, the 90L disposable bioreactor in this embodiment is used as a seed reactor to carry out microcarrier cell suspension culture; the 90L disposable digester is used as a place for the digestion reaction of the seed cells that have been cultivated to realize PK-15 cells After separation from the microcarriers, the free cells are transferred to a 300L disposable bioreactor for further culture. The working volumes of the 90L and 300L disposable bioreactors are 63L and 210L respectively. The height-to-diameter ratio of the reactor is 1.8:1.0. The disposable digester and the 10L disposable digester have geometric similarity characteristics. For other digestion reaction steps, refer to Example 2. The specific implementation flow chart is shown in Figure 6, and will not be repeated here.

The medium rich in free-suspending cells is pumped to a 300L disposable bioreactor, while the carrier is retained in a 90L disposable digester by the retention assembly. Data monitoring showed that the number of free cells was 0.48×10 ⁶ cells/mL. After 11 hours, the cells were attached to the new carrier, and the cell loss rate was only 3.98%.

Comparative example 1

Referring to shown in Fig. 1, the 10L multiple-time bioreactor (stainless steel fermenter) in this comparative example is used as seed reactor, carries out microcarrier cell suspension culture; 10L multiple-time digester is used as the seed cell digestion reaction that culture completes The place where PK-15 cells and microcarriers are separated, and the free cells are transferred to a 50L multiple-use bioreactor (stainless steel fermenter) for continued cultivation. Among them, the design of the 10L multiple-use digester is the same as that of the 10L multiple-use bioreactor. The working volumes of 10L and 50L multiple bioreactors are 7L and 35L respectively. The height-diameter ratio of the reactor is 1.8:1.0. The 1L bioreactor carrier medium used Cytodex-1 produced by GE Company, 3g/L, and the cell density was 2.5×10 ⁶ cells/mL. The implementation process of the digestion reaction is shown in Figure 1, and the specific implementation steps are as follows:

(i) Liquid preparation stage: Prepare 20L of cleaning solution, 1L of digestive solution, 5L of culture medium, 2L of acid solution (1N HCl), and 2L of lye solution (3N NaOH), wherein the culture medium contains 3% newborn bovine serum.

(ii) Digester installation stage: loosen the reactor cover and the screws at the joints of each pipeline, remove the upper cover of the reactor, remove the electrodes, hole plugs and cap heads, clean them with a rag, and rinse them with pure water. The joints, the inner wall of the reactor and the seal of the stirring device should be cleaned with pure water, and the residue and residual liquid should be washed away. Check whether the gaskets and aprons are aged and damaged, and replace them in time. After connecting all pipeline parts, connect the electrode and the central controller through the electrode wire, and perform zero point calibration and slope calibration for the pH value electrode and the dissolved oxygen electrode. Introduce air to maintain pressure, and check the airtightness of the reactor.

(iii) Digester sterilization stage: sterilize in air at 121°C for 30 minutes. Insert a pH electrode and a dissolved oxygen electrode, and put 7L of pure water in the digester, and sterilize at 121°C for 30 minutes.

(iv) Digester debugging stage: After the sterilization is completed, start the temperature control component, set the temperature at 37°C, and cool the system to the set temperature through the circulating water system; PID controls the heating of the heater and the cooling of the circulating water, and the temperature is maintained at 37°C.

(v) Digestion and cleaning stage: open the discharge valve to discharge the pure water from the tank. Close the digestive liquid discharge valve, open the cleaning liquid inlet valve, add 5L of cleaning liquid through the differential pressure method, and rinse the tank for 30 minutes; open the cleaning liquid discharge valve, and drain the cleaning liquid. Repeated cleaning 1-3 times, until the cleaning verification no residue. Settling the culture medium cultivated in the 10L multiple-use reactor, pumping the supernatant, and transferring the settled liquid to the 10L multiple-use digester through the bottom valve of the tank. Add 5L of cleaning solution to the digester for cleaning and sedimentation again. During cleaning, set the temperature at 37° C. and the rotation speed at 50 rpm. After cleaning, stop stirring, and drain the cleaning solution after settling.

(vi) Digestion start stage: After the cleaning stage is over, open the digestive liquid inlet valve and pump 0.6L of the digestive liquid into the digester, set the temperature at 37°C, the rotational speed at 25rpm, and the pH at 7.0. Samples were taken every 10 minutes to measure the number of free cells. When the number of cells no longer increased, the digestion process was considered complete. Turn off the stirring and pH, open the digestive juice discharge valve, and drain the digestive juice.

(vii) Digestion termination stage: After the digestion is completed, open the medium inlet valve, pump 5 L of medium into the digester, set the temperature at 37° C., and rotate at 100 rpm. Every 10 minutes, samples were taken to determine the number of free cells. When the number of cells no longer increased or the cell activity decreased, the digestion was considered to be terminated.

(viii) Cell transfer stage: after the digestion is terminated, the cell transfer valve is opened, and the cells are transferred to the 50L multiplex bioreactor by the differential pressure method. Sampling was taken to determine the number of free cells to be 0.3×10 ⁶ cells/mL. After 14 hours, the cells were attached to the new carrier, and the cell loss rate was 41.56%.

The multiple-use digester in Comparative Example 1 needs repeated installation and debugging, and needs to be disinfected and cleaned online, thus wasting time and manpower, and the cell loss rate of the multiple-use intelligent digester is as high as 41.56%, while the one-time digester provided by the utility model The disposable digester saves steps such as SIP (on-line disinfection) and CIP (on-line cleaning), repeated installation and debugging of equipment, simplifies the operation process, saves material and labor costs; and the loss rate of the disposable digester can reach as low as 2%. , greatly reducing the rate of cell digestion loss.

The above specific implementation is only to illustrate the technical concept and structural features of the present utility model, and the purpose is to allow relevant persons familiar with this technology to implement it accordingly, but the above-mentioned content does not limit the protection scope of the present utility model. Any equivalent changes or modifications made to the spirit of the present utility model shall fall within the protection scope of the present utility model.

Claims (27)

1. A disposable intelligent digester system for animal cell culture, characterized in that: it includes a disposable digester unit and an intelligent control unit, and the disposable digester unit includes at least fluid and A sterile closed chamber for digestive fluid, at least one feed port, at least one gas outlet, at least one air inlet, at least one digestive fluid inlet, at least one cleaning solution inlet, and at least one culture medium communicated with the closed chamber Inlet, at least one cleaning liquid and digestive liquid outlet, and at least one sampling port, and at least a retention component and a stirring component are provided in the closed chamber, and the retention component is at least used to remove the carrier in the fluid in the closed chamber Animal cells are retained and passed through, and the agitation assembly is at least used to mix the contents of the closed chamber. 2. The disposable intelligent digester system for animal cell culture according to claim 1, characterized in that: it comprises an airtight bag, the inner cavity of the airtight bag forms the closed chamber, and the airtight bag is provided with There are the feed inlet, gas outlet, air inlet, digestive fluid inlet, cleaning fluid inlet, and culture medium inlet, and the lower part of the airtight bag is also provided with the cleaning fluid and digestive fluid outlets and a sampling port. 3. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: the retaining component is arranged at the lower part of the inner cavity of the airtight bag, and is connected with the inner wall of the airtight bag to surround and seal , the airtight bag is also provided with at least one acid solution inlet and at least one alkali solution inlet. 4. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: at least one cell transfer inlet and at least one cell transfer outlet are also provided on the airtight bag. 5. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: the top and side of the airtight bag form a plurality of ports, and the plurality of ports are respectively used as the Feed inlet, gas outlet, air inlet, digestion solution inlet, cleaning solution inlet, culture medium inlet, cleaning solution and digestion solution outlet and sampling port. 6. The disposable intelligent digester system for animal cell culture according to claim 5, characterized in that: the port at least passes through a cap, a Luer connector, a hose barb connector, a one-way valve, a sampling device, Any of the sterile filters are isolated from the external environment. 7. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: at least one air intake pipe is inserted into the closed chamber through the air inlet, and the lower end of the air intake pipe is connected to the air The distributor communicates, the air distributor is located below the fluid level in the closed chamber, and at least one air outlet pipe is inserted into the closed chamber through the air outlet. 8. The disposable intelligent digester system for animal cell culture according to claim 7, characterized in that: the air distributor includes a ventilation ring, and a plurality of ventilation holes are evenly distributed on the ventilation ring, the The pore diameter of the vent hole is smaller than the diameter of the carrier. 9. The disposable intelligent digester system for animal cell culture according to claim 7, characterized in that: the air inlet pipe is also connected with a flow meter and a filter, and the air outlet pipe is also connected with a pressure gauge or A pressure sensor and a pressure control valve, the pressure in the closed chamber is maintained between 0.01-0.05Mpa. 10. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: the material of the airtight bag includes rigid, semi-rigid or flexible plastic. 11. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: the disposable digester unit includes a support structure, and the support structure is at least used to support the airtight bag. 12 . The disposable intelligent digester system for animal cell culture according to claim 11 , wherein the supporting structure includes fixing buckles extending outward from the upper and lower parts of the airtight bag. 13 . 13. The disposable intelligent digester system for animal cell culture according to claim 11, characterized in that: the support structure includes a support frame arranged outside the airtight bag, and the airtight bag is installed on the On or in the support frame, the support frame is a fixed support frame or a movable support frame. 14. The disposable intelligent digester system for animal cell culture according to claim 11, characterized in that: the supporting structure comprises a supporting frame arranged in the airtight bag. 15. The disposable intelligent digester system for animal cell culture according to claim 11, characterized in that: said supporting structure is at least selected from any one of metal barrels, plastic barrels, wooden barrels or glass barrels. 16. The disposable intelligent digester system for animal cell culture according to claim 11, characterized in that: the stirring assembly is in transmission connection with a driving mechanism, and the driving mechanism is connected with the support structure and is arranged on Outside or inside the airtight bag, the drive mechanism includes a motor. 17. The disposable intelligent digester system for animal cell culture according to claim 16, characterized in that: the stirring assembly includes a mechanical stirring mechanism and/or a magnetic stirring mechanism. 18. The disposable intelligent digester system for animal cell culture according to claim 17, characterized in that: the stirring assembly includes a stirring shaft and a stirring paddle, and the stirring paddle is fixedly connected to one end of the stirring shaft and arranged on In the closed chamber, the other end of the stirring shaft is located outside the closed chamber and is connected to the drive mechanism, and the joint between the stirring shaft and the wall of the closed chamber is passed through more than one sealing ring Sealed connection. 19. The disposable intelligent digester system for animal cell culture according to claim 18, characterized in that: the stirring paddles are at least selected from single-layer or multi-layer straight blade paddles, arrow blade paddles, and curved blade paddles , any one of the propellers. 20. The disposable intelligent digester system for animal cell culture according to claim 1, characterized in that: the retaining assembly includes at least one sieve plate or filter screen, and the aperture of the sieve plate or filter screen is larger than that of animals. The diameter of the cell is smaller than the diameter of the microcarrier. 21. The disposable intelligent digester system for animal cell culture according to claim 2, characterized in that: the intelligent control unit comprises: a temperature control assembly for controlling at least the temperature within the enclosed chamber; And/or, a pH control assembly, at least for monitoring the pH value of the fluid in the closed chamber; And/or, the dissolved oxygen component is at least used to detect the change of dissolved oxygen in the closed chamber; And/or, an online living cell sensor assembly, at least for monitoring the change in the number of living cells flowing away during the digestion process; And/or, a weighing assembly, at least for online monitoring of fluid ingress; And/or, the central controller is at least used to control the operating status of each component; The temperature control component, pH control component, dissolved oxygen component, online living cell sensor component and weighing component are all connected with the central controller and the closed chamber. 22. The disposable intelligent digester system for animal cell culture according to claim 21, characterized in that: the temperature control component, pH control component, dissolved oxygen component, online living cell sensor component and weighing component are all are disposable components. 23. The disposable intelligent digester system for animal cell culture according to claim 21, characterized in that: the intelligent control unit further comprises: a carbon dioxide sensing component, at least used to detect changes in carbon dioxide in the closed chamber; And/or, the mixing rate sensing component is at least used to detect the mixing condition of the fluid in the closed chamber; And/or, the photoelectric sensing component is at least used to detect the turbidity of the fluid in the closed chamber; The carbon dioxide sensing component, the mixing rate sensing component and the photoelectric sensing component are all connected to the central controller. 24. The disposable intelligent digester system for animal cell culture according to claim 21, characterized in that: the temperature control assembly includes a heater, a jacketed circulating water system, and plug-in or non-plug-in temperature electrodes; And/or, the pH control component includes a pH electrode; and/or, the dissolved oxygen component includes a dissolved oxygen electrode; and/or, the online live cell sensor component is installed in the plug-in or non-plug-in the closed chamber side, and the on-line live cell sensor assembly is close to the retention assembly; and/or the weighing assembly includes one or more weighing modules and a junction box supporting the disposable digester unit, The weighing module is electrically connected with the junction box. 25. The disposable intelligent digester system for animal cell culture according to claim 24, characterized in that: the weighing assembly includes 3-4 scales installed at the feet of the disposable digester unit Heavy modules. 26. The disposable intelligent digester system for animal cell culture according to claim 21, characterized in that: the intelligent control unit also includes a liquid pumping assembly, and the liquid pumping assembly includes a cleaning liquid pump, a digester Any one or a combination of two or more of liquid pumps, culture medium pumps, acid pumps, alkali pumps, cleaning liquid discharge pumps, digestive liquid discharge pumps and cell transfer pumps, the cleaning liquid pumps, digestive liquid pumps, medium The pumps, acid pumps, alkali pumps, cleaning liquid discharge pumps, digestive liquid discharge pumps and cell transfer pumps are respectively connected to the central controller. 27. The disposable intelligent digester system for animal cell culture according to claim 21, characterized in that: the central controller also includes a wireless communication module, and the central controller communicates with the remote via the wireless communication module Terminal wireless connection.