External circulation bioreactor and data monitoring method thereof

Abstract

The invention relates to an external circulation bioreactor and a data monitoring method thereof. The external circulation bioreactor comprises: a reactor tank for accommodating a mixture of cells and a liquid; the outer circulation pipeline is arranged outside the reactor tank body and is communicated with the reactor tank body; and the monitoring equipment is mounted on the outer circulating pipeline. According to the invention, the influence of bubbles on the detection accuracy of the bioreactor electrode is avoided, so that the measurement result of the bioreactor is more accurate.

Description

Technical Field

[0001] The invention relates to the field of bioreactors, and in particular to an external circulation bioreactor and a data monitoring method thereof. Background Art

[0002] With the development of existing biotechnology, optimizing the process by monitoring the changes in relevant parameters of the fermentation process is conducive to stabilizing the fermentation process and increasing the yield. Small bioreactors are often used for process exploration before large-scale fermentation due to their low production cost, small footprint, and easy control.

[0003] However, small bioreactors have fewer insertion holes and limited data that can be monitored in real time. In addition, the fermentation volume is small, and the bubbles generated by the gas at the bottom of the tank have a great impact on the electrode measurement, and the detected data fluctuates greatly. In addition, most of the existing reactor external circulation devices are powered by peristaltic pumps, which circulate the cell culture fluid in the pipeline, causing great damage to the cells.

[0004] There is a lack of suitable solutions to the above problems. Summary of the invention

[0005] Based on this, in order to solve the above problems, this paper proposes an external circulation bioreactor and its data monitoring method. The centrifugal force is generated by the rotation of the magnetic pump impeller, so that the cell culture fluid circulates in the pipeline, and the shear force on the cells is small. Multiple monitoring devices can be inserted into the circulation pipeline at the same time to monitor the fermentation process in real time, which is suitable for exploring small-scale fermentation process parameters.

[0006] According to a first aspect of the present invention, there is provided an external circulation bioreactor, comprising:

[0007] A reactor tank, the reactor tank being used to contain a mixture of cells and liquid;

[0008] An external circulation pipeline, which is arranged outside the reactor tank and communicated with the reactor tank;

[0009] Monitoring equipment, the monitoring equipment is installed on the external circulation pipeline.

[0010] In some embodiments, the external circulation circuit is powered by a pump, so that the mixture of cells and liquid circulates in the external circulation circuit.

[0011] In some embodiments, the pump is a magnetic pump.

[0012] In some embodiments, the monitoring device is a DO electrode.

[0013] In some embodiments, the measuring part of the monitoring device is located in the external circulation pipeline; when there are bubbles in the external circulation pipeline, the measuring part of the monitoring device is located in the external circulation pipeline so that the bubbles do not contact the measuring part of the monitoring device when flowing in the external circulation pipeline.

[0014] In some embodiments, the measuring part of the monitoring device is located at the lower part of the pipe in the external circulation pipeline (i.e., the position below the middle of the pipe). Preferably, the measuring part of the monitoring device is located at the horizontal section of the external circulation pipeline, in which the bubbles are located at the upper part of the pipe in the external circulation pipeline (i.e., the position above the middle of the pipe) due to gravity, and at this time, the bubbles do not contact the measuring part of the monitoring device.

[0015] In some embodiments, the inlet end of the external circulation pipeline connected to the reactor tank body is arranged upward (for example, the inlet end is connected by a bend pipe so that the downward inlet end becomes upward), so that the liquid in the reactor tank body enters the external circulation pipeline from top to bottom. Due to the influence of gravity, the bubbles in the reactor tank body move from bottom to top. The upward arrangement of the inlet end can avoid bubbles from following the liquid in the reactor tank body into the external circulation pipeline as much as possible.

[0016] In some embodiments, the DO electrode is installed from the lower side of the pipeline in the external circulation pipeline into the pipeline.

[0017] In some embodiments, a gas inlet is provided at the bottom of the reactor vessel.

[0018] According to a second aspect of the present invention, there is provided a data monitoring method for any of the above-mentioned external circulation bioreactors, comprising the following steps:

[0019] (1) Adding a mixture of cells and liquid into a reactor tank and setting the reaction temperature;

[0020] (2) introducing gas into the reactor tank and setting the gas introduction rate;

[0021] (3) providing power for the external circulation pipeline so that the mixture of cells and liquid circulates in the external circulation pipeline and the reactor tank;

[0022] (4) Obtain reaction parameters through monitoring equipment.

[0023] In some embodiments, the tank pressure of the reactor tank is 0 bar.

[0024] By implementing the above solution of the present invention, the following beneficial effects can be obtained:

[0025] 1. The centrifugal force generated by the rotation of the magnetic pump impeller is used as the driving force, and the shear force on the cells is relatively low;

[0026] 2. The external circulation pipeline can be installed with multiple measuring devices at the same time, which is suitable for parameter exploration and adjustment of small-scale fermentation process;

[0027] 3. The measuring part of the monitoring equipment is located at the lower part of the pipeline in the external circulation pipeline (i.e., the position below the middle part of the pipeline), so that the measuring part is not affected by the bubbles in the external circulation pipeline, and the measurement result is more accurate.

[0028] 4. The inlet end of the external circulation pipeline connected to the reactor tank body is arranged upward to prevent bubbles from entering the reactor tank body when the liquid in the reactor tank body enters the external circulation pipeline. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagrams of the structures of some embodiments of the external circulation bioreactor of the present invention;

[0030] Figure 2 is a diagram of the measurement results of the DO electrode in Example 1 of the present invention;

[0031] Figure 3 It is a measurement result diagram of the DO electrode in Comparative Example 1 of the present invention;

[0032] Figure 4 It is a measurement result diagram of the DO electrode in Comparative Example 2 of the present invention;

[0033] Figure 5 It is a graph showing changes in cell density and viability in the external circulation bioreactor of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Figure 1 Schematic diagrams showing the structures of some embodiments of the external circulation bioreactor of the present invention.

[0036] like Figure 1 As shown, the device comprises: a reactor tank body 6, which is used to contain a mixture of cells and liquid; an external circulation pipeline, which is arranged outside the reactor tank body and is connected to the reactor tank body through a discharge port 1 and a feed port 2; a monitoring device 3, which is installed on the external circulation pipeline; the external circulation pipeline is powered by a magnetic pump 5 through a pump head 4, so that the mixture of cells and liquid circulates in the external circulation pipeline.

[0037] In actual operation, the pump housing and the circulation pipe must be filled with cell culture fluid first, and then the motor is started to make the pump shaft drive the impeller and the cell culture fluid to rotate. Under the action of centrifugal force, the cell culture fluid is thrown to the outer edge of the impeller and flows into the feed inlet circulation pipeline through the side flow channel of the pump housing. At the center of the pump impeller, a vacuum is formed after the cell culture fluid is thrown out under the action of centrifugal force. The cell culture fluid in the reactor is pressed into the pump housing under the action of atmospheric pressure. The impeller rotates continuously, so that the cell culture fluid continuously flows in and out under the action of the impeller, achieving the purpose of circulation.

[0038] The external circulation pipeline is connected to the discharge port and feed port of the bioreactor. Multiple electrodes or other monitoring equipment can be installed on the pipeline to monitor the changes in the fermentation process in real time. The bubbles in the external circulation pipeline flow along the upper end of the cell culture fluid. The electrode is inserted upside down so that the measurement part is located at the lower part of the pipeline, eliminating the error caused by the bubble to the electrode detection. The centrifugal force generated by the rotation of the impeller of the magnetic pump makes the cell culture fluid in the pipeline circulate, and compared with the use of peristaltic pumps to pump liquids, the shear force of the centrifugal pump on the cells is small and can be ignored.

[0039] Example 1

[0040] An external circulation bioreactor, the structure and connection relationship of which are as follows Figure 1 shown.

[0041] The data monitoring method using this reactor is as follows: add 3.5L basal culture medium to a 5L bioreactor, set the temperature to 37°C, the speed to 85rpm, the tank pressure to 0bar, and introduce air at a constant flow rate of 1L / min. Turn on the centrifugal pump to circulate the cell culture fluid in the external pipe and the reactor tank. At this time, the measurement position of the DO electrode is located at the bottom of the pipeline, and the distance from the bottom of the pipeline is 1 / 3 of the distance from the top of the pipeline (that is, located in the lower 1 / 4 of the pipeline).

[0042] Comparative Example 1

[0043] The DO electrode was installed in the reactor tank in a conventional manner. Others were the same as in Example 1.

[0044] Comparative Example 2

[0045] The measuring position of the DO electrode is located at the upper part of the pipeline, and the distance from the top of the pipeline is 1 / 3 of the distance from the bottom of the pipeline (ie, located at the upper 1 / 4 of the pipeline).

[0046] Figure 2 The measurement results of the DO electrode in Example 1 of the present invention are shown. Figure 3 The measurement results of the DO electrode in Comparative Example 1 of the present invention are shown. Figure 4 The measurement results of the DO electrode in Comparative Example 2 of the present invention are shown.

[0047] from Figure 2 , Figure 3 , Figure 4 It can be seen that the value of the DO electrode in the tank fluctuates violently, while the value of the DO electrode in the circulation pipeline rises steadily with a small fluctuation amplitude, and the measurement result when the measurement location is located in the lower part of the pipeline is significantly better than that when the measurement location is located in the upper part of the pipeline.

[0048] The cell density and viability in the reactor were measured. The test method was as follows: cells were inoculated into a 5L reactor, and the magnetic pump was turned on to allow the cell culture fluid to circulate in the external pipe and fermenter. The control parameters were set according to the historical batch parameters, and the changes in cell density and viability were recorded every day.

[0049] Figure 5 The graph shows the changes in cell density and viability in the external circulation bioreactor of the present invention.

[0050] like Figure 5 As shown, A0012303 is historical data, and A0012304 is the measurement data of the above-mentioned external circulation bioreactor. From the data, it can be seen that the changes in cell density and viability are close to the historical data when the cells are circulated in the external pipe by using a magnetic pump, and there is no obvious difference. The historical data was obtained by stirring culture using a traditional bioreactor.

[0051] In addition, the external circulation bioreactor can also be connected to an online sampling device, an online biochemical analyzer, an online HPLC and other devices to achieve PAT parameter detection.

[0052] The present invention connects a circulation pipeline outside the reactor and uses a magnetic pump as a power to circulate the cell culture solution, thereby reducing the shear force on the cells and realizing the insertion of multiple monitoring devices.

[0053] In addition, the present invention can set the inlet end of the external circulation pipeline connected to the reactor tank body upward, so that the liquid in the reactor tank body enters the external circulation pipeline from top to bottom, and avoid bubbles following the liquid in the reactor tank body into the external circulation pipeline as much as possible.

[0054] Although some specific embodiments of the present invention have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It should be understood by those skilled in the art that the above embodiments may be modified or some technical features may be replaced by equivalents without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. An external circulation bioreactor, characterized in that: include: A reactor tank, the reactor tank being used to contain a mixture of cells and liquid; An external circulation pipeline, which is arranged outside the reactor tank and communicated with the reactor tank; Monitoring equipment, the monitoring equipment is installed on the external circulation pipeline.

2. The external circulation bioreactor according to claim 1, characterized in that: The external circulation pipeline is powered by a pump, so that the mixture of cells and liquid circulates in the external circulation pipeline.

3. The external circulation bioreactor according to claim 1, characterized in that: The monitoring device is a DO electrode.

4. The external circulation bioreactor according to claim 1, characterized in that: The measuring part of the monitoring device is located in the external circulation pipeline; When bubbles exist in the external circulation pipeline, the measuring part of the monitoring device is located in the external circulation pipeline so that the bubbles do not come into contact with the measuring part of the monitoring device when flowing in the external circulation pipeline.

5. The external circulation bioreactor according to claim 1, characterized in that: The measuring part of the monitoring device is located at the lower part of the pipeline in the external circulation pipeline.

6. The external circulation bioreactor according to claim 5, characterized in that: The measuring position of the monitoring device is located in the horizontal section of the external circulation pipeline.

7. The external circulation bioreactor according to claim 1, characterized in that: The inlet end of the external circulation pipeline communicating with the reactor tank body is arranged upward, so that the liquid in the reactor tank body enters the external circulation pipeline from top to bottom.

8. The external circulation bioreactor according to claim 1, characterized in that: A gas inlet is arranged at the bottom of the reactor tank.

9. The external circulation bioreactor according to claim 1, characterized in that: The reactor tank capacity is not higher than 50L.

10. A data monitoring method based on the external circulation bioreactor according to any one of claims 1 to 9, comprising the following steps: (1) Adding a mixture of cells and liquid into a reactor tank and setting the reaction temperature; (2) introducing gas into the reactor tank and setting the gas introduction rate; (3) providing power for the external circulation pipeline so that the mixture of cells and liquid circulates in the external circulation pipeline and the reactor tank; (4) Obtain reaction parameters through monitoring equipment.

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