Integrated continuous processing device for obtaining escherichia coli plasmids

Abstract

The utility model discloses an integrated continuous processing device for obtaining escherichia coli plasmids, the lower end of a circulating tank (1) is connected with a permeable pipeline (5), the upper end of the circulating tank (1) is connected with a liquid inlet pipeline (6) and two return pipelines, the permeable pipeline (5) is provided with a pump body, a flow meter, a mixing pipe, a pressure sensor and a coil pipe, the other end of the permeable pipeline (5) is provided with six branch pipelines, and the branch pipelines are communicated with the circulating tank (1). By controlling the switching of the valve, thalli are harvested in a tangential flow filtering manner, then the coiled tube type continuous alkali cracking process is switched to carry out cracking on the thalli, and then the clarification filtering process is switched to treat a large amount of floccules generated by cracking. According to the scheme, the processes of collecting, resuspending, alkali cracking and clarifying and filtering after escherichia coli fermentation can be stably and orderly connected in series, multiple processes are completed on one device to realize continuous production, the process of each step is stable, the number of equipment and the occupied area are reduced while manpower is saved, and the production efficiency of plasmids is improved.

Description

TECHNICAL FIELD

[0001] The utility model relates to the production equipment of escherichia coli fermentation after bacterial body harvest, alkali lysis and filtration, especially a kind of integrated continuous processing device for obtaining escherichia coli plasmid, belong to biological pharmaceutical purification separation technical field. BACKGROUND

[0002] With the rise of gene therapy and nucleic acid vaccine / drug, the use demand of GMP level plasmid significantly increases. Plasmid is generally produced and obtained in recombinant escherichia coli, and relevant process links include bacterial body fermentation amplification, bacterial body collection and resuspension, bacterial body lysis, clarification filtration, chromatography purification and concentration liquid change, etc., finally produce qualified plasmid product, and it is widely used in cell therapy and gene therapy (CGT).

[0003] However, each step of obtaining GMP level plasmid is independently carried out, its process is discontinuous, needs to consume a lot of manpower and equipment to carry out operation respectively, leads to that production efficiency is low, and process consistency between batches is difficult to control comprehensively. Therefore, it is necessary to provide a kind of integrated continuous processing device for obtaining escherichia coli plasmid to carry out the continuous production of multiple processes on one equipment, improve operation efficiency. SUMMARY

[0004] The utility model discloses a kind of integrated continuous processing devices for obtaining escherichia coli plasmid to solve the above problems, to solve the problem that plasmid production production efficiency is low, process cannot be controlled continuously, improve operation efficiency.

[0005] The utility model discloses a technical solution is: a kind of integrated continuous processing device of obtaining escherichia coli plasmid, including circulating tank, the lower end of the circulating tank is connected with the pipe line of showing, the upper end of the circulating tank is connected with liquid inlet pipe line, first return pipe line and second return pipe line respectively, its characteristics are: the first end of the pipe line of showing is connected with the showing end of the circulating tank, and first pump body, first flowmeter, first mixing pipe, pressure sensor and coil are sequentially arranged on the pipe line of showing, first showing pipe line valve is arranged between the first pump body and the circulating tank, neutralization collection liquid replenishing port, first injection water replenishing port and filtration balance liquid replenishing port are sequentially arranged between the first pump body and the first showing pipe line valve, the second end of the pipe line of showing is shunted with first branch pipe line, second branch pipe line, third branch pipe line, fourth branch pipe line, fifth branch pipe line and sixth branch pipe line, first branch pipe line is arranged between the first flowmeter and third showing pipe line valve, and first branch pipe line valve and deep filter are sequentially arranged on the first branch pipe line;First coil valve and coil are arranged on the third branch pipe line, and second coil valve is connected in series on both ends of the coil, and fourth showing pipe line valve is further arranged between the coil and the pressure sensor;Hollow fiber column is arranged on the fourth branch pipe line, and fifth showing pipe line valve and sixth showing pipe line valve are arranged on both ends of the hollow fiber column;The fourth branch pipe line and the fifth branch pipe line are connected and communicated with the circulating tank by the second return pipe line;The sixth branch pipe line is arranged between the fifth branch pipe line valve and the second mixing pipe, and sixth branch pipe line secondary valve, third flowmeter, third pump body and sixth branch pipe line main valve are sequentially arranged on the sixth branch pipe line.

[0006] Further, the above-mentioned integrated continuous processing device for obtaining escherichia coli plasmid, wherein: the first end of the liquid inlet pipe line is connected to the liquid inlet end of the circulating tank, and the liquid inlet pipe line is sequentially connected with a liquid inlet pipe line valve, a second flowmeter and a second pump body, and the second end of the liquid inlet pipe line is provided with an alkali solution replenishing port, a resuspension solution replenishing port, a second injection water replenishing port and a bacterial solution replenishing port.

[0007] Further, the above-mentioned integrated continuous processing device for obtaining escherichia coli plasmid, wherein: one end of the first return pipe line is connected to the first return end of the circulating tank, and the first return pipe line is sequentially connected with a metering control valve and a first detector group, and the other end of the first return pipe line is connected with the hollow fiber column.

[0008] Further, the integrated continuous processing device for obtaining E. coli plasmid, wherein: the second reflux pipeline is connected to the second reflux end of the circulating tank, the second reflux pipeline is sequentially connected with a reflux pipeline valve and a second detector group, and a waste discharge port, a lysis neutralization liquid collection port and a reserved port are sequentially arranged between the reflux pipeline valve and the second detector group.

[0009] Further, the integrated continuous processing device for obtaining E. coli plasmid, wherein: one end of the supplement pipeline is connected to the third end of the elution pipeline, the supplement pipeline is sequentially connected with a first supplement pipeline valve and a second supplement pipeline valve, and the other end of the supplement pipeline is connected to the third end of the liquid inlet pipeline.

[0010] Further, the integrated continuous processing device for obtaining E. coli plasmid, wherein: a second elution pipeline valve is arranged between the first flow meter and the first pump body, and a third elution pipeline valve is arranged between the first flow meter and the first mixing pipeline, and the solution is fully mixed through the first mixing pipeline.

[0011] Further, the integrated continuous processing device for obtaining E. coli plasmid, wherein: the second branch pipeline is provided with an atmospheric discharge port, so as to control the pressure change of each reaction.

[0012] Further, the integrated continuous processing device for obtaining E. coli plasmid, wherein: the fifth branch pipeline is provided with a fifth branch pipeline valve and a second mixing pipeline, and the solution after reaction is fully mixed through the second mixing pipeline.

[0013] The tangential flow filtration, alkaline lysis and clarification filtration integrated device provided by the technical scheme can realize automatic alkaline lysis, acid neutralization and other processes, and finally, a large amount of flocculent produced by lysis neutralization liquid is filtered out, so that a liquid with high clarity is obtained, and subsequent processing is facilitated.

[0014] Compared with the prior art, through switching and starting of different devices, the collection and resuspension of E. coli after fermentation, alkaline lysis and clarification filtration process are connected in a smooth and orderly manner, and multiple processes are completed on one device to realize continuous production, which can not only ensure stable operation of each step, but also greatly reduce the labor intensity of workers, save labor, reduce the number of devices and the occupied area, and improve the production efficiency of plasmid. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a structural schematic diagram of the whole device.

[0016] Figure 2 The tangential flow filtration process (recycle tank, first pump body, second pump body and hollow fiber column operating state) structural schematic view of the embodiment of the present application is shown in the figure.

[0017] Figure 3 The alkaline lysis process (recycle tank, first pump body, second pump body, third pump body and coil operating state) structural schematic view of the embodiment of the present application is shown in the figure.

[0018] Figure 4 The clarification filtration process (recycle tank, first pump body and deep layer filter operating state) structural schematic view of the embodiment of the present application is shown in the figure.

[0019] The meanings of the various reference signs in the figure are as follows: 1-recycle tank, 2-first pump body, 3-second pump body, 4-third pump body, 5-transmission pipeline, 51-first branch pipeline, 52-second branch pipeline, 53-third branch pipeline, 54-fourth branch pipeline, 55-fifth branch pipeline, 56-sixth branch pipeline, 6-liquid inlet pipeline, 7-first return pipeline, 8-second return pipeline, 9-supplement pipeline, 10-first transmission pipeline valve, 11-second transmission pipeline valve, 12-third transmission pipeline valve, 13-fourth transmission pipeline valve, 14-fifth transmission pipeline valve, 15-sixth transmission pipeline valve, 16-liquid inlet pipeline valve, 17-metering control valve, 18-return pipeline valve, 19-first supplement pipeline valve, 20-second supplement pipeline valve, 21-first branch pipeline valve, 22-first coil valve, 23-second coil valve, 24-fifth branch pipeline valve, 25-sixth branch pipeline secondary valve, 26-sixth branch pipeline primary valve, 27-first flowmeter, 28-pressure sensor, 29-second flowmeter, 30-first detector group, 31-second detector group, 32-third flowmeter, 33-first mixing pipe, 34-second mixing pipe, 35-deep layer filter, 36-coil, 37-hollow fiber column, 38-neutralization collection liquid supplement port, 39-first injection water supplement port, 40-filtration balance liquid supplement port, 41-normal pressure discharge port, 42-alkali liquid supplement port, 43-resuspension liquid supplement port, 44-second injection water supplement port, 45-bacterium liquid supplement port, 46-waste discharge port, 47-lysis neutralization liquid collection port, 48-reserved port. DETAILED DESCRIPTION

[0020] The technical scheme of the present application will be further described below in combination with the drawings so as to be more easily understood and mastered. The pump body, valve and sensor and other components involved are all commonly used by the ordinary skilled in the art and are not specially required in the present case.

[0021] As Figure 1The utility model provides a kind of integrated continuous processing device for obtaining escherichia coli plasmid, the lower end of circulating tank 1 is connected with the pipe 5 that shows, the upper end of circulating tank 1 is respectively connected with liquid inlet pipe 6, first reflux pipe 7 and second reflux pipe 8.

[0022] According to the technical solution of this utility model, the first end of the permeation pipe 5 is connected to the permeation end of the circulation tank 1, and the permeation pipe 5 is sequentially provided with a first pump body 2, a first flow meter 27, a first mixing pipe 33, a pressure sensor 28, and a coil 36. A first permeation pipe valve 10 is provided between the first pump body 2 and the circulation tank 1, a second permeation pipe valve 11 is provided between the first flow meter 27 and the first pump body 2, and a third permeation pipe valve 12 is provided between the first flow meter 27 and the first mixing pipe 33. 2. Between the first pump body 2 and the first permeation pipeline valve 10, a neutralization collection liquid replenishment port 38, a first injection water replenishment port 39, and a filter balance liquid replenishment port 40 are sequentially provided. The second end of the permeation pipeline 5 is divided into a first branch pipeline 51, a second branch pipeline 52, a third branch pipeline 53, a fourth branch pipeline 54, a fifth branch pipeline 55, and a sixth branch pipeline 56. The first branch pipeline 51 is located between the first flow meter 27 and the third permeation pipeline valve 12, and the first branch pipeline... The first branch pipe 51 is sequentially equipped with a first branch pipe valve 21 and a deep filter 35; the second branch pipe 52 has an atmospheric pressure discharge port 41; the third branch pipe 53 is equipped with a first coil valve 22 and a coil 36, with a second coil valve 23 connected in series at both ends of the coil 36, and a fourth permeation pipe valve 13 is provided between the coil 36 and the pressure sensor 28; the fourth branch pipe 54 is equipped with a hollow fiber column 37, with a fifth permeation pipe valve 14 and a first... The fifth branch pipeline 55 is equipped with a fifth branch pipeline valve 24 and a second mixing pipe 34; the fourth branch pipeline 54 intersects with the fifth branch pipeline 55 and is connected to the circulation tank 1 through the second return pipeline 8; the sixth branch pipeline 56 is located between the fifth branch pipeline valve 24 and the second mixing pipe 34, and the sixth branch pipeline 56 is sequentially equipped with a sixth branch pipeline secondary valve 25, a third flow meter 32, a third pump body 4, and a sixth branch pipeline main valve 26. The first end of the liquid inlet pipeline 6 is connected to the liquid inlet end of the circulation tank 1, and the liquid inlet pipeline 6 is sequentially connected with a liquid inlet pipeline valve 16, a second flow meter 29, and a second pump body 3; the second end of the liquid inlet pipeline 6 is provided with an alkali replenishment port 42, a resuspension replenishment port 43, a second injection water replenishment port 44, and a bacterial liquid replenishment port 45. One end of the first reflux pipeline 7 is connected to the first reflux end of the circulation tank 1. The first reflux pipeline 7 is connected in sequence to the metering control valve 17 and the first detector group 30. The other end of the first reflux pipeline 7 is connected to the hollow fiber column 37.The second reflux pipeline 8 is connected to the second reflux end of the circulation tank 1. A reflux pipeline valve 18 and a second detector group 31 are sequentially connected to the second reflux pipeline 8. A waste discharge port 46, a pyrolysis neutralization liquid collection port 47, and a reserved port 48 are sequentially provided between the reflux pipeline valve 18 and the second detector group 31. One end of the replenishment pipeline 9 is connected to the third end of the permeation pipeline 5. A first replenishment pipeline valve 19 and a second replenishment pipeline valve 20 are sequentially connected to the replenishment pipeline 9. The other end of the replenishment pipeline 9 is connected to the third end of the liquid inlet pipeline 6.

[0023] Preferably, in the above structure: the first detector group 30 includes, but is not limited to, a conductivity sensor, an ultraviolet sensor, a pH sensor and a pressure sensor (the types and quantities of sensors can be flexibly combined with each valve).

[0024] Preferably, in the above structure: the second detector group 31 includes, but is not limited to, a conductivity sensor, an ultraviolet sensor, a pH sensor and a pressure sensor (the types and quantities of sensors can be flexibly combined with each valve to integrate the flow path).

[0025] like Figure 2 As shown, when harvesting E. coli using tangential flow filtration, the automatic control software controls each valve to activate the following equipment: circulation tank 1, first pump body 2, second pump body 3, and hollow fiber column 37. The first pump body 2 is the feed pump, and the second pump body 3 is the replenishment pump. The E. coli fermentation broth is transferred to the circulation tank 1 through the inlet pipe 6 via the second pump body 3. The alkali replenishment port 42, resuspension replenishment port 43, second injection water replenishment port 44, and bacterial liquid replenishment port 45 can be switched to perform rinsing, balancing, top washing, or CIP internal cleaning treatment on the solution. The solution is concentrated through the first pump body 2 and hollow fiber column 37. After being concentrated to the standard volume, the first outlet pipe valve 10 of the second pump body 3 is switched to the alkali pyrolysis process solution (i.e., the resuspension is subjected to equal volume liquid exchange treatment), thereby realizing the harvesting of E. coli and the resuspension of bacterial sludge, and then the next step of alkali pyrolysis process can be carried out.

[0026] like Figure 3As shown, when using alkaline lysis to break up E. coli, the automatic control software controls each valve to activate the following equipment: circulation tank 1, first pump 2, second pump 3, third pump 4, and coil 36. This releases the target sample plasmid DNA. First pump 2 delivers the bacterial suspension to circulation tank 19 through a replenishment pipeline. Second pump 3 simultaneously delivers alkaline solution and activates first mixing tube 33 to mix the solution, initiating the lysis reaction. Then, second coil valve 23 controls the reaction solution to enter coil 36. The lysis reaction time is controlled by the volume of the inner cavity of coil 36 and the total flow rate. After the lysate exits coil 36, third pump 4 delivers acid (i.e., neutralization solution) to neutralize the lysate, terminating the lysis reaction. The mixing effect is then enhanced by second mixing tube 34. The neutralized solution sample can be collected in an independent container or returned to circulation tank 1 through return pipeline valve 18 for subsequent clarification and filtration processing.

[0027] like Figure 4 As shown, when the clarification and filtration process is used to treat the collected liquid, the automatic control software controls each valve to open the following equipment: circulation tank 1, first pump body 2 and depth filter 35. The pyrolysis and neutralization liquid contains a large number of flocculent impurities and needs to be clarified and filtered to obtain the supernatant. The neutralized solution sample in circulation tank 1 is filtered through the depth filter 35 by the first pump body 2. Different solutions can be selected for rinsing, balancing and top washing operations by switching the neutralized collected liquid replenishment port 38, the first injection water replenishment port 39 and the filter equilibration liquid replenishment port 40.

[0028] The key technology of this invention is the integration of tangential flow filtration, alkali pyrolysis, and clarification filtration into a continuous production process. Figure 1 The focus of the exhibition is on the components and flow path structures involved in the continuous production processes of tangential flow filtration, alkali pyrolysis, and clarification filtration. Figures 2 to 4 The examples demonstrate specific flow path structures for switching between different processes using automated control software to control various valves. This effectively scales up production, reduces batch-to-batch variability, ensures process consistency, and thus improves process robustness. For components such as pumps, valves, and sensors, those skilled in the art can perform conventional setups based on existing technology; this application does not have special requirements regarding model selection or combination.

[0029] Thus, this utility model provides an integrated continuous production device for tangential flow filtration, alkaline lysis, and clarification filtration. The device harvests bacterial cells using tangential flow filtration, followed by continuous alkaline lysis of the bacterial cells using a coil-type process to release plasmid DNA. This allows for automated alkaline lysis and acid neutralization processes. Finally, clarification filtration removes a large amount of flocculent material generated from the lysis and neutralization liquid, resulting in a highly clear liquid for subsequent processing (e.g., purification).

[0030] As can be seen from the above description, compared with the prior art, after adopting the technical solution of this utility model, the collection, resuspension, alkaline lysis and clarification filtration processes of E. coli fermentation are connected in a smooth and orderly manner by switching and starting different equipment. Multiple processes are completed on one device, realizing the automation of the production process, reducing manual operation, achieving continuous production, controllable process parameters, ensuring stable operation of each step, greatly reducing the labor intensity of operators, saving manpower, reducing the number of equipment and floor space, improving plasmid production efficiency and reducing production costs.

[0031] The technical solution, working process and implementation effect of this utility model have been described in detail above. It should be noted that the described example is only a typical example of this utility model. In addition, this utility model may have many other specific implementation methods. All technical solutions formed by equivalent substitution or equivalent transformation fall within the scope of protection claimed by this utility model.

Claims

1. An integrated continuous processing device for obtaining E. coli plasmid, comprising a circulating tank (1), a through pipe (5) connected to the lower end of the circulating tank (1), a liquid inlet pipe (6), a first return pipe (7) and a second return pipe (8) connected to the upper end of the circulating tank (1) respectively, characterized in that: The first end of the permeation pipeline (5) is communicated with the permeation end of the circulating tank (1), and the permeation pipeline (5) is sequentially provided with a first pump body (2), a first flow meter (27), a first mixing pipe (33), a pressure sensor (28) and a coil pipe (36), a first permeation pipeline valve (10) is arranged between the first pump body (2) and the circulating tank (1), and a neutralization collection liquid supplement opening (38), a first injection water supplement opening (39) and a filter balance liquid supplement opening (40) are sequentially arranged between the first pump body (2) and the first permeation pipeline valve (10), the second end of the permeation pipeline (5) is branched to be provided with a first branch pipeline (51), a second branch pipeline (52), a third branch pipeline (53), a fourth branch pipeline (54), a fifth branch pipeline (55) and a sixth branch pipeline (56), the first branch pipeline (51) is arranged between the first flow meter (27) and a third permeation pipeline valve (12), and the first branch pipeline (51) is sequentially provided with a first branch pipeline valve (21) and a deep filter (35); the third branch pipeline (53) is provided with a first coil pipe valve (22) and a coil pipe (36), both ends of the coil pipe (36) are further connected in series with a second coil pipe valve (23), and the coil pipe (36) is further provided with a fourth permeation pipeline valve (13) between the coil pipe (36) and the pressure sensor (28); the fourth branch pipeline (54) is provided with a hollow fiber column (37), both ends of the hollow fiber column (37) are provided with a fifth permeation pipeline valve (14) and a sixth permeation pipeline valve (15) respectively; the fourth branch pipeline (54) and the fifth branch pipeline (55) are connected and communicated with the circulating tank (1) through the second return pipeline (8); the sixth branch pipeline (56) is arranged between a fifth branch pipeline valve (24) and a second mixing pipe (34), and the sixth branch pipeline (56) is sequentially provided with a sixth branch pipeline secondary valve (25), a third flow meter (32), a third pump body (4) and a sixth branch pipeline main valve (26).

2. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: The first end of the liquid inlet pipeline (6) is communicated and connected to the liquid inlet end of the circulating tank (1), the liquid inlet pipeline (6) is sequentially connected with a liquid inlet pipeline valve (16), a second flow meter (29) and a second pump body (3), and the second end of the liquid inlet pipeline (6) is provided with a lye supplement opening (42), a resuspension liquid supplement opening (43), a second injection water supplement opening (44) and a bacteria liquid supplement opening (45).

3. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: One end of the first return pipeline (7) is communicated and connected to the first return end of the circulating tank (1), the first return pipeline (7) is sequentially connected with a metering control valve (17) and a first detector group (30), and the other end of the first return pipeline (7) is connected with the hollow fiber column (37).

4. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: The second reflux pipeline (8) is communicated with the second reflux end of the circulating tank (1), and a reflux pipeline valve (18) and a second detector group (31) are sequentially connected to the second reflux pipeline (8), and a waste discharge port (46), a cracking neutralization liquid collecting port (47) and a reserved port (48) are sequentially arranged between the reflux pipeline valve (18) and the second detector group (31).

5. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: A third end of the liquid inlet pipeline (6) and a third end of the permeation pipeline (5) are further provided with a supplement pipeline (9), and a first supplement pipeline valve (19) and a second supplement pipeline valve (20) are sequentially connected to the supplement pipeline (9).

6. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: A second permeation pipeline valve (11) is arranged between the first flow meter (27) and the first pump body (2), and a third permeation pipeline valve (12) is arranged between the first flow meter (27) and the first mixing pipeline (33).

7. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: The second branch pipeline (52) is provided with an atmospheric pressure discharge port (41).

8. The integrated continuous processing apparatus for the isolation of E. coli plasmid according to claim 1, wherein: The fifth branch pipeline (55) is provided with a fifth branch pipeline valve (24) and a second mixing pipeline (34).

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