Plasmid cracking and clarifying device
By designing a plasmid cleavage and clarification device including lysis, flocculation and filtration modules, the problem of plasmid cleavage and clarification in the prior art requires separate operation, and automated operation is achieved, and efficiency and product stability are improved.
Patent Information
- Application Number
- CN202421519450.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-28
- Publication Date
- 2025-05-30
- Estimated Expiration
- 2034-06-28
AI Technical Summary
The existing plasmid lysis and clarification require the use of two equipment to operate separately, which have problems such as large liquid volume, long batch processing, unstable flux, complex equipment and inconvenient maintenance.
A plasmid cleavage and clarification device is provided, including a cleavage module, a flocculation module and a filtration module. It realizes automated operation through a connected setting, uses flocculant to promote solid-liquid separation, reduces solids entering the filter, and delays the rise of filtration pressure.
The automation of plasmid cleavage and clarification is achieved, saving time and space, improving efficiency and product stability, and reducing equipment maintenance difficulties.
Smart Images
Figure CN222923118U_ABST
Abstract
Description
Technical Field
[0001] The embodiments of the present application relate to the technical field of biological product equipment, and in particular to a plasmid lysis and clarification device. Background Technique
[0002] A plasmid is a nucleic acid macromolecule that is independent of the cell chromosome, can carry certain genetic information, and can replicate itself. Plasmids are widely present in prokaryotic and eukaryotic cells and are an important class of gene carriers. Except for RNA plasmids found in yeast, other plasmids are circular, double-stranded DNA macromolecules. Plasmids are vectors for foreign genes in Escherichia coli and can be directly used as drugs for gene therapy. They are indispensable intermediates for gene therapy and cell therapy products. Whether it is to construct genetically engineered recombinant strains, develop gene therapy drugs, develop nucleic acid vaccines, or establish personalized cell therapy drugs, it is necessary to first prepare DNA plasmids.
[0003] Using recombinant Escherichia coli fermentation to produce DNA plasmids is the main means of plasmid production. The construction of Escherichia coli, cell fermentation, and plasmid expression belong to the upstream production process, while cell lysis and plasmid purification belong to the downstream production process. The progress of upstream strain construction technology and fermentation process has increased the production capacity of plasmids to several hundred to several thousand milligrams per liter of fermentation broth. In the downstream purification scheme, the second step is always cell lysis, and multiple chromatography units are required subsequently to further remove impurities such as RNA, host proteins, and endotoxins. In plasmid production, cell lysis usually adopts chemical lysis methods, among which alkaline lysis is the most widely used chemical lysis method.
[0004] Existing plasmid lysis and clarification need to be operated using two devices separately, and there are the following deficiencies: 1. The volume of the lysate is large, and when the production scale is large, the floor area of the storage tank is large; 2. Both operation steps are batch processes, and the total time consumption is long; 3. For a device based on simple depth filtration, the flux is unstable, the loading capacity is low, and the solids in the lysate are easy to block the filter components; 4. For a device based on microfiltration clarification, the equipment is relatively complex, inconvenient to maintain and use, and difficult to clean and verify. Summary of the Utility Model
[0005] In view of this, the embodiments of the present application provide a plasmid lysis and clarification device to solve the technical problems raised in the background technique.
[0006] The embodiments of the present application provide a plasmid lysis and clarification device, which includes a lysis module, a flocculation module, and a filtration module. The lysis module, the flocculation module, and the filtration module are connected in series.
[0007] Among them, the cracking module includes a feed pump assembly, and the feed pump assembly is connected with a cracking coil through a pipeline; the flocculation module includes a flocculation tank, and a liquid pump is arranged between the cracking coil and the flocculation tank, and the liquid pump is used to pump flocculant or top washing liquid into the flocculation tank;
[0008] The filtration module includes a filter assembly, and the filter assembly is connected with the flocculation tank through a pipeline.
[0009] In some embodiments that may include the above embodiments, the cracking module further includes a static mixer and a neutralization coil, and the cracking coil is sequentially connected with the static mixer and the neutralization coil through a pipeline.
[0010] In some embodiments that may include the above embodiments, the feed pump assembly includes an acid pump, a base pump and a resuspension liquid pump. The branch pipelines of the base pump and the resuspension liquid pump converge into one path and then are connected with the cracking coil through a pipeline, and the acid pump is connected with the static mixer through a pipeline.
[0011] In some embodiments that may include the above embodiments, the cracking module further includes a dynamic mixer. The branch pipelines of the base pump and the resuspension liquid pump converge into one path and then are connected with the dynamic mixer through a pipeline, and the dynamic mixer is connected with the cracking coil through a pipeline.
[0012] In some embodiments that may include the above embodiments, the flocculation module further includes a liquid level sensor assembly and an exhaust valve. The liquid level sensor assembly is arranged on the outer wall surface of the flocculation tank and is used to monitor the water level in the flocculation tank; the exhaust valve is arranged at the top of the flocculation tank and is used to discharge the gas in the flocculation tank.
[0013] In some embodiments that may include the above embodiments, the liquid level sensor assembly includes a lower liquid level sensor and an upper liquid level sensor. The lower liquid level sensor is arranged below the outer wall surface of the flocculation tank, and the upper liquid level sensor is arranged above the outer wall surface of the flocculation tank, and the lower liquid level sensor is directly below the upper liquid level sensor.
[0014] In some embodiments that may include the above embodiments, the filter assembly includes a first filter and a second filter. The first filter and the second filter are arranged in parallel, and valves are respectively arranged on the first filter and the second filter, and the flocculation tank is respectively connected with the first filter and the second filter through pipelines.
[0015] In some embodiments that may include the above embodiments, a pressure detector is further arranged between the flocculation tank and the filter assembly, and the pressure detector is used to monitor the pressure during filtration.
[0016] In some embodiments that may include the above embodiments, the branch pipelines of the first filter and the second filter converge into one path and are connected to a clarified liquid outlet through a pipeline.
[0017] Compared with the prior art, the embodiments of the present application have the following beneficial effects:
[0018] 1. The plasmid lysis clarification device in the embodiments of the present application is a highly automated device that automatically completes the steps of flocculation and clarification of the lysate;
[0019] 2. The plasmid lysis clarification device in the embodiments of the present application does not require additional lysate storage tanks and flocculant storage tanks, saving hardware costs;
[0020] 3. The plasmid lysis clarification device in the embodiments of the present application can continuously complete the two steps of lysis and clarification, saving time, improving efficiency, and reducing the risk of product stability;
[0021] 4. The plasmid lysis clarification device in the embodiments of the present application uses a flocculant to promote the solid-liquid separation of the lysate, reduces the entry of solids into the filter assembly, delays the rise of clarification pressure and the blockage of the filter assembly, and improves the loading capacity of the filter assembly;
[0022] 5. After all the lysate in the plasmid lysis clarification device in the embodiments of the present application enters, the liquid in the flocculation tank can be top-washed, the solids can be washed, and the filter of the filtration module can be top-washed, improving the product recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0024] Figure 1 It is a schematic structural diagram of the plasmid lysis clarification device according to the embodiment of the present application.
[0025] Description of the reference numerals:
[0026] 1. Acid pump; 2. Alkali pump; 3. Resuspension liquid pump; 4. Dynamic mixer; 5. Lysis coil; 6. Static mixer; 7. Neutralization coil; 8. Liquid pump; 9. Lower liquid level sensor; 10. Upper liquid level sensor; 11. Exhaust valve; 12. Flocculation tank; 13. Pressure detector; 14. First filter; 15. Second filter; 16. Clarified liquid outlet. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of this application without creative efforts shall fall within the protection scope of this application.
[0028] First of all, those skilled in the art should understand that these embodiments are only used to explain the technical principles of this application and are not intended to limit the protection scope of this application. Those skilled in the art can make adjustments according to needs to adapt to specific application scenarios.
[0029] Secondly, it should be noted that in the description of this application, the terms indicating directions or positional relationships such as "upper", "lower", "left", "right", "front", "rear", "inner", "outer", etc. are based on the directions or positional relationships shown in the accompanying drawings. This is only for convenience of description and does not indicate or imply that the device or component must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to this application.
[0030] In the related art, the existing plasmid lysis and clarification need to be operated using two devices separately, and there are the following deficiencies: 1. The volume of the lysis solution is large, and when the production scale is large, the floor area of the storage tank is large; 2. Both operation steps are batch processes, and the total time consumption is long; 3. Based on a simple depth filtration device, the flux is unstable, the loading capacity is low, and the solids in the lysis solution are easy to block the filter assembly; 4. Based on a microfiltration clarification device, the equipment is relatively complex, inconvenient to maintain and use, and difficult to clean and verify.
[0031] To solve the above technical problems, as Figure 1 shown, the embodiments of this application provide a plasmid lysis and clarification device, which includes a lysis module, a flocculation module, and a filtration module. The lysis module, the flocculation module, and the filtration module are connected in sequence.
[0032] Among them, the lysis module includes a feed pump assembly, and the feed pump assembly is connected through a pipeline to a lysis coil 5; the flocculation module includes a flocculation tank 12, and a liquid pump 8 is arranged between the lysis coil 5 and the flocculation tank 12. The liquid pump 8 is used to pump the flocculation liquid or the top washing liquid into the flocculation tank 12; the filtration module includes a filter assembly, and the filter assembly is connected to the flocculation tank 12 through a pipeline.
[0033] The lysate and the flocculant enter the flocculation tank 12 for reaction simultaneously. Under the action of the flocculant, the solids in the lysate flocculate to the upper layer of the liquid, and the lower clear liquid enters the filtration module for clarification. After all the lysate enters the flocculation tank 12, the top washing buffer solution can be added using the liquid pump 8 to completely displace the lysate in the flocculation tank 12 and wash the flocculated solids in the upper layer, and the clarification of the lysate is completed.
[0034] In some specific embodiments, the cracking module further includes a static mixer 6 and a neutralization coil 7, and the cracking coil 5 is connected to the static mixer 6 and the neutralization coil 7 in sequence through pipelines.
[0035] Specifically, the feed pump assembly includes an acid pump 1, a base pump 2, and a resuspension liquid pump 3. The branch pipelines of the base pump 2 and the resuspension liquid pump 3 converge into one path and then are connected to the cracking coil 5 through a pipeline, and the acid pump 1 is connected to the static mixer 6 through a pipeline.
[0036] In some specific embodiments, the cracking module further includes a dynamic mixer 4. The branch pipelines of the base pump 2 and the resuspension liquid pump 3 converge into one path and then are connected to the dynamic mixer 4 through a pipeline, and the dynamic mixer 4 is connected to the cracking coil 5 through a pipeline.
[0037] In some specific embodiments, the flocculation module further includes a liquid level sensor assembly and an exhaust valve 11. The liquid level sensor assembly is arranged on the outer wall surface of the flocculation tank 12 for monitoring the water level in the flocculation tank 12; the exhaust valve 11 is arranged at the top of the flocculation tank 12 for discharging the gas in the flocculation tank 12.
[0038] Specifically, the liquid level sensor assembly includes a lower liquid level sensor 9 and an upper liquid level sensor 10. The lower liquid level sensor 9 is arranged below on the outer wall surface of the flocculation tank 12, and the upper liquid level sensor 10 is arranged above on the outer wall surface of the flocculation tank 12, and the lower liquid level sensor 9 is directly below the upper liquid level sensor 10.
[0039] When the liquid level in the flocculation tank 12 reaches the upper liquid level sensor 10, the exhaust valve 11 is closed; when the liquid level in the flocculation tank 12 reaches the lower liquid level sensor 9, the exhaust valve 11 is opened to discharge the gas generated during flocculation, and the liquid level in the flocculation tank 12 is maintained between the lower liquid level sensor 9 and the upper liquid level sensor 10.
[0040] Specifically, the filter assembly includes a filter one 14 and a filter two 15. The filter one 14 and the filter two 15 are arranged in parallel, and valves are respectively arranged on the filter one 14 and the filter two 15. The flocculation tank 12 is connected to the filter one 14 and the filter two 15 respectively through pipelines. The filter one 14 and the filter two 15 are used separately, not simultaneously. When the pressure reaches a certain value, the filter one 14 and the filter two 15 will be switched, and the filter two 15 is a backup setting for the filter one 14.
[0041] Exemplarily, the branch pipelines of filter one 14 and filter two 15 converge and then are connected to a clarified liquid outlet 16 through a pipeline.
[0042] Furthermore, a pressure detector 13 is also provided between the flocculation tank 12 and the filter assembly, and the pressure detector 13 is used to monitor the pressure during filtration.
[0043] The specific working process of the plasmid lysis clarification device in this embodiment:
[0044] (1) Empty the preservation liquid in the device, then inject a cleaning liquid into the device for cleaning. After cleaning, rinse with purified water. After cleaning, assemble the filter assembly onto the device and discharge the air bubbles in the device; (2) Fill the pipeline of the lysis module with purified water, fill the pipeline of the flocculation module with buffer solution, inject a small amount of buffer solution into the flocculation tank 12, and discharge the air bubbles in the filtration module; (3) Start the resuspension liquid pump 3 and the alkali pump 2. When the lysis liquid reaches the static mixer 6 after passing through the neutralization coil 4 and the lysis coil 5 in sequence, turn on the acid pump 1 to neutralize the lysis solution, then open the exhaust valve 11. The lysis liquid enters the flocculation tank 12 through the neutralization coil 7. At the same time, turn on the liquid pump 8 to inject the flocculation liquid. When the lysis liquid level reaches the upper liquid level sensor 10, close the exhaust valve 11, open filter one 14 and its valve, and the lower layer of clarified liquid enters the filtration module for filtration and clarification. During the clarification process, when the liquid level in the flocculation tank 12 is lower than the lower liquid level sensor 9, open the exhaust valve 11 to promote the liquid level to rise; (4) During the clarification process, when the pressure of the pressure detector 12 exceeds the set value, switch to the pipeline of filter two 15 and open its valve for filtration and clarification. After all the lysis liquid enters the filtration module, turn off the acid pump 1, the alkali pump 2, and the resuspension liquid pump 3, and it is necessary to close the pipeline connecting the neutralization coil 7 to the flocculation module. The clarified liquid is discharged from the clarified liquid outlet 16; (5) Inject a top washing liquid by the liquid pump 8 for the top washing operation of the device. After the top washing is completed, empty the device, then inject purified water into the device for cleaning, then inject a cleaning liquid into the device for cleaning, inject purified water into the device for cleaning again, and finally use the preservation liquid to preserve the device.
[0045] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.
Claims
1. A plasmid lysis and clarification device, characterized in that: The plasmid lysis and clarification device comprises a lysis module, a flocculation module and a filtration module, wherein the lysis module, the flocculation module and the filtration module are connected in sequence; The cracking module comprises a feed pump assembly, and the feed pump assembly is connected to a cracking coil (5) via a pipeline; The flocculation module comprises a flocculation tank (12), and a liquid pump (8) is provided between the lysis coil (5) and the flocculation tank (12), and the liquid pump (8) is used to pump flocculation liquid or top wash liquid into the flocculation tank (12); The filtration module comprises a filter assembly, and the filter assembly is connected to the flocculation tank (12) via a pipeline.
2. The plasmid lysis and clarification device according to claim 1, characterized in that: The cracking module further comprises a static mixer (6) and a neutralizing coil (7), and the cracking coil (5) is connected to the static mixer (6) and the neutralizing coil (7) in sequence through a pipeline.
3. The plasmid lysis and clarification device according to claim 2, characterized in that: The feed pump assembly comprises an acid pump (1), an alkali pump (2) and a resuspension pump (3); the branch pipelines of the alkali pump (2) and the resuspension pump (3) are connected to the cracking coil (5) through a pipeline after being merged into one pipeline; and the acid pump (1) is connected to the static mixer (6) through a pipeline.
4. The plasmid lysis and clarification device according to claim 3, characterized in that: The lysis module further comprises a dynamic mixer (4), wherein the branch pipelines of the alkali pump (2) and the resuspension pump (3) are connected to the dynamic mixer (4) through a pipeline after being converged into one pipeline, and the dynamic mixer (4) is connected to the lysis coil (5) through a pipeline.
5. The plasmid lysis and clarification device according to claim 1, characterized in that: The flocculation module further comprises a liquid level sensor assembly and an exhaust valve (11); the liquid level sensor assembly is arranged on the outer wall surface of the flocculation tank (12) and is used to monitor the water level in the flocculation tank (12); the exhaust valve (11) is arranged on the top of the flocculation tank (12) and is used to discharge the gas in the flocculation tank (12).
6. The plasmid lysis and clarification device according to claim 5, characterized in that: The liquid level sensor assembly comprises a lower liquid level sensor (9) and an upper liquid level sensor (10), wherein the lower liquid level sensor (9) is arranged below the outer wall surface of the flocculation tank (12), and the upper liquid level sensor (10) is arranged above the outer wall surface of the flocculation tank (12), and the lower liquid level sensor (9) is located directly below the upper liquid level sensor (10).
7. The plasmid lysis and clarification device according to claim 1, characterized in that: The filter assembly comprises a filter 1 (14) and a filter 2 (15), wherein the filter 1 (14) and the filter 2 (15) are arranged in parallel, and valves are arranged on the filter 1 (14) and the filter 2 (15), respectively, and the flocculation tank (12) is connected to the filter 1 (14) and the filter 2 (15) through pipelines.
8. The plasmid lysis and clarification device according to claim 1, characterized in that: A pressure detector (13) is also provided between the flocculation tank (12) and the filter assembly, and the pressure detector (13) is used to monitor the pressure during filtration.
9. The plasmid lysis and clarification device according to claim 7, characterized in that: The branch pipelines of the filter 1 (14) and the filter 2 (15) are connected to one channel and then connected to a clarified liquid outlet (16) through a pipeline.