Continuous flow controllable parameter plasmid extraction device

Abstract

The utility model discloses a kind of continuous flow controllable parameter plasmid extraction devices, belong to biological pharmaceutical technical field, including power control module, power control module is connected with first peristaltic pump, second peristaltic pump, third peristaltic pump by circuit, first peristaltic pump is connected with first container, is connected with first static distributor, second peristaltic pump is connected with premixing tank, is connected with first static distributor, first static distributor is connected with alkali lysis tank, second peristaltic pump is connected with second container, is connected with second static distributor, alkali lysis tank is connected with second static distributor, second static distributor is connected with clarification tank, clarification tank is connected with sample tank, alkali lysis tank and clarification tank are all double-layer structure tank body with interlayer;The utility model uses above-mentioned structure a kind of continuous flow controllable parameter plasmid extraction device, realizes to plasmid extraction key parameter accurate control under full closed condition, significantly improves product superhelix proportion, production efficiency and process stability.

Description

Technical Field

[0001] This utility model relates to the field of biomedical technology, and in particular to a continuous flow controllable parameter plasmid extraction device. Background Technology

[0002] In recent years, with the commercialization of CAR-T / mRNA vaccines, increased investment in university / biomedical R&D, and the expansion of CDMOs, the demand for GMP-grade plasmids has increased significantly. Therefore, facilities for large-scale fermentation production of high-purity and high-quality DNA plasmids have become increasingly important.

[0003] Plasmid DNA has three conformations: supercoiled, open circular, and linear. The supercoiled conformation is a state where the two DNA strands are covalently closed; this conformation is the most stable and efficient in gene transcription and replication. The supercoil ratio refers to the proportion of supercoiled DNA in the plasmid. The supercoil ratio of a plasmid significantly affects transfection efficiency and is crucial for the integrity of gene therapy and in vitro transcription (IVT) products, as well as the purity of mRNA. Plasmid DNA with a high supercoil ratio can improve RNA yield and expression efficiency. If the supercoil ratio of the plasmid is too low, the impurities it contains can severely affect the formation of the transfection complex, further impacting subsequent in vivo therapeutic effects.

[0004] Alkaline lysis is widely used in plasmid production processes, but it faces many challenges in practical applications. For example, during lysis, solution temperature, pH, and mixing intensity all affect the supercoil ratio of plasmids. Therefore, precise control of lysis parameters is necessary.

[0005] Chinese patent CN114933960A discloses a continuous flow bacterial disruption and clarification device that allows for precise control of lysis and clarification times, linearly scales up production, and maintains stable production efficiency and product quality during scale-up, facilitating fully automated operation. However, it does not mention the control of temperature and pH values, or data on the subsequent supercoil ratio.

[0006] Chinese patent CN119120453A discloses a purification method that can increase the supercoil ratio of plasmids and reduce the open-ring ratio and aggregate ratio. This purification method is more suitable for large-scale industrial plasmid purification. However, no pyrolysis equipment capable of being scaled up for production was found in the key steps.

[0007] In summary, current large-scale lysis devices lack online monitoring of lysis parameters such as temperature and pH, making it difficult to accurately optimize the lysis parameters of different plasmids, obtain a stable and high plasmid supercoil ratio, and control environmental pollution. Utility Model Content

[0008] The purpose of this invention is to provide a continuous flow controllable parameter plasmid extraction device to solve the problems mentioned in the background art. It can achieve precise control of the lysis temperature and pH of different plasmids, linearly scale up production, and ensure controllable supercoiling ratio of plasmids during scale-up. It also achieves fully automated operation.

[0009] To achieve the above objectives, this utility model provides a continuous flow controllable parameter plasmid extraction device, including a power control module. The power control module is connected to a first peristaltic pump, a second peristaltic pump, and a third peristaltic pump via circuitry. The inlet of the first peristaltic pump is connected to a first container, and the outlet is connected to a first static distributor. The inlet of the second peristaltic pump is connected to a premixing tank, and the outlet is connected to the first static distributor. The outlet of the first static distributor is connected to an alkaline pyrolysis tank. The inlet of the second peristaltic pump is connected to a second container, and the outlet is connected to the second static distributor. The outlet of the alkaline pyrolysis tank is connected to the inlet of the second static distributor. The outlet of the second static distributor is connected to a clarification tank. The outlet of the clarification tank is connected to a sample tank. Both the alkaline pyrolysis tank and the clarification tank are double-layered tanks with jackets.

[0010] Preferably, the power control module is equipped with a switch and a display screen.

[0011] Preferably, a chiller interface is provided on the interlayer of both the alkali pyrolysis tank and the clarification tank.

[0012] Preferably, the alkaline pyrolysis tank is equipped with a temperature probe and a pH electrode, and the top of the alkaline pyrolysis tank is equipped with an alkaline solution inlet.

[0013] Preferably, the temperature probe and the pH electrode are connected to the power control module.

[0014] Therefore, the continuous flow controllable parameter plasmid extraction device of the present invention, which adopts the above structure, has the following beneficial effects: (1) By integrating a temperature probe and a pH electrode into the alkaline pyrolysis vessel and connecting them to the power control system module, the device can monitor and regulate the temperature and pH value during the pyrolysis process in real time. Combined with the jacket structure and external chiller, it ensures that the pyrolysis reaction is always carried out within the optimal parameter range (such as temperature 20-25℃ and pH value 12.0-12.5), thereby effectively improving the supercoil ratio of plasmids and the quality of products.

[0015] (2) The use of a static distributor enables rapid and uniform mixing of bacterial solution and alkali solution, lysis solution and neutralization solution, avoiding the problem of local over-lysis or low efficiency caused by uneven mixing in traditional operation; the device adopts a modular continuous flow design, which can easily be linearly scaled up, smoothly transitioning from laboratory level to industrial production scale, and can continuously ensure the consistency of product quality and high yield during the scale-up process.

[0016] (3) The entire pyrolysis and clarification process is carried out continuously in fully enclosed pipelines and tanks, forming a closed-loop operating system. This greatly reduces the generation of aerosols and exposure to harmful chemical reagents, lowers the risk of environmental pollution, and complies with the strict hygiene and safety regulations of biopharmaceutical production workshops.

[0017] (4) The device integrates a jacketed clarification tank, which can separate impurities by standing at low temperature and accelerate the separation by adding additives. The whole process realizes continuous operation in a low temperature environment, effectively preventing the degradation of plasmids during long-term processing and ensuring the bioactivity and stability of the finally collected plasmid samples.

[0018] The technical solutions in the embodiments of this utility model will now be clearly and completely described with reference to the accompanying drawings. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the overall structure of a continuous flow controllable parameter plasmid extraction device according to the present invention; Reference numerals: 1. Power control module; 2. Premixing tank; 3. First container; 4. Second container; 5. Switch; 6. First static distributor; 7. Second static distributor; 8. Alkali pyrolysis tank; 9. Clarification tank; 10. Sample tank; 11. First peristaltic pump; 12. Second peristaltic pump; 13. Third peristaltic pump; 14. Alkali inlet; 15. pH electrode; 16. Temperature probe; 17. Display screen; 18. Chiller interface. Detailed Implementation

[0020] The technical solution of this utility model will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this utility model shall have the ordinary meaning understood by one of ordinary skill in the art to which this utility model pertains. The terms "first," "second," and similar terms used in this utility model do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0022] Example like Figure 1 As shown, a continuous flow controllable parameter plasmid extraction device includes a power control module 1, which is equipped with a switch 5 and a display screen 17. The power control module 1 is connected to a first peristaltic pump 11, a second peristaltic pump 12, and a third peristaltic pump 13 via circuitry. The inlet of the first peristaltic pump 11 is connected to a first container 3, which is used to store alkali solution, and its outlet is connected to a first static distributor 6. The inlet of the second peristaltic pump 12 is connected to a premixing tank 2, which is used to suspend and disperse bacterial sludge to obtain bacterial solution, and its outlet is connected to the first static distributor 6. The outlet of the first static distributor 6 is connected to an alkali pyrolysis tank 8. The system is connected as follows: the first static distributor 6 mixes the bacterial solution and the alkaline solution and delivers them to the alkaline lysis tank 8; the inlet of the second peristaltic pump 12 is connected to the second container 4, which is used to store the neutralization solution, and the outlet of the second container 4 is connected to the second static distributor 7; the outlet of the alkaline lysis tank 8 is connected to the inlet of the second static distributor 7; the outlet of the second static distributor 7 is connected to the clarification tank 9; the second static distributor 7 receives the reaction solution from the alkaline lysis tank 8 and adds neutralizing reagent through the third peristaltic pump 13 for mixing; the outlet of the clarification tank 9 is connected to the sample container 10; the clarification tank 9 can separate impurities at low temperature; and the sample container 10 is used for lysis collection.

[0023] Both the alkali pyrolysis tank 8 and the clarification tank 9 are double-layered structures with jackets. Each tank has a chiller interface 18 on its jacket, allowing for precise control of the reaction temperature through a circulating cooling medium. The alkali pyrolysis tank 8 is equipped with a temperature probe 16 and a pH electrode 15 for real-time monitoring and control of key parameters in the pyrolysis process. An alkali inlet 14 is also located at the top of the alkali pyrolysis tank 8 for adding reagents when necessary. Both the temperature probe 16 and the pH electrode 15 are connected to the power control module 1, transmitting monitoring signals to the control center in real time, forming a closed-loop control circuit.

[0024] Working principle: After the bacterial sludge is stirred in the premixing tank 2 to form a uniform bacterial solution, it is pumped into the first static distributor 6 at the same flow rate by the first peristaltic pump 11 and the second peristaltic pump 12 to achieve instantaneous uniform mixing, and then enters the alkaline pyrolysis tank 8. In the alkaline pyrolysis tank 8, the built-in temperature probe 16 and pH electrode 15 are used for real-time monitoring, and the pyrolysis conditions are precisely controlled at a temperature of 20-25℃ and a pH value of 12.0-12.5 by the external chiller connected to the jacket, and the reaction is carried out under these conditions for 3-5 minutes. After the reaction is completed, the bottom valve of the alkaline pyrolysis tank 8 is opened, so that the pyrolysis liquid is mixed with the neutralized liquid from the second container 4 pumped in by the third peristaltic pump 13 in the second static distributor 7, and then enters the clarification tank 9. In the clarification tank 9, the external chiller connected to the jacket is immediately turned on to cool down, so that the temperature inside the tank is maintained at about 4℃, and after standing for 3-5 minutes until the liquid level is stable, 5g / L NaHCO3 is added to accelerate the separation. Continue to let it stand for 10-20 minutes until the solid-liquid separation is stable and the impurities float to the top. Then, open the discharge valve of the clarification tank 9 to allow the lower clear liquid to slowly flow into the sample tank 10 by gravity. The collected clear liquid can then be used for subsequent purification processes.

[0025] To verify the significant effect of this embodiment in increasing the supercoil ratio of plasmids, a comparative experiment was conducted using three different plasmid samples (plasmid 1, plasmid 2, and plasmid 3).

[0026] Example group: The continuous flow controllable parameter plasmid extraction device described in this example was used to precisely control the lysis process parameters at a temperature of 25°C and a pH of 12.5.

[0027] Comparative group: The samples were processed using conventional equipment or batch methods that do not have online temperature and pH monitoring and feedback control functions, and the pyrolysis conditions were uncontrolled.

[0028] After the experiment, the supercoil ratio of plasmids in each group was detected by methods such as agarose gel electrophoresis or HPLC. The results are shown in the table below: Table 1 Results of supercoiling ratio of plasmid 1

[0029] Table 2 Results of plasmid 2 supercoil ratio

[0030] Table 3 Results of plasmid supercoil ratio

[0031] The data above show that, under the condition of precise control of lysis parameters, the supercoiling ratio of the three plasmids in this embodiment remained stable at over 94%, significantly higher than that of the comparative group. This fully demonstrates that, by precisely controlling the temperature and pH during the lysis process, this embodiment can effectively avoid plasmid damage during lysis, greatly ensuring and improving the quality of the final product, and solving the technical problem of low supercoiling ratio and unstable product quality caused by parameter malfunction in traditional methods.

[0032] Therefore, the present invention provides a continuous flow controllable parameter plasmid extraction device with the above-mentioned structure. By integrating an online monitoring and jacket temperature control system and combining it with a static distributor to achieve efficient mixing, it realizes precise control of key parameters of plasmid extraction under fully enclosed conditions, which significantly improves the product superspiral ratio, production efficiency and process stability.

[0033] Finally, it should be noted that the above embodiments are only preferred embodiments of the present utility model, but the protection scope of the present utility model is not limited thereto. Any equivalent substitutions or changes made by those skilled in the art within the technical scope disclosed in the present utility model, based on the technical solution and the inventive concept of the present utility model, should be covered within the protection scope of the present utility model.

Claims

1. A continuous flow controllable parameter plasmid extraction device, characterized in that: The system includes a power control module, which is connected via circuitry to a first peristaltic pump, a second peristaltic pump, and a third peristaltic pump. The inlet of the first peristaltic pump is connected to a first container, and its outlet is connected to a first static distributor. The inlet of the second peristaltic pump is connected to a premixing tank, and its outlet is connected to the first static distributor. The outlet of the first static distributor is connected to an alkaline pyrolysis tank. The inlet of the second peristaltic pump is connected to a second container, and its outlet is connected to the second static distributor. The outlet of the alkaline pyrolysis tank is connected to the inlet of the second static distributor, and the outlet of the second static distributor is connected to a clarification tank. The outlet of the clarification tank is connected to a sample container. Both the alkaline pyrolysis tank and the clarification tank are double-layered tanks with a jacket.

2. The continuous flow controllable parameter plasmid extraction device according to claim 1, characterized in that: The power control module is equipped with a switch and a display screen.

3. The continuous flow controllable parameter plasmid extraction device according to claim 1, characterized in that: Both the alkali pyrolysis tank and the clarification tank are equipped with chiller interfaces on their interlayers.

4. The continuous flow controllable parameter plasmid extraction device according to claim 1, characterized in that: The alkaline pyrolysis tank is equipped with a temperature probe and a pH electrode, and an alkaline solution inlet is provided at the top of the alkaline pyrolysis tank.

5. The continuous flow controllable parameter plasmid extraction device according to claim 4, characterized in that: The temperature probe and the pH electrode are connected to the power control module.

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