Cell lysis buffer clarification and separation device for plasmid production
By designing a cell lysate clarification and separation device for plasmid production, the problem of low separation efficiency of cell debris and RNA residues in large-scale plasmid production is solved, and the effect of efficient separation and reduction of purification costs is achieved.
Patent Information
- Application Number
- CN202421691804.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-17
- Publication Date
- 2025-06-10
- Estimated Expiration
- 2034-07-17
AI Technical Summary
The prior art is difficult to effectively isolate cell debris and RNA residues after alkaline cleavage in large-scale plasmid production, resulting in low separation efficiency and high purification cost.
A cell lysate clarification and separation device for plasmid production is designed, including a phase separation container, an intake valve and an exhaust valve. By standing layering and gas blowing, the clarification layer in the cell lysate is efficiently separated.
It improves the separation efficiency of plasmids, reduces purification costs, effectively removes RNA residues, is suitable for large-scale plasmid production, and the final product meets the GMP standard.
Smart Images
Figure CN222961399U_ABST
Abstract
Description
Technical Field
[0001] The utility model belongs to the technical field of plasmid production, and relates to a clarification and separation device, in particular to a cell lysate clarification and separation device for plasmid production. Background Art
[0002] In recent years, pDNA (plasmid DNA) has attracted great interest in gene therapy, vaccine development, mRNA template preparation, etc. Its production process generally includes high-density fermentation after purification. The most commonly used pDNA purification process includes cell acquisition, alkaline lysis, cell debris removal, chromatography purification, and ultrafiltration. At a small scale, pDNA is relatively easy to produce. However, to expand the production scale, many technical obstacles need to be overcome. For example, cell debris after alkaline lysis can be easily removed by small-scale centrifugation, but it is not convenient to handle large volumes (such as 200 L), which will inevitably require extremely long processing times. In addition, too low centrifugation efficiency is not sufficient to clarify the cell lysate, which is a rate-limiting step in large-scale downstream purification. Therefore, those skilled in the art urgently need to develop relevant technologies for large-scale pDNA purification.
[0003] Currently, existing pDNA purification technologies include continuous flow centrifuge centrifugation, tangential flow filtration (TFF), and flotation separation methods based on CO 2 bubbles. Among them, continuous flow centrifuge centrifugation is difficult to achieve industrial-scale pDNA purification; for tangential flow filtration, cell debris easily clogs the TFF membrane, and the flux decreases to avoid high pressure and shear force, requiring a long operation time, and the separation efficiency is extremely low; based on CO 2 bubbles, the flotation separation method is relatively friendly to scale-up, so it has been widely used in industrial production, but it cannot effectively separate RNA residues, and there is still much room for improvement.
[0004] Therefore, how to provide a cell lysate clarification and separation device for plasmid production, realize large-scale production of plasmids, improve the separation efficiency while reducing the purification cost, and effectively separate RNA residues has become an urgent problem to be solved by those skilled in the art at present. Summary of the Utility Model
[0005] Aiming at the deficiencies of the existing technology, the purpose of the utility model is to provide a cell lysate clarification and separation device for plasmid production. The cell lysate clarification and separation device is particularly suitable for large-scale production of plasmids, improves the separation efficiency while reducing the purification cost, effectively separates RNA residues, and is conducive to large-scale popularization and application.
[0006] To achieve this purpose, the utility model adopts the following technical solutions:
[0007] The present utility model provides a cell lysate clarification and separation device for plasmid production. The cell lysate clarification and separation device includes a phase separation container, at least one air inlet valve disposed at the bottom of the phase separation container, and at least one liquid discharge valve disposed on the side wall of the phase separation container.
[0008] The phase separation container is used for static stratification of the cell lysate.
[0009] The air inlet valve is used to blow air at the bottom of the phase separation container.
[0010] The liquid discharge valve is used to discharge the clarified layer of the cell lysate after static stratification.
[0011] In the present utility model, the number of the air inlet valves is at least one, for example, it can be 1, 2, 3, 4 or 5, and further preferably 1; the number of the liquid discharge valves is at least one, for example, it can be 1, 2, 3, 4 or 5, and further preferably at least 3, but is not limited to the listed values, and other unlisted values within this value range are equally applicable.
[0012] In the present utility model, an air inlet valve is disposed at the bottom of the phase separation container, and a liquid discharge valve is disposed on the side wall, and it is used as a cell lysate clarification and separation device for plasmid production. The structure is simple and easy to process, suitable for large-scale production of plasmids, improving the separation efficiency while reducing the purification cost, and is conducive to large-scale popularization and application.
[0013] Preferably, the side wall of the phase separation container is made of a transparent material, or a transparent observation window is provided on the side wall of the phase separation container.
[0014] In the present utility model, the transparent material can be polymethyl methacrylate (PMMA), but is not limited thereto, as long as the distribution of the cell lysate in the container can be observed, so the specific type of the transparent material is not particularly limited herein.
[0015] Preferably, the shape of the phase separation container is any one of a cylindrical shape, a frustum shape, a prismatic shape or a frustum of a pyramid shape, and further preferably a cylindrical shape.
[0016] Preferably, the volume of the phase separation container is 5 - 300 L, for example, it can be 5 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 80 L, 100 L, 120 L, 140 L, 160 L, 180 L, 200 L, 220 L, 240 L, 260 L, 280 L or 300 L, but is not limited to the listed values, and other unlisted values within this value range are equally applicable.
[0017] Preferably, the top of the phase separation container is in an open form.
[0018] Preferably, a detachable isolation net is provided at the top of the phase separation container.
[0019] By providing a detachable isolation net at the top of the phase separation container, the present utility model can prevent impurities from falling into the cell lysate in the container during the static layering process, and at the same time, the detachable structure facilitates the addition of the solution.
[0020] Preferably, a support base is provided at the bottom of the phase separation container.
[0021] Preferably, the support base is equipped with fixable rollers to facilitate the flexible transfer and fixation of the container at any time.
[0022] Preferably, the intake valve is connected to an air compressor pump.
[0023] Preferably, the air compressor pump includes any one of a piston type compressor pump, a screw type compressor pump, a centrifugal type compressor pump or a sliding vane type compressor pump.
[0024] Using the cell lysate clarification and separation device provided by the present utility model for cell lysate clarification and separation includes the following steps:
[0025] (1) First, the cell lysate clarification and separation device is alkali-washed with a 0.1 - 1 mol / L sodium hydroxide solution for 0.5 - 2 h. After emptying, it is washed with purified water until the inner wall of the device reaches a neutral pH, and then the device is emptied;
[0026] (2) Pour the cell lysate into the phase separation container, add a 1 - 6 mol / L calcium chloride solution until the concentration of calcium chloride in the cell lysate reaches 0.5 - 1 mol / L, open the intake valve, and blow air at the bottom of the phase separation container for 2 - 4 min;
[0027] (3) After stopping blowing air, let the cell lysate stand and layer for 2 - 8 h;
[0028] (4) Open the drain valve, discharge the clarified layer of the cell lysate after standing and layering for clarified filtration treatment, and the filter element material used includes any one of polypropylene, polyethersulfone or glass fiber.
[0029] Using the cell lysate clarification and separation device provided by the present utility model in combination with phase separation treatment, finally, the cell debris and supernatant in the cell lysate are efficiently separated, the load capacity of subsequent clarified filtration is improved, the production scale of pDNA is significantly expanded, and the addition of calcium chloride solution fully removes RNA residues, improving the purification efficiency of pDNA. The final product meets GMP standards.
[0030] Compared with the prior art, the beneficial effects of the present utility model are:
[0031] (1) The present utility model provides an air inlet valve at the bottom of the phase separation container and a liquid discharge valve on the side wall, which is used as a cell lysate clarification and separation device for plasmid production. The structure is simple and easy to process, suitable for large-scale plasmid production, improving the separation efficiency while reducing the purification cost, and facilitating large-scale popularization and application.
[0032] (2) By using the cell lysate clarification and separation device provided by the present utility model in combination with phase separation treatment, cell debris and supernatant in the cell lysate are finally efficiently separated, improving the load capacity of subsequent clarification and filtration, significantly expanding the production scale of pDNA, and the addition of calcium chloride solution fully removes RNA residues, improving the purification efficiency of pDNA. The final product meets GMP standards. Description of the Drawings
[0033] Figure 1 It is a schematic structural diagram of the cell lysate clarification and separation device provided by the present utility model.
[0034] Wherein: 1 - phase separation container; 2 - air inlet valve; 3 - liquid discharge valve; 4 - support base. Detailed Embodiments
[0035] It should be understood that in the description of the present utility model, the orientation or positional relationships indicated by the terms "center", "longitudinal", "transverse", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationships shown in the drawings, and are only for the convenience of describing the present utility model and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation of the present utility model.
[0036] It should be noted that in the description of the present utility model, unless otherwise clearly specified and limited, the terms "set", "connected", "connected to" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present utility model can be understood according to specific circumstances.
[0037] The technical solution of the present utility model will be further described below with reference to the drawings and through specific embodiments.
[0038] Embodiment 1
[0039] This embodiment provides a cell lysate clarification and separation device for plasmid production, as Figure 1As shown in the figure, the cell lysate clarification and separation device includes a phase separation container 1, one air inlet valve 2 provided at the bottom of the phase separation container 1, and three liquid discharge valves 3 provided on the side wall of the phase separation container 1; the phase separation container 1 is used for static stratification of the cell lysate; the air inlet valve 2 is used to blow air into the bottom of the phase separation container 1; the liquid discharge valve 3 is used to discharge the clarified layer of the cell lysate after static stratification.
[0040] In this embodiment, the side wall of the phase separation container 1 is made of transparent polymethyl methacrylate, and the distribution of the cell lysate in the container can be observed; the shape of the phase separation container 1 is cylindrical, with a volume of 200 L, the top is in an open form, and a support seat 4 is provided at the bottom; the air inlet valve 2 is connected to a piston-type air compressor (not shown in the figure).
[0041] Example 2
[0042] This embodiment provides a cell lysate clarification and separation device for plasmid production. Except that the number of air inlet valves 2 is changed to 2 and the number of liquid discharge valves 3 is changed to 4, the rest of the structure and conditions are the same as those in Example 1, so they will not be elaborated here.
[0043] Example 3
[0044] This embodiment provides a cell lysate clarification and separation device for plasmid production. Except that the side wall of the phase separation container 1 is changed to opaque polyethylene material and a transparent observation window is provided on the side wall, and the transparent observation window is in a vertical long strip shape, the rest of the structure and conditions are the same as those in Example 1, so they will not be elaborated here.
[0045] Example 4
[0046] This embodiment provides a cell lysate clarification and separation device for plasmid production. Except that the shape of the phase separation container 1 is changed to an inverted frustum shape and the volume is 5 L, the rest of the structure and conditions are the same as those in Example 1, so they will not be elaborated here.
[0047] Example 5
[0048] This embodiment provides a cell lysate clarification and separation device for plasmid production. Except that the shape of the phase separation container 1 is changed to a triangular prism shape and the volume is 300 L, the rest of the structure and conditions are the same as those in Example 1, so they will not be elaborated here.
[0049] Example 6
[0050] This embodiment provides a cell lysate clarification and separation device for plasmid production. Except that a detachable isolation net is provided at the top of the phase separation container 1 and a fixed roller is installed on the support seat 4, the rest of the structure and conditions are the same as those in Example 1, so they will not be elaborated here.
[0051] Application Example 1
[0052] In this application example, the cell lysate clarification and separation device provided in Example 1 is used for cell lysate clarification and separation. The specific method includes the following steps:
[0053] (1) First, the cell lysate clarification and separation device is alkali-washed with 0.5 mol / L sodium hydroxide solution for 1 h. After emptying, it is washed with purified water until the inner wall of the device reaches pH neutral, and then the device is emptied;
[0054] (2) Pour the cell lysate into the phase separation container 1, add 3 mol / L calcium chloride solution until the calcium chloride concentration in the cell lysate reaches 0.8 mol / L. Open the air inlet valve 2 and blow air at the bottom of the phase separation container 1 for 3 min;
[0055] (3) After stopping blowing, let the cell lysate stand and separate into layers for 5 h;
[0056] (4) Open the drain valve 3 to discharge the clarified layer of the cell lysate after standing and separating for clarification and filtration treatment, and the filter element material used is polypropylene.
[0057] Application Example 2
[0058] In this application example, the cell lysate clarification and separation device provided in Example 2 is used for cell lysate clarification and separation. The specific method refers to Application Example 1, so it will not be elaborated here.
[0059] Application Example 3
[0060] In this application example, the cell lysate clarification and separation device provided in Example 3 is used for cell lysate clarification and separation. The specific method refers to Application Example 1, so it will not be elaborated here.
[0061] Application Example 4
[0062] In this application example, the cell lysate clarification and separation device provided in Example 4 is used for cell lysate clarification and separation. The specific method refers to Application Example 1, so it will not be elaborated here.
[0063] Application Example 5
[0064] In this application example, the cell lysate clarification and separation device provided in Example 5 is used for cell lysate clarification and separation. The specific method refers to Application Example 1, so it will not be elaborated here.
[0065] Application Example 6
[0066] In this application example, the cell lysate clarification and separation device provided in Example 6 is used for cell lysate clarification and separation. The specific method refers to Application Example 1, so it will not be elaborated here.
[0067] The results show that in Application Examples 1-6, a cell lysate clarification and separation device with a specific structure is adopted and combined with phase separation treatment. Finally, cell debris and supernatant in the cell lysate are efficiently separated, the load capacity of subsequent clarification filtration is improved, the production scale of pDNA is significantly expanded, and the addition of calcium chloride solution fully removes RNA residues, improving the purification efficiency of pDNA. The final product meets GMP standards.
[0068] Thus, it can be seen that the present utility model sets an air inlet valve at the bottom of the phase separation container and a liquid discharge valve on the side wall, and uses it as a cell lysate clarification and separation device for plasmid production. It has a simple structure, is easy to process, is suitable for large-scale plasmid production, improves the separation efficiency while reducing the purification cost, and is conducive to large-scale popularization and application.
[0069] The applicant declares that the above is only the specific implementation manner of the present utility model, but the protection scope of the present utility model is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present utility model fall within the protection scope and the disclosure scope of the present utility model.
Claims
1. A cell lysate clarification and separation device for plasmid production, characterized in that: The cell lysate clarification and separation device for plasmid production comprises a phase separation container, at least one air inlet valve arranged at the bottom of the phase separation container, and at least one liquid discharge valve arranged on the side wall of the phase separation container; The phase separation container is used for static stratification of the cell lysate; The air inlet valve is used to blow air toward the bottom of the phase separation container; The drain valve is used to discharge the clarified layer of the cell lysate after static stratification.
2. The cell lysate clarification and separation device for plasmid production according to claim 1, characterized in that: The side wall of the phase separation container is made of a transparent material, or the side wall of the phase separation container is provided with a transparent observation window.
3. The cell lysate clarification and separation device for plasmid production according to claim 1, characterized in that: The phase separation container is in any one of a cylindrical, truncated cone, prism or truncated cone shape.
4. The cell lysate clarification and separation device for plasmid production according to claim 1, characterized in that: The volume of the phase separation container is 5-300L.
5. The cell lysate clarification and separation device for plasmid production according to claim 1, characterized in that: The top of the phase separation container is in an open form.
6. The cell lysate clarification and separation device for plasmid production according to claim 5, characterized in that: A detachable isolation net is arranged on the top of the phase separation container.
7. The cell lysate clarification and separation device for plasmid production according to claim 1, characterized in that: A supporting seat is arranged at the bottom of the phase separation container.
8. The cell lysate clarification and separation device for plasmid production according to claim 7, characterized in that: The support seat is provided with a fixable roller.
9. The cell lysate clarification and separation device for plasmid production according to claim 1, characterized in that: The air inlet valve is connected to an air compression pump.
10. The cell lysate clarification and separation device for plasmid production according to claim 9, characterized in that: The air compression pump includes any one of a piston compression pump, a screw compression pump, a centrifugal compression pump or a vane compression pump.