WO2022237338A1 - Device for extracting plasmid dna in bacteria - Google Patents

Abstract

The present application relates to the technical field of biomedicines, and discloses a device for extracting plasmid DNA in bacteria. The device comprises a first mixing assembly and a second mixing assembly; the first mixing assembly and the second mixing assembly are connected by means of a cracking spiral tube; and at least one liquid inlet is provided on a connecting pipeline between the cracking spiral tube and the second mixing assembly. The present application has the advantages that: by means of series connection between the mixing assemblies, the cracking and neutralization process is in a closed environment, thereby reducing the probability of environmental pollution, facilitating the cleaning after use, achieving continuous processing, and improving the production efficiency; and the device is simple, and is low in design and production cost.

Description

Apparatus for extracting plasmid DNA from bacteria

Cross References to Related Applications

This disclosure claims the priority of the Chinese patent application with application number "CN 202110506985.5" entitled "Apparatus for Extracting Plasmid DNA from Bacteria" filed with the Chinese Patent Office on May 10, 2021, the entire contents of which are hereby incorporated by reference In this disclosure.

technical field

The disclosure relates to the technical field of biomedicine, in particular to a device for extracting plasmid DNA from bacteria.

Background technique

In recent years, due to the clinical success of gene therapy and DNA vaccines, the demand for industrial-scale plasmid fermentation production has become very urgent.

The current large-scale plasmid production technology mainly includes the following steps: vector construction, bacterial fermentation, cell lysis, solid-liquid separation and clarification, and plasmid purification. Although the current plasmid production process can produce plasmids that meet pharmaceutical quality standards and meet clinical requirements, there are still some difficult bottlenecks in these processes. For example, it is difficult to scale the output (kilogram level), the copy number and stability of the carrier, the DNA denaturation during the lysis process, the removal of HCD residues, the difficulty of solid-liquid separation, and the residue of endotoxin.

Plasmid DNA for biopharmaceuticals is mainly produced in Escherichia coli, and alkaline lysis is the most widely used method for preparing plasmid DNA. Cells are lysed under alkaline conditions, and chromosomal DNA undergoes irreversible denaturation at the same time, and plasmid DNA can be refolded when the pH returns to neutral to separate plasmids from chromosomal DNA. The first and most critical step in plasmid renaturation is cell lysis. How to completely lyse cells, completely co-precipitate chromosomal DNA, and remove most of the RNA has become the core issue of cell lysis. The main problems in the current industrial-scale plasmid extraction process are: 1. There is no automation equipment for alkaline lysis or the equipment capacity cannot meet the requirements of sufficient mixing; 2. A large amount of organic solvents and acids are used, which increases safety risks in large-scale industrial production. And it has high requirements for plant equipment; 3. After mixing with neutralizing liquid (solution Ⅲ), there is no automatic mixing equipment or mixing equipment can not meet the mixing requirements of uniform and low shear; 4. Solid-liquid separation production of neutralization reaction liquid High cost; 5. High host DNA residue; 6. Low RNA removal rate, which affects downstream purification. 7. It is difficult to clean reusable pipeline equipment, which is not conducive to CIP cleaning, and the cost of disposable consumable equipment is relatively high.

Chinese patent CN205710712U discloses a cracking device in plasmid extraction, including a frame, at least one accommodation chamber rotatably arranged on the frame, and the accommodation chamber has a cavity for placing at least one bottle filled with culture fluid. The driver is used to drive the storage compartment to rotate, and the controller to control the drive to drive the storage compartment to drive the bottle body to rotate at the required speed, direction and number of turns. The above-mentioned technical solution is a relatively traditional processing method, which belongs to laboratory production. Although the structure is relatively simple, the production speed is relatively slow and continuous operation cannot be performed.

Chinese patent CN111733060A discloses a plasmid DNA alkali lysis equipment, including bacteria liquid pipeline; The circulation pipeline is provided with a pump and a valve; an alkali lysate pre-distribution pipeline; a cell lysis reactor; a neutralization reactor; an acid pipeline; and a collection device. The above-mentioned technical device realizes continuous cracking, and cooperates with the corresponding filtering device, which can effectively improve the production level. However, the reaction device of this technical solution still needs to be ventilated for foaming and mixing, the structure of the device is complex, and the pipelines are arranged in a staggered manner, which is not conducive to pipeline cleaning.

Chinese patent CN111808716A discloses a plasmid extraction device, including a lysis container, a precipitation container, an eluent container, a collection container, and a chromatography column. The lysis container and the precipitation container are communicated through a first connecting pipe, and the precipitation container It communicates with the chromatographic column through the second connecting pipe, and the eluent container communicates with the chromatographic column through the third connecting pipe. The first connecting pipe, the second connecting pipe and the third connecting pipe are all A valve is provided, the collection container is arranged under the chromatographic column, and the chromatographic column is connected with a vibrating mechanism. The above-mentioned technical solution adopts a vibrating structure, and the processing process is discontinuous, so the processing efficiency needs to be further improved, and this method has the problem of high host DNA residue, which needs further improvement.

Although we have previously developed a method for lysing, neutralizing and extracting plasmid DNA in bacteria by mixing chamber vibrations in order to solve the defects in the current method for large-scale production of plasmid DNA (see: 200610114061.6, a continuous large-scale extraction of plasmid method), but it is not easy to amplify, and the efficiency of extracted plasmid DNA is low; in order to solve this problem, we have developed a device for extracting plasmid DNA through a bubble mixer (see: 202011120617.9, used to extract Plasmid DNA bubble generating device), which can make the bacteria liquid and lysate mix uniformly and fully, and the bubble mixing can effectively reduce the shear force and effectively improve the yield and quality. However, this device still has the problem that it is not easy to scale up. Customize bubble mixers of different sizes, and explore amplification conditions such as ventilation volume and flow rate.

Therefore, we have publicly designed a new extraction equipment and method for plasmid DNA. In addition to effectively controlling the shear force in the mixing process, it is easier to realize the scale-up process. Compared with the method of using a bubble mixer (bubble generating device), it can Effectively improve the mixing efficiency of the cracking and neutralization process and increase the yield; by adjusting the mixing parameters in an easy-to-control way, the shear force can be effectively controlled and the quality of the plasmid can be improved. Moreover, the device is simple in principle and can be precisely regulated. Therefore, shortening the time for exploring amplification conditions and further improving work efficiency will greatly promote the development of continuous and large-scale extraction of plasmid DNA, which is of great significance.

Contents of the invention

In view of this, the present disclosure provides a device for extracting plasmid DNA from bacteria, which can solve the above problems.

The present disclosure provides a device for extracting plasmid DNA in bacteria, comprising: a first mixing component and a second mixing component;

The first mixing component is connected to the second mixing component through the cracking helical tube; at least one liquid inlet is provided on the connecting pipeline between the cracking helical tube and the second mixing component;

After the resuspended bacteria solution flows into the first mixing component and mixes, it is lysed through the lysis spiral tube to obtain a lysate, and then passed into the second mixing component to neutralize with solution III to obtain a neutralization reaction solution, and the lysate is passed through The liquid inlet enters the second mixing component.

In some embodiments, the structure of the first mixing component and the second mixing component can be one of stirring type, emulsifying type and centrifugal type.

In some embodiments, the structure of the first mixing component is stirring or emulsifying or centrifugal; the second mixing component is of centrifugal structure; preferably, both the first mixing component and the second mixing component are Mixing pump or agitator; more preferably, the first mixing component and the second mixing component are respectively the first mixing pump and the second mixing pump, and the impellers of the first mixing pump and the second mixing pump are preferably half Closed impeller. By adopting the form of a mixing pump, the cracking and neutralization process is carried out in a closed environment, reducing the chance of polluting the environment, and it is convenient to carry out CIP and SIP after use.

In some embodiments, the impellers of the first mixing pump and the second mixing pump may include a back cover; a plurality of guide posts are evenly distributed on the back cover, and the guide posts are at least along the impeller. The outer surface in the direction of rotation is arc-shaped; the guide column is preferably a combination of one or more of a cylinder, a circular truncated column or a fan-shaped column.

In some embodiments, the diameter of the guide column is 0.5mm-40mm, preferably 2mm-10mm; the guide column is preferably a cylinder, or the cross-sectional area of the guide column is the largest in the middle, and the middle The cross-sectional area gradually decreases to both ends. Multiple diversion columns are evenly distributed, and the diameter is within an appropriate range, which can reduce shear force, prevent host DNA from contaminating the product, and enable lysis and neutralization to be automated.

In some embodiments, the cross-sectional area of the guide post is the largest in the middle, and the cross-sectional area gradually decreases from the middle to both ends. In specific implementation, the structure of the guide post may be spindle-shaped.

In some embodiments, the internal diameter of the cracking spiral tube is 0.5cm-15cm, preferably 0.5cm-9cm, and the diameter of the pump head of the first mixing pump and the second mixing pump can be 2cm-100cm, preferably 4cm -30cm; the ratio range of the pump cavity volume of the first mixing pump and the second mixing pump to the rated feed volume per minute of a single mixing pump can be 1:6-1:1, and the preferred ratio is 1:6- 1:3; or the volumes of the pump chambers of the first mixing pump and the second mixing pump are both the volume of the feed liquid flowing through the pump chamber for 10s-60s, preferably the volume of the feed liquid flowing through the pump chamber for 10s-20s.

In some embodiments, the length of the guide posts is related to the distribution position, the length of each guide post decreases from the center of the rear cover to the outer edge, and the vertices of each guide post are located on the same paraboloid superior.

In some embodiments, both the liquid inlets of the first mixing pump and the second mixing pump can be arranged coaxially with the liquid outlet; the liquid inlet is located at the center of the pump casing, and the liquid outlet is located at the pump casing. center of the seat. In this way, when the fluid enters the pump chamber, it needs to pass through the center of the rear cover plate to the edge, and then it can be discharged after going around to the rear, so that it can fully contact the guide column, achieve the purpose of uniform mixing, and improve the quality of neutralization reaction.

In some embodiments, the device further includes a filter assembly, the liquid outlet end of the second mixing assembly is connected to the liquid inlet end of the filter assembly, and the neutralization reaction liquid is filtered through the filter assembly.

In some embodiments, the resuspension liquid includes solution I and bacteria containing plasmid DNA, and the resuspension liquid is mixed and transported to the first mixing component by the first delivery pump, and transported to the first mixing component by the second delivery pump. After the solution II of the first mixing component is mixed, it is passed into the cracking spiral tube for cracking.

In some embodiments, the structure of the filter assembly is one or more combinations of screen type, depth filter type, and centrifugal filter type.

In some embodiments, the structure of the filter assembly is a screen mesh or depth filter structure; the filter pore size is 0.2 μm-800 μm, specifically 0.2 μm-200 μm; the filter material includes but is not limited to cellulose, diatomaceous earth, Activated charcoal, polypropylene fibers and silicone.

In some embodiments, the filter material includes, but is not limited to, one or more combinations of cellulose, diatomaceous earth, activated carbon, polypropylene fiber, silica gel, polyethersulfone, nylon, and polyvinylidene fluoride.

In some embodiments, the structure of the filter assembly is a centrifugal structure; the centrifugal force is 1000g-20000g, the centrifugation time is 2min-60min, and the temperature is 2°C-40°C.

The present disclosure also provides a method for extracting plasmid DNA in bacteria through the above-mentioned device, which realizes lysis and neutralization in the production process of the plasmid in two series-connected mixing components, specifically comprising the following steps:

Step S1: mixing;

Step S2: cracking;

Step S3: neutralization;

Wherein, step S1 is completed in the first mixing assembly, step S2 is completed in the pyrolysis spiral tube, step S3 is completed in the second mixing assembly, and the first mixing assembly, the cracking spiral tube and the second mixing assembly are sequentially connected in series.

In some embodiments, in step S2, lysis is performed for 2 min-10 min, preferably 5 min.

In some embodiments, the rotation speed of the first mixing component is 50rpm-1500rpm, preferably 200rpm-500rpm; the rotation speed of the second mixing component is 20rpm-1000rpm, preferably 150rpm-500rpm.

In some embodiments, the method for extracting plasmid DNA in bacteria specifically includes the following steps:

Step S1: After resuspending the bacterial cells with solution I, the resuspended bacterial liquid is obtained, and then the resuspended bacterial liquid and solution II are introduced into the first mixing component for mixing to obtain a bacterial cell mixed liquid;

Step S2: The bacterial cell mixture flows out from the first mixing component, enters the lysis spiral tube for lysis, and obtains a lysate after lysis;

Step S3: After the lysate and solution III enter the second mixing component, a neutralization reaction is carried out (or the lysate and solution III are premixed first, and then passed into the second mixing component) to obtain a neutralization reaction solution;

In a typical embodiment, after obtaining the neutralized reaction liquid, it further includes the steps of solid-liquid separation and purification.

in,

In step S1, the volume-to-mass ratio of the resuspended bacteria liquid to the bacteria is 3-20:1 (L:kg), more preferably 7:1 (L:kg).

In step S1, the volume ratio of solution I to solution II is 1:0.5-1:3, more preferably 1:1. Through different pipeline thickness and length, the alkali lysis time is controlled to 2min-10min to ensure the complete lysis of the bacteria and the lysis effect.

In step S1, the solution I includes Tris-HCl and EDTA-2Na, further preferably, the Tris-HCl concentration is 2mmol/L-100mmol/L, and the EDTA-2Na concentration is 0.1mmol/L-50mmol/L, Solution I had a pH range of 6.0-9.0.

In step S1, the solution II includes NaOH and SDS, further preferably, the concentration of NaOH is 0.02mol/L-5mol/L, and the concentration of SDS is 0.1%-10%.

In step S2, the lysis time is 2 min-10 min, more preferably 5 min.

In step S3, the solution III includes KAc and NH ₄ Ac, further preferably, the concentration of KAc is 0.1 mol/L-6 mol/L, and the concentration of NH ₄ Ac is 0.2 mol/L-10 mol/L.

In step S3, the volume ratio of the lysate to the solution III is 1:0.3-5, more preferably 1:1. The lysis and neutralization effects are controlled by the above conditions to ensure the precipitation of host DNA and the removal effect of host RNA.

The present disclosure has the following advantages:

This disclosure discloses the alkaline lysis and neutralization link in the plasmid production process, pioneeringly adopts the form of mixing components in series, and combines the delivery pump to make the lysis and neutralization process in a closed environment, reducing the chance of polluting the environment. It is convenient for CIP and SIP, and realizes continuous processing, which improves production efficiency and facilitates the expansion of production scale. Compared with the current mainstream production system of Airmix, it is easier to scale up and does not need to be scaled up. Customized bubble mixers of different sizes shortened the time to explore the amplification conditions and improved work efficiency;

Furthermore, the disclosed equipment is simple, easy to operate, and low in cost, does not require professional customization and expensive equipment, is easy to scale up in production, and has low production cost; The full mixing of the liquid can ensure the gentle mixing and neutralization of the neutralization liquid, avoiding the use of complex low-shear neutralization equipment, sufficient mixing and short mixing time during lysis, mild and uniform conditions during neutralization, and after lysis and neutralization, The host DNA and RNA residues are lower than the effect of the foaming mixer, and the product quality is good; at the same time, the size of the pump chamber is optimized, so that the time and shear force of lysis and neutralization are suitable for product production, and the proportion of supercoiled plasmid after lysis is higher , the host DNA and RNA residues are less; in addition, there is no need to use a complex multi-stage membrane filtration system, and there is no need for overnight precipitation after lysis. The equipment can be cleaned directly with CIP, which meets the production specifications of pharmaceutical production and saves Process time, reduce cost.

Further, by optimizing the size of the pump chamber and adjusting the ratio of the pump chamber and flow rate, the cracking neutralization time and shear force are suitable for product production, and at the same time, it is convenient to expand the production scale; the shape and size of the mixing pump head are optimized, Using 3D printing technology, the design and customization of the pump head can reduce the shear force under the premise of ensuring the mixing effect, prevent the host DNA from contaminating the product, and enable the lysis and neutralization to be automated.

In addition, no high-risk animal-derived ingredients are added during the preparation process, such as RNase, lysozyme, proteinase K, etc., and the production process does not use toxic organic solvents such as isopropanol, phenol, absolute ethanol, and other mutagens. The reagents can use general reagents or meet the pharmaceutical grade, do not use acid to neutralize, have low requirements on plant equipment, and are suitable for large-scale production.

Description of drawings

In order to illustrate the embodiments of the present disclosure more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only one or several embodiments of the present disclosure. Those of ordinary skill in the art can also obtain other drawings based on these drawings without any creative effort.

Fig. 1 is the schematic diagram of the device for extracting plasmid DNA in bacteria of the present disclosure;

Fig. 2 is a perspective view of the impeller of the first mixing assembly in Embodiment 1 of the present disclosure;

FIG. 3 is a perspective view of a second mixing component in Embodiment 1 of the present disclosure;

Figure 4 is an exploded view of the second mixing assembly in Figure 3;

Fig. 5 is a structural diagram of the second mixing assembly in Fig. 3 after the pump casing is removed;

Fig. 6 is a perspective view of the impeller of the second mixing assembly in Fig. 3;

Fig. 7 is a comparison chart of product electrophoresis results in Example 1 of the present disclosure, wherein, lane 1: Marker, lane 2: centrifuged supernatant, lane 3: control;

Fig. 8 is a side view of the impeller of the second mixing assembly in Embodiment 2 of the present disclosure;

Fig. 9 is a perspective view of a second mixing component in Embodiment 3 of the present disclosure;

10 is a perspective view of the second mixing component in Embodiment 3 of the present disclosure;

Figure 11 is a comparison chart of the electrophoresis results of Examples 1, 4, and 5 of the present disclosure, wherein, lane 1: the supernatant of the neutralization reaction solution of Example 5, lane 2: the supernatant of the neutralization reaction solution of Example 1, and lane 3 : the neutralization reaction liquid supernatant of embodiment 4, swimming lane 4: standard substance, swimming lane 5: Marker;

Fig. 12 is the electrophoresis result graph of comparative example 1 of the present disclosure, wherein, swimming lane 1: the neutralization reaction liquid supernatant of embodiment 1, swimming lane 2: the neutralization reaction liquid supernatant of comparative example 1, swimming lane 3: standard, swimming lane 4: Marker;

13 is a perspective view of the impeller of the second mixing assembly in Comparative Example 2 of the present disclosure;

14 is a perspective view of the impeller of the second mixing assembly in Comparative Example 3 of the present disclosure;

Figure 15 is a comparison chart of the electrophoresis results of Example 1 and Comparative Example 3 of the present disclosure, wherein, lane 1: the supernatant of the neutralization reaction solution of Example 1, lane 2: the supernatant of the neutralization reaction solution of Comparative Example 3, and lane 3: Standard, lane 4: Marker;

Fig. 16 is a perspective view of the diversion column in Embodiment 6 of the present disclosure; wherein, in Figs. 1-6, 9-10 and 16:

1-first mixing assembly; 2-second mixing assembly; 202-pump base; 203-sealing ring; 204-impeller; 205-pump casing; 2021-annular groove; 3-lysis spiral tube; 4-filter assembly; 5-resuspended bacteria solution; 6-solution II; 7-solution III; 201-spindle.

Detailed ways

The concept, specific structure and technical effects of the present disclosure will be clearly and completely described below in conjunction with the embodiments and drawings, so as to fully understand the purpose, scheme and effect of the present disclosure. It should be noted that, in the case of no conflict, features in the embodiments of the present application may be combined with each other.

It should also be noted that, in the description of the present disclosure, unless otherwise clearly stipulated and limited, the terms "setting", "installation", "connection", "connection" and the like should be interpreted in a broad sense, for example, it may be a fixed connection , can also be detachably connected, or integrally connected; can be mechanically connected, can also be electrically connected; can be directly connected, can also be indirectly connected through an intermediary, and can be internal communication between two components. Those skilled in the art can understand the specific meanings of the above terms in the present disclosure according to specific situations.

Unless otherwise specified, "lysing solution" herein refers to "solution II".

Unless otherwise specified, "neutralization solution" herein refers to "solution III".

The present disclosure will be further described below in conjunction with the accompanying drawings.

As shown in FIG. 1 , the disclosed device for extracting plasmid DNA from bacteria includes: a first mixing component 1 , a second mixing component 2 and a filter component 4 . The two mixing components are distinguished by function, the first mixing component 1 can be a cracking mixing component, and the second mixing component 2 is a neutralizing mixing component.

The first mixing component 1 and the second mixing component 2 are connected in series; a cracking spiral tube 3 is also connected in series between the two mixing components. Specifically, the solution I required for the lysis reaction is mixed with bacteria containing plasmid DNA to form a resuspended bacteria solution 5, and the flow rate and flow rate are controlled by the first delivery pump, and then delivered to the first mixing component 1, that is, the lysis mixing pump. During specific implementation, a first three-way joint (that is, a "Y" connector) is connected in series on the delivery pipeline, and the resuspended bacteria solution 5 and solution II 6 can be sent to the second delivery pump through the first delivery pump and the second delivery pump respectively. A three-way joint is passed into the first mixing component 1 for mixing to obtain a bacterial cell mixture.

The liquid outlet end of the first mixing assembly 1 is connected to the liquid inlet end of the cracking spiral tube 3 ; the liquid outlet end of the cracking spiral tube 3 is connected to the liquid inlet end of the second mixing assembly 2 . Wherein, a liquid inlet is provided on the pipeline between the cracking spiral tube 3 and the second mixing assembly 2, which is specifically the second three-way joint in the series connection pipeline, that is, a "Y"-shaped connector, and the second three-way joint One end is also connected to the container of solution III7 through the third delivery pump.

Preferably, the first mixing component 1 and the second mixing component 2 used in this embodiment can be agitators or mixing pumps, specifically the first mixing pumps and the second mixing pumps respectively, including but not limited to stirring pumps, emulsifying pumps , centrifugal pumps, etc., among which the stirring paddles of the stirring pump can be selected from paddle stirrer, pusher stirrer, turbine stirrer, anchor stirrer, frame stirrer, screw stirrer; the rotor and stator of the emulsification pump include But not limited to: coarse teeth, medium teeth, fine teeth. Through a pump head with a certain regular shape, the solution is fully mixed, and the shear force is low, which ensures that the chromosomal DNA does not undergo a large number of breaks, and can be carried out in a closed environment without pollution. Further because the first mixing assembly 1 is used for cracking reaction, preferably the first mixing assembly 1 structure is preferably one of agitation type or emulsification type or centrifugal type, specifically emulsification pump can be selected, and its blade structure can be as shown in Figure 2 ( Can also be the form of other emulsification pumps in the prior art, here only show it with Fig. 2); The second mixing assembly 2 is used for neutralization reaction, can choose centrifugal structure, as shown in Fig. 3,4, the second The second mixing assembly 2 mainly includes a main shaft 201 , a pump base 202 , a sealing ring 203 , an impeller 204 , and a pump casing 205 . The fluid delivery route is shown by the arrow in Figure 3. The fluid enters the pump through the liquid inlet end at the center of the pump casing 205. After centrifugal mixing, it can flow out through the liquid outlet end of the pump casing 205. The inner tube of the liquid outlet The path is tangent to the pump lumen. One end of the main shaft 201 is connected to the output end of the external motor, and the other end passes through the center of the pump base 202 through the sealing device, and is fixedly connected with the impeller 204. The contact area between the pump base 202 and the pump casing 205 is processed with an annular groove for installing the seal. Circle 203. The impeller 204 is preferably a semi-closed impeller; however, the traditional impeller has a big disadvantage, that is, the shear force is relatively large, so in this embodiment, the impeller 204 is designed as shown in Figures 5 and 6, including a rear cover 2041 . A plurality of guide columns 2042 are evenly distributed on the rear cover 2041 , a total of 32 pieces, which surround the center in three layers, and the guide columns 2042 are perpendicular to the surface of the rear cover 2041 . Further, in order to better reduce the generated shearing force, the shape of the guide column 2042 can be one or more combinations of cylinder, circular truncated or fan-shaped truncated, preferably cylindrical. The diameter of the guide post 2042 is in the range of 0.5mm-40mm; after testing, a better effect can be obtained when the diameter is preferably in the range of 2mm-10mm. By optimizing the design, the shear force can be reduced, the host DNA can be prevented from contaminating the product, and the lysis and neutralization can be automated. Through experiments, it can be seen that the second mixing component 2 can control the mixing effect and shear force of different scales by setting a certain speed range, combined with the first mixing component 1, it can realize the automatic cracking and neutralization of different scales of bacterial liquid, so as to realize continuous , large-scale production.

In a specific implementation, the structure of the first mixing component 1 may also preferably be the same as that of the second mixing component 1 .

Specifically, when the impeller speed of the second mixing assembly 2 is 20rpm-1000rpm, a better mixing effect is produced. It is also possible to change the pump head properties, size, and rotating speed for the second mixing assembly 2 to control the neutralization effect; the pump head diameter of the second mixing pump is 2cm-100cm, preferably 4cm-30cm, and the rotating speed is controlled at 20rpm-1000rpm, preferably 150rpm-500rpm, the ratio of the volume of the pump chamber to the rated feed volume per minute of the mixing pump is in the range of 1:6-1:1, preferably 1:6-1:3; or the volume of the pump chamber is designed so that the feed liquid flows through the pump The volume of 10s-60s in the cavity is preferably the volume of 10s-20s when the feed liquid flows through the pump cavity; it ensures complete neutralization and produces low shear force, reduces the breakage of chromosomal DNA, and improves the quality of plasmid DNA.

The liquid outlet end of the second mixing assembly 2 is connected to the liquid inlet end of the filter assembly 4 .

Preferably, the structure of the filter assembly 4 is one or more combinations of screen type, depth filter type and centrifugal filter type. Specifically, the structure of the filter assembly 4 in this embodiment is a deep filter structure; the filter pore size is 0.2 μm-800 μm; the optional filter pore size is between 0.1 μm-200 μm. Secondary clarification of the neutralized supernatant is carried out by means of deep filtration, and the filtered material components include but not limited to cellulose, diatomaceous earth, activated carbon, polypropylene fiber, silica gel and their combination products. The envelope area of the deep filter membrane is between 0.01m ² -2m ² .

The working process is as follows:

Step S1: Solution Ⅰ and bacteria containing plasmid DNA are premixed to obtain a resuspended bacteria solution. The resuspended bacteria solution 5 and solution II 6 can be pumped to the first three-way joint through the first delivery pump for pre-preparation in the pipeline. Mixing, and then entering the first mixing component 1 for stirring and mixing to obtain a bacterial cell mixture;

Step S2: Stir and mix the bacterial cell mixture into the lysis spiral tube 3 for lysis reaction, and then flow out to obtain the lysis solution;

Step S3: After the lysate flowing out of the lysis spiral tube 3 is pre-mixed with solution III7, it is then transported to the second mixing component 2 through the second three-way joint (or the lysate flowing out of the lysis spiral tube 3 flows through the second delivery pump The solution III is delivered to the second three-way joint, and then passed into the second mixing component 2), a neutralization reaction occurs, and the neutralized reaction solution after neutralization is obtained;

Step S4: After the neutralized neutralization reaction liquid flows out, it enters the filter assembly 4 for filtration, and performs solid-liquid separation and purification.

in,

In step S1, the volume-to-mass ratio of the resuspended bacteria liquid to the bacteria is 3-20:1 (v:m), more preferably 7:1 (v:m).

In step S1, the volume ratio of solution I to solution II is 1:0.5-1:3, more preferably 1:1.

In step S1, solution I includes Tris-HCl and EDTA-2Na, further preferably, Tris-HCl concentration is 2mmol/L-100mmol/L, EDTA-2Na concentration is 0.1mmol/L-50mmol/L, the pH of solution I The range is 6.0-9.0.

In step S1, the solution II includes NaOH and SDS, further preferably, the concentration of NaOH is 0.02mol/L-5mol/L, and the concentration of SDS is 0.1%-10%.

In step S2, the lysis time is 2 min-10 min, more preferably 5 min.

In step S3, solution III includes KAc and NH ₄ Ac, further preferably, the concentration of KAc is 0.1 mol/L-6 mol/L, and the concentration of NH ₄ Ac is 0.2 mol/L-10 mol/L.

In step S3, the volume ratio of the lysate flowing out of the lysis spiral tube 3 to the solution III is 1:0.3-5, more preferably 1:1. The lysis and neutralization effects are controlled by the above conditions to ensure the precipitation of chromosomal DNA and the removal of host RNA.

In step S4, the solid-liquid separation method includes but is not limited to one or more combinations of filtration, depth filtration, centrifugation and other methods. Preferably, when filtering is selected, the filtering material includes but not limited to one or more of cellulose, diatomaceous earth, activated carbon, polypropylene fiber and silica gel, and the filter pore size is between 0.2 μm and 800 μm; preferably, when deep filtration is selected , the depth filter material includes but not limited to one or more of cellulose, diatomaceous earth, activated carbon, polypropylene fiber and silica gel, the depth filter pore size is 0.1μm-200μm, and the depth filter membrane package area is 0.01m ² -2m ² ; Preferably, when selecting centrifugation, the centrifugation method includes but is not limited to the selection of a desktop centrifuge, a tube centrifuge or a disc centrifuge, the centrifugal force is 1000g-20000g, the centrifugation time is 2min-60min, and the centrifugation temperature is 2°C-40°C .

The extraction device in the above-mentioned embodiments can be used in the following examples, if there is any difference, it will be shown specifically, and the specific method for extracting plasmid DNA from bacteria can be as follows:

Embodiment 1: 50L fermentation scale processing example

In the device for extracting plasmid DNA from bacteria in Example 1, the diameter of the pump head is 10 cm, the impellers of the pump heads of the two pumps are shown in Figure 6, and the diameter of the diversion column is 5 mm.

(1) Escherichia coli (E.coli) containing plasmid A was fermented at a high density. The OD ₆₀₀ measured by a spectrophotometer was 84.2, and 23.3 L of the fermentation broth was centrifuged to harvest 3684 g of bacterial cells with a wet weight of 15.8%. 3684g of cells were resuspended in a pH 8.0 resuspension (Solution I) composed of 25mM Tris-HCl and 10mM EDTA-2Na to obtain a resuspension liquid, the volume of which was 25.8L after resuspension (bacteria and resuspension The volume-to-volume ratio is 1:7).

(2) Pump the resuspended bacteria to the lysis mixing pump at a speed of 140ml/min, and at the same time pump the lysis solution (solution II) composed of 0.2M NaOH and 1% SDS to the lysis mixing pump at a speed of 140ml/min Pump (first mixing component). Adjust the rotational speed of the lysis mixing pump to 200rpm, start lysis and mixing, and obtain the bacterial cell mixture. Among them, the pump chamber volume of the cracking mixing pump is 1:3 in ratio to the rated feed volume per minute of a single mixing pump.

(3) After being pumped out from the lysis mixing pump, the bacterium mixed solution enters the lysis spiral tube 3, which has an inner diameter of 1.9 cm and a length of 5 m. The lysis time in the lysis spiral tube 3 is 5 minutes to obtain a lysate.

(4) The lysate after lysis enters the neutralization mixing pump (second mixing component), and the other end of the neutralization mixing pump is composed of 1MKAc and 7M NH ₄ Ac solution III (pre-cooled at 2-8°C) at 280ml/min The speed enters, and the speed of the neutralization mixing pump is set to 250rpm, wherein the diameter of the guide column on the neutralization mixing pump impeller is 5mm, the shape of the guide column is a cylinder, and the diameter of the pump head of the neutralization mixing pump is 8.5cm; wherein, The pump chamber volume of the neutralizing mixing pump is 1:4 in ratio to the rated feed volume per minute of the single mixing pump.

(5) After the neutralization is completed, the neutralization reaction solution is collected and entered into the filter assembly 4, and the centrifugal filtration method is selected, and the supernatant is collected by centrifugation for 20 minutes with a centrifugal force of 8000g for the next step of purification.

Result detection:

The plasmid concentration measured in the resuspension solution was 545mg/L (measured by QIAGEN's plasmid mini-extraction kit), and the total amount of plasmid was 14.06g.

After neutralization, 100L of neutralization reaction solution was obtained, and 82L of supernatant was obtained after centrifugation, and the plasmid concentration recorded by HPLC quantitative method was 121.8mg/L (HPLC model: Waters 2695; chromatographic column model: TOSOH, Tskgel DNA-NPR 4.6mm *7.5cm2.5μm, the HPLC measurement conditions in the following examples are the same). The cracking yield was 71%.

The electrophoresis diagram is shown in Figure 7. From Figure 7, it can be seen that the purity of the plasmid in the supernatant lysed by the device and method of the present application is relatively high, and the RNA and host DNA are less.

The plasmid DNA prepared by the above method was detected by HPLC test and pharmacopoeia method, and the results showed that the plasmid was the target plasmid, and the purity was high, the supercoiled ratio was greater than 95%, and the ring-opening ratio was less.

Example 2

On the basis of Embodiment 1, the length of the guide column 2042 in this embodiment is related to the distribution position, the length of each guide column 2042 is gradually decreased from the center of the rear cover 2041 to the outer edge, and each guide column 2042 The vertices of are all located on the same paraboloid, as shown by the dotted line in Figure 8. Correspondingly, the inner surface of the pump housing 205 is also designed as a paraboloid, which is matched with the guide post 2042 .

The structure of the filter assembly 4 is a centrifugal structure, which can be selected as a desktop, tube or disc centrifuge connected in series; the centrifugal force is 1000g-20000g, the centrifugation time is 2min-60min, and the temperature is 2°C-40°C.

Example 3

On the basis of Embodiment 1, the liquid inlet and liquid outlet of the second mixing assembly 2 in this embodiment are arranged coaxially. As shown in Figures 9 and 10, the liquid inlet is arranged at the center of the pump casing 205, and The liquid outlet is not located on the peripheral side of the pump casing 205. In order to further increase the mixing effect and avoid the situation where the liquid inlet and outlet are adjacent to each other and cause uneven mixing, the liquid outlet is set at the center of the pump base 202, that is, the rear cover At the rear of the plate 2041, an annular groove 2021 is processed around the rotating shaft, and the bottom of the groove or the side opening of the groove can be used as a liquid outlet, so that the fluid entering the pump chamber needs to pass through the center to the edge of the rear cover plate 2041, and then go around to the rear. It is discharged through the annular groove 2021, so that it can fully contact the guide column 2042, achieve the purpose of uniform mixing, and improve the quality of neutralization reaction.

Example 4

The difference from Example 1 is that the rotation speed of the first mixing pump is 400 rpm, the rotation speed of the second mixing pump is 500 rpm, the diameter of the cracking spiral tube is 1.9 cm; the diameter of the pump head of the second mixing pump is 10 cm. The guide post is a cylinder with a diameter of 1mm. Everything else is the same.

Then the neutralized reaction solution after neutralization is carried out to agarose nucleic acid electrophoresis, the test results are as shown in Figure 11, and it is found that the host DNA and RNA in the lysed supernatant in Example 4 are higher than the lysed supernatant in Example 1, proving that the rotation speed When it is high, there will be more impurities.

Example 5

Different from Example 1, the bacterial suspension is 2.5L, the rotating speed of the first mixing pump is 100rpm, the rotating speed of the second mixing pump is 50rpm, and the diameter of the cracking spiral tube is 1.9cm; the pump head of the second mixing pump The diameter is 10cm. The guide post is a cylinder with a diameter of 1mm. Everything else is the same.

Then obtain neutralization reaction solution 10L after neutralization, centrifuge to obtain supernatant 7.8L altogether, record supernatant plasmid concentration and be 96mg/L (HPLC measures), cleavage yield 55.0%, electrophoresis test result is shown in Fig. 11 As shown, it can be seen from FIG. 11 that when the rotating speeds of the first mixing pump and the second mixing pump are low, the mixing and neutralization are insufficient, and the yield of plasmid DNA is lower than that of Example 1.

Example 6

Different from Embodiment 1, the diversion column 2042 in this embodiment is designed with a variable cross-section. The purpose is to further reduce the influence of shearing on the neutralization process. Through fluid motion analysis, as shown by the arrow in Figure 16, a single During the rotation of the root guide column, the flow velocity distribution of the fluid relative to the main body; that is, it decreases from the middle layer to both sides. The reason is that the upper and lower sides of the fluid are respectively subjected to the viscous resistance of the pump casing, that is, the pump seat, and the velocity is distributed in a gradient. In order to maintain a relatively consistent shear force generated by a single diversion column on the genetic material in the fluid, it is designed as a variable cross-section structure. Specifically, taking the cylinder in Example 1 as an example, the cross-section of a single guide column increases first and then decreases from the side of the pump casing to the side of the pump seat, forming a "spindle-shaped" structure, see Figure 16 for details. In the above design, although the relative velocity at the center of the diversion column 2042 is relatively high and the impact is strong, combined with the large radius of curvature and force-bearing area, it can effectively reduce the shearing effect on the plasmid and increase the yield of the plasmid to a certain extent.

Example 7

Same as in Example 1, the result of detecting residual host DNA (HCD) was 5.87 μg/mg (E. coli residual DNA detection kit). As shown in Table 1, the plasmid DNA was detected by HPLC test and Pharmacopoeia method, and the results showed that the plasmid was the target plasmid, and the purity was high, the supercoiled ratio was 95.92%, and the ring-opening ratio was less.

Table 1 is sample plasmid and purity detection HPLC peak result table in embodiment 5

sample name		Impurities 1,%	open loop, %	Supercoil, %	linear, %		Impurity 2,%
After centrifugation	ND	1.15	95.92	ND	2.93

Note: "ND" means not detected. Impurity 1 and Impurity 2 are unknown states of the plasmid.

Example 8

Same as in Example 4, the result of detecting residual host DNA (HCD) was 18.7 μg/mg (E. coli residual DNA detection kit).

Example 9

The difference from Example 1 is that in this example, after the lysis and neutralization, the solid-liquid separation is carried out by filter bag filtration and depth filtration. The purpose is to increase the processing capacity and increase the production efficiency in the enlarged production process, and at the same time reduce the mechanical shearing effect of the continuous centrifuge in production, reduce the generation of impurities and the destruction of the target plasmid. Use polypropylene filter bags with a filter area of 0.5m ² and a pore size of 100μm and 200μm for primary filtration, and then use a filter bag with a filter pore size of 0.2-2μm and a composite material of cellulose and inorganic filter aid for secondary filtration. level filtering. The impurity removal rate after primary filtration can reach 86.2%, the turbidity before and after filtration is 43NTU and 10.7NTU respectively, and the purity of the plasmid has not changed significantly after filtration. After secondary filtration, the filtrate is clearer and the turbidity can be reduced to below 3NTU, which can be directly purified downstream. As shown in Table 2, the plasmid DNA was detected by HPLC, and the results showed that the plasmid was the target plasmid, and the purity was high. Before filtration, the supercoiled ratio reached 96.08%. After filtration with a 100 μm filter bag, the supercoiled ratio was 97.6%. After filtration with a 200 μm filter bag Finally, the supercoil ratio is 97.55%.

Table 2 shows the results of HPLC detection of plasmid purity in the supernatant before and after filtration with 100 μm and 200 μm filter bags

sample name		Impurities 1,%	open loop, %	Supercoil, %	linear, %		Impurity 2,%
Before filtering	0.59	0.74	96.08	ND	2.59
After 100μm filtration	ND	0.28	97.60	ND	2.12
After 200μm filtration	ND	0.34	97.55	ND	2.11

Note: "ND" means not detected. The samples were centrifuged before HPLC injection, and the supernatant was taken for injection.

Comparative example 1

Different from Example 1, the neutralization step of Comparative Example 1 does not use a pump, but is carried out in a bubble mixer, and the specific steps are as follows:

(1) Escherichia coli containing plasmid A is fermented at a high density, and the OD600 measured by a spectrophotometer is 78.9. 23.5 L of the fermented liquid was taken and centrifuged to harvest 3603 g of bacterial cells with a wet weight of 15.3%. The cell resuspension of 3603g is in the pH 8.0 resuspension liquid (solution I) that is made of 25mM Tris-HCl and 10mM EDTA-2Na, obtains resuspension liquid, and volume is 25.2L (thalline and solution I mass volume The ratio is 1:7).

(2) The resuspended bacteria liquid is pumped to one side of the "Y" shape connector at a speed of 140ml/min, and the lysis solution (solution II) composed of 0.2M NaOH and 1% SDS is pumped at a speed of 140ml/min Speed pumps to the other side of the "Y" connector. Connect the "Y" connector to the lysis mixing pump (the first mixing component), adjust the rotation speed to 200 rpm, and start lysis and mixing to obtain a bacterial cell mixture. Wherein, the volume ratio of solution I to solution II is 1:1.

(3) After being pumped out from the lysis mixing pump, the bacterial cell mixture enters the lysis helical tube. The inner diameter of the lysis helical tube is 1.9 cm, the length is 5 m, and the lysis time in the lysis helical tube is 5 minutes to obtain the lysate.

(4) The lysed lysate enters another "Y"-shaped connector, and the solution III (pre-cooled at 2-8°C) composed of 1M KAc and 7M NH ₄ Ac at the other end of the connector enters at a speed of 280ml/min. Enter the bubble mixer through the "Y" connector, and set the compressed air flow rate of the bubble mixer to 1.2L/min. The volume ratio of lysate and solution III is 1:1.

(5) After the neutralization is completed, the neutralization reaction solution is collected, centrifuged at 8000 g for 20 min, and the comparison supernatant is collected for further purification.

Detected by the microplate reader method, the results showed that the plasmid concentration measured in the resuspended bacteria liquid was 570mg/L (calculated by extracting with the plasmid mini-extraction kit), and the total amount of the plasmid was 14.36g.

After neutralization, 101 L of neutralization reaction solution was obtained, and a total of 79.3 L of supernatant was obtained by centrifugation. The comparison supernatant plasmid concentration was measured to be 116.3 mg/L (as determined by HPLC), and the cleavage yield was 64.2%. The results of electrophoresis are shown in Figure 12. It can be seen from Figure 12 that the plasmid DNA obtained by using the bubble generator method in the neutralization process of this comparative example is compared with the plasmid DNA obtained by the method in Example 1. The plasmid concentration is equivalent, but the host RNA More, indicating that the preparation method of the present application is better, and it is easier to scale up and easy to operate.

Comparative example 2

The difference from Example 1 is that the centrifugal pump head impeller used in the second mixing pump in Comparative Example 2 is shown in Figure 13, and the rest are the same.

80L of neutralization reaction solution was obtained after neutralization by a microplate reader, and a total of 66L of supernatant was obtained by centrifugation. The plasmid concentration of the supernatant was measured to be 106.6mg/L (as determined by HPLC), and the cleavage yield was 64.5%. Cracking yield is lower than embodiment 1.

Comparative example 3

The difference from Example 1 is that the impeller of the centrifugal pump head used in the second mixing pump in Comparative Example 3 is shown in FIG. 14 . The rest of the settings are the same.

Compared with the results of Example 1, the test electropherogram is shown in Figure 15. It can be seen from the figure that the content of host DNA and RNA in the lysed supernatant of the pump head in this comparative example is relatively high, which is not conducive to plasmid purification.

Based on the results of the above examples, it can be seen that the extraction device of the present disclosure fully mixes the final product when it is lysed and the mixing time is short, and the conditions are mild and uniform when neutralized. Effect, the product quality is better, and when the speed is moderate, the extracted plasmid DNA has less impurities and high yield.

In the alkaline lysis and neutralization link in the plasmid production process, this disclosure pioneered the use of a series of mixing components in the form of series, combined with a delivery pump to make the lysis and neutralization process in a closed environment, reducing the chance of polluting the environment, and convenient after use. Carry out CIP and SIP, and achieve continuous processing, improve production efficiency, and facilitate the expansion of production scale. Compared with the current mainstream bubble mixer Airmix production system, it is easier to scale up and does not need to be customized according to scale Bubble mixers of different sizes shorten the time to explore the amplification conditions and improve work efficiency; the equipment is simple, easy to operate, and low in cost. It does not require professional customization and expensive equipment. It is easy to enlarge in production and low in production costs. ; The two mixing components used can not only fully mix the bacterial solution and lysate, but also ensure the neutralization solution (solution Ⅲ) is mixed and neutralized gently, avoiding the use of complex low-shear neutralization equipment, and fully mixed during lysis And the mixing time is short, the conditions are mild and uniform during neutralization, after lysis and neutralization, the host DNA and RNA residues are lower than the effect of the foaming mixer, and the product quality is good; at the same time, the size of the pump chamber is optimized, so that the lysis and neutralization The time and shearing force are suitable for product production, the proportion of supercoiled plasmid after lysis is high, and the host DNA and RNA are less residual; in addition, there is no need to use complex multi-stage membrane filtration systems, and no steps such as overnight precipitation after lysis , The equipment can be cleaned directly with CIP, which conforms to the production specifications of pharmaceutical production, and at the same time saves process time and reduces costs.

Further, by optimizing the size of the mixing pump chamber and adjusting the ratio of the pump chamber and flow rate, the cracking and neutralization time and shear force are suitable for product production, and at the same time facilitate the expansion of the production scale; optimize the shape and size of the mixing pump head , using 3D printing technology to design and customize the pump head. Under the premise of ensuring the mixing effect, the shear force is reduced, the host DNA is prevented from contaminating the product, and the lysis and neutralization can be automated.

In addition, no high-risk animal-derived ingredients are added during the preparation process, such as RNase, lysozyme, proteinase K, etc., and the production process does not use toxic organic solvents such as isopropanol, phenol, absolute ethanol, and other mutagens. The reagents can use general reagents or meet the pharmaceutical grade, do not use acid to neutralize, have low requirements on plant equipment, and are suitable for large-scale production.

The above specific embodiments are only used to illustrate the technical solutions of the present disclosure and not to limit them. Although the present disclosure has been described in detail with reference to examples, those skilled in the art should understand that the disclosed technical solutions can be modified or equivalently replaced without Any deviation from the spirit and scope of the technical solutions of the present disclosure shall be included in the scope of the claims of the present disclosure.

Claims (10)

1. The device for extracting plasmid DNA in bacteria is characterized in that it includes a first mixing assembly and a second mixing assembly; the first mixing assembly is connected to the second mixing assembly through a cracking spiral tube; the cracking spiral tube At least one liquid inlet is provided on the connecting pipeline with the second mixing component; after the resuspended bacteria liquid flows into the first mixing component and mixes, it is lysed by the lysis spiral tube to obtain a lysate, and then passed into the second mixing component. The second mixing component is neutralized with solution III to obtain a neutralized reaction solution, and the lysate enters the second mixing component through the liquid inlet.
2. The device for extracting plasmid DNA in bacteria according to claim 1, wherein the structure of the first mixing assembly and the second mixing assembly is one of stirring, emulsifying, and centrifugal, preferably Preferably, the first mixing component and the second mixing component are both mixing pumps or agitators, and the first mixing component and the second mixing component may be preferably the first mixing pump and the second mixing pump respectively.
3. The device for extracting plasmid DNA in bacteria according to claim 2, wherein the impellers of the first mixing pump and the second mixing pump all include a back cover; A guide column; the outer surface of the guide column at least along the rotation direction of the impeller is arranged on an arc surface.
4. The device for extracting plasmid DNA in bacteria according to claim 3, wherein the diversion column is a combination of one or more of cylinders, circular truncated columns or fan-shaped columns.
5. The device for extracting plasmid DNA in bacteria according to claim 3, wherein the maximum cross-sectional width of the guide column is 0.5mm-40mm, preferably 2mm-10mm; the guide column is preferably It is a cylinder, or the cross-sectional area of the guide post is the largest in the middle, and the cross-sectional area of the guide post gradually decreases from the middle to both ends.
6. The device for extracting plasmid DNA in bacteria according to claim 3, wherein the internal diameter of the cracking helical tube is 0.5cm-15cm, preferably 0.5cm-9cm; the first mixing pump and the second mixing The diameter of the pump head of the pump is 2cm-100cm, preferably 4cm-30cm; the ratio range of the pump cavity volume of the first mixing pump and the second mixing pump to the rated feed volume per minute of a single mixing pump is 1:6 -1:1, the preferred ratio is 1:6-1:3; or the pump chamber volumes of the first mixing pump and the second mixing pump are designed to be the volume of the feed liquid flowing through the pump chamber for 10s-60s, preferably It is the volume of the feed liquid flowing through the pump cavity for 10s-20s.
7. According to the device for extracting plasmid DNA in bacteria described in claim 3, it is characterized in that the liquid inlet ends of the first mixing pump and the second mixing pump are coaxially arranged with the liquid outlet; the liquid inlet The outlet end is located at the center of the pump casing, and the outlet end is located at the center of the pump base.
8. The device for extracting plasmid DNA in bacteria according to any one of claims 1 to 7, wherein the device also includes a filter assembly, and the liquid outlet of the second mixing assembly is connected to the filter The liquid inlet end of the component, the neutralization reaction liquid is filtered through the filter component.
9. The device for extracting plasmid DNA in bacteria according to claim 8, wherein the resuspended bacteria liquid includes solution I and bacteria containing plasmid DNA, and the resuspended bacteria liquid is mixed by the first delivery pump It is sent to the first mixing component, mixed with the solution II delivered to the first mixing component by the second delivery pump, and then passed into the cracking spiral tube for cracking.
10. The device for extracting plasmid DNA in bacteria according to claim 8, wherein the filter assembly structure is one or more combinations of screen mesh, depth filter, centrifugal filter; preferably, The structure of the filter assembly is a screen mesh or deep filter structure; the filter aperture is 0.2 μm-800 μm; the filter material is cellulose, diatomaceous earth, activated carbon, polypropylene fiber or silica gel.

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