WO2022237337A1 - 用于提取细菌中质粒dna的方法 - Google Patents

用于提取细菌中质粒dna的方法 Download PDF

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WO2022237337A1
WO2022237337A1 PCT/CN2022/082698 CN2022082698W WO2022237337A1 WO 2022237337 A1 WO2022237337 A1 WO 2022237337A1 CN 2022082698 W CN2022082698 W CN 2022082698W WO 2022237337 A1 WO2022237337 A1 WO 2022237337A1
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mixing
pump
plasmid dna
assembly
lysis
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PCT/CN2022/082698
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English (en)
French (fr)
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杨鸿凯
吴宗圣
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艾棣维欣(苏州)生物制药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F29/00—Mixers with rotating receptacles
    • B01F29/25—Mixers with rotating receptacles with material flowing continuously through the receptacles from inlet to discharge
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/40—Mixers using gas or liquid agitation, e.g. with air supply tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/33—Disintegrators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/08—Apparatus for tissue disaggregation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the disclosure belongs to the technical field of biopharmaceuticals, and in particular relates to a method for extracting plasmid DNA from bacteria.
  • Gene therapy is to introduce foreign genes into target cells, express genes in patient cells to treat or prevent diseases, and its ultimate goal is to cure genetic and acquired diseases by adding, correcting or replacing genes.
  • gene therapy vectors There are two main types of gene therapy vectors to achieve these goals, namely viral vectors based on inactivated viruses and non-viral vectors based on plasmid DNA.
  • DNA vaccine also known as nucleic acid vaccine or gene vaccine, refers to the direct injection of a recombinant eukaryotic expression vector encoding a certain protein antigen into the body, so that the foreign gene is expressed in vivo, and the antigen produced activates the immune system of the body, thereby inducing specific Sexual humoral and cellular immune responses.
  • DNA vaccine is called the "third generation vaccine" after inactivated vaccine, attenuated vaccine and subunit vaccine, and has broad development prospects, and plasmid is a common carrier of DNA vaccine. Therefore, the large-scale production technology of plasmids is very important for the development of gene therapy and DNA vaccines.
  • 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.
  • 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 used in biopharmaceuticals is mainly produced in Escherichia coli.
  • 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.
  • Plasmid DNA at pH The principle of refoldability when neutrality is restored, separating the plasmid from the chromosomal DNA.
  • the first and most critical step in plasmid preparation is cell lysis. How to completely lyse cells, completely co-precipitate chromosomal DNA, and remove most of the RNA becomes the core issue of cell lysis.
  • the main problems in the current industrial-scale plasmid extraction process are: 1.
  • 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.
  • the present disclosure provides a method for extracting plasmid DNA from bacteria, which requires simple equipment, convenient operation, and low cost, and does not require professional customized and expensive equipment. A large amount of impurities in the cell lysis process can be removed, the ingredients are safe, and automatic continuous lysis can be realized, which is beneficial to industrial production.
  • the present disclosure provides a method for extracting plasmid DNA in bacteria, which realizes lysis and neutralization in the process of plasmid production in two series-connected mixing components, specifically comprising the following steps:
  • step (1) is completed in the first mixing assembly
  • step (2) is completed in the cracking spiral tube
  • step (3) is completed in the second mixing assembly, the first mixing assembly, the cracking spiral tube and the second mixing assembly sequentially connected in series.
  • 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.
  • step (2) lysis is performed for 2 min-10 min, preferably 5 min.
  • the structures of the first mixing component and the second mixing component are each independently selected from any one of stirring type, emulsifying type, and centrifugal type, and the first mixing component and the second mixing component are both It is preferably a mixing pump or an agitator; preferably, the first mixing component is stirring or emulsifying or centrifugal, and the second mixing component is centrifugal.
  • the first mixing assembly and the second mixing assembly are respectively a first mixing pump and a second mixing pump, and the pump chamber volumes of the first mixing pump and the second mixing pump are equal to the rated volume of a single mixing pump per
  • the ratio range of the minute feed volume is 1:6-1:1, preferably 1:6-1:3; or the pump cavity volumes of the first mixing pump and the second mixing pump are both feed liquid flowing through the pump
  • the volume in the chamber for 10s-60s is preferably the volume of the feed liquid flowing through the pump chamber for 10s-20s.
  • the impellers of the first mixing pump and the second mixing pump are preferably semi-closed impellers.
  • the internal diameter of the cracking spiral tube is 0.5cm-15cm, preferably 0.5cm-6cm; the pump head diameters of the first mixing pump and the second mixing pump are both 2cm-100cm, preferably 4cm-6cm. 30cm.
  • the impellers of the first mixing pump and the second mixing pump both include a back cover; a plurality of guide posts are evenly distributed on the back cover, and the guide posts rotate at least along the impeller
  • the outer surface of the direction is provided as an arc surface.
  • the guide column is one or more combinations of a cylinder, a circular truncated column, or a fan-shaped column.
  • the cross-sectional width of the guide column is 0.5mm-40mm, preferably 2mm-10mm.
  • the guide post is preferably a cylinder, or the cross-sectional area of the guide post is the largest in the middle, and the cross-sectional area gradually becomes smaller from the middle to both ends.
  • the structure may be spindle-shaped.
  • the plasmid preparation process specifically includes the following steps:
  • the bacterium mixed liquid flows out from the first mixing component, enters the cracking spiral tube for cracking, and obtains the lysate;
  • the step of solid-liquid separation and purification is also included.
  • 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).
  • step (1) the volume ratio of solution I to solution II is 1:0.5-1:3, more preferably 1:1.
  • the alkali lysis time is controlled to 2min-10min to ensure the complete lysis of the bacteria and the lysis effect.
  • 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 L, the pH range of solution I is 6.0-9.0.
  • the solution II includes NaOH and SDS, further preferably, the concentration of NaOH is 0.02-5mol/L, and the concentration of SDS is 0.1-10%.
  • step (2) the lysis time is 2min-10min, more preferably 5min.
  • 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.
  • step (3) the volume ratio of the lysate to 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 solid-liquid separation is performed through a filter assembly, and the solid-liquid separation method includes but not limited to one or more combinations of filtration, depth filtration, centrifugation and the like.
  • the solid-liquid separation is carried out through a filter assembly, and the structure of the filter assembly is one or more combinations of screen type, depth filter type, and centrifugal filter type; further preferably, the filter assembly is a sieve Mesh or deep filter structure; filter pore size is 0.2 ⁇ m-800 ⁇ m, preferably 0.1 ⁇ m-200 ⁇ m; filter material includes cellulose, diatomaceous earth, activated carbon, polypropylene fiber and silica gel.
  • 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.
  • 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 device for extracting plasmid DNA in bacteria by the above method, 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;
  • 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.
  • the internal diameter of the cracking spiral tube is 0.5 cm to 15 cm, preferably 0.5 cm to 6 cm; the diameter of the pump head of the first mixing pump and the second mixing pump can be 2 cm to 100 cm, preferably 4 cm ⁇ 30cm.
  • 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.
  • 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 at the center of the pump seat.
  • 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.
  • 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.
  • This disclosure innovatively adopts the form of mixing components (which can be pumps) in the alkali lysis and neutralization link in the plasmid production process, so that the lysis and neutralization process is in a closed environment, reducing the chance of polluting the environment, It is convenient to carry out CIP and SIP after use, and realizes continuous processing, improves production efficiency, and is low in cost, does not require professional customization and expensive equipment, is easy to scale up in production, and has low production cost; fully mixed during cracking 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 shear force are suitable for product production, and it is also convenient to expand the production scale. Compared with the production system of the current mainstream bubble mixer Airmix, it is easier to scale up, and there is no need to customize different sizes of bubble mixers according
  • the equipment used in the extraction method of the plasmid DNA is simple and easy to operate.
  • the two mixing components used can not only fully mix the bacterial solution and the lysate, but also ensure the gentle mixing and neutralization of the neutralizing solution, avoiding complicated use.
  • Advanced low-shear neutralization equipment the proportion of supercoiled plasmid after lysis is high, and host DNA and RNA residues are less; in addition, using a complex multi-stage membrane filtration system, there is no need for overnight precipitation and other steps after lysis, and the equipment can Cleaning directly with CIP is in line with the production specifications of pharmaceutical production, and at the same time saves process time and reduces costs; does not use complex multi-stage membrane filtration systems, and does not require steps such as overnight precipitation after lysis, and the proportion of supercoiled plasmids after lysis is relatively high High, host DNA and RNA residues are less, the equipment can be directly cleaned by CIP, which meets the production specifications of pharmaceutical production, saves process time, reduces costs, and is easy to
  • the cracking neutralization time and shear force are suitable for product production, and it is also convenient for the expansion of the production scale; the properties and dimensions of the mixing pump head are adjusted.
  • Optimizing, using 3D printing technology, designing and customizing 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.
  • 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 component of the device in the embodiment of the present disclosure
  • Fig. 3 is a perspective view of the second mixing component of the device in the embodiment of the present disclosure, and the arrow indicates the flow direction of the feed liquid;
  • 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 the electrophoresis results of Example 1, wherein, swimming Lane 1 is Marker, swimming Lane 2 is the supernatant of the lysis neutralization reaction solution of Example 1, and swimming Lane 3 is a standard;
  • Fig. 8 is the comparison chart of the electrophoresis results of Comparative Example 1, wherein, swimming lane 1 is the supernatant of the neutralization reaction solution of Example 1, swimming lane 2 is the supernatant of the neutralization reaction solution of Comparative Example 1, swimming lane 3 is the standard product, and swimming lane 4 is the supernatant of the neutralization reaction solution of Comparative Example 1. Marker;
  • Fig. 9 is the schematic diagram of the used impeller of comparative example 2.
  • Fig. 10 is the schematic diagram of the used impeller of comparative example 3.
  • Fig. 11 is a comparison chart of the electrophoresis results of Comparative Example 3, wherein, swimming Lane 1 is the supernatant of the neutralization reaction solution of Example 1, swimming Lane 2 is the supernatant of the neutralization reaction solution of Comparative Example 3, swimming Lane 3 is a standard, and swimming Lane 4 is Marker;
  • Figure 12 is a comparison chart of the electrophoresis results of Examples 1, 2, and 3, wherein, swimming Lane 1 is the supernatant of the neutralization reaction solution of Example 3, swimming Lane 2 is the supernatant of the neutralization reaction solution of Example 1, and swimming Lane 3 is the supernatant of the neutralization reaction solution of Example 1.
  • the supernatant of the neutralization reaction solution of 2 is the standard, and the lane 5 is the Marker;
  • Fig. 13 is a schematic diagram of the three-dimensional structure of the diversion column in embodiment 4; among the above-mentioned Figs. 1-6:
  • 1-first mixing assembly 2-second mixing assembly; 201-main shaft; 202-pump seat; 203-sealing ring; 204-impeller; 205-pump casing; Diversion column; 3-cleavage spiral tube; 4-filter assembly; 5-resuspended bacteria solution; 6-solution II; 7-solution III.
  • neutralization solution herein refers to "solution III”.
  • FIG. 1 it mainly includes: a first mixing assembly 1 , a second mixing assembly 2 and a filter assembly 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.
  • the solution I is mixed with bacteria containing plasmid DNA to form a resuspended bacteria solution 5, and the flow and velocity are controlled by the first delivery pump, and then delivered to the first mixing component 1, that is, the lysis mixing pump.
  • a first three-way connector i.e. a "Y" connector
  • the resuspended bacteria liquid and solution II6 are sent to the first three-way connection through the first delivery pump and the second delivery pump respectively.
  • 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 .
  • a liquid inlet is provided on the pipeline between the cracking spiral tube 3 and the second mixing assembly 2, specifically the second three-way joint in the series connection pipeline, and one end of the second three-way joint is also passed through the third delivery pump Connect the container of solution III7.
  • 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 pump and the second mixing pump, including but not limited to stirring pumps, emulsifying pumps and centrifugal pumps Etc., wherein the agitator of the agitation pump can be selected from paddle agitator, pusher agitator, turbine agitator, anchor agitator, frame agitator, screw agitator; the rotor and stator of the emulsification pump include but are not limited to : Coarse teeth, medium teeth, fine teeth.
  • the first mixing assembly 1 is used for the cracking reaction, preferably the first mixing assembly 1 structure is selected as one of stirring type or emulsifying type, specifically an emulsifying pump can be selected, and its blade (or impeller) structure is as shown in Figure 2 ( It can also be in the form of other emulsification pumps in the prior art, which are only shown in Figure 2 here);
  • the second mixing assembly 2 is used for the neutralization reaction, and a centrifugal structure can be selected.
  • 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 .
  • 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.
  • the shear force can be reduced, the host DNA can be prevented from contaminating the product, and the lysis and neutralization can be automated.
  • 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.
  • the structure of the first mixing component 1 is also preferably the same as that of the second mixing component 1 .
  • 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 properties, size and rotating speed of the pump head for the second mixing component 2 to control the neutralization effect; the diameter of the pump head 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 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 to ensure complete neutralization and generate low shear force, reduce the breakage of chromosomal DNA, and improve 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 .
  • the structure of the filter assembly 4 is one or more combinations of screen type, depth filter type and centrifugal filter type.
  • the structure of the filter assembly 4 in this embodiment is a deep filter structure; the filter pore size is 0.2 ⁇ m-800 ⁇ m; the optional filter pore size is between 0.1 ⁇ m-200 ⁇ m.
  • Secondary clarification of the neutralized supernatant is carried out by 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 .
  • Embodiment 1 50L fermentation scale processing
  • the diameter of the pump head is 10 cm
  • the impellers of the pump heads of the two pumps are shown in Figure 6
  • the diameter of the diversion column is 5 mm.
  • the high-density fermented bacterium liquid of Escherichia coli containing the plasmid A, the OD600 measured by a spectrophotometer is 84.2, 23.3 L of the fermented liquid is taken and centrifuged, and 3684 g of bacterial cells are harvested, with a wet weight of 15.8%.
  • the cell resuspension of 3684g is in the pH 8.0 resuspension (solution I) that is made of 25mM Tris-HCl and 10mM EDTA-2Na, obtains resuspension liquid, and volume is 25.8L (thalline and solution I mass volume The ratio is 1:7 (kg:L)).
  • the bacterial cell mixture is pumped out from the first mixing assembly (lysis mixing pump), it 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 min. lysate.
  • the lysed lysate enters another "Y"-shaped connector, and the other end of the connector is composed of 1M KAc and 7M NH4Ac solution III (pre-cooled at 2-8°C) at a speed of 280ml/min, through the " The Y" connector goes to the neutralizing mixing pump (second mixing assembly) which is set at 250 rpm.
  • the volume ratio of lysate and solution III is 1:1.
  • the diameter of the diversion column on the impeller of the neutralization mixing pump is 5mm
  • the shape of the diversion column is a cylinder
  • the diameter of the pump head of the neutralization mixing pump is 8.5cm; wherein, the volume of the pump cavity of the neutralization mixing pump is equal to that of a single
  • the mixing pump is rated for a ratio of 1:4 feed volume per minute.
  • the plasmid concentration measured in the resuspended bacteria liquid was 545 mg/L (measured by QIAGEN plasmid mini-extraction kit), and the total amount of plasmid was 14.06 g.
  • 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.
  • Embodiment 1 The difference from Embodiment 1 is that the rotational speed of the first mixing pump is 400 rpm, and the rotational speed of the second mixing pump is 500 rpm.
  • the guide post is a cylinder with a diameter of 1mm. Everything else is the same.
  • Example 1 The difference from Example 1 is that the bacteria suspension is 2.5 L, the rotation speed of the first mixing pump is 100 rpm, the rotation speed of the second mixing pump is 50 rpm, and the diversion column is a cylinder with a diameter of 1 mm. Everything else is the same.
  • the electrophoretic graph is shown in Figure 12. It can be seen from Figure 12 that when the speed of the mixing pump is low, the mixing and neutralization will be insufficient, and the yield of plasmid DNA is lower than that of Example 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 13, 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.
  • 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 13 for details.
  • the above-mentioned design although the relative velocity at the center of the diversion column is relatively high and the impact is strong, but combined with the large radius of curvature and the force-bearing area, it can effectively reduce the shearing effect on the plasmid and increase the yield of the plasmid to a certain extent.
  • HCD residual host DNA
  • E. coli residual DNA detection kit E. coli residual DNA detection kit.
  • 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
  • Impurity 1 and Impurity 2 are unknown states of the plasmid.
  • HCD residual host DNA
  • Example 2 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.
  • 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.
  • the filtrate is clearer and the turbidity can be reduced to below 3NTU, which can be directly purified downstream.
  • 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.
  • the supercoiled ratio reached 96.08%, and after filtration with a 100 ⁇ m filter bag, the supercoiled ratio was 97.6%. 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 ⁇ m and 200 ⁇ m filter bags
  • 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:
  • 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 (solution I) that is made of 25mM Tris-HCl and 10mM EDTA-2Na, obtains resuspension liquid, and volume is 25.2L (thalline and the mass volume of solution I) The ratio is 1:7).
  • the bacterial cell mixture 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.
  • the lysed lysate enters another "Y" 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 was 1:1.
  • the results showed that the plasmid concentration measured in the resuspended bacteria liquid was 570mg/L (calculated by QIAGEN's plasmid mini-extraction kit), and the total amount of the plasmid was 14.36g.
  • Embodiment 1 The difference from Embodiment 1 is that the impeller of the centrifugal pump head used in the second mixing pump in this embodiment is shown in FIG. 9 , and the rest are the same.
  • Embodiment 1 The difference from Embodiment 1 is that the centrifugal pump head impeller used in the second mixing pump in this embodiment is shown in Figure 10, and the rest of the settings are the same.
  • the electrophoresis graph is shown in Figure 11. It can be seen from Figure 11 that compared with the results in Example 1, using the pump head shown in Figure 10, the content of host DNA and RNA in the lysed supernatant is more, which is not conducive to plasmid purification.
  • the extraction device and extraction method of the present disclosure the final product is fully mixed and the mixing time is short when lysed, and the conditions are mild and uniform when neutralized. After lysed and neutralized, the host DNA and RNA residues are lower than the foaming The effect of the mixer, the product quality is better, and the extracted plasmid DNA has less impurities and high yield when the speed is moderate.
  • the present disclosure innovatively adopts the form of a mixing component (which can be a pump), so that the lysis and neutralization process can be carried out in a closed environment, reducing the chance of polluting the environment and making it convenient after use.
  • a mixing component which can be a pump
  • the equipment used in this method is simple and easy to operate.
  • the two mixing components used can not only fully mix the bacterial solution and the lysate, but also ensure the gentle mixing and neutralization of the neutralizing solution, avoiding the use of complex low-shear Cut neutralization equipment, the proportion of supercoiled plasmid after lysis is higher, and the host DNA and RNA residues are less CIP cleaning is in line with the production specifications of pharmaceutical production, and at the same time saves process time and reduces costs; does not use complex multi-stage membrane filtration systems, and does not require steps such as overnight precipitation after lysis, and the proportion of supercoiled plasmids after lysis is high.
  • the time and shear force of cracking and neutralization are suitable for product production, and it is also convenient for the expansion of 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.

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Abstract

提供了一种用于提取细菌中质粒DNA的方法，在两个串联的混合组件中实现质粒生产过程中的裂解和中和，包括以下步骤：(1)混合，(2)裂解，(3)中和；其中，步骤(1)在第一混合组件中完成，步骤(2)在裂解螺旋管中完成，步骤(3)在第二混合组件中完成，第一混合组件、裂解螺旋管和第二混合组件依次串联。该质粒制备工艺中的设备简单，操作方便，成本低廉，不需要专业的定制化和价格高昂的设备，可去除细胞裂解过程中的大量杂质，成分安全，实现自动化连续裂解，利于工业化生产。

Description

用于提取细菌中质粒DNA的方法
相关申请的交叉引用
本公开要求于2021年5月10日提交中国专利局的申请号为“CN 202110505674.7”名称为“用于提取细菌中质粒DNA的方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于生物制药技术领域，具体涉及一种用于提取细菌中质粒DNA的方法。
背景技术
近年来，由于基因治疗和DNA疫苗临床上的成功，人们对工业化规模的质粒发酵生产需求已经非常迫切。基因治疗是将外源基因导入靶细胞，在患者细胞中表达基因来治疗或预防疾病，其最终目标是通过添加、纠正或替换基因来治愈遗传和后天疾病。实现这些目标的基因治疗载体主要有两种，即以灭活病毒为基础的病毒载体和以质粒DNA为基础的非病毒载体。DNA疫苗又称核酸疫苗或基因疫苗，是指将编码某种蛋白质抗原的重组真核表达载体直接注射到体内，使外源基因在活体内表达，产生的抗原激活机体的免疫系统，从而诱导特异性的体液免疫和细胞免疫应答。DNA疫苗被称为继灭活疫苗、弱毒疫苗和亚单位疫苗之后的“第三代疫苗”，具有广阔的发展前景，而质粒为DNA疫苗的常用载体。所以质粒的大规模生产技术对于基因治疗和DNA疫苗的发展至关重要。
当前规模化质粒的生产技术主要包括以下几道工序：载体构建、细菌发酵、菌体裂解、固液分离及澄清、质粒纯化。虽然目前的质粒生产工艺可以生产出符合药学质量标准的质粒，满足临床要求，但在这些工艺中还存在一些难以克服的瓶颈。如产量规模化(千克级)较难，载体拷贝数、稳定性问题，裂解过程中导致DNA变性、HCD残留去除，固液分离较难，内毒素残留等难题。
用于生物制药的质粒DNA主要在大肠杆菌中生产，碱裂解法是一种应用最为广泛的制备质粒DNA的方法，利用碱性条件将细胞裂解，同时染色体DNA发生不可逆变性，而质粒DNA在pH恢复中性时可复性的原理，将质粒与染色体DNA分离。质粒制备的第一步也是最关键的一步是细胞裂解，如何将细胞彻底 裂解，染色体DNA完全共沉淀，去除大部分的RNA成为细胞裂解的核心问题。目前工业规模的质粒提取工艺存在的主要问题有：1、碱裂解无自动化设备或设备能力满足不了充分混合的要求；2、使用大量有机溶剂及酸液，这在大规模工业化生产中增加安全风险及对厂房设备要求较高；3、使用中和液(溶液Ⅲ)混合后，无自动化的混合设备或混合设备满足不了均匀低剪切的混合要求；4、中和混合液固液分离生产成本较高；5、宿主DNA残留较高；6、RNA去除率较低，影响下游纯化；7、重复用管路设备清洗困难，不利于CIP清洗，而一次性耗材设备成本较高。
中国专利CN111808716A公开了一种质粒提取装置，包括裂解容器、沉淀容器、洗脱液容器、收集容器和层析柱，所述裂解容器与沉淀容器之间通过第一连接管连通，所述沉淀容器与层析柱之间通过第二连接管连通，所述洗脱液容器与层析柱之间通过第三连接管连通，所述第一连接管、第二连接管和第三连接管上均设置有阀门，所述收集容器设置在层析柱的下方，所述层析柱连接有振动机构。上述技术方案采取了振动式结构，加工过程不连续，需要进一步提升加工效率，并且该种方式存在宿主DNA残留较高的问题，还需要进一步改进。
虽然我们为了解决目前大规模生产质粒DNA的方法存在的缺陷，先前研发了一种通过混合腔震荡的方式进行裂解、中和提取细菌中质粒DNA的方法(参见：200610114061.6，一种连续大量提取质粒的方法)，但是其不容易放大，提取的质粒DNA的效率较低；为解决这一问题，我们又研发了通过气泡混合器提取质粒DNA的装置(参见：202011120617.9，用于提取细菌中质粒DNA的气泡发生装置)，其使菌液与裂解液能均一且充分混合，气泡混合有效降低剪切力，有效提高收率与质量，但是其仍存在不太容易放大的问题，需要根据规模定制不同大小的气泡混合器，摸索通气量，流速等放大条件。
因此，我们又公开设计了一种新的质粒DNA的提取方法，除了能有效控制混合过程剪切力，更容易实现放大工艺，相比通过气泡混合器(气泡发生装置)的方式，能有效提高裂解及中和过程的混合效率，增加收率；通过易控地调节混合参数，能有效控制剪切力，提高质粒质量。并且此工艺所用的装置原理简单，可精准调控，因此缩短放大条件摸索的时间，进一步提高工作效率，会大大促进连续地、大规模提取质粒DNA相关研究的发展，具有重要意义。
公开内容
本公开针对现有技术中存在的问题，提供一种用于提取细菌中质粒DNA的方法，其所需设备简单，操作方便，且成本低廉，不需要专业的定制化、价格高昂的设备，就可以去除细胞裂解过程中的大量杂质，成分安全，可以实现自动化连续裂解，有利于工业化生产。
一方面本公开提供一种用于提取细菌中质粒DNA的方法，在两个串联的混合组件中实现质粒生产过程中的裂解和中和，具体包括以下步骤：
(1)混合；
(2)裂解；
(3)中和；
其中，步骤(1)在第一混合组件中完成，步骤(2)在裂解螺旋管中完成，步骤(3)在第二混合组件中完成，第一混合组件、裂解螺旋管和第二混合组件依次串联。优选地，所述第一混合组件的转速为50rpm-1500rpm，优选为200rpm-500rpm；所述第二混合组件的转速为20rpm-1000rpm，优选为150rpm-500rpm。
在一些实施方式中，在步骤(2)中，裂解2min-10min，优选5min。
在一些实施方式中，所述第一混合组件和第二混合组件的结构各自独立地选自搅拌式、乳化式、离心式中的任一种，所述第一混合组件和第二混合组件均优选为混合泵或搅拌器；优选地，所述第一混合组件为搅拌式或乳化式或离心式，所述第二混合组件为离心式。
在一些实施方式中，所述第一混合组件和第二混合组件分别为第一混合泵和第二混合泵，所述第一混合泵和第二混合泵的泵腔体积与单个混合泵额定每分钟进料体积的比值范围均为1：6-1:1，优选为1:6-1:3；或所述第一混合泵和第二混合泵的泵腔体积均为料液流经泵腔内10s-60s的体积，优选为料液流经泵腔内10s-20s的体积。所述第一混合泵和第二混合泵的叶轮均优选为半闭式叶轮。通过采用混合泵的形式，使得裂解和中和过程在密闭的环境中，降低污染环境的几率，使用后方便进行CIP和SIP。
在一些实施方式中，所述裂解螺旋管内径为0.5cm-15cm，优选为0.5cm-6cm；所述第一混合泵和第二混合泵的泵头直径均为2cm-100cm，优选为4cm-30cm。
在一些实施方式中，所述第一混合泵和第二混合泵的叶轮均包括后盖板；所 述后盖板上均匀分布有多个导流柱，所述导流柱上至少沿叶轮旋转方向的外侧面呈弧面设置。
在一些实施方式中，所述导流柱为圆柱、圆台或扇形柱的一种或多种组合。
在典型的实施方式中，所述导流柱的横截面宽度为0.5mm-40mm，优选为2mm-10mm。通过导流柱多个均匀分布，且直径在合适的范围内，能够减小剪切力，防止宿主DNA污染产品，使得裂解中和可以自动化进行。
在典型的实施方式中，所述导流柱优选为圆柱，或所述导流柱的截面积中间最大，且由中间至两端的截面积逐渐变小，具体实施时，所述导流柱的结构可为纺锤形。
在一些实施方式中，所述质粒制备工艺具体包括以下步骤：
(1)用溶液I将菌体重悬后，得到重悬菌液，再将重悬菌液、溶液II导入第一混合组件混合，得到菌体混合液；
(2)所述菌体混合液从第一混合组件中流出，进入裂解螺旋管裂解，得到裂解液；
(3)所述裂解液与溶液III进入第二混合组件后，进行中和反应(或所述裂解液与溶液III混合后，再通入第二混合组件)，中和完成后得到中和反应液；
优选地，得到中和反应液后，还包括将其进行固液分离和纯化的步骤。
其中，
步骤(1)中，重悬菌液与菌体的体积质量比为3-20:1(L:kg)，进一步优选为7:1(L:kg)。
步骤(1)中，溶液I与溶液II的体积比为1:0.5-1:3，进一步优选为1:1。通过不同的管路粗细和长度，控制碱裂解时间为2min-10min，保证菌体裂解完全及裂解效果。
步骤(1)中，所述溶液I包括Tris-HCl和EDTA-2Na，进一步优选地，所述Tris-HCl浓度为2mmol/L-100mmol/L，EDTA-2Na浓度为0.1mmol/L-50mmol/L，溶液I的pH范围为6.0-9.0。
步骤(1)中，所述溶液II包括NaOH和SDS，进一步优选地，所述NaOH浓度为0.02-5mol/L，SDS浓度为0.1-10％。
步骤(2)中，所述裂解的时间为2min-10min，进一步优选为5min。
步骤(3)中，所述溶液III包括KAc和NH 4Ac，进一步优选地，所述KAc浓度为0.1mol/L-6mol/L，NH 4Ac浓度为0.2mol/L-10mol/L。
步骤(3)中，裂解液与溶液III的体积比为1:0.3-5，进一步优选为1:1。通过上述条件来控制裂解、中和效果，保证宿主DNA的沉淀和宿主RNA去除效果。
在一些实施方式中，通过过滤组件进行固液分离，所述固液分离方式包括但不限于过滤、深层过滤、离心等方式中的一种或多种组合。
在一些实施方式中，通过过滤组件进行固液分离，所述过滤组件结构为筛网式、深层过滤式、离心过滤式中的一种或多种组合；进一步优选地，所述过滤组件为筛网式或深层过滤式结构；过滤孔径为0.2μm-800μm，优选0.1μm-200μm；过滤材质包括纤维素、硅藻土、活性炭、聚丙烯纤维和硅胶。
在一些实施方式中，过滤材质包括但不限于纤维素、硅藻土、活性炭、聚丙烯纤维、硅胶、聚醚砜、尼龙、聚偏氟乙烯的一种或多种组合。
在一些实施方式中，所述过滤组件结构为离心式结构；离心力为1000g～20000g，离心时间为2min～60min，温度为2℃～40℃。
另外一方面，本公开还提供一种通过上述方法用于提取细菌中质粒DNA的装置，包括：第一混合组件和第二混合组件；
所述第一混合组件与所述第二混合组件通过所述裂解螺旋管相连接；所述裂解螺旋管与所述第二混合组件的连接管路上设有至少一个进液口；
重悬菌液流入所述第一混合组件混合后，通过所述裂解螺旋管裂解得到裂解液，再通入所述第二混合组件与溶液Ⅲ中和得到中和反应液，所述裂解液通过所述进液口进入所述第二混合组件。
在一些实施方式中，所述裂解螺旋管内径为0.5cm～15cm，优选为0.5cm～6cm；所述第一混合泵和第二混合泵的泵头直径均可为2cm～100cm，优选为4cm～30cm。
在一些实施方式中，所述导流柱的长度与分布位置关联，各导流柱的长度由所述后盖板的中心处向外缘依次递减，且各导流柱的顶点均位于同一抛物面上。
在一些实施方式中，所述第一混合泵和第二混合泵的进液端均可与出液端同轴设置；所述进液端位于泵壳的中心处，所述出液端位于所述泵座的中心处。这 样流体进入泵腔内需沿后盖板中心至边缘的顺序经过，绕至后方才可排出，使其充分接触导流柱，达到均匀混合的目的，提升中和反应质量。
在一些实施方式中，所述装置还包括过滤组件，所述第二混合组件的出液端连接至所述过滤组件的进液端，所述中和反应液通过所述过滤组件过滤。
在一些实施方式中，所述重悬菌液包括溶液Ⅰ和含有质粒DNA的菌体，所述重悬菌液通过第一输送泵混合输送至第一混合组件，与通过第二输送泵输送至第一混合组件的溶液Ⅱ混合后通入裂解螺旋管裂解。
和现有技术相比，本公开的有益效果是：
(1)本公开在质粒生产过程中的碱裂解和中和环节创新性的采用的混合组件(可为泵)的形式，使得裂解和中和过程在密闭的环境中，降低污染环境的几率，使用后方便进行CIP和SIP，且实现了连续的加工，提升了生产效率，且成本低廉，不需要专业的定制化、价格高昂的设备，易于在生产中放大，生产成本低；裂解时混合充分且混合时间较短，中和时条件温和均一，裂解中和后，宿主DNA和RNA残留均低于起泡混合器的效果，产品质量好；同时优化了泵腔的大小，使得裂解中和的时间和剪切力适合产品生产，同时也方便生产规模的放大，相较于目前主流的气泡混合器Airmix的生产体系来讲，比较容易放大，不需要根据规模定制不同大小的气泡混合器，缩短了放大条件摸索的时间，提高了工作效率。
(2)该质粒DNA的提取方法所用到的设备简单，操作方便，使用的两台混合组件既可以使菌液和裂解液充分混合又可以保证和中和液温和地混合中和，避免使用复杂的低剪切中和设备，裂解后的质粒超螺旋比例较高，宿主DNA、RNA残留较少；此外，使用复杂的多级的膜过滤系统，裂解后也不需要过夜沉淀等步骤，设备可直接用CIP清洗，符合药物生产的生产规范，同时节省了工艺时间，降低成本；不使用复杂的多级的膜过滤系统，裂解后也不需要过夜沉淀等步骤，裂解后的质粒超螺旋比例较高，宿主DNA、RNA残留较少，设备可直接用CIP清洗，符合药物生产的生产规范，同时节省了工艺时间，降低成本，操作方便，不需要专业的定制化、价格高昂的设备，易于在生产中放大，生产成本低。
(3)质粒DNA的提取过程中不添加高风险的动物来源成分，如RNase、溶菌酶、蛋白酶K等，生产工艺不使用有毒害的有机溶剂如异丙醇、酚、无水乙醇 和其他诱变剂等，所用的试剂可以使用一般的试剂或满足药用级别，不使用酸液中和，对厂房设备要求较低，适合大规模生产。
(4)通过优化混合泵腔的大小，结合调整泵腔和流速的比例，使得裂解中和的时间和剪切力适合产品生产，同时也方便生产规模的放大；对混合泵头的性状尺寸进行优化，使用3D打印技术，对泵头进行设计和定制在保证混合效果的前提下，降低了剪切力，防止宿主DNA污染产品，使得裂解中和可以自动化进行。
附图说明
图1为本公开的用于提取细菌中质粒DNA的装置的示意图；
图2为本公开实施例中装置第一混合组件叶轮立体图；
图3为本公开实施例中装置第二混合组件立体图，箭头表示料液流动方向；
图4为图3中的第二混合组件爆炸图；
图5为图3中第二混合组件去除泵壳后结构图；
图6为图3中第二混合组件叶轮立体图；
图7为实施例1电泳结果对比图，其中，泳道1为Marker，泳道2为实施例1裂解中和反应液上清，泳道3为标准品；
图8为对比例1电泳结果对比图，其中，泳道1为实施例1的中和反应液上清，泳道2为对比例1的中和反应液上清，泳道3为标准品，泳道4为Marker；
图9为对比例2所用叶轮的示意图；
图10为对比例3所用叶轮的示意图；
图11为对比例3电泳结果对比图，其中，泳道1为实施例1的中和反应液上清，泳道2为对比例3的中和反应液上清，泳道3为标准品，泳道4为Marker；
图12为实施例1、2、3电泳结果对比图，其中，泳道1为实施例3的中和反应液上清，泳道2为实施例1的中和反应液上清，泳道3为实施例2的中和反应液上清，泳道4为标准品，泳道5为Marker；
图13为实施例4中的导流柱的立体结构的示意图；上述图1-6中：
1-第一混合组件；2-第二混合组件；201-主轴；202-泵座；203-密封圈；204-叶轮；205-泵壳；2021-环形槽；2041-后盖板；2042-导流柱；3-裂解螺旋管；4-过滤组件；5-重悬菌液；6-溶液Ⅱ；7-溶液Ⅲ。
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面的理解本公开，但不以任何方式限制本公开。下述内容仅仅是对本申请要求保护的范围的示例性说明，本领域技术人员可以根据所公开的内容对本申请的公开作出多种改变和修饰，而其也应当属于本申请要求保护的范围之中。下面描述中的附图仅是本公开的一个或几个实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
下面以具体实施例的方式对本公开作进一步的说明。本公开实施例中所使用的各种化学试剂如无特殊说明均通过常规商业途径获得。若无特殊说明，浓度百分数均为质量百分数。
除非特别指明，本文中的“裂解溶液”均指“溶液Ⅱ”。
除非特别指明，本文中的“中和液”均指“溶液Ⅲ”。
下述实施例中，所述下一步纯化可选择本领域常规纯化手段。
本公开所用装置：
如图1所示，主要包括：第一混合组件1、第二混合组件2和过滤组件4。两混合组件按功能区分，第一混合组件1可为裂解混合组件，第二混合组件2为中和混合组件。
优选地，第一混合组件1与第二混合组件2串联连接；两混合组件之间还串接有裂解螺旋管3。具体地，溶液Ⅰ与含质粒DNA的菌体配比后形成重悬菌液5，通过第一输送泵控制流量与流速，向第一混合组件1即裂解混合泵输送。具体实施时，输送管路上还串接有一个第一三通接头(即“Y”形连接器，重悬菌液与溶液Ⅱ6分别通过第一输送泵、第二输送泵送至第一三通接头处，再通入第一混合组件1混合，得到菌体混合液。
第一混合组件1的出液端连接裂解螺旋管3的进液端；裂解螺旋管3的出液端连接第二混合组件2的进液端。其中，裂解螺旋管3与第二混合组件2之间的管路上设有一个进液口，具体为串接管路中的第二三通接头，第二三通接头的一端还通过第三输送泵连接溶液Ⅲ7的容器。
本实施例所用的第一混合组件1和第二混合组件2可为搅拌器或混合泵，具体地可分别为第一混合泵和第二混合泵，包括不限于搅拌泵、乳化泵和离心泵等， 其中搅拌泵的搅拌桨可选用桨式搅拌器、推进式搅拌器、涡轮式搅拌器、锚式搅拌器、框式搅拌器、螺旋式搅拌器；乳化泵的转子和定子包括但不限于：粗齿、中齿、细齿。通过一定规则形状的泵头，实现了溶液的充分混合，且剪切力较低，保证染色体DNA不发生大量断裂，且可以在密闭环境下进行，没有污染。进一步由于第一混合组件1用于裂解反应，优选第一混合组件1结构选为搅拌式或乳化式其中一种，具体可选取乳化泵，其叶片(或叶轮)结构如图2中所示(还可为现有技术中的其他乳化泵的形式，此处仅以图2示之)；
第二混合组件2用于中和反应，可选取离心式结构，如图3、4所示，第二混合组件2主要包括主轴201、泵座202、密封圈203、叶轮204、泵壳205。流体输送路线如图3中箭头所示方向，流体通过泵壳205中心处的进液端进入泵内部，经过离心混合后可通过泵壳205侧向设置的出液端流出，出液端内部管路与泵内腔相切。其中主轴201一端连接外部电机的输出端，另一端通过密封装置穿过泵座202的中心处，并与叶轮204固定连接，泵座202与泵壳205接触区域加工有环形槽，用于安装密封圈203。叶轮204优选为半闭式叶轮；但是传统的叶轮存在较大缺点，即剪切力比较大，故本实施例中，叶轮204如图5、6所示设计，包括后盖板2041。后盖板2041上均匀分布有多个导流柱2042，共计32根，分三层环绕中心处，且导流柱2042垂直于后盖板2041的表面。进一步，为了更好的降低产生的剪切力，导流柱2042的形状可为圆柱、圆台或扇形台的一种或多种组合，优选为圆柱。导流柱2042的直径范围为0.5mm-40mm；经检验，优选直径为2mm-10mm时可获得较佳的效果。通过优化设计可以减小剪切力，防止宿主DNA污染产品，使得裂解中和可以自动化进行。经过实验可知第二混合组件2通过设定一定转速范围，控制不同规模的混合效果及剪切力的大小，结合第一混合组件1可实现不同规模的菌液自动裂解、中和，从而实现连续化、大规模生产。
在具体实施例中，第一混合组件1的结构还可优选地与第二混合组件1的结构相同。
具体地，第二混合组件2的叶轮转速为20rpm-1000rpm时产生较好的混合效果。还可针对第二混合组件2改变泵头性状、大小、转速以控制中和效果；第二混合泵的泵头直径为2cm-100cm，优选为4cm～30cm，转速控制在20rpm-1000rpm， 优选为150rpm～500rpm，泵腔体积与该混合泵额定每分钟进料体积的比值范围为1：6～1:1，优选为1:6～1:3；或泵腔体积设计为料液流经泵腔内10s-60s的体积，优选为料液流经泵腔内10s-20s的体积，保证中和完全且产生较低的剪切力，减少染色体DNA的断裂，提高质粒DNA的质量。
第二混合组件2的出液端连接过滤组件4的进液端。
优选地，过滤组件4结构为筛网式、深层过滤式、离心过滤式其中一种或多种组合。具体地，本实施例中过滤组件4结构为深层过滤式结构；过滤孔径为0.2μm-800μm；具体可选过滤孔径在0.1μm-200μm之间。通过深层过滤的方式对中和后上清液进行二次澄清，过滤的材质成分包括不限于纤维素、硅藻土、活性炭、聚丙烯纤维、硅胶及其组合产品。深层过滤膜包膜面积在0.01m 2-2m 2之间。
上述实施例中的提取装置均可用于以下实施例，如有不同具体会示出，具体从细菌中提取质粒DNA的方法如下：
实施例1 50L发酵规模处理
实施例1的用于提取细菌中质粒DNA的装置，泵头直径为10cm，两个泵的泵头叶轮均如附图6所示，导流柱直径为5mm。
(1)将含有质粒A的大肠杆菌高密度发酵菌液，分光光度计测定OD600为84.2，取该发酵液23.3L离心，收获菌体3684g，湿重为15.8％。将3684g的细胞重悬于由25mM Tris-HCl和10mM EDTA-2Na构成的pH为8.0重悬液(溶液I)中，得重悬菌液，体积为25.8L(菌体与溶液I的质量体积比为1:7(kg:L))。
(2)将重悬菌液以140ml/min的速度泵送至“Y”形连接器的一侧，同时将由0.2M NaOH和1％SDS构成的裂解溶液(溶液II)以140ml/min的速度泵送至“Y”形连接器的另一侧。将“Y”形连接器接入裂解混合泵(第一混合泵)，调节转速为200rpm，开始裂解混合，得到菌体混合液。其中，溶液I与溶液II的体积比为1:1，其中，裂解混合泵的泵腔体积为与单个混合泵额定每分钟进料体积的比值1:3。
(3)菌体混合液从第一混合组件(裂解混合泵)中泵出后，进入裂解螺旋管，裂解螺旋管内径为1.9cm，长度为5m，在裂解螺旋管中裂解时间为5min，得到裂解液。
(4)裂解后的裂解液进入另一“Y”形连接器，连接器另一端由1M KAc和7M NH4Ac组成的溶液III(预冷2-8℃)以280ml/min的速度进入，通过“Y”形连接器进入中和混合泵(第二混合组件)，混合泵设置转速250rpm。裂解液和溶液III的体积比为1:1。其中，中和混合泵叶轮上的导流柱的直径为5mm，导流柱的形状为圆柱，中和混合泵的泵头直径为8.5cm；其中，中和混合泵的泵腔体积为与单个混合泵额定每分钟进料体积的比值1:4。
(5)中和完成后，收集中和反应液，以8000g离心力离心20min，收集到上清液，可进行下一步纯化。
结果检测：
重悬菌液测得质粒浓度为545mg/L(QIAGEN的质粒小提试剂盒测得)，质粒总量为14.06g。
中和后得到中和反应液100L，离心得到上清液共82L，以HPLC定量法测得上清液质粒浓度为121.8mg/L(HPLC机型:Waters 2695；色谱柱型号:TOSOH，Tskgel DNA-NPR 4.6mm*7.5cm 2.5um，下述实施例中的HPLC测定条件相同)，裂解收率71％。
电泳结果如图7所示，从图7可以看出通过本申请的方法裂解后的上清中质粒纯度较高，RNA及宿主DNA较少。
通过HPLC试验以及药典方法检测上述方法制得的质粒DNA，结果表明质粒为目的质粒，且纯度较高，超螺旋比例大于95％，开环比例较少。
实施例2
与实施例1不同的是，第一混合泵的转速为400rpm，第二混合泵的转速为500rpm。导流柱为圆柱，其直径为1mm。其余皆相同。
然后中和后的中和反应液进行琼脂糖核酸电泳，电泳图如图12所示。可以看出实施例2裂解上清中宿主DNA和RNA均高于实施例1裂解上清，转速偏高时，杂质会较多。
实施例3
与实施例1不同的是，菌体重悬液为2.5L，第一混合泵的转速为100rpm，第二混合泵的转速为50rpm，导流柱为圆柱，其直径为1mm。其余皆相同。
通过HPLC检测中和后得到中和反应液10L，离心得到上清液共7.8L，测得 上清液质粒浓度为96mg/L(HPLC测定)，裂解收率55.0％。
电泳图如图12所示，由图12可以看出混合泵转速较低时会导致混合和中和不充分，质粒DNA收率较实施例1低。
实施例4
与实施例1不同的是，本实施例中导流柱2042为变截面设计，目的是为了进一步降低剪切了对中和过程的影响，通过流体运动分析，如图13中箭头所示，单根导流柱转动中，流体相对主体的流速分布情况；即从中间层向两边减小，原因分析为流体上下两侧分别受到泵壳即泵座的粘滞阻力，速度呈梯度分布，由此为了保持单根导流柱对流体中遗传物质产生的剪切力较为一致，故将其设计为变截面结构。具体地，以实施例1中圆柱体为例，单根导流柱的截面从泵壳一侧至泵座一侧依次为先增加后减小，形成“纺锤形”结构，具体参阅图13。上述设计，虽然导流柱中心处相对速度较大，冲击较强，但是结合较大曲率半径及受力面积，可以有效减少对质粒的剪切作用，一定程度上提升了质粒产率。
实施例5
同实施例1，检测宿主DNA残留(HCD)结果为5.87μg/mg(E.coli残留DNA检测试剂盒)。如表1所示，通过HPLC试验以及药典方法检测质粒DNA，结果表明质粒为目的质粒，且纯度较高，超螺旋比例为95.92％，开环比例较少。
表1为实施例5中样品质粒及纯度检测HPLC峰结果表
样品名称 杂质1，％ 开环，％ 超螺旋，％ 线性，％ 杂质2，％
离心后 ND 1.15 95.92 ND 2.93
备注：“ND”表示未检出。杂质1和杂质2为质粒的未知状态。
实施例6
同实施例2，进一步检测宿主DNA残留(HCD)结果为18.7μg/mg(E.coli残留DNA检测试剂盒)。
实施例7
与实施例1不同的是，本实施例中裂解中和后，采用滤袋过滤和深层过滤的方式进行固液分离。目的是提高放大生产过程中的处理量和提高生产效率，同时减少生产上连续式离心机的机械剪切作用，减少杂质产生和目的质粒被破坏。采用过滤面积为0.5m 2，孔径大小分别为100μm和200μm聚丙烯材质的滤袋进行 初级过滤，之后采用过滤孔径为0.2-2μm，材质为纤维素和无机助滤剂复合材质的深层过滤进行二级过滤。初级过滤后杂质去除率可达86.2％，过滤前后的浊度分别为43NTU和10.7NTU，且过滤后质粒纯度未发生明显改变。二级过滤后，滤液更加澄清，浊度可降低至3NTU以下，可直接进行下游纯化。如表2所示，通过HPLC检测质粒DNA，结果表明质粒为目的质粒，且纯度较高，过滤前，超螺旋比例达96.08％，100μm滤袋过滤后，超螺旋比例97.6％，200μm滤袋过滤后，超螺旋比例97.55％。
表2为100μm和200μm滤袋过滤前后清液中质粒纯度HPLC检测结果
Figure PCTCN2022082698-appb-000001
备注：“ND”表示未检出。HPLC进样前对均会对样品进行离心，取上清液进样。
对比例1 气泡混合器处理
与实施例1不同的是，对比例1的中和步骤不使用泵，在气泡混合器中进行，具体步骤如下：
(1)将含有质粒A的大肠杆菌高密度发酵菌液，分光光度计测定OD600为78.9。取该发酵液23.5L离心，收获菌体3603g，湿重为15.3％。将3603g的细胞重悬于由25mM Tris-HCl和10mM EDTA-2Na构成的pH为8.0重悬液(溶液I)中，得重悬菌液，体积为25.2L(菌体与溶液I的质量体积比为1:7)。
(2)将重悬菌液与以140ml/min的速度泵送至“Y”形连接器的一侧，同时将由0.2M NaOH和1％SDS构成的裂解溶液(溶液II)以140ml/min的速度泵送至“Y”形连接器的另一侧。将“Y”形连接器接入裂解混合泵(第一混合泵)，调节转速为200rpm，开始裂解混合，得到菌体混合液。其中，溶液I与溶液II的体积比为1:1。
(3)菌体混合液从裂解混合泵中泵出后，进入裂解螺旋管，裂解螺旋管内径为1.9cm，长度为5m，在裂解螺旋管中裂解时间为5min，得到裂解液。
(4)裂解后的裂解液进入另一“Y”形连接器，连接器另一端由1M KAc和7M NH 4Ac组成的溶液III(预冷2-8℃)以280ml/min的速度进入，通过“Y”形 连接器进入气泡混合器，气泡混合器设置压缩空气流速为1.2L/min。裂解液和溶液III的体积比为1:1。
(5)中和完成后，收集中和反应液，以8000g离心力离心20min，收集到对比上清液，可进行下一步纯化。
通过酶标仪检测，结果表明，重悬菌液测得质粒浓度为570mg/L(通过QIAGEN的质粒小提试剂盒计算得出)，质粒总量为14.36g。
中和后得到中和反应液101L，离心得到上清液共79.3L，测得对比上清液质粒浓度为116.3mg/L(HPLC测定)，裂解收率64.2％，低于实施例1。
电泳结果如图8所示，从图8可以看出本对比例中和过程使用气泡发生器的方法得到质粒DNA与实施例1的方法得到的质粒DNA相比，质粒浓度相当，但是其宿主RNA较多，说明本申请的制备方法更优，并且更容易放大，操作简单。
对比例2
与实施例1不同的是，该实施例中第二混合泵所用离心泵头叶轮如图9所示，其余皆相同。
通过酶标仪检测：
中和后得到中和反应液80L，离心得到上清液共66L，测得上清液质粒浓度为106.6mg/L(HPLC测定)，裂解收率64.5％。裂解收率低于实施例1。
对比例3
与实施例1不同的是，该实施例中第二混合泵所用离心泵头叶轮如图10所示，其余设置均相同。
电泳图如图11所示，从图11可以看出，与实施例1结果相比，使用图10所示泵头，裂解上清中宿主DNA和RNA含量均较多，不利于质粒纯化。
基于上述实施例结果可知，本公开的提取装置和提取方法，最终产品裂解时混合充分且混合时间较短，中和时条件温和均一，裂解中和后，宿主DNA和RNA残留均低于起泡混合器的效果，产品质量较好，且速度适中时提取的质粒DNA杂质少，收率高。
本公开在质粒生产过程中的碱裂解和中和环节创新性的采用的混合组件(可为泵)的形式，使得裂解和中和过程在密闭的环境中，降低污染环境的几率，使 用后方便进行CIP和SIP，且实现了连续的加工，提升了生产效率，且成本低廉，不需要专业的定制化、价格高昂的设备，易于在生产中放大，生产成本低；裂解时混合充分且混合时间较短，中和时条件温和均一，裂解中和后，宿主DNA和RNA残留均低于起泡混合器的效果，产品质量好；同时优化了泵腔的大小，使得裂解中和的时间和剪切力适合产品生产，同时也方便生产规模的放大，相较于目前主流的气泡混合器Airmix的生产体系来讲，比较容易放大，不需要根据规模定制不同大小的气泡混合器，缩短了放大条件摸索的时间，提高工作效率。
(2)该方法所用到的设备简单，操作方便，使用的两台混合组件既可以使菌液和裂解液的充分混合又可以保证和中和液温和地混合中和，避免使用复杂的低剪切中和设备，裂解后的质粒超螺旋比例较高，宿主DNA、RNA残留较少[；此外，使用复杂的多级的膜过滤系统，裂解后也不需要过夜沉淀等步骤，设备可直接用CIP清洗，符合药物生产的生产规范，同时节省了工艺时间，降低成本；不使用复杂的多级的膜过滤系统，裂解后也不需要过夜沉淀等步骤，裂解后的质粒超螺旋比例较高，宿主DNA、RNA残留较少，设备可直接用CIP清洗，符合药物生产的生产规范，同时节省了工艺时间，降低成本，操作方便，不需要专业的定制化、价格高昂的设备，易于在生产中放大，生产成本低。
(3)过程中不添加高风险的动物来源成分，如RNase、溶菌酶、蛋白酶K等，生产工艺不使用有毒害的有机溶剂如异丙醇、酚、无水乙醇和其他诱变剂等，所用的试剂可以使用一般的试剂或满足药用级别，不使用酸液中和，对厂房设备要求较低，适合大规模生产。
(4)通过优化泵腔的大小，结合调整泵腔和流速的比例，使得裂解中和的时间和剪切力适合产品生产，同时也方便生产规模的放大；对混合泵头的性状尺寸进行优化，使用3D打印技术，对泵头进行设计和定制在保证混合效果的前提下，降低了剪切力，防止宿主DNA污染产品，使得裂解中和可以自动化进行。
以上所述仅为本公开的较佳实施例而已，并不用以限制本公开，凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (10)

  1. 一种用于提取细菌中质粒DNA的方法，其特征在于，在两个串联的混合组件中实现质粒DNA生产过程中的裂解和中和，具体包括以下步骤：
    (1)混合；
    (2)裂解；
    (3)中和；
    其中，步骤(1)在第一混合组件中完成，步骤(2)在裂解螺旋管中完成，步骤(3)在第二混合组件中完成，所述第一混合组件、裂解螺旋管和第二混合组件依次串联。
  2. 根据权利要求1所述的用于提取细菌中质粒DNA的方法，其特征在于，所述第一混合组件的转速为50rpm-1500rpm，优选为200rpm-500rpm；所述第二混合组件的转速为20rpm-1000rpm，优选为150rpm-500rpm。
  3. 根据权利要求1所述的用于提取细菌中质粒DNA的方法，其特征在于，所述第一混合组件和第二混合组件的结构各自独立地选自搅拌式、乳化式和离心式中的任一种，所述第一混合组件和第二混合组件均优选为混合泵或搅拌器；优选地，所述第一混合组件为搅拌式或乳化式或离心式，所述第二混合组件为离心式。
  4. 根据权利要求3所述的用于提取细菌中质粒DNA的方法，其特征在于，所述第一混合组件和第二混合组件分别为第一混合泵和第二混合泵，所述第一混合泵和第二混合泵的泵腔体积与单个混合泵额定每分钟进料体积的比值范围均为1：6-1:1，优选为1:6-1:3；或
    所述第一混合泵和第二混合泵的泵腔体积均为料液流经泵腔内10s-60s的体积，优选为料液流经泵腔内10s-20s的体积。
  5. 根据权利要求4所述的用于提取细菌中质粒DNA的方法，其特征在于，所述裂解螺旋管内径为0.5cm-15cm，优选为0.5cm-6cm；所述第一混合泵和第二混合泵的泵头直径均为2cm-100cm，优选为4cm-30cm。
  6. 根据权利要求1至5中任一项所述的用于提取细菌中质粒DNA的方法，其特征在于，在步骤(2)中，所述裂解的时间为2min-10min，优选5min。
  7. 根据权利要求3所述的用于提取细菌中质粒DNA的方法，其特征在于，所述第一混合泵和第二混合泵的叶轮均包括后盖板；所述后盖板上均匀分布有多 个导流柱，所述导流柱上至少沿叶轮旋转方向的外侧面呈弧面设置。
  8. 根据权利要求7所述的用于提取细菌中质粒DNA的方法，其特征在于，所述导流柱为圆柱、圆台或扇形柱的一种或多种组合；优选地，所述导流柱的横截面宽度为0.5mm-40mm，优选为2mm-10mm；所述导流柱优选为圆柱，或所述导流柱的截面积中间最大，且由中间至两端的截面积逐渐变小。
  9. 根据权利要求1-8任一项所述的用于提取细菌中质粒DNA的方法，其特征在于，具体包括以下步骤：
    (1)用溶液I重悬菌体后，得到重悬菌液，再将重悬菌液、溶液II导入第一混合组件混合，得到菌体混合液；
    (2)所述菌体混合液从第一混合组件中流出，进入裂解螺旋管裂解，裂解后得到裂解液；
    (3)所述裂解液与溶液III导入第二混合组件后(或所述裂解液与溶液III混合后，再通入第二混合组件)，进行中和反应，中和完成后得到中和反应液；
    优选地，得到中和反应液后，还包括将其进行固液分离和纯化的步骤。
  10. 根据权利要求9所述的用于提取细菌中质粒DNA的方法，其特征在于，步骤(1)中，溶液I与菌体的体积质量比为3-20:1(L:kg)，优选为7:1(L:kg)，溶液I与溶液II的体积比为1:0.5-3，优选为1:1；或
    步骤(3)中，裂解液与溶液III的体积比为1:0.3-5，优选为1:1；
    优选地，通过过滤组件进行固液分离，所述过滤组件结构为筛网式、深层过滤式、离心过滤式其中一种或多种组合；
    进一步优选地，所述过滤组件为筛网式或深层过滤式结构；过滤孔径为0.2μm-800μm；过滤材质包括纤维素、硅藻土、活性炭、聚丙烯纤维和硅胶。
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