Method for purifying plasmid DNA

Abstract

The invention discloses a method for purifying plasmid DNA (deoxyribonucleic acid). The method sequentially comprises the following steps: firstly, splitting thalli containing DNA plasmids, and precipitating to obtain a thalli splitting precipitation solution; then clarifying the thallus cracking precipitation solution to obtain a plasmid clarified solution; then carrying out composite chromatography on the plasmid clarified liquid through a chromatographic column filled with Capto Core700 multi-mode chromatographic filler, and collecting a flow-through liquid with an A260 ultraviolet absorption value higher than a baseline; finally, carrying out hydrophobic chromatography on the flow-through liquid, and collecting an eluent, namely a hydrophobic chromatography eluent; the hydrophobic chromatography eluent is a plasmid DNA solution. The plasmid DNA obtained by purification through the method provided by the invention has a higher quality level, not only has higher superhelix purity, but also has a lower residual value of host impurities, is suitable for large-scale production of plasmids within a scale of nearly 100L, greatly improves the benefit output of enterprises, and has a higher industrial application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for purifying plasmid DNA. Background Art

[0002] The application of plasmid DNA in clinical trials mainly includes two ways: 1) direct use as a therapeutic drug; 2) used as a starting raw material (such as lentivirus or adeno-associated virus (AAV)) for production. Whether it is used as a therapeutic drug or as a raw material, the production of plasmids must meet GMP requirements, and the detection and release must be in accordance with clinical grade requirements. Plasmid DNA used in clinical trials needs to remove bacterial host cell contaminants (including chromosomal DNA, RNA and endotoxins) as well as open-circular plasmids and linear plasmids. These contaminants have similar properties to the target molecule, so a complete purification strategy must be developed. At present, the FDA has set acceptable standards for the level of plasmid DNA contaminants: the proportion of chromosomal DNA, RNA and protein in the total mass should be <1%, the endotoxin in each mg of plasmid should be <40EU, and the supercoiling ratio of pDNA should be >80%. The normal expectation of the supercoiling ratio of plasmids in the industry is usually >90%, and can even be as high as 95%. Since the constructed pDNA is highly variable in terms of plasmid size, plasmid backbone, and gene insert, the established plasmid purification platform must be highly robust in terms of contaminant removal, and due to the market's expectations for product value, the purification process should also minimize process costs.

[0003] At present, the methods for extracting small amounts of plasmids at the laboratory level include: using lysis methods to lyse the bacteria and removing the precipitate in the lysate by physical or chemical means; cetyltrimethylammonium bromide (CTAB) combined with silica absorption and ultrafiltration non-chromatographic plasmid purification methods. In large-scale preparation of plasmid purification methods include: the lysate is usually treated and purified through several chromatographic steps to further remove contaminants; chromatographic separation is usually based on a combination of ion exchange (IEX), hydrophobic interaction (including ion pair interaction) and steric exclusion (SEC) chromatography. A large number of contaminants such as RNA, host cell proteins and endotoxins can be removed by these techniques. However, due to the chemical similarity between host DNA fragments and open circular plasmids and target supercoiled plasmids, it is difficult to achieve the removal of host chromosomal DNA fragments and open circular plasmids, and the sources and properties of these contaminants are poorly understood, making analysis complicated. In addition, the generation of small fragments of host chromosomal DNA that are not precipitated during alkaline lysis is believed to be affected by many factors, such as enzymatic reactions during lysis and mechanical degradation. The level of host DNA varies significantly depending on the host strain and the characteristics of the constructed plasmid. There is also limited understanding of why higher levels of open circular plasmid forms are produced in certain production systems. While fermentation conditions can be manipulated to reduce open circular plasmids, the levels of isoforms vary between plasmids. Larger plasmids are significantly more susceptible to open circularity. Therefore, separation of various plasmid constructs on an industrial scale requires a highly selective process.

[0004] At present, there are two main plasmid purification process platforms, one is a three-step chromatography process (represented by Cytiva), and the other is a monolithic column two-step chromatography process (represented by Sarturius). Figure 1 The chromatographic fillers used in the above two plasmid purification methods (Sepharose 6Fast Flow, PlasmidSelectXtra, SOURCE 30Q, Capto Q ImpRes, DEAE and C4) is widely used in various plasmid development process routes. However, the above-mentioned chromatography methods still have many problems in practical application. For example, molecular sieve chromatography (steric exclusion) can efficiently remove host DNA fragments and RNA, but the molecular sieve chromatography loading and process flow rate are limited. The increase in loading and the increase in flow rate will reduce the separation effect of impurities, which creates obstacles for large-scale production of plasmids after process amplification. In addition, ion exchange and hydrophobic chromatography processes have high requirements for solution ionic strength. It is difficult to avoid the dilution or replacement process of the solution during the connection process, resulting in cumbersome and complicated process operations. Therefore, the selection of new fillers to develop a more concise and effective plasmid purification process that is conducive to later process amplification has gradually become a core issue of concern in the process of plasmid process development.

[0005] Capto Core series of fillers are new composite mode fillers designed by Cytiva (formerly GE Healthcare) for the intermediate and fine purification steps of viruses and other biomolecules. The octylamine ligand enables Capto Core to have the dual functions of separation of large and small molecules and adsorption chromatography of small molecules in one chromatographic medium. These features make Capto Core an excellent alternative to gel filtration (GF), which is commonly used in the production of biological products. Gel filtration is often regarded as a bottleneck for process scale-up due to its limitations of low flow rate and small sample load in fine purification steps, while Capto Core provides a series of performance points that go beyond gel concerns. The key performance characteristics of the Capto Core series of fillers include: (1) Compared with molecular sieve chromatography, the sample load of up to 10-100 times the same column volume and significantly higher flow rate ensure a significant improvement in production efficiency; (2) The activated ligand is located inside the filler shell, which can effectively capture small molecule impurities that enter the shell while allowing the target macromolecule to flow through the external water volume for collection; (3) Due to stable performance and convenient condition optimization, the application range is wider. Summary of the invention

[0006] The object of the present invention is to protect a method for purifying plasmid DNA.

[0007] The present invention firstly protects a method for purifying plasmid DNA, which may include the following steps in sequence:

[0008] (1) Lysing the bacterial cells containing the DNA plasmid, precipitating, and obtaining a bacterial cell lysis precipitate;

[0009] (2) clarifying the bacterial cell lysis precipitate to obtain a plasmid clarified solution;

[0010] (3) passing the plasmid clarified solution or the concentrated replacement solution of the plasmid clarified solution through a chromatography column filled with CaptoCore700 multi-mode chromatography filler for composite chromatography, and collecting the flow-through with an A260 ultraviolet absorbance value higher than the baseline;

[0011] (4) subjecting the flow-through to hydrophobic chromatography and collecting the eluate, i.e., the hydrophobic chromatography eluate;

[0012] The hydrophobic chromatography eluate or the concentrated replacement solution of the hydrophobic chromatography eluate is a plasmid DNA solution.

[0013] In the above method, in the step (1), lysis is performed using Buffer II; the solute and concentration of the Buffer II are 0.2-0.5M (e.g., 0.2-0.3M, 0.3-0.4M, 0.4-0.5M, 0.2M, 0.3M, 0.4M or 0.5M) NaOH and 0.8-1.2% (e.g., 0.8-1.0%, 1.0-1.2%, 0.8%, 1.0% or 1.2%) SDS, and the solvent is water.

[0014] In the above method, in step (1), the precipitation can be performed by precooling Buffer III and then standing.

[0015] The solute and concentration of the Buffer III are 2-4M (such as 2-3M, 3-4M, 2M, 3M or 4M) CH3COOK (potassium acetate) and 2-3M (such as 2-3M, 2M or 3M) CH3COOH (glacial acetic acid), the solvent is water, and the pH value is 5.4-5.6 (such as 5.4-5.5, 5.5-5.6, 5.4, 5.5 or 5.6).

[0016] In the above method, in step (1), the preparation method of the bacterial body containing the DNA plasmid is: fermenting and culturing the bacteria containing the DNA plasmid, and then centrifuging to collect the bacterial body. The bacteria containing the DNA plasmid can be obtained by transforming the DNA plasmid into bacterial competent cells, and then culturing in a small amount on a shaking table or fermenting in a microbial reactor.

[0017] In the above method, in step (2), clarification can be performed using a depth filter. The depth filter can be 3MZeta Plus TM Depth filter 60SP02A.

[0018] In the above method, in step (3), the concentrated replacement solution of the plasmid clarified solution can be obtained by concentrating and replacing the plasmid clarified solution through an ultrafiltration membrane package. The ultrafiltration membrane package can be a Millipore 50cm 2 PES ultrafiltration membrane package. The Millipore 50cm2 The pore size of the PES ultrafiltration membrane package can be specifically 100KD.

[0019] In the above method, in step (4), "subjecting the flow-through to hydrophobic chromatography and collecting the eluate" may be subjecting the flow-through to hydrophobic chromatography through a CIMmultus C4 HLD Monolithic Column and collecting the eluate.

[0020] In the above method, the "passing the flow-through liquid through a CIMmultusC4 HLD Monolithic Column for hydrophobic chromatography and collecting the eluate" can be loading the flow-through liquid through a CIMmultusC4 HLD Monolithic Column, then first using Buffer A to balance the column, then using Buffer A to rinse the CIMmultusC4 HLD Monolithic Column for more than 3 column volumes (such as 3 column volumes, 4 column volumes, 5 column volumes), then using a mixture of Buffer A (80%) and Buffer B (20%) to rinse the CIMmultusC4 HLD Monolithic Column for more than 3 column volumes (such as 3 column volumes, 4 column volumes, 5 column volumes), and finally using Buffer B to elute the CIMmultusC4HLD Monolithic Column.

[0021] The solute and concentration of the Buffer A are 40-60mM (such as 40-50mM, 50-60mM, 40mM, 50mM or 60mM) Tris, 8-12mM (such as 8-10mM, 10-12mM, 8mM, 10mM or 12mM) EDTA and 1.5-1.7M (such as 1.5-1.6M, 1.6-1.7M, 1.5M, 1.6M or 1.7M) (NH4)2SO4, the solvent is water, and the pH value is 7.0-7.4 (such as 7.0-7.2, 7.2-7.4, 7.0, 7.2 or 7.4).

[0022] The solute and concentration of the Buffer B are 40-60mM (such as 40-50mM, 50-60mM, 40mM, 50mM or 60mM) Tris, 8-12mM (such as 8-10mM, 10-12mM, 8mM, 10mM or 12mM) EDTA and 0.5-0.9M (such as 0.5-0.7M, 0.7-0.9M, 0.5M, 0.7M or 0.9M) (NH4)2SO4, the solvent is water, and the pH value is 7.0-7.4 (such as 7.0-7.2, 7.2-7.4, 7.0, 7.2 or 7.4).

[0023] In the above method, in step (4), the concentrated replacement solution of the hydrophobic chromatography eluate can be obtained by concentrating and replacing the hydrophobic chromatography eluate through an ultrafiltration membrane package. The ultrafiltration membrane package can be a Millipore 50cm 2 PES material ultrafiltration membrane package.

[0024] In the above method, the residual RNA in the plasmid DNA solution is ≤200 ng / mg and the supercoil ratio of the plasmid DNA is ≥90%.

[0025] The present invention also protects a kit for purifying plasmid DNA, which may include Capto Core 700 multi-mode chromatography filler and / or CIMmultus C4 HLD Monolithic Column.

[0026] The kit for purifying plasmid DNA may specifically consist of Capto Core 700 multi-mode chromatography filler and / or CIMmultus C4 HLD Monolithic Column.

[0027] The kit for purifying plasmid DNA may further include at least one of any of the above-mentioned Buffer II, any of the above-mentioned Buffer III, any of the above-mentioned Buffer A and any of the above-mentioned Buffer B.

[0028] The kit for purifying plasmid DNA can specifically be composed of Capto Core700 multi-mode chromatography filler, CIMmultusC4 HLD Monolithic Column, any one of the above-mentioned Buffer II, any one of the above-mentioned Buffer III, any one of the above-mentioned Buffer A and any one of the above-mentioned Buffer B.

[0029] Experiments have shown that compared with the plasmid quality test results after purification by other plasmid purification processes, the plasmid DNA obtained by the method provided by the present application has a higher quality level in the plasmid DNA solution, not only the supercoil purity is higher, but also the host impurity residual value is lower, and it is suitable for large-scale production of plasmids within a scale of nearly 100L, and has a high industrial application value. The method provided by the present application adopts composite chromatography technology and hydrophobic chromatography, and purifies plasmid DNA by two-step chromatography. Not only is the purification process more streamlined, but the production cost is more controllable and cheap, which can improve the benefit output of the enterprise and meet the needs of large-scale production of plasmids. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This is a flow chart of the two existing plasmid purification processes.

[0031] Figure 2 The plasmid supernatant was subjected to complex chromatography using a chromatography column filled with gel filtration medium Sepharose 6FF.

[0032] Figure 3 The plasmid clear solution was subjected to composite chromatography using a chromatography column filled with Capto Core 700 multi-mode chromatography filler, wherein CIP was a 30% isopropanol solution and the solvent was 1 M NaOH.

[0033] Figure 4 for Figure 2 The flow through peak 1 (6FF-1) and Figure 3 Agarose gel electrophoresis results of the target peak in . Among them, L1 is Figure 2 The flow through peak 1 (6FF-1) in the Figure 3 The target peak in. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1. Establishment of a method for purifying plasmid DNA

[0037] The inventors of the present invention have established a novel method for purifying plasmid DNA that can be scaled up after a large number of experiments. The specific process flow is as follows:

[0038] 1. Bacteria collection

[0039] The bacteria containing the DNA plasmid are cultured by fermentation, and then centrifuged to collect the bacteria. The bacteria containing the DNA plasmid can be obtained by transforming the DNA plasmid into bacterial competent cells and then culturing them in small quantities on a shaking platform.

[0040] 2. Bacterial lysis

[0041] (1) The cells collected in step 1 were resuspended in Buffer I (solutes and their concentrations were 50 mM Tris, 50 mM glucose and 10 mM EDTA, solvent was water, and pH was 7.5) to obtain a resuspension.

[0042] (2) After completing step (1), the resuspension solution and Buffer II (solute and its concentration are an aqueous solution containing 0.4 M NaOH and 1.0% SDS) are mixed in equal volumes and lysed to obtain a bacterial lysate.

[0043] (3) After completing step (2), add an equal volume of 4-8°C pre-cooled Buffer III (solute and its concentration are 3M CH3COOK and 2M CH3COOH, solvent is water, pH value is 5.5) to the bacterial lysis solution, let it stand, and obtain the bacterial lysis precipitate.

[0044] The purpose of adding Buffer III is to precipitate the complex between cell debris and SDS.

[0045] 3. Clarification of bacterial solution

[0046] The bacterial lysis precipitate obtained in step 2 is taken and clarified by a depth filter to obtain a plasmid clarified solution.

[0047] Among them, the depth filter can be 3M Zeta Plus TM Depth filter 60SP02A. 3M Zeta Plus TM The depth filter 60SP02A is a product of 3M Company, and the product catalog number is E0340FSA60SP02A.

[0048] 4. Concentration replacement

[0049] The plasmid clarified liquid obtained in step 3 is taken and concentrated and replaced by an ultrafiltration membrane package to obtain a plasmid concentrated replacement liquid.

[0050] Among them, the ultrafiltration membrane package can be Millipore 50cm 2 PES ultrafiltration membrane package. Millipore 50cm 2 The PES ultrafiltration membrane package is a product of Millipore Corporation, and the product catalog number is PXB100C50.

[0051] 5. Composite chromatography

[0052] The plasmid clarification liquid or the plasmid concentrated replacement liquid was subjected to composite chromatography using a chromatography column filled with Capto Core700 multi-mode chromatography filler (product of Cytiv, catalog number 17548103). After balancing to the baseline with a buffer solution (solute and its concentration are 50mM Tris, 10mM EDTA and 0.4M NaCl, the solvent is water, and the pH value is 7.2), the plasmid concentrated replacement liquid was then loaded, and the loading load was ≤1g bacteria / ml column volume. After the loading was completed, the buffer solution was continued to be rinsed to the baseline. This chromatography is a flow-through mode, and the flow-through liquid with an A260 ultraviolet absorbance value higher than the baseline is collected, that is, the composite chromatography collection liquid.

[0053] 6. Hydrophobic chromatography

[0054] Use Buffer A (solutes and their concentrations are 50mM Tris, 10mM EDTA and 1.5M (NH4)2SO4, the solvent is water, and the pH value is 7.2) to fully balance the CIMmultusC4 HLD Monolithic Column (a product of Sartorius, product catalog number is 611.8136-2); use 4M (NH4)2SO4 solution to adjust the concentration of the composite chromatography collection liquid to 2.1M, then load the sample, and continue to use Buffer A to rinse at least 3 column volumes after loading; then use a buffer solution (mixed by 4 volumes of Buffer A and 1 volume of Buffer B) to rinse at least 3 column volumes; then use Buffer B (solutes and their concentrations are 50mM Tris, 10mM EDTA and 0.7M (NH4)2SO4, the solvent is water, and the pH value is 7.2) for elution, and collect the eluate, which is the hydrophobic chromatography collection liquid.

[0055] 7. Replacement solution

[0056] The hydrophobic chromatography collection liquid obtained in step 6 is taken and concentrated and replaced by an ultrafiltration membrane package (the solute and its concentration of the plasmid prescription solution are 10mM Tris and 1mM EDTA, the solvent is water, and the pH value is 8.0). The concentrated and replaced liquid is then sterilized and filtered to obtain a plasmid DNA solution.

[0057] Among them, the ultrafiltration membrane package can be Millipore 50cm 2 PES ultrafiltration membrane package. Millipore 50cm 2 The PES ultrafiltration membrane package is a product of Millipore Corporation, and the product catalog number is P2B100C01.

[0058] Example 2: Development of a plasmid DNA purification process - screening of composite chromatography fillers

[0059] The traditional gel filtration process is often regarded as a bottleneck for process scale-up due to its limitations of low flow rate and small sample loading in the purification step. The octylamine ligand of the Capto core series composite mode filler enables Capto Core to have the dual functions of separation of large and small molecules and adsorption chromatography of small molecules in a chromatographic medium. These characteristics make Capto Core an excellent alternative to gel filtration (GF) commonly used in the production of biological products. In addition, the Capto Core series removes residual RNA in plasmids mainly by utilizing the different molecular weights of plasmid DNA and RNA. RNA can enter the nuclear pores of Capto Core and adsorb to the ligand, while plasmid DNA cannot enter the nuclear pores due to its large molecular weight, thus completely excluding it and can be collected by flow-through. In addition, host DNA, host proteins and endotoxins with smaller molecular weights can enter the pores and adsorb to the ligands, thereby removing part of the host DNA, host proteins and endotoxins.

[0060] 1. Bacteria collection

[0061] Same as step 1 in Example 1.

[0062] 2. Bacterial lysis

[0063] Same as step 2 in Example 1.

[0064] 3. Clarification of bacterial solution

[0065] Same as step 3 in Example 1.

[0066] The plasmid clarified liquid obtained at this time contains not only plasmid DNA but also a large amount of RNA.

[0067] 4. According to the method of step 5 in Example 1, the plasmid clarified liquid is subjected to composite chromatography using a chromatography column filled with Capto Core 700 multimodal chromatography filler; then, hydrophobic chromatography and solution replacement are performed according to the methods of steps 6 and 7 in Example 1 to obtain a plasmid DNA solution.

[0068] 5. According to the method of step 5 in Example 1, the Capto Core 700 multimodal chromatography filler was replaced with the gel filtration filler Sepharose 6FF, and the other steps remained unchanged, and composite chromatography was performed; then, hydrophobic chromatography and solution replacement were performed according to the methods of steps 6 and 7 in Example 1 to obtain a plasmid DNA solution.

[0069] 6. Check the volume and concentration of the plasmid DNA solution obtained in steps 4 and 5, the residual host RNA, and the supercoil ratio.

[0070] The test results are shown in Table 1.

[0071] The results of the composite chromatography of the plasmid clear solution using a chromatography column filled with gel filtration filler Sepharose 6FF are shown in Figure 2 .

[0072] The results of the complex chromatography of the plasmid clarification using a column filled with Capto Core 700 multimodal chromatography medium are shown in Figure 3 .

[0073] Will Figure 2 The flow through peak 1 (6FF-1) and Figure 3 The target peak in the sample was subjected to agarose gel electrophoresis. The results are shown in Figure 4 .

[0074] The results showed that the plasmid clarification solution was separated into two peaks by chromatography using a chromatography column filled with gel filtration filler Sepharose 6FF. Flow-through peak 1 (6FF-1) was mainly composed of plasmid DNA components, and flow-through peak 2 (6FF-2) contained a large amount of RNA and some plasmid DNA. The nucleic acid yield and supercoil ratio of the two fillers were basically the same, and the supercoil purity was in line with expectations. However, the RNA removal effect of chromatography using a chromatography column filled with Capto Core700 multi-mode chromatography filler was significantly better than that of gel filtration filler Sepharose 6FF.

[0075] Table 1

[0076]

[0077]

[0078] Example 3. Development of a Plasmid DNA Purification Process—Optimization of Hydrophobic Chromatography

[0079] Hydrophobic chromatography is a key step in the plasmid purification process to remove open plasmids and host DNA. The CIMmultusC4 HLD Monolithic Column based on the platform process was optimized. Among them, the wash buffer (i.e., Buffer A) and the elution buffer (i.e., Buffer B) play a key role in removing host DNA and improving the purity of supercoils. The experiment optimized the ammonium sulfate concentration in the wash buffer and the elution buffer.

[0080] The experiment used washing buffer containing different concentrations of ammonium sulfate, and the elution conditions remained unchanged. The residual E. coli host DNA in each washing component was detected to determine the optimal washing conditions. Some test results are shown in Table 2.

[0081] Table 2

[0082]

[0083] The results showed that the E. coli host DNA residues were mainly enriched in the 1.5M ammonium sulfate component, and the E. coli host DNA residues of the samples under the 1.4M, 1.3M, and 1.2M ammonium sulfate washing conditions were lower than the limit value of 2μg / mg. In summary, the hydrophobic chromatography process washing increased the 1.5M ammonium sulfate concentration washing ≥3CV, and the E. coli host DNA residues of the plasmid after purification were qualified, and the yield was maintained at ≥50%.

[0084] The experiment used Buffer A (solutes and their concentrations were 50mM Tris, 10mM EDTA and 1.5M (NH4)2SO4, solvent was water, pH value was 7.2) for washing, and then eluted with elution buffer containing different concentrations of ammonium sulfate, and the residual E. coli host DNA in each elution component was detected to determine the best elution conditions. The results showed that the optimal concentration of ammonium sulfate in the elution buffer was 0.7M.

[0085] Example 4: Pilot scale-up of the plasmid DNA purification process established in this application

[0086] When the plasmid DNA purification process established in this application is used for pilot-scale production, the upstream fermentation scale is 70L; after the bacterial resuspension, lysis, neutralization, precipitation, clarification and filtration, the ultrafiltration concentration is no more than 20 times, and the sample volume is estimated to be about 5-6L. Load the composite chromatography, the concentration of the collected liquid can be determined by A260, and then hydrophobic chromatography is performed. A 4000mL hydrophobic chromatography monolithic column is selected, and finally the concentration of the hydrophobic chromatography collected liquid is determined by A260, and finally the ultrafiltration and preparation liquid are replaced, and the liquid is replaced with the plasmid prescription liquid, and the final concentration is controlled at 0.8-1.2mg / mL; finally, the stock solution is formed after 0.22μm sterilization filtration. The quality test results of the plasmid after pilot-scale batch purification are shown in Table 3.

[0087] Table 3. Results of quality test of three batches of plasmids

[0088]

[0089]

[0090] It can be seen that compared with the plasmid quality test results after purification by other plasmid purification processes, the method provided in the present application has a higher quality level, not only with higher supercoil purity, but also with lower host impurity residual value, and is suitable for large-scale production of plasmids within a scale of nearly 100L, and has high industrial application value.

[0091] Compared with the traditional three-step chromatography plasmid purification method (gel filtration chromatography, affinity (hydrophobic) chromatography, anion exchange chromatography), the method provided by the present application adopts composite chromatography technology and hydrophobic chromatography, and purifies plasmid DNA by two-step chromatography, which is a more streamlined purification process, and obtains plasmid products whose quality parameters meet the quality standards of the current "Chinese Pharmacopoeia". The production cost of the method provided by the present application is more controllable and low, which can improve the efficiency output of enterprises and meet the needs of large-scale production of plasmids.

[0092] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for purifying plasmid DNA, comprising the following steps in sequence: (1) lysing the bacterial cells containing the DNA plasmid, precipitating, and obtaining a bacterial cell lysis precipitate; (2) clarifying the bacterial cell lysis precipitate to obtain a plasmid clarified solution; (3) passing the plasmid clarified liquid or the concentrated replacement liquid of the plasmid clarified liquid through a chromatography column filled with Capto Core 700 multi-mode chromatography filler for composite chromatography, and collecting the flow-through liquid with an A260 ultraviolet absorbance value higher than the baseline; (4) subjecting the flow-through to hydrophobic chromatography and collecting the eluate, i.e., the hydrophobic chromatography eluate; The hydrophobic chromatography eluate or the concentrated replacement solution of the hydrophobic chromatography eluate is a plasmid DNA solution.

2. The method according to claim 1, characterized in that: In the step (1), The lysis is performed using Buffer II; the solute and concentration of the Buffer II are 0.2-0.5M NaOH and 0.8-1.2% SDS, and the solvent is water; The precipitation is performed by precooling Buffer III and then standing still; the solute and concentration of the Buffer III are 2-4M CH3COOK and 2-3M CH3COOH, the solvent is water, and the pH value is 5.4-5.

6.

3. The method according to claim 1, characterized in that: In the step (2), clarification is performed using a depth filter.

4. The method according to claim 1, characterized in that: In the step (3), the concentrated replacement solution of the plasmid clarified liquid is obtained by concentrating and replacing the plasmid clarified liquid through an ultrafiltration membrane package.

5. The method according to claim 1, characterized in that: In the step (4), the "subjecting the flow-through to hydrophobic chromatography and collecting the eluate" means subjecting the flow-through to hydrophobic chromatography through a CIMmultus C4 HLD Monolithic Column and collecting the eluate.

6. The method according to claim 5, characterized in that: The “passing the flow-through through a CIMmultusC4HLD Monolithic Column for hydrophobic chromatography and collecting the eluate” means loading the flow-through through a CIMmultusC4HLD Monolithic Column, then first using Buffer A to balance the column, then using Buffer A to rinse the CIMmultusC4 HLD Monolithic Column for more than 3 column volumes, then using a mixture of Buffer A and Buffer B to rinse the CIMmultusC4 HLD Monolithic Column for more than 3 column volumes, and finally using Buffer B to elute the CIMmultusC4 HLD Monolithic Column; The solute and concentration of the buffer A are 40-60 mM Tris, 8-12 mM EDTA and 1.5-1.7 M (NH4)2SO4, the solvent is water, and the pH value is 7.0-7.4; The solutes and concentrations of the Buffer B are 40-60 mM Tris, 8-12 mM EDTA and 0.5-0.9 M (NH4)2SO4, the solvent is water, and the pH value is 7.0-7.

4.

7. The method according to claim 1, characterized in that: In the step (4), the concentrated replacement solution of the hydrophobic chromatography eluate is obtained by concentrating and replacing the hydrophobic chromatography eluate through an ultrafiltration membrane package.

8. The method according to any one of claims 1 to 7, characterized in that: The residual RNA in the plasmid DNA solution is ≤200 ng / mg and the supercoil ratio of the plasmid DNA is ≥90%.

9. A kit for purifying plasmid DNA, comprising Capto Core 700 multi-mode chromatography filler and / or CIMmultus C4 HLD Monolithic Column.

10. The kit according to claim 9, characterized in that: The kit further comprises at least one of Buffer II, Buffer III, Buffer A and Buffer B; The solute and concentration of the Buffer II are 0.2-0.5M NaOH and 0.8-1.2% SDS, and the solvent is water; The solute and concentration of the Buffer III are 2-4M CH3COOK and 2-3M CH3COOH, the solvent is water, and the pH value is 5.4-5.6; The solute and concentration of the buffer A are 40-60 mM Tris, 8-12 mM EDTA and 1.5-1.7 M (NH4)2SO4, the solvent is water, and the pH value is 7.0-7.4; The solutes and concentrations of the Buffer B are 40-60 mM Tris, 8-12 mM EDTA and 0.5-0.9 M (NH4)2SO4, the solvent is water, and the pH value is 7.0-7.4.

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