Purification method and application of plasmid DNA

Abstract

The invention relates to the technical field of biology, in particular to a purification method and application of plasmid DNA. The plasmid DNA purification method comprises the following steps: mixing an inorganic salt and / or an organic solvent with plasmid DNA to obtain a sample; after balancing the chromatographic column by the equilibrium buffer solution, loading the sample obtained in the step S1 onto the chromatographic column; and performing primary elution on the chromatographic column by using the first buffer solution, and performing secondary elution on the chromatographic column by using the second buffer solution to obtain the purified superhelix plasmid DNA. According to the method, the plasmid DNA crude product is pretreated by adopting the inorganic salt and / or the organic solvent, so that the solubility of the plasmid DNA in the mobile phase in the purification process is reduced, the binding efficiency of a target object is further improved, and meanwhile, the loss of the plasmid DNA caused by flow-through of the plasmid DNA is avoided. By optimizing the purification process, the purpose of purifying the plasmid DNA only through one-step chromatography is achieved.

Description

Technical Field

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

[0002] Plasmids are DNA molecules other than chromosomes (or nucleoids) in organisms such as bacteria, yeast and actinomycetes. They are generally present in the cytoplasm and have the ability to replicate autonomously, so that they can maintain a constant copy number in daughter cells and express the genetic information they carry. The genetic information carried by plasmids can give the host bacteria certain biological traits, which is beneficial for the survival of bacteria under specific environmental conditions. The property of plasmid DNA molecules that can be modified can be used to study specific genes. In recent years, plasmid DNA has made great progress in the research of gene therapy, biotechnology, gene expression and cell analysis, especially as an important vector in gene therapy and nucleic acid vaccines. Plasmid DNA mainly introduces exogenous normal genes into target cells by acting as a gene carrier and infecting T cell nuclear delivery system. Therefore, the quality of plasmid DNA is the key to ensuring the effectiveness and biosafety of gene therapy and nucleic acid vaccines.

[0003] There are three different conformations of plasmids: supercoiled plasmids, linear plasmids, and open-circular plasmids. Due to the low efficiency of transducing cells, the content of open-circular plasmids needs to be strictly controlled in the finished plasmid product, while supercoiled plasmids are considered to be the most effective plasmid form to improve transfection efficiency and target gene expression. In the research of gene therapy and DNA vaccines, clear requirements are made on the supercoiled content of medicinal plasmids (FDA: >80%, NMPA: ≥90%, SMPA: >85%). Therefore, the supercoiled content in plasmid DNA has become one of the key indicators for judging the quality of plasmid DNA.

[0004] The prior art discloses a GMP-grade plasmid DNA large-scale simplified production method, which purifies the plasmid DNA through the steps of salting out, filtration, and anion chromatography. However, salting out can cause the plasmid DNA to precipitate, resulting in a decrease in yield, and it is impossible to separate the supercoiled plasmid and the open-circular plasmid, resulting in low purity of the plasmid DNA. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art in that the steps for purifying plasmid DNA are complicated, open-circular plasmid DNA and supercoiled plasmid DNA cannot be separated, resulting in low purity and yield of plasmid DNA, thereby providing a method for purifying plasmid DNA and its application.

[0006] In one aspect, the present invention provides a method and application of purifying plasmid DNA, comprising the following steps:

[0007] S1, mixing an inorganic salt and / or an organic solvent with plasmid DNA to obtain a sample;

[0008] S2, after the chromatography column is equilibrated with the equilibration buffer, the sample obtained in step S1 is loaded onto the chromatography column;

[0009] S3, after the chromatography column is eluted once with the first buffer, the chromatography column is eluted twice with the second buffer to obtain purified supercoiled plasmid DNA.

[0010] The filler of the chromatography column is a hydrophobic ion exchange resin.

[0011] The inorganic salt in step S1 is at least one of calcium chloride, ammonium sulfate, ammonium bicarbonate and sodium chloride.

[0012] The organic solvent is at least one of ethanol and isopropanol. The inorganic salt concentration in the sample is 0.1M-1M. The mass fraction of the organic solvent is 1%-15%.

[0013] The inorganic salt is ammonium sulfate. The organic solvent is isopropanol.

[0014] The equilibration buffer and / or the first buffer and / or the second buffer comprises sodium chloride at a concentration of 0.1-2M.

[0015] The equilibration buffer and / or the first buffer and / or the second buffer further comprise an inorganic salt, an organic solvent and EDTA.

[0016] The equilibration buffer and / or the first buffer and / or the second buffer is at least one of a Tris hydrochloride buffer, a phosphate buffer, a 2-morpholineethanesulfonic acid buffer and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer;

[0017] Optionally, the inorganic salt concentration in the equilibration buffer and / or the first buffer and / or the second buffer is 0.1-2M;

[0018] Optionally, the mass fraction of the organic solvent is 1%-15%;

[0019] Optionally, the EDTA concentration is 5 mM-20 mM.

[0020] The concentration of each buffer is 0.1-0.5 M, and the pH value is 6.5-7.5.

[0021] The inorganic salt in each buffer solution is at least one of calcium chloride, ammonium sulfate, ammonium bicarbonate, and sodium chloride, wherein the organic solvent is at least one of ethanol or isopropanol.

[0022] The sodium chloride concentration in the equilibration buffer is 0.1-1M.

[0023] The concentration of sodium chloride in the first buffer is 0.5-1M.

[0024] The concentration of sodium chloride in the second buffer is 1-2M.

[0025] The flow rate of loading in step S2 is 0.1 mL / min-200 mL / min.

[0026] In step S3, the elution flow rate of the first buffer and / or the second buffer is 0.1 mL / min-200 mL / min.

[0027] On the other hand, the plasmid DNA purification method provided by the present invention and the plasmid obtained by the purification method can be used in vaccine preparation.

[0028] The technical solution of the present invention has the following advantages:

[0029] 1. The method for purifying plasmid DNA provided by the present invention comprises the following steps: mixing inorganic salts and / or organic solvents with plasmid DNA to obtain a sample; after the chromatographic column is balanced by a balancing buffer, the sample obtained in step S1 is loaded onto the chromatographic column; after the chromatographic column is eluted once by a first buffer, the chromatographic column is eluted twice by a second buffer to obtain purified supercoiled plasmid DNA. The present invention uses inorganic salts and / or organic solvents to pre-treat the crude plasmid DNA product, reduces the solubility of the plasmid DNA in the mobile phase during the purification process, thereby improving the binding efficiency of the target, and avoiding the flow-through of the plasmid DNA. The present invention achieves the purification of plasmid DNA by only one-step chromatography through the optimization of the purification process, improves the removal efficiency of RNA and other impurities in the crude product, and simultaneously achieves the separation of open-circular plasmids and supercoiled plasmids.

[0030] 2. The method for purifying plasmid DNA provided by the present invention, wherein the equilibration buffer and / or the first buffer and / or the second buffer comprise sodium chloride at a concentration of 0.1-2M. The present invention adds a certain concentration of sodium chloride to each buffer. On the one hand, the optimized equilibration buffer can reduce the interaction force between RNA and other impurities in the chromatographic medium to cause flow-through during the loading stage, and at the same time, improve the interaction force between plasmid DNA and the chromatographic medium so that the plasmid DNA is bound to the chromatographic medium to avoid plasmid DNA loss. On the other hand, the optimized first buffer can ensure that the chromatographic medium stably binds to the supercoiled plasmid DNA, and effectively elutes the open-circular plasmid DNA and RNA. In addition, the optimized buffer can effectively elute the supercoiled plasmid DNA from the chromatographic medium. Therefore, the present invention effectively removes RNA in the crude product by optimizing the equilibration, washing and elution buffers, and can separate the open-circular plasmid DNA and the supercoiled plasmid DNA, thereby improving the purity of the purified plasmid DNA.

[0031] 3. The plasmid DNA purification method provided by the present invention can be applied to a variety of plasmids and can match the buffer commonly used in the current prior art, significantly improving the quality of large-scale production of purified plasmid DNA, shortening the purification time, and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 It is a chromatogram of each eluent in the purification process of plasmid DNA in Example 1 of the present invention, wherein the flow-through peak is the sample to be tested in the loading process, the impurity peak is the first eluent, and the elution peak is the second eluent;

[0034] Figure 2 This is a gel electrophoresis diagram of the crude plasmid DNA, the sample to be tested, the first eluate and the second eluate in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0036] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0037] The present invention does not limit the preparation method of the crude plasmid DNA, as long as the plasmid DNA can be obtained. The present invention provides a method for preparing a crude plasmid DNA, and the specific steps are as follows:

[0038] The E. coli fermentation broth containing plasmid DNA was centrifuged, and after removing the supernatant, a cell resuspension solution (50 mM glucose, 25 mM Tris, 10 mM EDTA, pH 8.0) was injected into the bacterial sludge, wherein the mass volume ratio of the bacterial sludge to the cell resuspension solution was 1:10, g / mL. After thorough mixing, a lysis solution (0.2 M NaOH, 1% SDS) twice the volume of the cell resuspension solution was slowly added, and gently stirred for 5 min for thorough lysis. The neutralization solution (3 M KAc, 2 M HAc, pH 8.0) was added according to a volume ratio of the cell resuspension solution to the neutralization solution of 1:1. 5.5) adding the obtained product to the fully lysed solution, gently mixing, and standing at 4°C for 30 min to obtain a crude product, filtering the crude product through a 10 μm filter to obtain a filtrate, filtering the filtrate through a 0.45 μm capsule filter to obtain a clarified liquid, and concentrating the clarified liquid through ultrafiltration using a hollow fiber to obtain a concentrated liquid, and diluting the concentrated liquid to a certain concentration to obtain a crude plasmid DNA product, wherein the buffer system used for ultrafiltration and concentration is 0.1 M Tris-HCl, 10 mM EDTA, pH 7.5.

[0039] The chromatographic columns of the embodiments of the present invention and the comparative examples are composite mode chromatographic columns, which can be commercially available. The embodiments of the present invention and the comparative examples use composite mode chromatographic columns provided by Xi'an Lanxiao Technology New Materials Co., Ltd. The filler is Pd oneS composite chromatographic medium provided by Xi'an Lanxiao Technology New Materials Co., Ltd. The ligand type is a hydrophobic-anionic composite mode. The chromatographic medium is a porous microsphere cross-linked with a polystyrene-divinylbenzene copolymer, the particle size of the microsphere is 10-1000 μm, the average pore size is 100-1000 nm, and the specific surface area is 10-100 m 2 / g, true density is 0.95-1.15g / mL. Column temperature is room temperature, column volume is 5mL.

[0040] Example 1

[0041] This embodiment provides a method for purifying plasmid DNA, and the specific steps and parameters are as follows:

[0042] A Tris-HCl buffer having a pH value of 7.5 and a concentration of 100 mM containing 0.5 M sodium chloride, 0.5 M ammonium sulfate, 5% (mass percentage) isopropanol, and 10 mM EDTA is prepared as an equilibrium buffer;

[0043] A Tris-HCl buffer having a pH value of 7.5 and a concentration of 100 mM containing 0.8 M sodium chloride, 0.5 M ammonium sulfate, and 10 mM EDTA is prepared as the first buffer;

[0044] A Tris-HCl buffer having a pH value of 7.5 and a concentration of 100 mM containing 1.5 M sodium chloride, 0.5 M ammonium sulfate, and 10 mM EDTA is prepared as the second buffer;

[0045] Ammonium sulfate and isopropanol were added to the crude plasmid DNA prepared by conventional alkaline lysis method, and the mixture was gently stirred at room temperature so that the final concentration of ammonium sulfate in the crude plasmid DNA was 0.5 M and the mass fraction of isopropanol was 5%. The sample was allowed to stand for 30 min to obtain a sample. The plasmid concentration was 0.5 mg / mL using HPLC. The plasmid DNA in this example was pET30a-GFP.

[0046] The column was balanced with 5CV of equilibrium buffer until the conductivity, pH value, and absorbance at 260nm of the column were balanced. Then, 10mL of sample was loaded onto the column at a flow rate of 1mL / min. After the loading was completed, a sample was taken and recorded as the sample to be tested.

[0047] Load 5CV of the first buffer solution onto the loaded chromatography column at a flow rate of 3mL / min to elute the chromatography column once, and take the eluate from the elution once as the first eluate;

[0048] Load 5 CV of the second buffer at a flow rate of 3 mL / min onto the chromatography column after the first elution to perform a second elution on the chromatography column, and take the eluate from the second elution as the second eluate;

[0049] The second eluate is the purified pure supercoiled plasmid DNA.

[0050] The elution process of this embodiment was detected by an ultraviolet detector, and the ultraviolet absorbance wavelength was 260nm, see Figure 1 As shown, it can be seen that the separation degree between the separated peaks in this embodiment is good.

[0051] The crude plasmid DNA, the sample to be tested, the first eluate and the second eluate were subjected to 1% agarose gel electrophoresis (with a sample load of 10 μg). The test results are shown in Figure 2 In this embodiment, the size of the open circular plasmid DNA is 6600 kb, and the size of the supercoiled plasmid DNA is about 5500 kb. Figure 2 It can be seen that the purification method of this embodiment can effectively separate the open-circular plasmid DNA, RNA and supercoiled plasmid DNA in the crude plasmid DNA.

[0052] Example 2

[0053] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1. The only difference is that the inorganic salt used in the pretreatment of the plasmid DNA is calcium chloride, the final concentration of calcium chloride in the crude plasmid DNA is 0.5 M, and the mass fraction of isopropanol is 5%.

[0054] Example 3

[0055] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1. The only difference is that the inorganic salt used in the pretreatment of the plasmid DNA is sodium chloride, the final concentration of sodium chloride in the crude plasmid DNA is 0.5 M, and the mass fraction of isopropanol is 5%.

[0056] Example 4

[0057] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1. The only difference is that only organic solvents are used in the pretreatment of plasmid DNA without inorganic salts, that is, the mass fraction of isopropanol in the crude plasmid DNA is 5%.

[0058] Example 5

[0059] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1. The only difference is that the organic solvent used in the pretreatment of the plasmid DNA is ethanol, the final concentration of ammonium sulfate in the crude plasmid DNA is 0.5 M, and the mass fraction of ethanol is 5%.

[0060] Example 6

[0061] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the final concentration of ammonium sulfate in the crude plasmid DNA is 0.1 M, and the mass fraction of isopropanol is 5%.

[0062] Example 7

[0063] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the final concentration of ammonium sulfate in the crude plasmid DNA is 1 M, and the mass fraction of isopropanol is 5%.

[0064] Example 8

[0065] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the final concentration of ammonium sulfate in the crude plasmid DNA is 0.5 M, and the mass fraction of isopropanol is 1%.

[0066] Example 9

[0067] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the final concentration of ammonium sulfate in the crude plasmid DNA is 0.5 M, and the mass fraction of isopropanol is 15%.

[0068] Example 10

[0069] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the equilibration buffer is 0.1 M.

[0070] Embodiment 11

[0071] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the equilibration buffer is 0.25 M.

[0072] Example 12

[0073] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the equilibrium buffer is 0.75 M.

[0074] Example 13

[0075] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the equilibration buffer is 1 M.

[0076] Embodiment 14

[0077] This embodiment provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the first buffer is 1 M.

[0078] Embodiment 15

[0079] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the second buffer is 2M.

[0080] Example 16

[0081] This embodiment provides a method for purifying plasmid DNA, and the specific steps and parameters are as follows:

[0082] A phosphate buffer with a pH value of 7.5 and a concentration of 100 mM containing 0.5 M sodium chloride, 0.5 M ammonium sulfate, 5% (mass percentage) isopropanol, and 5 mM EDTA is prepared as an equilibrium buffer;

[0083] A phosphate buffer having a pH value of 7.5 and a concentration of 100 mM containing 0.8 M sodium chloride, 0.5 M ammonium sulfate, and 10 mM EDTA is prepared as the first buffer;

[0084] A phosphate buffer solution with a pH value of 7.5 and a concentration of 100 mM containing 1 M sodium chloride and 20 mM EDTA is prepared as the second buffer solution;

[0085] Ammonium sulfate and isopropanol were added to the crude plasmid DNA prepared by conventional alkaline lysis method, and the mixture was gently stirred at room temperature so that the final concentration of ammonium sulfate in the crude plasmid DNA was 0.5 M and the mass fraction of isopropanol was 5%. The sample was allowed to stand for 30 min to obtain a sample. The plasmid concentration was 0.5 mg / mL using HPLC. The plasmid DNA in this example was pET30a-GFP.

[0086] The column was balanced with 5CV of equilibrium buffer until all parameters of the chromatograph were balanced, and then 10mL of sample was loaded onto the column at a flow rate of 0.1mL / min. After the loading was completed, a sample was taken and recorded as the sample to be tested;

[0087] Load 5 CV of the first buffer onto the loaded chromatography column at a flow rate of 0.1 mL / min to elute the chromatography column once, and take the eluate from the elution once as the first eluate;

[0088] Load 5 CV of the second buffer at a flow rate of 20 mL / min onto the chromatography column after the first elution to perform a second elution on the chromatography column, and take the eluate from the second elution as the second eluate;

[0089] The second eluate is the purified supercoiled plasmid DNA.

[0090] Embodiment 17

[0091] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that:

[0092] A MES buffer with a pH value of 7.5 and a concentration of 100 mM containing 0.5 M sodium chloride, 0.5 M ammonium sulfate, 5% (mass percentage) isopropanol, and 10 mM EDTA was prepared as an equilibrium buffer;

[0093] A MES buffer having a pH value of 7.5 and a concentration of 100 mM containing 0.5 M sodium chloride, 0.5 M ammonium sulfate, and 10 mM EDTA is prepared as the first buffer;

[0094] A MES buffer having a pH value of 7.5 and a concentration of 100 mM containing 1.25 M sodium chloride and 10 mM EDTA was prepared as the second buffer.

[0095] Embodiment 18

[0096] This example provides a method for purifying plasmid DNA. The specific steps and parameters are the same as those in Example 1, except that:

[0097] A HEPES buffer with a pH value of 7.5 and a concentration of 100 mM containing 0.5 M sodium chloride, 0.5 M ammonium sulfate, 5% (mass percentage) isopropanol, and 10 mM EDTA was prepared as an equilibrium buffer;

[0098] A HEPES buffer having a pH value of 7.5 and a concentration of 100 mM containing 0.8 M sodium chloride, 0.5 M ammonium sulfate, and 10 mM EDTA is prepared as the first buffer;

[0099] A HEPES buffer having a pH value of 7.5 and a concentration of 100 mM containing 1.5 M sodium chloride and 10 mM EDTA was prepared as the second buffer.

[0100] Comparative Example 1

[0101] This comparative example provides a method for purifying plasmid DNA, and the specific steps and parameters are the same as those of Example 1, except that the step of pre-treatment of plasmid DNA is not included, that is, the crude plasmid DNA is directly loaded onto the equilibrated chromatography column, and no inorganic salt and organic solvent are added to the plasmid DNA. The experimental results show that 55% of the plasmid DNA flows through, resulting in the loss of plasmid DNA.

[0102] Comparative Example 2

[0103] This comparative example provides a method for purifying plasmid DNA, and the specific steps and parameters are the same as those in Example 1, except that the final concentration of ammonium sulfate in the crude plasmid DNA is 2M, and the mass fraction of isopropanol is 5%. The experimental results show that 2M causes some nucleic acids in the sample to precipitate, ultimately affecting the yield of plasmid DNA.

[0104] Comparative Example 3

[0105] This comparative example provides a method for purifying plasmid DNA, and the specific steps and parameters are the same as those in Example 1, except that the mass fraction of isopropanol in the crude plasmid DNA is 20%, and the concentration of ammonium sulfate is 0.5 M. The experimental results show that the addition of 20% isopropanol by mass will cause part of the RNA to be deposited in the chromatographic medium, affecting the separation effect of RNA and plasmid DNA. In addition, isopropanol greater than 15% does not substantially improve the purification effect.

[0106] Comparative Example 4

[0107] This comparative example provides a method for purifying plasmid DNA, and the specific steps and parameters are the same as those in Example 1, except that the equilibration buffer does not contain sodium chloride. The experimental results show that no plasmid DNA flows through during the loading process, but the RNA residue is serious, resulting in excessive RNA residue in the obtained supercoiled plasmid DNA sample.

[0108] Comparative Example 5

[0109] This comparative example provides a plasmid DNA purification method, the specific steps and parameters are the same as those of Example 1, except that the first buffer does not contain sodium chloride. The experimental results show that the effective separation of RNA, open plasmid and supercoiled plasmid DNA cannot be achieved during the washing process.

[0110] Comparative Example 6

[0111] This comparative example provides a method for purifying plasmid DNA, and the specific steps and parameters are the same as those in Example 1, except that the second buffer does not contain sodium chloride. The experimental results show that plasmid DNA cannot be eluted from the chromatography medium during the elution process, and the yield is almost zero.

[0112] Comparative Example 7

[0113] This embodiment provides a method for purifying plasmid DNA, and the specific steps and parameters are the same as those in Example 1, except that the concentration of sodium chloride in the second buffer is 2.5 M. The experimental results show that high salt concentrations can cause sedimentation of plasmid DNA samples, which is not conducive to sample preservation. In addition, high salt concentration elution does not substantially improve the recovery rate of supercoiled plasmid DNA.

[0114] Experimental Example 1

[0115] Plasmid DNA flow-through detection was performed on the samples to be tested in Examples 1-9 and Comparative Example 1, and the detection method was as follows:

[0116] Opened plasmid DNA: 1% agarose gel electrophoresis and HPLC were used to detect the content of the opened plasmid DNA.

[0117] Supercoiled plasmid DNA: 1% agarose gel electrophoresis and HPLC were used to detect the content of supercoiled plasmid DNA.

[0118] The test results are shown in Table 1.

[0119] Table 1 Results of plasmid DNA flow-through detection of the samples to be tested

[0120]

[0121]

[0122] Note: Plasmid DNA flow-through, % = plasmid DNA content of the sample to be tested / plasmid DNA content of the crude plasmid DNA. The plasmid DNA content is the sum of the open-circular plasmid DNA and supercoiled plasmid DNA.

[0123] It can be seen that after the alkaline-lysed plasmid DNA sample contains at least one of 0.05M-1M ammonium sulfate, calcium chloride, sodium chloride and / or at least one of 1%-15% ethanol or isopropanol, the amount of plasmid DNA flow-through during the loading process is small, especially for the plasmid DNA sample containing 0.5M ammonium sulfate and 5% isopropanol, no plasmid DNA flow-through occurs during the loading process, while Comparative Example 1 does not use organic solvents or inorganic salts to pre-treat the plasmid DNA, resulting in a large amount of plasmid DNA flow-through, affecting the yield of the plasmid DNA.

[0124] Experimental Example 2

[0125] The open-circle plasmid DNA flowthrough and RNA flowthrough detection were performed on the samples to be tested in Examples 1, 10-13 and Comparative Example 4, and the detection method was as follows:

[0126] Opened plasmid DNA: 1% agarose gel electrophoresis and HPLC were used to detect the content of the opened plasmid DNA.

[0127] Supercoiled plasmid DNA: 1% agarose gel electrophoresis and HPLC were used to detect the content of supercoiled plasmid DNA.

[0128] RNA content: 1% agarose gel electrophoresis and enzyme immunoassay (ELISA).

[0129] The test results are shown in Table 2.

[0130] Table 2 Open-circle plasmid DNA flow-through and RNA flow-through detection results of the samples to be tested during the loading process

[0131]

[0132]

[0133] Note: Open-circular plasmid DNA flow-through, % = open-circular plasmid DNA content of the sample to be tested / open-circular plasmid DNA content of the crude plasmid DNA;

[0134] RNA flow-through, % = RNA flow-through amount of the sample to be tested / total RNA amount in the crude plasmid DNA.

[0135] It can be seen that compared with comparative example 4 in which no sodium chloride is added to the equilibration buffer, the equilibration buffers containing different concentrations of sodium chloride used in Examples 10-13 can effectively flow through the RNA of the plasmid DNA test sample, while avoiding the flow through of the open-circular plasmid DNA. In particular, the equilibration buffer containing 0.5 M sodium chloride can not only allow most of the RNA to flow through, but also ensure that the plasmid DNA structure is not destroyed.

[0136] Experimental Example 3

[0137] The contents of open-circular plasmid DNA, supercoiled plasmid DNA and RNA in the first eluate in Examples 1, 14 and Comparative Example 5 were detected by the following method:

[0138] Opened plasmid DNA: 1% agarose gel electrophoresis and HPLC were used to detect the content of the opened plasmid DNA.

[0139] Supercoiled plasmid DNA: 1% agarose gel electrophoresis and HPLC were used to detect the content of supercoiled plasmid DNA.

[0140] RNA content: 1% agarose gel electrophoresis and enzyme immunoassay (ELISA).

[0141] The test results are shown in Table 3.

[0142] Table 3 Test results of samples collected by the first eluent

[0143]

[0144] Note: Plasmid DNA flow-through, % = the content of open-circular plasmid DNA or supercoiled plasmid DNA in the first elution solution / the crude plasmid DNA content of plasmid DNA. The crude plasmid DNA content is the content of open-circular plasmid DNA or supercoiled plasmid DNA in the crude product. RNA flow-through, % = the total flow-through amount of RNA in the sample loading process and the first buffer elution process / the total amount of RNA in the crude product.

[0145] It can be seen that the use of a buffer containing 0.8M-1M sodium chloride in Example 1 and 14 of the present invention to remove impurities from the plasmid DNA on the chromatographic column can effectively remove RNA and open-circular plasmid DNA that cannot be removed by the equilibrium buffer. Combined with the data in Table 2, it can be seen that the use of a first buffer containing 0.8M sodium chloride in Example 1 of the present invention can effectively remove 99% of RNA and effectively separate open-circular plasmid DNA and supercoiled plasmid DNA. If the first buffer containing 1M sodium chloride is used, a small amount of supercoiled plasmid DNA will flow through, and as shown in Comparative Example 5, the first buffer without sodium chloride can basically not achieve effective separation of RNA, open-circular plasmid DNA and supercoiled plasmid DNA, and the purpose of purification cannot be achieved.

[0146] Experimental Example 4

[0147] The contents of open-circular plasmid DNA, supercoiled plasmid DNA and RNA in the second eluate in Examples 1, 15 and Comparative Examples 1-7 were detected by the same detection method as that in Experimental Example 3. The loading amount of the crude plasmid DNA as described in Example 1 was 5 mg (excluding the amount of RNA), and the detection results are shown in Table 4.

[0148] Table 4 Quality test results of purified samples obtained from the second eluent

[0149]

[0150]

[0151] Note: The content of collected purified plasmid DNA / mg = the content of supercoiled plasmid DNA in the purified plasmid DNA.

[0152] It can be seen that the second buffer containing 1M-2M sodium chloride in the present invention can elute the supercoiled plasmid DNA bound to the chromatography column, especially the second buffer containing 1.5M sodium chloride can elute most of the supercoiled plasmid DNA. In Comparative Example 1, a large amount of plasmid DNA flows through during the purification process without inorganic salt and organic solvent pretreatment, resulting in a decrease in the recovery rate of plasmid DNA. Comparative Example 2 uses a high concentration of inorganic salts, and Comparative Example 3 uses a high content of organic solvents, which will cause plasmid DNA loss and affect the total yield. When balancing the resin column in Comparative Example 4, the balancing buffer used does not contain sodium chloride, and RNA cannot flow through, resulting in low purity of the plasmid DNA. The first buffer containing no sodium chloride used in Comparative Example 5 affects the elution of the open-circular plasmid DNA, resulting in low purity of the finally purified plasmid DNA. The second buffer containing no sodium chloride in Comparative Example 6 cannot effectively elute the supercoiled plasmid DNA in the resin column, resulting in extremely low purity and recovery rate. Although the second buffer solution containing 2.5 M sodium chloride in Comparative Example 7 has little effect on the recovery rate and purity, the high salt concentration will cause the sedimentation of the plasmid DNA sample, which is not conducive to the preservation of the sample.

[0153] Experimental Example 5

[0154] The purified plasmid DNA obtained in Example 1 was subjected to quality inspection. The quality inspection content, inspection method, evaluation criteria and inspection results are shown in Table 5.

[0155] Table 5 Plasmid DNA quality test results

[0156] Test items Detection Methods standard Test results Supercoiled plasmid DNA purity High performance liquid chromatography More than 80% 95％ Residual RNA Enzyme immunoassay (ELISA) Less than 1% 0.06％ Host protein residues Q-PCR Less than 1% 0.02％ Host DNA residue RT-QPCR Less than 1% 0.03％ endotoxin Endotoxin Detection Kit Less than 10EU / mg 6.8EU / mg

[0157] It can be seen that the plasmid DNA purification method provided in the embodiment of the present invention can effectively improve the supercoil purity of plasmid DNA and ensure that important indicators such as RNA residues, host protein residues and endotoxins meet the quality requirements of nucleic acid drug raw materials.

[0158] Application Examples

[0159] Crude plasmid DNA containing about 100 mg of pUC19-GFP, pET30a-GFP, pBR322-GFP, pKD46-GFP, pGEX6P-GFP, pQE30-GFP, pCMV-GFP, pMAL-GFP, pcDNA-GFP, and pFastBac1-GFP was prepared respectively, and purified using the steps and parameters of Example 1. The supercoiled plasmid content of the purified plasmid DNA was detected by gel electrophoresis and HPLC. The detection results are shown in Table 6.

[0160] Table 6 Purification results of different plasmids

[0161]

[0162] It can be seen that the plasmid DNA purification method of the embodiment of the present invention can be applied to a variety of plasmids, and the purity and recovery rate of supercoiled plasmids are high.

[0163] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for purifying plasmid DNA, characterized in that: The following steps are included: S1, mixing an inorganic salt and / or an organic solvent with plasmid DNA to obtain a sample; S2, after the chromatography column is equilibrated with the equilibration buffer, the sample obtained in step S1 is loaded onto the chromatography column; S3, after the chromatography column is eluted once with the first buffer, the chromatography column is eluted twice with the second buffer to obtain purified supercoiled plasmid DNA.

2. The method for purifying plasmid DNA according to claim 1, characterized in that The inorganic salt in step S1 is at least one of calcium chloride, ammonium sulfate, ammonium bicarbonate and sodium chloride; and / or, The organic solvent is at least one of ethanol and isopropanol.

3. The method for purifying plasmid DNA according to claim 2, characterized in that: The inorganic salt concentration in the sample is 0.1M-1M; and / or, The mass fraction of the organic solvent is 1%-15%; and / or, The inorganic salt is ammonium sulfate; and / or, The organic solvent was isopropyl alcohol.

4. The method for purifying plasmid DNA according to claim 3, characterized in that: The equilibration buffer and / or the first buffer and / or the second buffer comprises sodium chloride at a concentration of 0.1-2M.

5. The method for purifying plasmid DNA according to claim 4, characterized in that: The equilibration buffer and / or the first buffer and / or the second buffer further comprise an inorganic salt, an organic solvent and EDTA.

6. The method for purifying plasmid DNA according to any one of claims 1 to 5, characterized in that: The equilibration buffer and / or the first buffer and / or the second buffer is at least one of a Tris hydrochloride buffer, a phosphate buffer, a 2-morpholineethanesulfonic acid buffer and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer; Optionally, the inorganic salt concentration in the equilibration buffer and / or the first buffer and / or the second buffer is 0.1-2M; Optionally, the mass fraction of the organic solvent is 1%-15%; Optionally, the EDTA concentration is 5 mM-20 mM.

7. The method for purifying plasmid DNA according to claim 6, characterized in that: The concentration of each of the buffers is 0.1-0.5M.

8. The method for purifying plasmid DNA according to claim 4, characterized in that: The sodium chloride concentration in the equilibration buffer is 0.1-1M; and / or, The concentration of sodium chloride in the first buffer is 0.5-1M; and / or, The concentration of sodium chloride in the second buffer is 1-2M.

9. A plasmid DNA obtained by the method for purifying plasmid DNA according to any one of claims 1 to 8.

10. A method for purifying the plasmid DNA according to any one of claims 1 to 8 or use of the plasmid DNA according to claim 9 in vaccine preparation.

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