Plasmid purification method for improving superhelix ratio

Abstract

The invention relates to a plasmid purification method for improving a superhelix ratio. The method comprises the following steps: (1) carrying out flocculation impurity removal, microfiltration and ultrafiltration concentration on a pyrolysis neutralization solution of fermentation thalli to obtain a plasmid crude extraction concentrated solution; (2) carrying out affinity chromatography purification on the plasmid crude extraction concentrated solution, wherein a chromatography filler adopted by the affinity chromatography purification is Capto Plasmid Select produced by Cytiva Company; and (3) carrying out anion chromatography purification on the plasmid solution after the affinity chromatography purification, wherein the chromatography filler adopted by the anion chromatography purification is Capto Q ImpRes produced by Cytiva Company. The plasmid purification method disclosed by the invention can be used for effectively removing polymers generated by fermentation, reducing the open-loop proportion and improving the superhelix monomer proportion of the plasmid, the pressure of upstream development of thalli with high expression quantity and high superhelix proportion is greatly relieved, and the plasmid purification method disclosed by the invention is simple and efficient, can be effectively enlarged to a production scale and has a relatively high practical value.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a plasmid purification method for improving supercoil ratio. Background Art

[0002] With the continuous development of the biopharmaceutical industry, the research and development and application of cell and gene therapy in my country are accelerating. High-quality and high-purity plasmid DNA is a key component in the process of cell and gene therapy. There are three configurations of plasmid DNA: supercoiled, open-circular and linear. The supercoiled configuration is a state in which two DNA chains are covalently closed. This configuration is the most stable and effective in gene transcription and replication. The supercoil ratio refers to the ratio of supercoiled configurations in plasmid DNA. The supercoil ratio of the plasmid will seriously affect the efficiency of transfection, which is crucial for the integrity of gene therapy and in vitro transcription (IVT) products. Plasmid DNA with a high supercoil ratio can increase the yield and expression efficiency of RNA. If the supercoil ratio of the plasmid is too low, the impurities it contains will seriously affect the formation of the transfection complex and further affect the subsequent in vivo efficacy. In addition, during the fermentation and expansion culture process, the homologous regions between plasmids produce aggregates through homologous recombination, but in the current purification process, there is no more effective way to remove aggregates. In order to promote the application of plasmids in the medical field, it is necessary to develop a purification method that can increase the supercoil ratio and reduce the aggregates produced during the fermentation process. Summary of the invention

[0003] The technical problem solved by the present invention is to overcome the deficiencies of the prior art and provide a purification method capable of increasing the supercoil ratio of plasmids and reducing the open ring ratio and the aggregate ratio. The purification method is more suitable for large-scale industrial plasmid purification processing.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] The present invention provides a plasmid purification method for improving the supercoil ratio, and the plasmid purification method comprises the following steps:

[0006] (1) The lysed and neutralized solution of the fermented bacteria is subjected to flocculation, impurity removal, microfiltration and ultrafiltration to obtain a plasmid crude extract concentrate;

[0007] (2) Purifying the crude plasmid concentrate by affinity chromatography, wherein the chromatographic filler used in the affinity chromatography purification is Capto Plasmid Select produced by Cytiva;

[0008] (3) The plasmid solution purified by affinity chromatography was purified by anion chromatography, wherein the chromatography filler used in the anion chromatography purification was Capto Q ImpRes produced by Cytiva.

[0009] In the embodiment of the present invention, in the step (2), the affinity chromatography purification process is: equilibration, loading, re-equilibration, washing and elution.

[0010] Specifically, the balancing and rebalancing use Buffer B, the formula of which is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 1.8-2.2 M (NH 4 ) 2 SO 4 , balance water.

[0011] The sample loading is a mixture of the plasmid crude extract concentrate and Buffer K, wherein the formula of Buffer K is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 3.2-3.8 M (NH 4 ) 2 SO 4 , balance water.

[0012] The washing method uses a mixture of Buffer A and Buffer E in a volume ratio of (8-10):1, wherein the formula of Buffer A is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 2.0-2.15 M (NH 4 ) 2 SO 4 , the balance is water; the formula of the Buffer E is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 0.8-1.2 M NaCl, and the balance is water.

[0013] The elution adopts Buffer C, and the formula of Buffer C is: 90-110mM Tris, 8-12mM EDTA·2Na, 0.2-0.4M NaCl, 1.5-1.8M (NH 4 ) 2 SO 4 , balance water.

[0014] According to some specific embodiments of the present invention, the column height of the chromatography column used in the affinity chromatography is 15-18 cm, the column diameter is 25-28 cm, the HETP is 1400-1500, and the upper limit of the sample loading capacity is 1.60 mg / mL.

[0015] Furthermore, the step (2) is specifically as follows:

[0016] Balance: Use the Buffer B to balance the chromatography column for 2 to 5 CV, with a linear flow rate of 80 to 100 cm / h;

[0017] Loading: Load the mixture of the plasmid crude concentrate and Buffer K at a flow rate of 80-100 cm / h;

[0018] Re-equilibration: flush the chromatography column with the Buffer B for 8 to 12 CV at a linear flow rate of 80 to 100 cm / h;

[0019] Washing: Use the mixture of Buffer A and Buffer E to wash the chromatography column for 2 to 5 CV at a linear flow rate of 50 to 70 cm / h;

[0020] Elution: Use the Buffer C to rinse the chromatography column at a linear flow rate of 50 to 70 cm / h. Observe the change of UV260 during the elution process. Start collecting when UV260 rises to 1000 mAu, pause for 10 to 30 min when UV260 drops to 500 mAu, and then continue eluting until UV260 drops to 200 mAu and stop collecting.

[0021] In an embodiment of the present invention, in the step (3), the anion chromatography purification process is: equilibration, loading, re-equilibration, washing and gradient elution.

[0022] Specifically, the balance and the re-balance use Buffer D, and the formula of Buffer D is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 0.2-0.5 M NaCl, and the balance is water.

[0023] The washing adopts a mixed solution of Buffer D and Buffer E in a volume ratio of 1: (1.1-1.5), and the formula of the Buffer E is: 90-110mM Tris, 8-12mM EDTA·2Na, 0.8-1.2M NaCl, and the balance water; the linear elution adopts a mixed linear gradient elution of Buffer D and the Buffer E, the proportion of the Buffer D increases from 46% to 53%, and the proportion of the Buffer E decreases from 54% to 47%.

[0024] According to some specific embodiments of the present invention, the column height of the chromatography column used for the anion chromatography purification is 30-32 cm, the column diameter is 1.5-1.8 cm, the HETP is 4800-4900, and the upper limit of the sample loading capacity is 1.0 mg / mL.

[0025] Furthermore, the step (3) is specifically as follows:

[0026] Balance: Use the Buffer D to balance the chromatography column for 2-5 CV, with a linear flow rate of 80-100 cm / h;

[0027] Loading: The eluate purified by affinity chromatography is used for loading, and the loading flow rate is 80-100 cm / h;

[0028] Re-equilibration: using the Buffer D to equilibrate the chromatography column for 4-8 CV, with a linear flow rate of 80-100 cm / h;

[0029] Washing: The mixture of Buffer D and Buffer E is used to wash the chromatography column for 2 to 5 CV at a linear flow rate of 80 to 100 cm / h;

[0030] Gradient elution: using the Buffer D and the Buffer E mixed linear gradient elution, the gradient is Buffer D (46%-53%): Buffer E (54%-47%), the elution volume is 10-15CV, the change of UV260 is observed during the elution process, and the collection starts at 0.3CV after UV260 rises to 100mAu, and the collection is terminated after the collection volume reaches 2.1±0.1CV.

[0031] In an embodiment of the present invention, in the step (1), the flocculant for flocculation and impurity removal is calcium chloride.

[0032] In an embodiment of the present invention, in the step (1), the microfiltration includes pre-filtration using a 45-55 μm capsule filter and secondary filtration using a 0.4-0.6 μm capsule filter.

[0033] In the embodiment of the present invention, in the step (1), the ultrafiltration concentration includes first using a 90-110KD hollow fiber column to concentrate 30-60 times, and then using a 10×TE solution to replace the solution and concentrate 5-6 times.

[0034] In an embodiment of the present invention, the preparation method of the lysis and neutralization liquid of the fermented bacteria is: using a 700-800KD hollow fiber column to ultrafilter, concentrate and diafilter the fermented bacterial liquid, adding a heavy suspension to mix to obtain a bacterial suspension, mixing the bacterial suspension and the lysis liquid for alkaline lysis, and then adding a neutralizing liquid for acid neutralization.

[0035] Specifically, the formula of the resuspension is: 8-10 g / kg glucose, 3.5-4 g / kg edetate disodium, 6.05 g / kg tromethamine, and the rest is water.

[0036] Specifically, the formula of the lysate is: 7.5-8.5 g / kg sodium hydroxide, 9.5-10 g / kg sodium dodecyl sulfate, and the rest is water.

[0037] Specifically, the formula of the neutralizing solution is: 250-255 g / kg potassium acetate, 270-275 g / kg glacial acetic acid, and the rest is water.

[0038] In the embodiment of the present invention, the fermentation cell is an Escherichia coli cell containing the target plasmid.

[0039] In the embodiment of the present invention, the size of the target plasmid is 3Kb to 7Kb.

[0040] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0041] The plasmid purification method of the present invention can effectively remove polymers produced by fermentation, reduce the ratio of open rings, and increase the ratio of plasmid supercoiled monomers, which greatly alleviates the pressure of upstream development of bacteria with high expression levels and high supercoiled ratios. The method of the present invention is simple and efficient, can be effectively scaled up to production, and has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the AEX-HPLC detection spectrum of the purified plasmid in Example 1;

[0043] Figure 2 This is the AEX-HPLC detection spectrum of the purified plasmid of Comparative Example 1;

[0044] Figure 3 This is the AEX-HPLC detection spectrum of the purified plasmid of Comparative Example 2. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0046] In the present invention, unless otherwise specified, the instruments, raw materials and reagents used can be obtained from commercial sources.

[0047] The preparation method of the crude plasmid concentrate in the following examples and comparative examples comprises the following steps:

[0048] (1) Fermentation and expansion culture

[0049] The Escherichia coli containing the target plasmid (size of about 5002 bp) was cultured in a fermenter. The initial culture medium for Escherichia coli fermentation was LB culture medium supplemented with 6 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1.5 g / L anhydrous magnesium sulfate, 12.6 g / L glucose, 2 g / L ammonium sulfate, and 100 μL / L defoamer. The fermentation time was 30 h, and the feeding strategy was OD 600Feeding began when OD was ≥15, and the feeding was continued until OD recovered: 3ml / L / h; dissolved oxygen recovered until 18h of culture: 6ml / L / h; cultured for 24h: 8ml / L / h, and after 24h of culture: 12ml / L / h. The fermentation process was stable, and the expression level was about 108mg / L. The plasmid obtained by the plasmid extraction kit (DP103)-TIENGEN after the fermentation process was extracted, and the gel electrophoresis results showed that the supercoil ratio was 75.8%.

[0050] (2) Preparation of cleavage neutralization solution for subsequent purification

[0051] Use 750KD hollow fiber column to ultrafiltration, concentration and diafiltration of fermentation broth, membrane loading ≤50kg / m 2 , concentrated 4 times, and diafiltered twice with a heavy suspension (9.00 g / kg glucose, 3.72 g / kg disodium edetate, 6.05 g / kg tromethamine, the rest water). The shear rate during concentration and diafiltration was ≤4500 s -1 , TMP≤0.8bar, add 19 times the volume of heavy suspension (9.00g / kg glucose, 3.72g / kg disodium edetate, 6.05g / kg tromethamine, the rest is water), stir evenly to obtain bacterial suspension. The bacterial suspension and lysis solution (7.97g / kg sodium hydroxide, 9.96g / kg sodium dodecyl sulfate, the rest is water) are mixed evenly for alkaline lysis, and after lysis for 4-5min, neutralization solution (254.25g / kg potassium acetate, 272.02g / kg glacial acetic acid, the rest is water) is added for acid neutralization to obtain lysis neutralization solution, wherein the ratio of bacterial suspension, lysis solution and neutralization solution is 1:1.3(±0.15):1.3(±0.15).

[0052] Example 1

[0053] This embodiment provides a plasmid purification method for increasing the supercoil ratio, and the steps are as follows:

[0054] 1. Preparation of Plasmid Crude Extract Concentrate

[0055] Add 0.5 mol of calcium chloride per kilogram of cleavage neutralization liquid, stir evenly and let stand for 120 minutes. After standing, slowly add 60g of 10% ammonium bicarbonate solution to the bottom of the cleavage neutralization liquid. After adding, let stand for no less than 15 minutes to allow the flocculants and cleavage neutralization liquid to be clearly separated. After standing, extract the separated cleavage neutralization liquid. Use a 50μm capsule filter to pre-filter the cleavage neutralization liquid, and then use a 0.45μm capsule filter for clarification filtration, with a membrane load of ≤90kg / m 2, filtration pressure ≤1.5bar. Use 100KD hollow fiber column to ultrafiltration concentrate the clarified cleavage neutralization solution. During the ultrafiltration concentration process, the shear rate is ≤5000S -1 , TMP≤0.5bar, membrane load≤120kg / m 2 The clarified lysate neutralization solution was concentrated 50 times and then replaced with 10×TE buffer (3.70 g / kg disodium edetate, 12.06 g / kg tromethamine, the rest was water, pH=7.5) for 5 times concentration, and the concentrate was collected. The hollow fiber column was rinsed with 1 column volume of 10×TE buffer and the pipeline was emptied, and the recovery solution was collected. The concentrate was mixed with the recovery solution to obtain a plasmid crude extract concentrate.

[0056] 2. Affinity chromatography

[0057] The plasmid crude concentrate was mixed with Buffer K (100 mM Tris, 10 mM EDTA·2Na, 3.5 M (NH 4 ) 2 SO 4 , the balance water, pH 7.5) were mixed in a volume ratio of 1:1.5 as the loading sample.

[0058] Information of the chromatography column used: column height: 16 cm; column diameter: 26 mm; symmetry: 1.06, HETP: 1418.9, filler: Capto PlasmidSelect.

[0059] The operation process is as follows:

[0060] Balance: Use Buffer B (100mM Tris, 10mM EDTA·2Na, 2.0M (NH 4 ) 2 SO 4 , balance water, pH 7.5) equilibrated the column for 3 CV, with a linear flow rate of 90 cm / h;

[0061] Sample loading: The upper limit of sample loading is 1.60 mg / mL, and the sample loading flow rate is 90 cm / h;

[0062] Re-equilibration: Use Buffer B (100 mM Tris, 10 mM EDTA·2Na, 2.0 M (NH 4 ) 2 SO 4 , balance water, pH 7.5) to rinse the column for 10CV, with a linear flow rate of 90 cm / h;

[0063] Wash: Buffer A (100 mM Tris, 10 mM EDTA·2Na, 2.1 M (NH 4 )2 SO 4 , balance water, pH 7.5; 90%) & Buffer E (100 mM Tris, 10 mM EDTA·2Na, 1 M NaCl, balance water, pH 7.5; 10%) was used to wash the column for 3 CV at a linear flow rate of 60 cm / h;

[0064] Elution: Use Buffer C (100 mM Tris, 10 mM EDTA·2Na, 0.3 M NaCl, 1.7 M (NH 4 ) 2 SO 4 The column was rinsed with 1% 4% iodine (20% ethanol, balance water, pH 7.5) at a linear flow rate of 60 cm / h. The changes in UV260 were observed during the elution process. Collection began when UV260 rose to 1000 mAu, and was paused for 20 min when UV260 dropped to 500 mAu. Elution was then continued until UV260 dropped to 200 mAu, and collection was stopped.

[0065] 3. Anion chromatography

[0066] The solution collected by affinity chromatography was mixed with water at a volume ratio of 1:2 and used as the sample for anion chromatography.

[0067] Information of the chromatography column used: column height: 31.7 cm; column diameter: 1.6 mm; symmetry: 0.81; HETP: 4828.8, filler: Capto Q ImpRes.

[0068] The operation process is as follows:

[0069] Balance: Use Buffer D (100 mM Tris, 10 mM EDTA·2Na, 0.4 M NaCl, balance water, pH 7.5) to balance the column for 3 CV, linear flow rate 90 cm / h;

[0070] Sample loading: The upper limit of sample loading is 1.0 mg / mL, and the sample loading flow rate is 90 cm / h;

[0071] Re-equilibration: Use Buffer D (100 mM Tris, 10 mM EDTA·2Na, 0.4 M NaCl, balance water, pH 7.5) to equilibrate the column for 5 CV, with a linear flow rate of 90 cm / h;

[0072] Wash: 3CV of Buffer D (100mM Tris, 10mM EDTA·2Na, 0.4M NaCl, balance water, pH 7.5); 44%) & Buffer E (100mM Tris, 10mM EDTA·2Na, 1M NaCl, balance water, pH 7.5; 56%) mixed by double pump, linear flow rate 90cm / h;

[0073] Gradient elution: use Buffer D (100mM Tris, 10mM EDTA·2Na, 0.4M NaCl, balance water, pH7.5) & Buffer E (100mM Tris, 10mM EDTA·2Na, 1M NaCl, balance water, pH 7.5) mixed linear gradient elution, the gradient is Buffer D (46%-53%): Buffer E (54%-47%), and the elution volume is 12CV; observe the change of UV260 during the elution process, start collecting 0.3CV after UV260 rises to 100mAu; end collection after the collection volume reaches 2.1±0.1CV.

[0074] Comparative Example 1

[0075] This comparative example provides a plasmid purification method. This comparative example performs purification according to the method provided by the chromatography column manufacturer. The difference from the purification method of Example 1 is that there is no washing operation during the affinity chromatography purification process, no washing operation during the anion chromatography purification process, and the elution method is different. The steps of this comparative example are as follows:

[0076] 1. Prepare a plasmid crude concentrate in the same manner as in Example 1.

[0077] 2. Affinity chromatography

[0078] The plasmid crude extract concentrate was mixed with Buffer K (100 mM Tris, 10 mM EDTA·2Na, 3.5 M (NH 4 ) 2 SO 4 , the balance water, pH 7.5) were mixed in a volume ratio of 1:1.5 as the loading sample.

[0079] Information of the chromatography column used: column height: 16 cm; column diameter: 26 mm; symmetry: 1.06, HETP: 1418.9, filler: Capto PlasmidSelect.

[0080] The operation process is as follows:

[0081] Balance: Use Buffer B (100mM Tris, 10mM EDTA·2Na, 2.0M (NH 4 ) 2 SO 4 , balance water, pH 7.5) equilibrated the column for 3 CV, with a linear flow rate of 90 cm / h;

[0082] Sample loading: The upper limit of sample loading is 1.60 mg / mL, and the sample loading flow rate is 90 cm / h;

[0083] Re-equilibration: Use Buffer B (100 mM Tris, 10 mM EDTA·2Na, 2.0 M (NH 4 ) 2 SO 4 , balance water, pH 7.5) to rinse the column for 10CV, with a linear flow rate of 90 cm / h;

[0084] Elution: Use Buffer C (100 mM Tris, 10 mM EDTA·2Na, 0.3 M NaCl, 1.7 M (NH 4 ) 2 SO 4 The column was rinsed with 1% 4% iodine (20% ethanol, balance water, pH 7.5) at a linear flow rate of 60 cm / h. The changes in UV260 were observed during the elution process. Collection began when UV260 rose to 1000 mAu, and was paused for 20 min when UV260 dropped to 500 mAu. Elution was then continued until UV260 dropped to 200 mAu, and collection was stopped.

[0085] 3. Anion chromatography

[0086] The solution collected by affinity chromatography was mixed with water at a volume ratio of 1:2 and used as the sample for anion chromatography.

[0087] Information of the chromatography column used: column height: 31.7 cm; column diameter: 1.6 mm; symmetry: 0.81; HETP: 4828.8, filler: Capto Q ImpRes.

[0088] The operation process is as follows:

[0089] Balance: Use Buffer D (100 mM Tris, 10 mM EDTA·2Na, 0.4 M NaCl, balance water, pH 7.5) to balance the column for 3 CV, linear flow rate 90 cm / h;

[0090] Sample loading: The upper limit of sample loading is 1.0 mg / mL, and the sample loading flow rate is 90 cm / h;

[0091] Re-equilibration: Use Buffer D (100 mM Tris, 10 mM EDTA·2Na, 0.4 M NaCl, balance water, pH 7.5) to equilibrate the column for 5 CV, with a linear flow rate of 90 cm / h;

[0092] Elution: Use Buffer E (100mM Tris, 10mM EDTA·2Na, 1M NaCl, balance water, pH 7.5) to wash the column, the elution volume is 12CV, and the flow rate is 60cm / h; observe the changes in UV260 during the elution process, start collecting when UV260 rises to 1000mAu, and stop collecting when UV260 drops to 200mAu or baseline.

[0093] Comparative Example 2

[0094] This comparative example provides a plasmid purification method, including a three-step chromatography method, including gel chromatography, affinity chromatography and anion chromatography performed in sequence, and the chromatography purification is performed in the manner provided by the chromatography column manufacturer, as follows:

[0095] 1. Gel chromatography

[0096] The lysis neutralization solution was used as the loading sample.

[0097] Information of the chromatography column used: column height: 30 cm; column diameter: 50 mm; symmetry: 1.02, HETP: 7872, filler: 6FF.

[0098] The operation process is as follows:

[0099] Balance: Use Buffer A (100mM Tris, 10mM EDTA·2Na, 2.1M (NH 4 ) 2 SO 4 , balance water, pH 7.5) equilibrated the column for 2CV, linear flow rate 60 cm / h;

[0100] Sample loading: The upper limit of sample loading is one quarter of the column volume, and the sample loading flow rate is 60 cm / h;

[0101] Elution: Use Buffer A (100mM Tris, 10mM EDTA·2Na, 2.1M (NH 4 ) 2 SO 4 The chromatography column was rinsed with 1% 4% iodine (2% water, balance water, pH 7.5) at a linear flow rate of 60 cm / h. The change of UV260 was observed during the elution process. Collection began when UV260 rose to 1000 mAu, and collection was stopped when UV260 dropped to 200 mAu or UV passed the peak value and began to rise again.

[0102] 2. Affinity chromatography

[0103] The solution collected by gel chromatography was used as the affinity chromatography loading sample.

[0104] Information of the chromatography column used: column height: 16 cm; column diameter: 26 mm; symmetry: 1.15; HETP: 6800. Filler: Cytiva PlasmidSelect Xtra.

[0105] The operation process is as follows:

[0106] Balance: Use Buffer B (100mM Tris, 10mM EDTA·2Na, 2.0M (NH 4 ) 2 SO 4 , balance water, pH 7.5) equilibrated the column for 2CV, with a linear flow rate of 90 cm / h;

[0107] Sample loading: The upper limit of sample loading is 1.60 mg / mL, and the sample loading flow rate is 90 cm / h;

[0108] Re-equilibration: Use Buffer B (100 mM Tris, 10 mM EDTA·2Na, 2.0 M (NH 4 ) 2 SO 4 , balance water, pH 7.5) to rinse the column for 10CV, with a linear flow rate of 90 cm / h;

[0109] Elution: Use Buffer C (100 mM Tris, 10 mM EDTA·2Na, 0.3 M NaCl, 1.7 M (NH 4 ) 2 SO 4 The column was rinsed with 1% iodine (20% by weight, 1% water, pH 7.5) at a flow rate of 60 cm / h. The change of UV260 was observed during the elution process. Collection was started when UV260 rose to 1000 mAu, and collection was stopped when UV260 dropped to 200 mAu.

[0110] 3. Anion chromatography

[0111] The solution collected by affinity chromatography was mixed with water at a volume ratio of 1:2 and used as the sample for anion chromatography.

[0112] Information of the chromatography column used: column height: 31.7 cm; column diameter: 1.6 mm; symmetry: 0.81; HETP: 4828.8. Packing material: Capto Q ImpRes.

[0113] The operation process is as follows:

[0114] Balance: Use Buffer D (100 mM Tris, 10 mM EDTA·2Na, 0.4 M NaCl, balance water, pH 7.5) to balance the column for 2CV, linear flow rate 90 cm / h;

[0115] Sample loading: The upper limit of sample loading is 1.0 mg / mL, and the sample loading flow rate is 90 cm / h;

[0116] Re-equilibration: Use Buffer D (100 mM Tris, 10 mM EDTA·2Na, 0.4 M NaCl, balance water, pH 7.5) to equilibrate the column for 3 CV, with a linear flow rate of 90 cm / h;

[0117] Elution: Use Buffer E (100mM Tris, 10mM EDTA·2Na, 1M NaCl, balance water, pH 7.5) to wash the column at a flow rate of 60cm / h; observe the changes in UV260 during the elution process, start collecting when UV260 rises to 1000mAu, and stop collecting when UV260 drops to 200mAu.

[0118] The samples collected in the above Example 1, Comparative Example 1 and Comparative Example 2 were respectively concentrated by ultrafiltration to adjust the concentration to 1.0±0.1 mg / mL, and then filtered through a 0.22 μm capsule filter, and then the ratios of supercoiled plasmid DNA, open-circular plasmid DNA and polymers were calculated by anion exchange high performance liquid chromatography (AEX-HPLC). Figure 1 This is the AEX-HPLC detection spectrum of the purified plasmid in Example 1, Figure 2 This is the AEX-HPLC detection spectrum of the purified plasmid in Comparative Example 1, Figure 3 This is the AEX-HPLC detection spectrum of the purified plasmid of Comparative Example 2.

[0119] The AEX-HPLC detection method is as follows: accurately measure 200 μL each of the blank solution (same as mobile phase A), the system suitability sample solution (take the qualified plasmid solution and dilute the sample to 200 ng / μL with the diluent), and the test solution (diluted to 200 ng / μL with the diluent), and inject them into the chromatograph in sequence for analysis.

[0120] Mobile phase A: 100 mM Tris + 0.3 GuHCl, pH 8.0

[0121] Mobile phase B: 100 mM + 0.3 GuHCl + 1 M NaCl, pH 8.0

[0122] Diluent: Same as mobile phase A

[0123] Blank solution: Take the diluent as the blank solution.

[0124] Chromatographic column: Proteomix SAX-NP5 4.6*250mm, 5μL.

[0125] The detection method parameter settings are shown in Table 1.

[0126] Table 1

[0127]

[0128]

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

[0130] Table 2

[0131] Supercoil ratio (%) Open loop ratio (%) Polymer ratio (%) Example 1 93.18 0.74 3.77 Comparative Example 1 80.91 4.44 13.11 Comparative Example 2 84.96 3.23 11.49

[0132] The AEX-HPLC test results show that the supercoil ratio of Example 1 is significantly higher than that of Comparative Examples 1 and 2, and the ring-opening ratio and polymer ratio are significantly lower than those of Comparative Examples 1 and 2. Figures 1 to 3 From the comparison of the peak areas in , it can be seen that the supercoiled plasmid content of Example 1 is greater than that of Comparative Example 2 and greater than that of Comparative Example 1.

[0133] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the protection scope of the present invention. The present invention is not limited to the above embodiments, and any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for purifying a plasmid for increasing the supercoil ratio, characterized in that: The plasmid purification method comprises the following steps: (1) The lysed and neutralized solution of the fermented bacteria is subjected to flocculation, impurity removal, microfiltration and ultrafiltration to obtain a plasmid crude extract concentrate; (2) Purifying the crude plasmid concentrate by affinity chromatography, wherein the chromatographic filler used in the affinity chromatography purification is Capto Plasmid Select produced by Cytiva; (3) The plasmid solution purified by affinity chromatography was purified by anion chromatography, wherein the chromatography filler used in the anion chromatography purification was Capto Q ImpRes produced by Cytiva.

2. The plasmid purification method according to claim 1, characterized in that: In step (2), the affinity chromatography purification process is: equilibration, loading, re-equilibration, washing and elution. The balance and the re-balance use Buffer B, and the formula of Buffer B is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 1.8-2.2 M (NH4)2SO4, and the balance is water. The sample loading is a mixture of the plasmid crude extract concentrate and Buffer K, wherein the formula of Buffer K is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 3.2-3.8 M (NH4)2SO4, and the balance is water. The washing method uses a mixture of Buffer A and Buffer E in a volume ratio of (8-10):

1. The formula of Buffer A is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 2.0-2.15 M (NH4)2SO4, and the balance water. The formula of Buffer E is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 0.8-1.2 M NaCl, and the balance is water. The elution adopts Buffer C, and the formula of Buffer C is: 90-110mM Tris, 8-12mM EDTA·2Na, 0.2-0.4M NaCl, 1.5-1.8M (NH4)2SO4, and the balance is water.

3. The plasmid purification method according to claim 2, characterized in that: The column height of the chromatographic column used in the affinity chromatography is 15-18 cm, the column diameter is 25-28 cm, the HETP is 1400-1500, and the upper limit of the sample loading capacity is 1.60 mg / mL.

4. The plasmid purification method according to claim 3, characterized in that: The step (2) is specifically: Balance: Use the Buffer B to balance the chromatography column for 2 to 5 CV, with a linear flow rate of 80 to 100 cm / h; Loading: Load the mixture of the plasmid crude concentrate and Buffer K at a loading flow rate of 80-100 cm / h; Re-equilibration: flush the chromatography column with the Buffer B for 8 to 12 CV at a linear flow rate of 80 to 100 cm / h; Washing: Use the mixture of Buffer A and Buffer E to wash the chromatography column for 2 to 5 CV at a linear flow rate of 50 to 70 cm / h; Elution: Use the Buffer C to rinse the chromatography column at a linear flow rate of 50 to 70 cm / h. Observe the change of UV260 during the elution process. Start collecting when UV260 rises to 1000 mAu, pause for 10 to 30 min when UV260 drops to 500 mAu, and then continue eluting until UV260 drops to 200 mAu and stop collecting.

5. The plasmid purification method according to claim 1, characterized in that: In step (3), the anion chromatography purification process is: equilibration, sample loading, re-equilibration, washing and gradient elution. The balance and re-balance use Buffer D, and the formula of Buffer D is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 0.2-0.5 M NaCl, and the balance is water. The washing method uses a mixture of Buffer D and Buffer E in a volume ratio of 1: (1.1-1.5). The formula of Buffer E is: 90-110 mM Tris, 8-12 mM EDTA·2Na, 0.8-1.2 M NaCl, and the balance is water. The linear elution adopts a mixed linear gradient elution of Buffer D and Buffer E, the proportion of Buffer D increases from 46% to 53%, and the proportion of Buffer E decreases from 54% to 47%.

6. The plasmid purification method according to claim 5, characterized in that: The column height of the chromatography column used for the anion chromatography purification is 30-32 cm, the column diameter is 1.5-1.8 cm, the HETP is 4800-4900, and the upper limit of the sample loading is 1.0 mg / mL.

7. The plasmid purification method according to claim 6, characterized in that: The step (3) is specifically: Balance: Use the Buffer D to balance the chromatography column for 2-5 CV, with a linear flow rate of 80-100 cm / h; Loading: The eluate purified by affinity chromatography is used for loading, and the loading flow rate is 80-100 cm / h; Re-equilibration: using the Buffer D to equilibrate the chromatography column for 4-8 CV, with a linear flow rate of 80-100 cm / h; Washing: The mixture of Buffer D and Buffer E is used to wash the chromatography column for 2 to 5 CV at a linear flow rate of 80 to 100 cm / h; Gradient elution: using the Buffer D and the Buffer E mixed linear gradient elution, the gradient is Buffer D (46%-53%): Buffer E (54%-47%), the elution volume is 10-15CV, the change of UV260 is observed during the elution process, and the collection starts at 0.3CV after UV260 rises to 100mAu, and the collection is terminated after the collection volume reaches 2.1±0.1CV.

8. The plasmid purification method according to claim 1, characterized in that: In the step (1), the flocculant for flocculation and impurity removal is calcium chloride; And / or, the microfiltration includes pre-filtration using a 45-55 μm capsule filter and secondary filtration using a 0.4-0.6 μm capsule filter; And / or, the ultrafiltration concentration includes first using a 90-110KD hollow fiber column to concentrate by 30-60 times, and then using a 10×TE solution to replace the solution and concentrate by 5-6 times.

9. The plasmid purification method according to claim 1, characterized in that: The preparation method of the lysed neutralized solution of the fermented bacteria is as follows: using a 700-800KD hollow fiber column to perform ultrafiltration concentration and diafiltration on the fermented bacterial solution, adding a heavy suspension solution to mix to obtain a bacterial suspension, mixing the bacterial suspension and the lysed solution to perform alkaline lysis, and then adding a neutralizing solution to perform acid neutralization. The formula of the resuspension is: 8-10 g / kg glucose, 3.5-4 g / kg disodium edetate, 6.05 g / kg tromethamine, and the rest is water; The formula of the lysate is: 7.5-8.5 g / kg sodium hydroxide, 9.5-10 g / kg sodium dodecyl sulfate, and the rest is water; The formula of the neutralizing solution is: 250-255 g / kg potassium acetate, 270-275 g / kg glacial acetic acid, and the rest is water.

10. The plasmid purification method according to claim 1, characterized in that: The fermentation bacteria are Escherichia coli bacteria containing the target plasmid; and / or the size of the target plasmid is 3Kb to 7Kb.

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