An industrial separation and purification method for plasmid DNA and its application

Abstract

The present invention provides a method for industrial separation and purification of plasmid DNA and its application. The method comprises the following steps: subjecting a bacterial lysate containing plasmid DNA to ion exchange chromatography and hydrophobic chromatography in sequence to obtain purified plasmid DNA. The present invention uses a two-step purification process of ion chromatography and hydrophobic chromatography for the purification of plasmid DNA products. It is not only simple and easy to scale up, reducing the number of operation steps, but also improves the total recovery rate of purified plasmids and meets the pharmaceutical standards. The separation and purification method of the present invention has good purification effect, strong repeatability, and simple operation, can meet the requirements of industrialized purification production, greatly reduces the production cost, and has important application value in the industrial separation and purification of plasmid DNA.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and particularly relates to a method for industrial separation and purification of plasmid DNA and its application. Background Art

[0002] Gene therapy has become an effective treatment strategy for some major diseases in the 21st century. Currently, recombinant plasmids carrying therapeutic genes have entered clinical research as gene drugs. Plasmid DNA gene therapy drugs are gene therapy products with plasmid DNA as the carrier. Natural plasmids are a class of genetic components independent of chromosomes present in the cytoplasm. Most of them are circular double-stranded DNA molecules, which can continuously and stably exist outside the chromosome, can replicate autonomously, and may also be reversibly integrated into the host chromosome under certain conditions and replicate with the replication of the chromosome. Therefore, plasmids are the most common vector molecules in gene cloning.

[0003] Plasmids themselves can carry exogenous target genes for the development of plasmid DNA gene therapy drugs, and are also the basic raw materials for cell and gene therapy drugs such as CAR-T and mRNA. Moreover, with the development of science and technology, plasmids have been widely used in the fields of medicine, agriculture, environmental protection, food, etc.

[0004] Although large-scale plasmid DNA production processes that meet pharmaceutical specifications have been established and can meet clinical needs. However, it is still relatively difficult to obtain a large amount of high-purity DNA, and the purity of supercoiled plasmid DNA needs to reach more than 90%. In the industrial preparation of plasmids, there are strict requirements for the final product in terms of endotoxin content, purity (HPLC), homogeneity (different forms of plasmid DNA), and residual host protein and nucleic acid, etc.

[0005] The traditional three-step chromatography method has been verified by many platforms and customers and can stably produce plasmid DNA on a scale from hundreds of grams to kilograms. Of course, with the continuous acceleration of the development of new therapies and the change of product quality requirements for different application directions, in the downstream process development of plasmid DNA, the two-step purification process is also expected and selected by many people.

[0006] Therefore, it has important application value to provide a two-step method for industrial separation and purification of plasmid DNA that can meet the requirements of industrialized purification production and greatly reduce the production cost. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for industrial separation and purification of plasmid DNA and its application. The present invention uses a two-step purification process of ion chromatography and hydrophobic chromatography for the purification of plasmid DNA products. It is not only simple to operate and easy to scale up, reducing the operation steps, but also improving the total recovery rate of the purified plasmid and meeting the pharmaceutical standards. The separation and purification method of the present invention has good purification effect, strong repeatability, simple operation, can meet the requirements of industrialized purification production, and greatly reduces the production cost.

[0008] To achieve the purpose of this invention, the following technical solutions are adopted:

[0009] In the first aspect, the present invention provides a method for industrial separation and purification of plasmid DNA, and the method comprises the following steps: subjecting a bacterial lysate containing plasmid DNA to ion exchange chromatography and hydrophobic chromatography in sequence to obtain purified plasmid DNA.

[0010] Preferably, the ion exchange chromatography packing material used in the ion exchange chromatography comprises UniGel-30DEAE.

[0011] Preferably, the hydrophobic chromatography packing material used in the hydrophobic chromatography comprises UniHR Phenyl-30L.

[0012] In the present invention, the two-step purification process of ion chromatography and hydrophobic chromatography is used for the purification of plasmid DNA products. It is not only simple to operate and easy to scale up, reducing the operation steps, but also improving the total recovery rate of the purified plasmid and meeting the pharmaceutical standards. The residual indexes are: HCP < 1 ppm, HCD < 10 μg / mg, endotoxin < 1 EU / mL.

[0013] The purification method of the present invention has good purification effect. The two chromatography packing materials used cooperate with each other. In the preliminary purification of plasmid, UniGel-30DEAE of NanoChromBio is used to remove HC-RNA, and then UniHR Phenyl-30L is used for further purification, which can effectively remove open circular DNA and trace impurities including endotoxin, etc. The purification method of the present invention has strong repeatability, simple operation, can meet the requirements of industrialized purification production, and greatly reduces the production cost.

[0014] Preferably, the method comprises the following steps:

[0015] (1) Lysing bacteria containing plasmid DNA, collecting the bacterial lysate containing plasmid DNA, centrifuging, and collecting the supernatant;

[0016] (2) Loading the supernatant obtained in step (1) onto an equilibrated ion exchange chromatography column, rinsing the ion exchange chromatography column with a washing buffer and an elution buffer in sequence, and collecting the eluate containing plasmid DNA;

[0017] (3) The eluate containing the plasmid DNA obtained in step (2) is loaded onto the equilibrated hydrophobic chromatography column, and the hydrophobic chromatography column is rinsed with a washing buffer and an elution buffer in sequence, and the eluate containing the plasmid DNA is collected.

[0018] Preferably, in step (1), the lysis method includes alkaline lysis.

[0019] Preferably, in step (1), the centrifugal speed is 8000-12000 rpm, for example, it can be 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm or 12000 rpm, etc., the centrifugal time is 10-15 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.; the centrifugal temperature is 0-4°C, for example, it can be 0°C, 2°C, 3°C or 4°C, etc.

[0020] Preferably, in step (2), the ion exchange chromatography column is equilibrated with 4-6 column volumes of equilibration buffer, for example, 4, 5 or 6 column volumes.

[0021] Preferably, the equilibration buffer comprises 48-52 mM Tris-HCl (for example, 48 mM, 49 mM, 50 mM, 51 mM or 52 mM, etc.) and 8-12 mM EDTA (for example, 8 mM, 9 mM, 10 mM, 11 mM or 12 mM, etc.) on a molar basis, a pH of 7.3-7.7 (for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.), and the solvent is water.

[0022] Preferably, the equilibration buffer comprises 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, a pH of 7.5, and the solvent is water.

[0023] Preferably, in step (2), the washing buffer comprises 48-52 mM Tris-HCl (for example, 48 mM, 49 mM, 50 mM, 51 mM or 52 mM, etc.) and 8-12 mM EDTA (for example, 8 mM, 9 mM, 10 mM, 11 mM or 12 mM, etc.) on a molar concentration basis, a pH of 7.3-7.7 (for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.), and the solvent is water.

[0024] Preferably, the washing buffer has a molar concentration of 50 mM Tris-HCl and 10 mM EDTA, a pH of 7.5, and the solvent is water.

[0025] Preferably, in step (2), the ion exchange chromatography column is washed with 4-6 column volumes of washing buffer, for example, 4, 5 or 6.

[0026] Preferably, in step (2), the elution buffer comprises elution buffer I and elution buffer II with gradually increasing salt concentrations in sequence.

[0027] Preferably, elution buffer I comprises, in terms of molar concentration, 48 - 52 mM Tris-HCl (such as 48 mM, 49 mM, 50 mM, 51 mM or 52 mM, etc.), 8 - 12 mM EDTA (such as 8 mM, 9 mM, 10 mM, 11 mM or 12 mM, etc.) and 0.8 - 1.2 M NaCl (such as 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM or 1.2 mM, etc.), with a pH of 7.3 - 7.7 (such as 7.3, 7.4, 7.5, 7.6 or 7.7, etc.), and the solvent is water.

[0028] Preferably, elution buffer I is 50 mM Tris-HCl, 10 mM EDTA and 1 M NaCl, with a pH of 7.5 and the solvent being water.

[0029] Preferably, elution buffer II comprises, in terms of molar concentration, 48 - 52 mM Tris-HCl (such as 48 mM, 49 mM, 50 mM, 51 mM or 52 mM, etc.), 8 - 12 mM EDTA (such as 8 mM, 9 mM, 10 mM, 11 mM or 12 mM, etc.) and 1.8 - 2.2 M NaCl (such as 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM or 2.2 mM, etc.), with a pH of 7.3 - 7.7 (such as 7.3, 7.4, 7.5, 7.6 or 7.7, etc.), and the solvent is water.

[0030] Preferably, elution buffer II is 50 mM Tris-HCl, 10 mM EDTA and 2 M NaCl, with a pH of 7.5 and the solvent being water.

[0031] Preferably, elution buffer I with 4 - 6 (such as 4, 5 or 6, etc.) column volumes and elution buffer II with 4 - 6 (such as 4, 5 or 6, etc.) column volumes are sequentially used to elute the ion exchange chromatography column, and the eluate containing plasmid DNA is collected.

[0032] Preferably, in step (3), the hydrophobic chromatography column is equilibrated with 4 - 6 column volumes of equilibration buffer, such as 4, 5 or 6, etc.

[0033] Preferably, the equilibration buffer contains, in terms of molar concentration, 1.8 - 2.2 M ammonium sulfate (such as 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, or 2.2 mM, etc.) and 18 - 22 mM Tris-HCl (such as 18 mM, 19 mM, 20 mM, 21 mM, or 22 mM, etc.), with a pH of 7.8 - 8.2 (such as 7.8, 7.9, 8.0, 8.1, or 8.2, etc.), and the solvent is water.

[0034] Preferably, the equilibration buffer contains, in terms of molar concentration, 2 M ammonium sulfate and 20 mM Tris-HCl, with a pH of 8.0, and the solvent is water.

[0035] Preferably, in step (3), the washing buffer contains, in terms of molar concentration, 1.8 - 2.2 M ammonium sulfate (such as 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, or 2.2 mM, etc.) and 18 - 22 mM Tris-HCl (such as 18 mM, 19 mM, 20 mM, 21 mM, or 22 mM, etc.), with a pH of 7.8 - 8.2 (such as 7.8, 7.9, 8.0, 8.1, or 8.2, etc.), and the solvent is water.

[0036] Preferably, the washing buffer contains, in terms of molar concentration, 2 M ammonium sulfate and 20 mM Tris-HCl, with a pH of 8.0, and the solvent is water.

[0037] Preferably, in step (3), 4 - 6 column volumes of the washing buffer are used to wash the hydrophobic chromatography column, such as 4, 5, or 6, etc.

[0038] Preferably, in step (3), the elution buffer includes a first elution buffer and a second elution buffer.

[0039] Preferably, the first elution buffer contains, in terms of molar concentration, 1.3 - 1.7 M trisodium citrate (such as 1.3 M, 1.4 M, 1.5 M, 1.6 M, or 1.7 M, etc.) and 18 - 22 mM Tris-HCl (such as 18 M, 19 M, 20 M, 21 M, or 22 M, etc.), with a pH of 7.8 - 8.2 (such as 7.8, 7.9, 8.0, 8.1, or 8.2, etc.), and the solvent is water.

[0040] Preferably, the second elution buffer contains, in terms of molar concentration, 1.8 - 2.2 M ammonium sulfate (such as 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM or 2.2 mM, etc.), 18 - 22 mM Tris-HCl (such as 18 mM, 19 mM, 20 mM, 21 mM or 22 mM, etc.), with a pH of 7.8 - 8.2 (such as 7.8, 7.9, 8.0, 8.1 or 8.2, etc.), and the solvent is water.

[0041] Preferably, the first elution buffer contains, in terms of molar concentration, 1.5 M trisodium citrate and 20 mM Tris-HCl, with a pH of 8.0, and the solvent is water.

[0042] Preferably, the hydrophobic chromatography column is rinsed with the elution buffer according to the following steps: first elution and second elution are carried out in sequence. The first elution is a linear gradient elution, which is carried out using the first elution buffer and / or the second elution buffer. During the linear gradient elution, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is carried out using the second elution buffer; the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm is collected.

[0043] Preferably, the first elution is carried out using 12 - 17 column volumes of the elution buffer, such as 12, 13, 14, 15, 16 or 17, etc. <{

[0044] Preferably, the second elution is carried out using 4 - 6 column volumes of the second elution buffer, such as 4, 5 or 6, etc.

[0045] As a preferred technical solution of the present invention, the method includes the following steps:

[0046] (1) Lysing bacteria containing plasmid DNA, collecting the bacterial lysate containing plasmid DNA, centrifuging the lysate at 8000 - 12000 rpm for 10 - 15 min at a temperature of 0 - 4°C, and collecting the supernatant;

[0047] (2) Equilibrating an ion exchange chromatography column with 4 - 6 column volumes of the equilibration buffer, loading the supernatant obtained in step (1) onto the ion exchange chromatography column, rinsing the ion exchange chromatography column with 4 - 6 column volumes of the washing buffer, and eluting the ion exchange chromatography column with 4 - 6 column volumes of the first elution buffer and 4 - 6 column volumes of the second elution buffer in sequence, and collecting the eluate containing plasmid DNA;

[0048] (3) Equilibrate the hydrophobic chromatography column with 4 - 6 column volumes of equilibration buffer, load the eluate containing plasmid DNA obtained in step (2) onto the hydrophobic chromatography column, and wash the hydrophobic chromatography column with 4 - 6 column volumes of washing buffer; perform the first elution and the second elution in sequence, and collect the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm.

[0049] The first elution is a linear gradient elution, which is carried out using the first elution buffer and / or the second elution buffer. During the linear gradient elution, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is carried out using the second elution buffer.

[0050] In a second aspect, the present invention provides the use of the method for industrial separation and purification of plasmid DNA described in the first aspect in the preparation of DNA drugs.

[0051] The numerical ranges described in the present invention not only include the above - listed point values, but also include any point values between the above - mentioned numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the ranges.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The method for separating and purifying the plasmid of the present invention only requires two - step chromatography to separate and purify the plasmid. The obtained product not only has high purity, high yield and stability, but also is simple and convenient to operate, and the purification cycle is relatively short, greatly reducing the cost. The purification recovery rate of the first - step ion - exchange chromatography is 80%, and the purification recovery rate of the second - step hydrophobic chromatography reaches 74%. The final sample is detected, and the detection results show that after two - step chromatography with UniGel - 30DEAE and UniHR Phenyl - 30L, the yield is 74%, and the residual indexes are: HCP < 1 ppm, HCD < 10 μg / mg, endotoxin < 1 EU / mL. The detection results in the present invention show that the two - step plasmid purification process can efficiently obtain high - quality plasmid DNA samples. Description of the Drawings

[0054] Figure 1 is the chromatogram of the ion - exchange chromatography packing UniGel - 30DEAE in Example 1.

[0055] Figure 2 is the chromatogram of the hydrophobic chromatography packing UniHR Phenyl - 30L in Example 1. Detailed Embodiments

[0056] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0057] For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through regular channels.

[0058] Example 1

[0059] This example provides a method for industrial separation and purification of plasmid DNA. The separation and purification method includes the following steps:

[0060] (1) Use the alkaline lysis method to lyse the bacteria containing plasmid DNA, collect the bacterial lysate containing plasmid DNA, centrifuge the lysate at 10,000 rpm for 12 min at a temperature of 4°C, and collect the supernatant.

[0061] (2) Equilibrate the ion exchange chromatography column with 5 column volumes of equilibration buffer. The ion exchange chromatography packing used in the ion exchange chromatography column is UniGel-30DEAE (NanoChrom Tech, part number 04083-030100). Load the supernatant obtained in step (1) onto the ion exchange chromatography column, wash the ion exchange chromatography column with 5 column volumes of washing buffer, and elute the ion exchange chromatography column successively with 5 column volumes of elution buffer I and 5 column volumes of elution buffer II, and collect the eluate containing plasmid DNA; the chromatogram of the ion exchange chromatography packing UniGel-30DEAE is as Figure 1 shown. It can be seen from Figure 1 that the ion exchange chromatography packing UniGel-30DEAE can effectively separate RNA from plasmid DNA.

[0062] The equilibration buffer is 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, with a pH of 7.5 and the solvent being water;

[0063] The washing buffer is 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, with a pH of 7.5 and the solvent being water;

[0064] The elution buffer I is 50 mM Tris-HCl, 10 mM EDTA and 1 M NaCl in terms of molar concentration, with a pH of 7.5 and the solvent being water;

[0065] The second elution buffer is 50 mM Tris-HCl, 10 mM EDTA and 2 M NaCl in terms of molar concentration, with a pH of 7.5 and water as the solvent.

[0066] (3) Equilibrate the hydrophobic chromatography column with 5 column volumes of the equilibration buffer. The hydrophobic chromatography packing material used in the hydrophobic chromatography column is UniHR Phenyl-30L (NanoChrom Technology, part number 06131-030100). Load the eluate containing plasmid DNA obtained in step (2) onto the hydrophobic chromatography column, and rinse the hydrophobic chromatography column with 5 column volumes of the washing buffer; perform the first elution with 15 column volumes of the buffer, and then perform the second elution with 5 column volumes of the buffer. Collect the effluent with a UV absorption peak greater than 50 mAU at a wavelength of 260 nm, and collect the eluate containing plasmid DNA;

[0067] The first elution is a linear gradient elution, which is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution process, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer.

[0068] The chromatogram of the hydrophobic chromatography packing material UniHR Phenyl-30L is as Figure 2 shown. It can be seen from Figure 2 that as the conductivity decreases, the plasmid DNA is gradually eluted.

[0069] The equilibration buffer is 2 M ammonium sulfate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent;

[0070] The washing buffer is 2 M ammonium sulfate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent;

[0071] The first elution buffer is 1.5 M trisodium citrate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent;

[0072] The second elution buffer is 2 M ammonium sulfate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent.

[0073] Example 2

[0074] This example provides a method for industrial separation and purification of plasmid DNA. The separation and purification method includes the following steps:

[0075] (1) Lyse the bacteria containing plasmid DNA using the alkaline lysis method, collect the bacterial lysate containing plasmid DNA, centrifuge the lysate at 8000 rpm for 15 min at a temperature of 4 °C, and collect the supernatant.

[0076] (2) Equilibrate the ion exchange chromatography column with 4 column volumes of equilibration buffer. The ion exchange chromatography packing used in the ion exchange chromatography column is UniGel-30DEAE (NanoChrom Tech, product number 04083-030100). Load the supernatant obtained in step (1) onto the ion exchange chromatography column, rinse the ion exchange chromatography column with 4 column volumes of washing buffer, and elute the ion exchange chromatography column successively with 4 column volumes of elution buffer I and 4 column volumes of elution buffer II, and collect the eluate containing plasmid DNA;

[0077] The equilibration buffer contains 48 mM Tris-HCl and 12 mM EDTA by molar concentration, with a pH of 7.3 and water as the solvent;

[0078] The washing buffer contains 48 mM Tris-HCl and 12 mM EDTA by molar concentration, with a pH of 7.3 and water as the solvent;

[0079] The elution buffer I contains 48 mM Tris-HCl, 12 mM EDTA and 0.8 M NaCl by molar concentration, with a pH of 7.3 and water as the solvent;

[0080] The elution buffer II contains 48 mM Tris-HCl, 12 mM EDTA and 1.8 M NaCl by molar concentration, with a pH of 7.3 and water as the solvent.

[0081] (3) Equilibrate the hydrophobic chromatography column with 4 column volumes of equilibration buffer. The hydrophobic chromatography packing used in the hydrophobic chromatography column is UniHR Phenyl-30L (NanoChrom Tech, product number 06131-030100). Load the eluate containing plasmid DNA obtained in step (2) onto the hydrophobic chromatography column, and rinse the hydrophobic chromatography column with 4 column volumes of washing buffer; perform the first elution with 15 column volumes of buffer, and then perform the second elution with 5 column volumes of buffer, and collect the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm, and collect the eluate containing plasmid DNA;

[0082] The first elution is a linear gradient elution, which is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer.

[0083] The balance buffer consists of 1.8 M ammonium sulfate and 22 mM Tris-HCl, with a pH of 8.2 and water as the solvent;

[0084] The washing buffer consists of 1.8 M ammonium sulfate and 22 mM Tris-HCl, with a pH of 8.2 and water as the solvent;

[0085] The first elution buffer consists of 1.7 M trisodium citrate and 22 mM Tris-HCl, with a pH of 8.2 and water as the solvent;

[0086] The second elution buffer consists of 1.8 M ammonium sulfate and 22 mM Tris-HCl, with a pH of 8.2 and water as the solvent.

[0087] Example 3

[0088] This example provides an industrial separation and purification method for plasmid DNA. The separation and purification method includes the following steps:

[0089] (1) Use the alkaline lysis method to lyse the bacteria containing plasmid DNA, collect the bacterial lysate containing plasmid DNA, centrifuge the lysate at 12000 rpm for 10 min at a temperature of 4 °C, and collect the supernatant.

[0090] (2) Equilibrate the ion exchange chromatography column with 6 column volumes of the balance buffer. The ion exchange chromatography packing used in the ion exchange chromatography column is UniGel-30DEAE (NanoChrom Tech, product number 04083-030100). Load the supernatant obtained in step (1) onto the ion exchange chromatography column, wash the ion exchange chromatography column with 6 column volumes of the washing buffer, and elute the ion exchange chromatography column successively with 6 column volumes of the first elution buffer and 6 column volumes of the second elution buffer, and collect the eluate containing plasmid DNA;

[0091] The balance buffer consists of 52 mM Tris-HCl and 8 mM EDTA, with a pH of 7.7 and water as the solvent;

[0092] The washing buffer consists of 52 mM Tris-HCl and 8 mM EDTA, with a pH of 7.7 and water as the solvent;

[0093] The first elution buffer consists of 52 mM Tris-HCl, 8 mM EDTA, and 1.2 M NaCl, with a pH of 7.7 and water as the solvent;

[0094] The second elution buffer is 52 mM Tris-HCl, 8 mM EDTA and 2.2 M NaCl in terms of molar concentration, with a pH of 7.7 and water as the solvent.

[0095] (3) Equilibrate the hydrophobic chromatography column with 6 column volumes of the equilibration buffer. The hydrophobic chromatography packing material used in the hydrophobic chromatography column is UniHR Phenyl-30L (NanoMicro Technologies, part number 06131-030100). Load the eluate containing plasmid DNA obtained in step (2) onto the hydrophobic chromatography column, and rinse the hydrophobic chromatography column with 6 column volumes of the washing buffer; perform a first elution with 15 column volumes of the buffer, and then perform a second elution with 5 column volumes of the buffer. Collect the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm, and collect the eluate containing plasmid DNA;

[0096] The first elution is a linear gradient elution, which is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution process, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer.

[0097] The equilibration buffer is 2.2 M ammonium sulfate and 18 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 and water as the solvent;

[0098] The washing buffer is 2.2 M ammonium sulfate and 18 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 and water as the solvent;

[0099] The first elution buffer is 1.2 M trisodium citrate and 18 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 and water as the solvent;

[0100] The second elution buffer is 2.2 M ammonium sulfate and 18 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 and water as the solvent.

[0101] Example 4

[0102] This example provides a method for industrial separation and purification of plasmid DNA. The difference between this separation and purification method and that of Example 1 is only that the ion exchange chromatography packing material used in the ion exchange chromatography column in this example is Q Agarose 6FF (Yeasen, part number 20547ES76), and the remaining steps refer to Example 1.

[0103] Example 5

[0104] This example provides a method for industrial separation and purification of plasmid DNA. The only difference between this separation and purification method and that of Example 1 is that in this example, the ion exchange chromatography packing material used in the ion exchange chromatography column is UniGel-80Q (NanoChrom Tech, part number 04084-080100), and the remaining steps refer to Example 1.

[0105] Example 6

[0106] This example provides a method for industrial separation and purification of plasmid DNA. The only difference between this separation and purification method and that of Example 1 is that in this example, the hydrophobic chromatography packing material used in the hydrophobic chromatography column is UniHR Butyl-30L (NanoChrom Tech, part number 06132-030100), and the remaining steps refer to Example 1.

[0107] Example 7

[0108] This example provides a method for industrial separation and purification of plasmid DNA. The only difference between this separation and purification method and that of Example 1 is that in this example, the hydrophobic chromatography packing material used in the hydrophobic chromatography column is the packing material Toyopearl Butyl-650S (manufacturer TOSOH, part number 043153), and the remaining steps refer to Example 1.

[0109] Comparative Example 1

[0110] This comparative example provides a method for industrial separation and purification of plasmid DNA. The method includes the following steps: subjecting the bacterial lysate containing plasmid DNA to hydrophobic chromatography and ion exchange chromatography treatments in sequence to obtain purified plasmid DNA.

[0111] (1) Lysing the bacteria containing plasmid DNA by the alkaline lysis method, collecting the bacterial lysate containing plasmid DNA, centrifuging the lysate at 10000 rpm for 12 min at a temperature of 4 °C, and collecting the supernatant.

[0112] (2) Equilibrating the hydrophobic chromatography column with 5 column volumes of equilibration buffer. The hydrophobic chromatography packing material used in the hydrophobic chromatography column is UniHR Phenyl-30L (NanoChrom Tech, part number 06131-030100). Loading the supernatant obtained in step (1) onto the hydrophobic chromatography column, and rinsing the hydrophobic chromatography column with 5 column volumes of washing buffer; performing a first elution with 15 column volumes of buffer, and then performing a second elution with 5 column volumes of buffer. Collecting the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm, and collecting the eluate containing plasmid DNA;

[0113] The first elution is a linear gradient elution, which is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer.

[0114] The equilibrium buffer is 2 M ammonium sulfate and 20 mM Tris-HCl at a pH of 8.0, with water as the solvent;

[0115] The washing buffer is 2 M ammonium sulfate and 20 mM Tris-HCl at a pH of 8.0, with water as the solvent;

[0116] The first elution buffer is 1.5 M trisodium citrate and 20 mM Tris-HCl at a pH of 8.0, with water as the solvent;

[0117] The second elution buffer is 2 M ammonium sulfate and 20 mM Tris-HCl at a pH of 8.0, with water as the solvent.

[0118] (3) Equilibrate the ion exchange chromatography column with 5 column volumes of the equilibrium buffer. The ion exchange chromatography packing used in the ion exchange chromatography column is UniGel-30DEAE (NanoChrom Tech, product number 04083-030100). Load the eluate containing plasmid DNA obtained in step (1) onto the ion exchange chromatography column. Wash the ion exchange chromatography column with 5 column volumes of the washing buffer. Elute the ion exchange chromatography column successively with 5 column volumes of elution buffer one and 5 column volumes of elution buffer two, and collect the eluate containing plasmid DNA;

[0119] The equilibrium buffer is 50 mM Tris-HCl and 10 mM EDTA at a pH of 7.5, with water as the solvent;

[0120] The washing buffer is 50 mM Tris-HCl and 10 mM EDTA at a pH of 7.5, with water as the solvent;

[0121] Elution buffer one is 50 mM Tris-HCl, 10 mM EDTA and 1 M NaCl at a pH of 7.5, with water as the solvent;

[0122] Elution buffer two is 50 mM Tris-HCl, 10 mM EDTA and 2 M NaCl at a pH of 7.5, with water as the solvent.

[0123] Comparative Example 2

[0124] This comparative example provides a method for industrial separation and purification of plasmid DNA. The separation and purification method includes the following steps: subjecting a bacterial lysate containing plasmid DNA to ion exchange chromatography and composite mode chromatography in sequence to obtain purified plasmid DNA.

[0125] The method includes the following specific steps:

[0126] (1) Lysing bacteria containing plasmid DNA by alkaline lysis method, collecting the bacterial lysate containing plasmid DNA, centrifuging the lysate at 10000 rpm for 12 min at a temperature of 4 °C, and collecting the supernatant.

[0127] (2) Equilibrating an ion exchange chromatography column with 5 column volumes of equilibration buffer. The ion exchange chromatography packing used in the ion exchange chromatography column is UniGel-30DEAE (NanoChrom Tech, product number 04083-030100). Loading the supernatant obtained in step (1) onto the ion exchange chromatography column, rinsing the ion exchange chromatography column with 5 column volumes of washing buffer, eluting the ion exchange chromatography column with 5 column volumes of elution buffer I and 5 column volumes of elution buffer II in sequence, and collecting the eluate containing plasmid DNA;

[0128] The equilibration buffer is 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, with a pH of 7.5 and water as the solvent;

[0129] The washing buffer is 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, with a pH of 7.5 and water as the solvent;

[0130] The elution buffer I is 50 mM Tris-HCl, 10 mM EDTA and 1 M NaCl in terms of molar concentration, with a pH of 7.5 and water as the solvent;

[0131] The elution buffer II is 50 mM Tris-HCl, 10 mM EDTA and 2 M NaCl in terms of molar concentration, with a pH of 7.5 and water as the solvent.

[0132] (3) Equilibrating a composite mode chromatography column with 5 column volumes of equilibration buffer. The composite chromatography packing used in the composite mode chromatography column is NW ROSE Viral L (NanoChrom Tech, product number 60053-548100). Loading the eluate containing plasmid DNA obtained in step (2) onto the composite mode chromatography column, rinsing the composite mode chromatography column with 5 column volumes of washing buffer, eluting the composite mode chromatography column with 5 column volumes of elution buffer, and collecting the eluate containing plasmid DNA;

[0133] The balance buffer is composed of 150 mM NaCl and 20 mM Tris-HCl, with a pH of 7.2 and water as the solvent;

[0134] The elution buffer is composed of 150 mM NaCl and 20 mM Tris-HCl, with a pH of 7.2 and water as the solvent.

[0135] Comparative Example 3

[0136] This comparative example provides a method for industrial separation and purification of plasmid DNA. The separation and purification method includes the following steps: subjecting the bacterial lysate containing plasmid DNA to affinity chromatography and hydrophobic chromatography in sequence to obtain purified plasmid DNA.

[0137] The method includes the following specific steps:

[0138] (1) Using the alkaline lysis method to lyse the bacteria containing plasmid DNA, collecting the bacterial lysate containing plasmid DNA, centrifuging the lysate at 10000 rpm for 12 min at a temperature of 4 °C, and collecting the supernatant.

[0139] (2) Equilibrating the affinity chromatography column with 5 column volumes of the balance buffer. The affinity chromatography packing used in the affinity chromatography column is NW Rose Plasmid (NanoMicro Technologies, product number 60043-402400). Loading the supernatant obtained in step (1) onto the affinity chromatography column, rinsing the affinity chromatography column with 5 column volumes of the washing buffer, eluting the affinity chromatography column with 5 column volumes of elution buffer I and 5 column volumes of elution buffer II in sequence, and collecting the eluate containing plasmid DNA; <(

[0140] The balance buffer is composed of 2.0 M ammonium sulfate, 10 mM EDTA and 100 mM Tris HCl, with a pH of 7.5 and water as the solvent;

[0141] The washing buffer is composed of 2.0 M ammonium sulfate, 10 mM EDTA and 100 mM Tris HCl, with a pH of 7.5 and water as the solvent;

[0142] The elution buffer is composed of 1.7 M ammonium sulfate, 0.3 M NaCl, 10 mM EDTA and 100 mM Tris HCl, with a pH of 7.5 and water as the solvent;

[0143] (3) Equilibrate the hydrophobic chromatography column with 5 column volumes of equilibration buffer. The hydrophobic chromatography packing material used in the hydrophobic chromatography column is UniHR Phenyl-30L (NanoMicro Technologies, part number 06131-030100). Load the eluate containing plasmid DNA obtained in step (2) onto the hydrophobic chromatography column, and rinse the hydrophobic chromatography column with 5 column volumes of washing buffer; perform the first elution with 15 column volumes of buffer, and then perform the second elution with 5 column volumes of buffer. Collect the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm, and collect the eluate containing plasmid DNA;

[0144] The first elution is a linear gradient elution, and the linear gradient elution is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution process, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer.

[0145] The equilibration buffer is 2 M ammonium sulfate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent;

[0146] The washing buffer is 2 M ammonium sulfate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent;

[0147] The first elution buffer is 1.5 M trisodium citrate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent;

[0148] The second elution buffer is 2 M ammonium sulfate and 20 mM Tris-HCl in terms of molar concentration, with a pH of 8.0 and water as the solvent.

[0149] Test Example 1

[0150] This test example detects the products in Examples 1-7 and Comparative Examples 1-3. The detection includes recovery rate detection, residue detection, and endotoxin detection.

[0151] The steps for recovery rate detection are as follows:

[0152] (1) Collect the eluate, measure the product concentration with NanoDrop, and calculate the product content

[0153] (2) Divide the amount of product in the eluate by the loading amount

[0154] The steps for residue detection are as follows:

[0155] (1) The HCP residue is referenced to the F550-1 ELISA Kit, manufactured by Cygnus, product number 231221D-1;

[0156] (2) The HCD residue is referenced to the CHO Host Cell DNA D550W Kit, manufactured by Cygnus, product number 281218-10;

[0157] The steps for endotoxin detection are as follows:

[0158] (1) Endotoxin detection is referenced to the PyroGene TM Recombinant Factor C Endotoxin Detection System Kit, manufactured by Lonza, product number 0001079736;

[0159] The test results are shown in Table 1.

[0160] Table 1

[0161]

[0162] From the results in Table 1, it can be seen that for the plasmid isolation and purification method in Examples 1-3, only two steps of chromatography are required to isolate and purify the plasmid. It not only has high purity, high yield and stability, but also is simple and convenient to operate, with a relatively short purification cycle, greatly reducing the cost. The recovery rate of the first-step anion UniGel-30DEAE purification is 76-80%, and the recovery rate of the second-step hydrophobic purification UniHR Phenyl-30L reaches 72-74%. For the final sample detection, the test results show that after two steps of chromatography with UniGel-30DEAE and UniHR Phenyl-30L, the recovery rate is 72-74%, and the residual indexes are: HCP < 1 ppm, HCD < 10 μg / mg, endotoxin < 1 EU / mL. The results indicate that the two-step plasmid purification process can efficiently obtain high-quality plasmid DNA samples.

[0163] From the comparison between Example 1 and Example 4 and Example 5, it can be seen that the separation effect of Q Agarose 6FF and UniGel-80Q is worse than that of the ion exchange chromatography packing UniGel-30DEAE in Example 1.

[0164] From the comparison between Example 1 and Example 6 and Example 7, it can be seen that the separation effect of UniHR Butyl-30L and Toyopearl Butyl-650S is worse than that of the hydrophobic chromatography packing UniHR Phenyl-30L in Example 1.

[0165] From the comparison between Example 1 and Comparative Example 1, it can be seen that changing the steps of two-step purification will affect the final separation effect. The separation and purification product obtained by first performing ion exchange chromatography and then hydrophobic chromatography has a higher yield, lower residue, and lower endotoxin content.

[0166] From the comparison between Example 1 and Comparative Examples 2 and 3, it can be seen that after replacing the hydrophobic chromatography treatment with composite mode chromatography, the yield decreases, the endotoxin content increases, and the content of trace impurities increases; after replacing the ion exchange chromatography with affinity chromatography, the yield decreases, the endotoxin content increases, and the content of trace impurities increases. This shows that the combination of ion exchange chromatography and hydrophobic chromatography has a better separation and purification effect, which is not only simple to operate and easy to scale up, reducing the operation steps, but also improving the total recovery rate of the purified plasmid and meeting the pharmaceutical standards.

[0167] In summary, the plasmid DNA industrial separation and purification method provided by the present invention is not only simple to operate and easy to scale up, reducing the operation steps, but also improving the total recovery rate of the purified plasmid and meeting the pharmaceutical standards. The separation and purification method of the present invention has good purification effect, strong repeatability, and simple operation, can meet the requirements of industrialized purification production, greatly reduces the production cost, and has important application value in the industrial separation and purification of plasmid DNA.

[0168] The applicant declares that the above description is only the specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived within the technical scope disclosed by the present invention by those skilled in the art all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An industrial separation and purification method for plasmid DNA, characterized in that, The method comprises the following steps: (1) Lysing bacteria containing plasmid DNA, collecting the bacterial lysate containing plasmid DNA, centrifuging, and collecting the supernatant; (2) Loading the supernatant obtained in step (1) onto an equilibrated ion exchange chromatography column, rinsing the ion exchange chromatography column successively with a washing buffer and an elution buffer, and collecting the eluate containing plasmid DNA; the ion exchange chromatography packing used for the ion exchange chromatography is UniGel-30DEAE; the ion exchange chromatography column is equilibrated with 4 - 6 column volumes of an equilibration buffer; The equilibration buffer contains 48 - 52 mM Tris-HCl and 8 - 12 mM EDTA in terms of molar concentration, with a pH of 7.3 - 7.7, and the solvent is water; The washing buffer contains 48 - 52 mM Tris-HCl and 8 - 12 mM EDTA in terms of molar concentration, with a pH of 7.3 - 7.7, and the solvent is water; The elution buffer comprises an elution buffer one and an elution buffer two with successively increasing salt concentrations; the elution buffer one contains 48 - 52 mM Tris-HCl, 8 - 12 mM EDTA, and 0.8 - 1.2 M NaCl in terms of molar concentration, with a pH of 7.3 - 7.7, and the solvent is water; the elution buffer two contains 48 - 52 mM Tris-HCl, 8 - 12 mM EDTA, and 1.8 - 2.2 M NaCl in terms of molar concentration, with a pH of 7.3 - 7.7, and the solvent is water; (3) Loading the eluate containing plasmid DNA obtained in step (2) onto an equilibrated hydrophobic chromatography column, rinsing the hydrophobic chromatography column successively with a washing buffer and an elution buffer, and collecting the eluate containing plasmid DNA; the hydrophobic chromatography packing used for the hydrophobic chromatography is UniHR Phenyl-30L; the hydrophobic chromatography column is equilibrated with 4 - 6 column volumes of an equilibration buffer; The equilibration buffer contains 1.8 - 2.2 M ammonium sulfate and 18 - 22 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 - 8.2, and the solvent is water; The washing buffer contains 1.8 - 2.2 M ammonium sulfate and 18 - 22 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 - 8.2, and the solvent is water; In step (3), the elution buffer comprises a first elution buffer and a second elution buffer; the first elution buffer contains 1.3 - 1.7 M trisodium citrate and 18 - 22 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 - 8.2, and the solvent is water; the second elution buffer contains 1.8 - 2.2 M ammonium sulfate and 18 - 22 mM Tris-HCl in terms of molar concentration, with a pH of 7.8 - 8.2, and the solvent is water.

2. The plasmid DNA industrial separation and purification method according to claim 1, characterized in that, In step (1), the lysis method includes the alkaline lysis method.

3. The plasmid DNA industrial separation and purification method according to claim 1, characterized in that, In step (1), the centrifugation speed is 8000 - 12000 rpm, the centrifugation time is 10 - 15 min; the centrifugation temperature is 0 - 4°C.

4. The plasmid DNA industrial separation and purification method according to claim 1, characterized in that, In step (2), wash the ion exchange chromatography column with 4 - 6 column volumes of impurity washing buffer.

5. The plasmid DNA industrial separation and purification method according to claim 1, characterized in that, Elute the ion exchange chromatography column successively with 4 - 6 column volumes of elution buffer I and 4 - 6 column volumes of elution buffer II, and collect the eluate containing plasmid DNA.

6. The plasmid DNA industrial separation and purification method according to claim 1, characterized in that In step (3), wash the hydrophobic chromatography column with 4 - 6 column volumes of impurity washing buffer.

7. The plasmid DNA industrial separation and purification method according to claim 1, characterized in that, The hydrophobic chromatography column is washed with the elution buffer by the following steps: Perform the first elution and the second elution successively. The first elution is a linear gradient elution, and the linear gradient elution is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer; collect the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm.

8. The plasmid DNA industrial separation and purification method according to claim 7, characterized in that, The first elution is performed using 12 - 17 column volumes of the elution buffer.

9. The plasmid DNA industrial separation and purification method according to claim 7, characterized in that, The second elution is performed using 4 - 6 column volumes of the second elution buffer.

10. The plasmid DNA industrial separation and purification method according to claim 1, wherein The method includes the following steps: (1) Lyse the bacteria containing plasmid DNA, collect the bacterial lysate containing plasmid DNA, centrifuge the lysate at 8000 - 12000 rpm for 10 - 15 min at a temperature of 0 - 4°C, and collect the supernatant. (2) Equilibrate the ion exchange chromatography column with 4 - 6 column volumes of equilibration buffer, load the supernatant obtained in step (1) onto the ion exchange chromatography column, wash the ion exchange chromatography column with 4 - 6 column volumes of impurity washing buffer, elute the ion exchange chromatography column successively with 4 - 6 column volumes of elution buffer I and 4 - 6 column volumes of elution buffer II, and collect the eluate containing plasmid DNA. (3) Equilibrate the hydrophobic chromatography column with 4 - 6 column volumes of equilibration buffer, load the eluate containing plasmid DNA obtained in step (2) onto the hydrophobic chromatography column, wash the hydrophobic chromatography column with 4 - 6 column volumes of impurity washing buffer; perform the first elution and the second elution successively, and collect the effluent with an ultraviolet absorption peak greater than 50 mAU at a wavelength of 260 nm. The first elution is a linear gradient elution, and the linear gradient elution is performed using the first elution buffer and / or the second elution buffer. During the linear gradient elution, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100% → 100%:0%; the second elution is performed using the second elution buffer.

11. Use of the method for industrial separation and purification of plasmid DNA according to any one of claims 1 - 10 in the preparation of DNA drugs.

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