Plasmid DNA purification method based on membrane chromatography

Abstract

The invention relates to the technical field of biology, in particular to a plasmid DNA purification method based on membrane chromatography. According to the method, a membrane chromatography technology is used, a packing experiment is not needed, the working flow speed is high, plasmid purification only needs 1-2 h once, the recovery rate of obtained DNA is 73% or above, the recovery rate of super-spiral DNA is 97% or above, and the proportion of the super-spiral DNA is 95% or above.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for purifying plasmid DNA based on membrane chromatography. Background Technology

[0002] Plasmid DNA is a crucial raw material for many gene therapies, such as a raw material for producing viral vectors, a template for in vitro transcription of mRNA, and an effective vector for the therapy itself. The increasing complexity of gene therapy has led to a greater demand for plasmid DNA. Plasmid purification is a technique that separates plasmid DNA from host DNA, RNA, host proteins, endotoxins, and non-supercoiled plasmid conformations. High-quality, high-purity plasmid DNA is a key component in cell and gene therapy processes. Plasmid purity significantly affects transfection efficiency; if the plasmid is impure, impurities can cause cytotoxicity or severely impair the formation of the transfection complex. Therefore, plasmid DNA purification is a critical step in its production process.

[0003] Plasmid production typically uses *E. coli* as the host cell, and plasmids are obtained by lysing the cells after fermentation. The lysed solution may contain various plasmid forms: supercoiled plasmid DNA, open circular DNA, linear DNA, and plasmid DNA aggregates, as well as impurities such as host proteins, host DNA, RNA, and endotoxins. These impurities can be removed using chromatography. Existing plasmid purification processes mainly include the following:

[0004] (1) The classic three-step process (molecular sieve + hydrophobic chromatography + anion exchange chromatography) is as follows: The first step uses gel filtration chromatography to separate plasmid DNA and RNA based on their molecular weight difference, quickly removing a large amount of RNA. This step can achieve a purity of about 80% for supercoiled plasmids and a yield of over 90%. It also replaces the buffer solution to facilitate the next step of plasmid affinity chromatography. In the plasmid affinity chromatography step, a packing material with 2-mercaptopyridine as the ligand is used. The main purpose is to remove non-circular DNA and achieve efficient separation of supercoiled plasmids. Through this step, the purity of supercoiled plasmids can be increased to over 85%. Finally, anion exchange chromatography is used for purification. This step is mainly aimed at removing residual endotoxins and trace impurities.

[0005] (2) Based on a simple improvement of the classic three-step method, calcium chloride or ammonium sulfate precipitation is used instead of molecular sieves for RNA removal, and the other steps are similar to the three-step method.

[0006] (3) A two-step process based on the composite mode Capto Core 700 and hydrophobic chromatography or plasmid affinity chromatography. Capto Core 700 is a composite mode packing material. The outer surface of the packing microspheres is an inert shell, and the pores contain strongly adsorbed octylamine groups. Molecules larger than 700KD are excluded from the outside, while smaller impurities enter the pores and bind to the octylamine groups. Under normal circumstances, plasmid DNA is excluded from the outside of the packing microspheres due to its large molecular size and is separated in a flow-through mode, while RNA, host proteins, host nucleic acids, and endotoxins enter the inside of the packing microspheres and bind to the octylamine groups to achieve separation. The second step of chromatography can use hydrophobic chromatography or plasmid affinity chromatography to increase the supercoil ratio of plasmids and purify them.

[0007] (4) Anion exchange chromatography + hydrophobic chromatography. Anion exchange chromatography can remove impurities such as RNA and endotoxins from plasmid samples by utilizing the difference in charge properties between plasmids and impurities. Hydrophobic chromatography or plasmid affinity chromatography can remove non-closed circular plasmids and purify the samples.

[0008] It is evident that in order to obtain plasmid DNA with high purity, existing technologies often require multi-step chromatography processes combined with other techniques, which are time-consuming, costly, and complex. Therefore, there is a need to develop a rapid plasmid purification process.

[0009] Membrane chromatography is a highly efficient separation technique that allows ligands to be coupled to microporous membranes made of materials such as PES and PP, enabling them to bind. Depending on the type of ligand used, membrane chromatography currently includes anion exchange chromatography membranes, cation exchange chromatography membranes, and hydrophobic chromatography membranes. Previously, membrane chromatography technology has been used in processes such as protein purification and virus purification, but it has not yet achieved good results in the separation and purification of DNA. Summary of the Invention

[0010] In view of this, the technical problem to be solved by the present invention is to provide a method for purifying plasmid DNA based on membrane chromatography.

[0011] The present invention provides a method for purifying plasmid DNA based on membrane chromatography, comprising:

[0012] The plasmid DNA solution was purified by affinity chromatography to obtain purified plasmid DNA;

[0013] The purification process includes sample loading, washing, and elution.

[0014] In this invention, plasmid DNA is purified by using a chromatographic membrane purification method. This method is faster, has a higher yield, and contains a higher proportion of DNA with supercoiled structures.

[0015] In the purification method provided by the present invention, the ligand of the affinity chromatography membrane is 2-mercaptopyridine. The affinity of 2-mercaptopyridine enables it to selectively bind to DNA in supercoiled form, while the open circular form is not easily bound to it. Therefore, compared with other ligands, 2-mercaptopyridine can improve the purification effect of plasmid DNA, especially supercoiled plasmid DNA.

[0016] In the purification method of this invention, the affinity chromatography membrane is a PES membrane. In order to further improve the purification effect, this invention screened the membrane material and the pore size of the membrane material, and finally found that the PES membrane with a pore size of 0.65 μm can obtain the best effect.

[0017] In this invention, the washing and elution steps play a decisive role in the final result. The washing step removes not only residual proteins and other impurities, but more importantly, it removes open-circular DNA molecules and linear DNA molecules. In some embodiments, the washing solution contains 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.3. For example, the washing solution consists of 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.3; or the washing solution consists of 1.8M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.3.

[0018] In the elution step, to obtain as much supercoiled DNA as possible, the elution buffer has been optimized and screened in this invention. In some embodiments, the elution solution contains 0.39M–1.51M (NH4)2SO4 + 0.6M–1.61M NaCl, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.3. Preferably, the elution solution contains 1.44M (NH4)2SO4 + 0.6M NaCl, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.3.

[0019] In this invention, the sample loading includes the steps of: equilibration, sample loading, and reequilibration;

[0020] The equilibrium solution comprises 1.8 M to 1.9 M (NH4)2SO4, 100 mM Tris-HCl, 10 mM EDTA and water, with a pH of 7.5 ± 0.3.

[0021] The sample loaded with the sample contained 5-500 ng / μL plasmid DNA, 1.8-1.9 M (NH4)2SO4, 100 mM Tris-HCl, 10 mM EDTA and water, with a pH of 7.5 ± 0.3.

[0022] The rebalancing solution comprises 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.3.

[0023] In this invention, the volume of the chromatography membrane is 1MV, where 1MV = 0.2–2000 mL. For example, 1MV = 0.2–100 mL, 100–200 mL, 200–300 mL, 300–500 mL, 500–1000 mL, or 1000–2000 mL. In some embodiments, 1MV = 0.2–30 mL, for example, it can be 0.2–1 mL. In a specific embodiment, 1MV = 0.3 mL.

[0024] In this invention, the flow rate of the solution during the purification step is 5–15 MV / min; preferably, the flow rate is 8–12 MV / min. In some embodiments, the flow rate of the solution is 8 MV / min, 9 MV / min, 10 MV / min, 11 MV / min, or 12 MV / min.

[0025] In the sample loading step of this invention, the equilibrium solution is 60-80 mV; for example, the volume of the equilibrium solution is 60 mV, 70 mV, or 80 mV.

[0026] In the sample loading step of this invention, the sample loading solution is 50–150 mV; for example, the volume of the sample loading solution is 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, 110 mV, 120 mV, 130 mV, 140 mV, or 150 mV. To obtain better purification results, the plasmid DNA content in the loaded sample should be ≤2 mg / mL membrane.

[0027] In the sample loading step of this invention, the reequilibration solution is 80-120 MV; for example, the volume of the reequilibration solution is 80 MV, 90 MV, 100 MV, 110 MV or 120 MV.

[0028] In the washing step of the present invention, the solution is 80-120 mV; for example, the volume of the washing solution is 80 mV, 90 mV, 100 mV, 110 mV or 120 mV.

[0029] In the elution step described in this invention, the solution volume is 100–150 mV. For example, the volume of the elution solution is 100 mV, 110 mV, 120 mV, 130 mV, 140 mV, or 150 mV.

[0030] In this invention, the method for purifying plasmid DNA based on membrane chromatography specifically includes: equilibrating the plasmid affinity chromatography membrane to 60-80 mV with a solution containing 1.8 M-1.9 M (NH4)2SO4 + 100 mM Tris-HCl + 10 mM EDTA, pH 7.5 ± 0.3; loading the sample to be separated within the sample loading range (≤2 mg / mL membrane); reequilibrating the plasmid affinity chromatography membrane to 80-120 mV with a solution containing 1.8 M-1.9 M (NH4)2SO4 + 100 mM Tris-HCl + 10 mM EDTA, pH 7.5 ± 0.3; and then equilibrating the membrane again with a solution containing 1.8 M-1.9 M (NH4)2SO4 + 100 mM Tris-HCl + 10 mM EDTA, pH 7.5 ± 0.3. The plasmid affinity chromatography membrane was washed with a solution of pH 7.5 ± 0.3 for 80–120 mV to elute plasmids of conformation other than the supercoiled plasmid. Then, the plasmid affinity chromatography membrane was washed with a solution containing 0.39 M–2 M (NH4)2SO4 + 0.4 M–2 M NaCl + 100 mM Tris-HCl + 10 mM EDTA at pH 7.5 ± 0.3 for 100–150 mV to elute the supercoiled plasmid.

[0031] In this invention, after elution, a cleaning step is further included, comprising: cleaning with cleaning solution 1 and cleaning solution 2, and then storing with a preservation solution.

[0032] In some embodiments, the cleaning solution 1 is an aqueous solution containing 1M NaOH and 1M NaCl.

[0033] In some embodiments, the cleaning solution 2 comprises 1.8M to 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH value of 7.5±0.3.

[0034] The preservation solution is a 0.1M NaOH aqueous solution.

[0035] The present invention also provides a purification reagent for plasmid DNA based on membrane chromatography, which includes a washing buffer and an elution buffer;

[0036] The washing solution contains 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5±0.3.

[0037] The eluent contains 0.39M–1.51M (NH4)2SO4 + 0.6M–1.61M NaCl, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5 ± 0.3.

[0038] Furthermore, the purification reagent also includes an equilibration solution and a loading solvent:

[0039] The equilibrium solution comprises: 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5±0.3.

[0040] The loading solvents include: 1.8M~1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5±0.3.

[0041] The plasmid purification method provided by this invention uses membrane chromatography, eliminates the need for column packing experiments, and has a relatively fast working flow rate. It has at least one of the following advantages:

[0042] Membrane chromatography can significantly shorten the process time. Plasmid purification by membrane chromatography takes only 1 to 2 hours per cycle, while plasmid purification by column chromatography (plasmid affinity chromatography) takes 3.5 to 4.5 hours per cycle. Therefore, using membrane chromatography can significantly shorten the process time.

[0043] Reduced process costs: Membrane materials are cheaper than chromatography packing materials, resulting in lower costs compared to traditional chromatography when used in single-use production processes. Therefore, using membrane chromatography technology can reduce process costs.

[0044] The recovery rate is high. The DNA recovery rate obtained by membrane chromatography is over 73%, the recovery rate of supercoiled DNA is over 97%, and the proportion of supercoiled DNA is over 95%, while the DNA recovery rate obtained by column chromatography is less than 60%, and the recovery rate of supercoiled DNA is less than 80%. Therefore, the plasmid recovery rate obtained by membrane chromatography is relatively high.

[0045] Membrane chromatography is simple to operate; it eliminates the need for column packing experiments and can be used once during GMP production without requiring cleaning validation. Therefore, it is relatively easy to operate. Attached Figure Description

[0046] Figure 1 The chromatograms under the conditions of Comparative Example 1 are shown;

[0047] Figure 2 Electrophoresis diagram under the conditions of Comparative Example 1;

[0048] Figure 3 The chromatograms under the conditions shown in Comparative Example 2 are as follows;

[0049] Figure 4 The electrophoresis diagram under the conditions shown in Comparative Example 2 is shown.

[0050] Figure 5 The chromatograms under the conditions shown in Comparative Example 3 are as follows;

[0051] Figure 6 The electrophoresis diagram under the conditions shown in Comparative Example 3 is shown.

[0052] Figure 7 The chromatograms under the conditions shown in Example 1 are as follows;

[0053] Figure 8 Electrophoresis diagram under the conditions of Example 1;

[0054] Figure 9 The chromatograms under the conditions shown in Example 2 are as follows;

[0055] Figure 10 Electrophoresis diagram under the conditions of Example 2;

[0056] Figure 11 The chromatograms under the conditions shown in Example 3 are as follows;

[0057] Figure 12 Electrophoresis diagram under the conditions of Example 3;

[0058] Figure 13 A comparison chart showing the process time of Comparative Example 1 and Example 3 is provided.

[0059] Figure 14 A comparison chart showing the process recovery rates of Comparative Example 1 and Example 3 is provided.

[0060] Figure 15 The graph shows a comparison of the process recovery rates between Comparative Example 2 and Example 3.

[0061] Figure 16 The results show that supercoiled plasmids with a supercoil ratio of >80% can be eluted in concentrations of (NH4)2SO4 ranging from 0.39M to 1.51M and NaCl ranging from 0.6M to 1.61M. Detailed Implementation

[0062] This invention provides a plasmid purification method based on membrane chromatography. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0063] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0064] In this invention, "and / or" describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone.

[0065] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0066] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions that are "composed of" or "as shown."

[0067] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0068] The embodiments and comparative examples of this invention describe some cases. These embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these cases. In fact, good plasmid DNA purification can be achieved at any concentration of each component between the two endpoint values ​​shown in the embodiments, with Example 3 showing the best results. The comparative examples only list some cases where the results were unsatisfactory in the experiments; many other attempts were made during the research and development process, which will not be elaborated here.

[0069] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0070] This invention does not limit the sequence or source of the plasmid, nor does it limit the method of preparing the plasmid before purification. Plasmids obtained by methods well known in the art can be purified using the method described in this invention. The preparation of samples conforming to the input sample properties in the technical solution, as described in the embodiments, includes:

[0071] Bacterial cells were lysed using an alkaline lysis method to obtain a crude plasmid extract. This extract was then preliminarily purified using TFF and clarification processes to remove some RNA, endotoxins, host proteins, and other impurities. Further purification was performed using Capto Core 700 chromatography, anion exchange chromatography, or molecular sieve chromatography to remove further impurities. The salt concentration was adjusted to the plasmid affinity membrane chromatography loading concentration (1.8M–1.9M (NH4)2SO4) by adding (NH4)2SO4 solution. The sample was then filtered using a filter with a pore size of 0.45 μm or smaller to obtain the sample to be separated. This sample must meet the conditions in the following table (enter sample attributes):

[0072] parameter scope Turbidity <5NTU plasmid concentration 5~500ng / μL Salt concentration <![CDATA[1.8M~1.9M(NH4)2SO4]]> Superspiral ratio ≥50％ RNA concentration ≤10μg / mL Endotoxin concentration ≤20 EU / mL Host protein concentration ≤10μg / mL

[0073] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0074] Comparative Example 1

[0075] 1) Sample preparation: Prepare a sample that conforms to the sample properties input in the technical solution, wherein the sample salt concentration is 2.0M (NH4)2SO4, and the sample is denoted as PS1.

[0076] 2) Plasmid affinity chromatography: The column was equilibrated with 2.0M (NH4)2SO4 solution (containing 2.0M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), loaded with PS1, and washed with 2.0M (NH4)2SO4 solution (containing 2.0M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3). Then, isocratic elution was performed with 1.7M (NH4)2SO4 + 0.3M NaCl + 100mM Tris-HCl, 10mM EDTA, and pH 7.5±0.3 to elute the supercoiled plasmid.

[0077] 3) Plasmid affinity chromatography (CIP): CIP was performed using water + 0.5M NaOH + water + pCh01 (30% isopropanol + 20mM sodium phosphate buffer) + water, and the column was finally preserved with 20% ethanol.

[0078] The recovery rate and the proportion of supercoiled DNA obtained are as follows:

[0079] Sample Description supercoiled DNA ratio DNA recovery rate Supercoiled DNA recovery rate Remark Load 66.8％ N / A N / A PS1 Elution 92.55％ 52.34％ 75.95％ N / A

[0080] The total duration of plasmid affinity chromatography experiments was 3.5–4.5 h. Chromatograms and electrophoresis results are shown below. Figures 1-2 As shown in the figure, the DNA recovery rate obtained by column chromatography was 52.34%, the supercoiled DNA recovery rate was 75.95%, and the proportion of supercoiled DNA was 92.55%.

[0081] Comparative Example 2

[0082] 1) Sample preparation: Prepare a sample that conforms to the sample properties input in the technical solution, wherein the sample salt concentration is 2.0M and the sample is denoted as PS1'.

[0083] 2) Plasmid affinity membrane chromatography (1MV = 0.3mL, flow rate: 10MV / min): Equilibrate the plasmid affinity membrane to 60MV using 2.0M (NH4)2SO4 solution (containing 2.0M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), load 34.4mL of PS1' (0.589mg / mL), reequilibrate the membrane to 100MV using 2.0M (NH4)2SO4 solution (containing 2.0M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), and then equilibrate the membrane again using 1.9M (NH4)2SO4 solution (containing 2.0M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3). The plasmid affinity chromatography membrane was washed for 106 mV with EDTA (pH 7.5 ± 0.3) to elute plasmids of conformation other than the supercoiled plasmid. Then, the membrane was washed for 244 mV with 1.5 M (NH4)2SO4 + 0.4 M NaCl solution containing 100 mM Tris-HCl, 10 mM EDTA, and pH 7.5 ± 0.3 to elute the supercoiled plasmid.

[0084] 3) Plasmid affinity membrane chromatography (CIP): CIP was performed using 1M NaOH + 1M NaCl, followed by reequilibration of the membrane using 1.9M to 2.0M (NH4)2SO4 solution, and finally the membrane was preserved using 0.1M NaOH.

[0085] The recovery rate and the proportion of supercoiled DNA obtained are as follows:

[0086]

[0087] Chromatograms and electrophoresis diagrams as follows Figures 3-4The chromatograms, electrophoresis results, recovery rates, and supercoiled DNA ratios show that the plasmid binding was strong under these loading conditions. The open-circular plasmid failed to elute during both loading and washing stages. Specifically, the plasmid binding was strong at 2.0 M ammonium sulfate concentration, and the open-circular plasmid could not be eluted at this salt concentration. Even using 1.9 M ammonium sulfate failed to elute the open-circular plasmid, and the plasmid DNA was not completely eluted during the elution stage. The total DNA recovery rate of the elution buffer was 35.77%, indicating that the loading, washing, and elution conditions need to be optimized.

[0088] Comparative Example 3

[0089] 1) Sample preparation: Prepare a sample that conforms to the sample properties input in the technical solution, wherein the sample salt concentration is 1.9M and the sample is denoted as PS1'.

[0090] 2) Plasmid affinity membrane chromatography (1MV = 0.3mL, flow rate: 10MV / min): Equilibrate the plasmid affinity membrane to 80MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), load 45mL of PS1' (2.49mg / mL), reequilibrate the membrane to 120MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), and then equilibrate the membrane again using 1.8M (NH4)2SO4 solution (containing 1.8M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3). The column was flushed with 120 mV of plasmid affinity chromatography at 7.5 ± 0.3 mV to elute plasmids of other conformations besides the supercoiled plasmid. The elution phase consisted of a gradient wash from 1.8 M (NH4)2SO4 + 0.6 M NaCl solution to 0.6 M NaCl solution, which also contained 100 mM Tris-HCl, 10 mM EDTA, and pH 7.5 ± 0.3. Eluted samples were collected in 3 mL tubes and electrophoresed at a volume of 150 mV to try to find a suitable ammonium sulfate concentration for eluting the supercoiled plasmid.

[0091] 3) Plasmid affinity membrane chromatography (CIP): CIP was performed using water, 1M NaOH + 1M NaCl, and water. Finally, the membrane was preserved using 0.1M NaOH.

[0092] The resulting proportions of supercoiled DNA and their corresponding salt concentrations are as follows:

[0093]

[0094] Chromatograms and electrophoresis diagrams as follows Figures 5-6The electrophoresis results, recovery rate, and supercoiled DNA ratio showed that under these loading conditions, supercoiled DNA flowed through during both the loading and washing stages. This indicates that the sample loading was excessive, exceeding the maximum capacity of the membrane chromatography. The excess DNA flowed through during the loading and washing stages, causing DNA loss and affecting the DNA recovery rate. Most of the open-circular plasmids eluted during the loading and rebalancing stages at 1.9M ammonium sulfate concentration, and a portion eluted during the washing stage at 1.8M ammonium sulfate concentration. The remaining small amount of open-circular plasmids eluted at the beginning of the elution. Starting from the second elution tube (Elution-2), samples of supercoiled plasmids with acceptable purity (supercoiled ratio greater than 80%) were obtained, with a total DNA recovery rate of 59.1%.

[0095] Example 1

[0096] 1) Sample preparation: Prepare a sample that conforms to the sample properties input in the technical solution, wherein the sample salt concentration is 1.9M and the sample is denoted as PS1'.

[0097] 2) Plasmid affinity membrane chromatography (1MV = 0.3mL, flow rate: 10MV / min): Equilibrate the plasmid affinity membrane to 80MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), load 45mL of PS1' (1.248mg / mL), reequilibrate the membrane to 100MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), and then equilibrate the membrane again using 1.8M (NH4)2SO4 solution (containing 1.8M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3). The plasmid affinity chromatography membrane was washed with EDTA (pH 7.5 ± 0.3) for 100 mV to elute plasmids of other conformations besides the supercoiled plasmid. The elution phase consisted of a gradient wash from 2.0 M (NH4)2SO4 solution to 2 M NaCl solution, which also included 100 mM Tris-HCl, 10 mM EDTA, and pH 7.5 ± 0.3. Eluted samples were collected in separate tubes (3 mL / tube) and electrophoresed. The elution volume was 120 mV, in an attempt to find a suitable salt concentration for eluting the supercoiled plasmid.

[0098] 3) Plasmid affinity membrane chromatography (CIP): CIP was performed using water, 1M NaOH + 1M NaCl, and water. Finally, the membrane was preserved using 0.1M NaOH.

[0099] The resulting proportions of supercoiled DNA and their corresponding salt concentrations are as follows:

[0100]

[0101]

[0102] Chromatograms and electrophoresis diagrams as follows Figures 7-8 .

[0103] In this embodiment, the concentrations of loading and washing salts were reduced compared to Comparative Example 2. The results showed that most open-circular plasmids could be eluted at concentrations of 1.9M and 1.8M ammonium sulfate. The removal rate of open-circular DNA from the flow-through was increased by 65.58% compared to Comparative Example 2. The remaining open-circular plasmids were eluted at the beginning of elution. The supercoiled plasmids were eluted starting at 1.4M (NH4)2SO4 + 0.6M NaCl. Starting from the first tube (Elution-1), samples of supercoiled plasmids with acceptable purity (supercoiled ratio greater than 80%) could be obtained, indicating that the salt concentrations corresponding to each tube after the first tube could be used to elute supercoiled plasmids. The salt concentration range was 0.39M to 1.40M (NH4)2SO4 + 0.60M to 1.61M NaCl. The total DNA recovery rate of the elution buffer was 82.28%.

[0104] The process conditions according to this embodiment allow for more complete plasmid elution and can remove most of the open-ring plasmids, thereby increasing the proportion of supercoiled plasmids.

[0105] Example 2

[0106] 1) Sample preparation: Prepare a sample that conforms to the sample properties input in the technical solution, wherein the sample salt concentration is 1.9M and the sample is denoted as PS1'.

[0107] 2) Plasmid affinity membrane chromatography (1MV = 0.3mL, flow rate: 10MV / min): Equilibrate the plasmid affinity membrane to 80MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), load 15mL of PS1' (1.335mg / mL), reequilibrate the membrane to 80MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), and then equilibrate the membrane again using 1.8M (NH4)2SO4 solution (containing 1.8M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3). The plasmid affinity chromatography membrane was washed with 80 mV of EDTA (pH 7.5 ± 0.3) column to elute plasmids of other conformations besides the supercoiled plasmid. The elution phase consisted of a gradient wash from 1.8 M (NH4)2SO4 + 0.6 M NaCl solution to 0.6 M NaCl solution, which also contained 100 mM Tris-HCl, 10 mM EDTA, and pH 7.5 ± 0.3. Eluted samples were collected in 3 mL tubes and electrophoresed. The elution volume was 150 mV. The aim was to find a suitable ammonium sulfate concentration for eluting the supercoiled plasmid.

[0108] 3) Plasmid affinity membrane chromatography (CIP): CIP was performed using water, 1M NaOH + 1M NaCl, and water. Finally, the membrane was preserved using 0.1M NaOH.

[0109] The resulting proportions of supercoiled DNA and their corresponding salt concentrations are as follows:

[0110]

[0111] Chromatograms and electrophoresis diagrams as follows Figures 9-10 .

[0112] In this embodiment, the open-circular plasmid was mainly eluted at ammonium sulfate concentrations of 1.9M and 1.8M. The removal rate of open-circular DNA in the flow-through was 66.82% higher than that in Comparative Example 2. The remaining small amount of open-circular plasmid was eluted at the beginning of elution. The supercoiled plasmid was eluted starting at 1.51M (NH4)2SO4 + 0.6M NaCl. Starting from the first tube (Elution-1), samples of supercoiled plasmid with qualified purity (supercoiled ratio greater than 80%) were obtained, indicating that the salt concentrations corresponding to each tube after the first tube can be used to elute supercoiled plasmids. That is, under the condition of NaCl concentration of 0.6M, the (NH4)2SO4 concentration in the range of 0.55 to 1.51M can elute supercoiled plasmids. The total DNA recovery rate of the elution buffer was 85.61%.

[0113] Compared with Comparative Example 3, this example changed the sample loading amount, while other conditions were the same as those in Comparative Example 3. The DNA recovery rate was increased by 44.87% compared with Comparative Example 3, and the open circular plasmid was mainly eluted during the washing stage, i.e., at a concentration of 1.8M ammonium sulfate.

[0114] The process conditions according to this embodiment allow for more complete plasmid elution, remove most of the open circular plasmids, increase the proportion of supercoiled plasmids, and ensure that the proportion of supercoiled DNA after mixing of eluted samples is still acceptable (>80%), with a high recovery rate.

[0115] Example 3

[0116] 1) Sample preparation: Prepare a sample that conforms to the sample properties input in the technical solution, wherein the sample salt concentration is 1.9M and the sample is denoted as PS1'.

[0117] 2) Plasmid affinity membrane chromatography (1MV = 0.3mL, flow rate: 10MV / min): Equilibrate the plasmid affinity membrane to 80MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), load 15mL of PS1' (1.16mg / mL), reequilibrate the membrane to 80MV using 1.9M (NH4)2SO4 solution (containing 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3), and then equilibrate the membrane again using 1.8M (NH4)2SO4 solution (containing 1.8M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, pH 7.5±0.3). The plasmid affinity chromatography membrane was washed with EDTA (pH 7.5 ± 0.3) column for 80 mV to elute plasmids of conformation other than the supercoiled plasmid. Then, the plasmid affinity chromatography membrane was eluted isocratically for 150 mV with 1.8 M (NH4)2SO4 + 0.6 M NaCl + 100 mM Tris-HCl, 10 mM EDTA, pH 7.5 ± 0.3 (80%) and 0.6 M NaCl + 100 mM Tris-HCl, 10 mM EDTA, pH 7.5 ± 0.3 (20%) solutions to elute the supercoiled plasmid.

[0118] 3) Plasmid affinity membrane chromatography (CIP): CIP was performed using 1M NaOH + 1M NaCl, followed by reequilibration of the membrane using 1.9M to 2.0M (NH4)2SO4 solution, and finally the membrane was preserved using 0.1M NaOH.

[0119] The recovery rate and the proportion of supercoiled DNA obtained are as follows:

[0120]

[0121]

[0122] Effect Analysis:

[0123] The total time for plasmid affinity membrane chromatography experiments was 1–2 hours, as shown in the comparison chart of process times between Comparative Example 1 and Example 3. Figure 13 Compared with Comparative Example 1, the process time in this embodiment is reduced by 62.5%.

[0124] The recovery rates of the examples and comparative examples are shown in the figure below. Figures 14-15 As shown in the figure, the open-circular plasmids were basically eluted at concentrations of 1.9M and 1.8M ammonium sulfate. The removal rate of open-circular DNA from the flow-through was 88.9% higher than that of Comparative Example 2. The proportion of supercoiled DNA obtained by elution under the conditions of 1.44M (NH4)2SO4 + 0.6M NaCl was 95.7%, the DNA recovery rate was 73.31%, and the supercoiled DNA recovery rate was 97.99%. As shown in the comparison graph of the recovery rates of Comparative Example 1 and Example 3, compared with Comparative Example 1, the DNA recovery rate of Example 3 increased by 40.07%, and the supercoiled DNA recovery rate increased by 29.02%. As shown in the comparison graph of the recovery rates of Comparative Example 2 and Example 3, compared with Comparative Example 2, the DNA recovery rate of Example 3 increased by 104.95%, and the supercoiled DNA recovery rate increased by 231.29%, and the proportion of supercoiled DNA reached the qualified level.

[0125] The process conditions described in Example 3 can remove the open-ring plasmid, allowing the supercoiled plasmid to be completely eluted, resulting in supercoiled plasmids with high recovery rate and high purity. Therefore, the process conditions in Example 3 are the optimal process conditions.

[0126] In summary, (1) when loading the sample using 2.0M (NH4)2SO4, the plasmid binding is relatively strong, and the open-ring plasmid cannot be eluted. However, when loading the sample using 1.9M (NH4)2SO4, the open-ring plasmid can be eluted, but the supercoiled plasmid cannot. Under the condition of 1.8M (NH4)2SO4, the supercoiled plasmid is also not eluted, but the open-ring plasmid is eluted in large quantities. Therefore, the loading conditions can be selected as 1.8M~1.9M (NH4)2SO4; (2) Using 1.8M (NH4)2SO4 for washing, a large amount of open-ring plasmids can be eluted. Under 1.9M (NH4)2SO4 conditions, open-ring plasmids can also be partially eluted. Therefore, the washing conditions can be selected as 1.8M~1.9M (NH4)2SO4; (3) Optimal conditions: using 1.9M (NH4)2SO4 for loading, 1.8M (NH4)2SO4 for washing, and 1.44M (NH4)2SO4 + 0.6M NaCl for elution, the proportion of supercoiled plasmids obtained is above 95%, the DNA recovery rate is above 73%, the supercoiled DNA recovery rate is above 97%, and the process time is 1~2h; (4) Based on the results of the above examples, it can be concluded that, Figure 16 As shown, supercoiled plasmids with a supercoil ratio of >80% can be eluted in concentrations of (NH4)2SO4 ranging from 0.39M to 1.51M and NaCl ranging from 0.6M to 1.61M.

[0127] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for purifying plasmid DNA based on membrane chromatography, comprising: The plasmid DNA solution was purified by affinity chromatography to obtain purified plasmid DNA; The purification process includes sample loading, washing, and elution.

2. The purification method according to claim 1, characterized in that, The ligand of the affinity chromatography membrane is 2-mercaptopyridine.

3. The purification method according to claim 1, characterized in that, The washing solution contains 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5 ± 0.

3.

4. The purification method according to claim 1, characterized in that, The elution solution contains 0.39M–1.51M (NH4)2SO4 + 0.6M–1.61M NaCl, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5 ± 0.

3.

5. The purification method according to claim 4, characterized in that, The elution solution contained 1.44M (NH4)2SO4 + 0.6M NaCl, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5 ± 0.

3.

6. The purification method according to claim 1, characterized in that, The sample loading includes the steps of equilibration, sample loading, and reequilibration. The equilibrium solution comprises 1.8 M to 1.9 M (NH4)2SO4, 100 mM Tris-HCl, 10 mM EDTA and water, with a pH of 7.5 ± 0.

3. The sample loaded with plasmid DNA contained 5–500 ng / μL, 1.8 M–1.9 M (NH4)2SO4, 100 mM Tris-HCl, 10 mM EDTA and water, with a pH of 7.5 ± 0.

3. The rebalancing solution comprises 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA, and water, with a pH of 7.5 ± 0.

3.

7. The purification method according to any one of claims 1 to 6, characterized in that, The flow rate of the solution during the purification step is 10 mV / min; During the sample loading step, the equilibrium solution is 60–80 mV; In the sample loading step, the sample loading solution is 50-150 mV; During the sample loading step, the reequilibration solution is 80–120 mV; In the washing step, the solution concentration is 80–120 mV; In the elution step, the solution is 100-150 mV.

8. The purification method according to any one of claims 1 to 6, characterized in that, After elution, the process also includes a cleaning step, which includes cleaning with cleaning solution 1 and cleaning solution 2, and then storing with a preservation solution. The cleaning solution 1 is an aqueous solution containing 1M NaOH and 1M NaCl; The cleaning solution 2 comprises 1.8M to 1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH value of 7.5±0.

3. The preservation solution is a 0.1M NaOH aqueous solution.

9. A purification reagent for plasmid DNA based on membrane chromatography, comprising washing buffer and elution buffer; The washing solution contains 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5±0.

3. The eluent contains 0.39M–1.51M (NH4)2SO4 + 0.6M–1.61M NaCl, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5 ± 0.

3.

10. The purification reagent according to claim 9, characterized in that, It also includes the equilibration solution and the loading solvent: The equilibrium solution comprises: 1.8M–1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5±0.

3. The loading solvents include: 1.8M~1.9M (NH4)2SO4, 100mM Tris-HCl, 10mM EDTA and water, with a pH of 7.5±0.3.

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