Plasmid DNA preparation and production method independent of chromatographic process

Abstract

The invention provides a plasmid DNA preparation and production method independent of a chromatographic process, and the method comprises the following steps: treating a plasmid DNA sample subjected to alkali lysis to remove most RNA, and then removing residual RNA and endotoxin through an anionic membrane in a filtering manner to finally obtain plasmid DNA with relatively high purity and low endotoxin content. According to the method, tedious chromatographic process operation is not needed, the workload is greatly reduced, the process time is shortened, and meanwhile, the plasmid production yield is increased. The method is suitable for preparing different types of plasmid DNAs, has good repeatability, and is beneficial for being amplified to an industrial production scale.

Description

Technical Field

[0001] This application relates to the field of molecular biology, specifically to a method for preparing and producing plasmid DNA that does not rely on chromatography. Background Technology

[0002] With the development of gene therapy, large-scale production of plasmid DNA has attracted the attention and research of the biopharmaceutical industry. The main method for plasmid preparation currently involves fermenting recombinant E. coli to produce DNA plasmids, followed by harvesting via alkaline lysis. However, this method faces many challenges in practical applications. For example, the host RNA released during E. coli lysis is a major impurity in subsequent purification processes.

[0003] Digestion with RNases is a common method for removing these RNA impurities, but this method is time-consuming and introduces new impurities while digesting RNA. Molecular sieve chromatography is a common laboratory practice for RNA removal, but it has limited sample throughput and is difficult to scale up industrially. Furthermore, using industrially common chromatographic methods (such as three-step or two-step chromatography) to purify plasmids can also be cumbersome and result in low recovery rates.

[0004] It should be noted that the methods described in this section are not necessarily methods that had been previously conceived or adopted. Unless otherwise specified, no method described in this section should be assumed to be prior art simply because it is included in this section. Similarly, unless otherwise specified, the issues mentioned in this section should not be considered to be accepted in any prior art. Summary of the Invention

[0005] To address the aforementioned problems, this application provides a method for purifying plasmid DNA, comprising the following steps: sequentially concentrating and changing the buffer of the plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; and eluting the anion exchange membrane with an elution buffer to obtain purified plasmid DNA.

[0006] The novel purification method provided in this application uses anion exchange membrane filtration instead of traditional chromatography methods (such as anion exchange column chromatography), and is used in conjunction with concentration and buffer replacement and RNA precipitation treatment steps, which can effectively remove residual RNA, HCP and endotoxins and other impurities from plasmid DNA samples.

[0007] Traditional chromatography methods (such as anion exchange chromatography) are cumbersome and require more sophisticated chromatography equipment. Therefore, while chromatography is generally considered to achieve higher sample purity than simple filtration, it also suffers from lower sample recovery rates and is expensive for industrial applications. The purification method provided in this application, which does not rely on chromatography, combines simple anion exchange membrane filtration with concentration and buffer replacement, and RNA precipitant treatment. This method not only achieves sample purity comparable to chromatography but also further improves sample recovery rates. Furthermore, it eliminates the need for fixed assets such as chromatography equipment and columns, simplifying the operation and significantly reducing processing time.

[0008] In some embodiments, the RNA precipitant includes at least one of calcium chloride or ammonium sulfate.

[0009] In some embodiments, the concentration and fluid exchange are performed via a tangential flow filtration membrane.

[0010] In some embodiments, after the step of filtering the plasmid DNA sample to be purified through an anion exchange membrane and before the step of eluting the anion exchange membrane with an elution buffer, a step of washing the anion exchange membrane with a washing solution is included.

[0011] This application, based on the application of anion exchange membrane filtration, uses a tangential flow filtration membrane to concentrate and change the sample before filtration, and further pre-treats it with RNA precipitants such as calcium chloride or ammonium sulfate. Additionally, washing and elution steps can be added after filtration to further remove impurities from the plasmid DNA sample, achieving better purification results.

[0012] In some embodiments, before the step of filtering the plasmid DNA sample to be purified through an anion exchange membrane and after the step of treating the plasmid DNA sample to be purified with an RNA precipitant, a step of diluting the plasmid DNA sample to be purified is included to make the conductivity of the plasmid DNA sample to be purified 29 to 31 mS / cm.

[0013] In this application, controlling the conductivity of the plasmid DNA sample to be purified within the above-mentioned range can further improve the effect of the anion exchange membrane in capturing plasmid DNA, thereby further improving the purification effect.

[0014] According to one embodiment of this application, a purified plasmid DNA is also provided, which is obtained using the purification method described in this application.

[0015] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0016] The accompanying drawings exemplify embodiments and form part of the specification, serving together with the textual description to explain exemplary implementations of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0017] Figure 1 The anion chromatography spectrum of the plasmid sample in Comparative Example 1 of this application;

[0018] Figure 2 This is a 1% agarose electrophoresis pattern of the plasmid sample from Comparative Example 1 of this application during the anion exchange chromatography process. Lane 1 is the DNA marker, lane 2 is the anion exchange chromatography loading solution for plasmid 102, lane 3 is the anion exchange chromatography flow-through solution for plasmid 102, and lane 4 is the anion exchange chromatography elution solution for plasmid 102.

[0019] Figure 3 The hydrophobic chromatography pattern of the plasmid sample in Comparative Example 1 of this application;

[0020] Figure 4 This is a 1% agarose electrophoresis pattern of the plasmid samples from Example 2 and Comparative Example 1 of this application, obtained through anion exchange membrane purification and chromatography. Lane 1 is the DNA marker, lane 2 is the anion exchange chromatography loading buffer for plasmid 102 from Comparative Example 1, lane 3 is the anion exchange membrane elution buffer for plasmid 102 from Example 2, and lane 4 is the hydrophobic chromatography elution buffer for plasmid 102 from Comparative Example 1.

[0021] Figure 5 The results are obtained by HPLC-AEX analysis of the eluent sample from the anion exchange membrane purification of the plasmid sample in Example 2 of this application.

[0022] Figure 6 The results of HPLC-AEX analysis of the eluent sample from the hydrophobic chromatography of the plasmid sample in Comparative Example 1 of this application are shown.

[0023] Figure 7 This is a 1% agarose electrophoresis pattern of the anion exchange membrane purification and chromatography process of the plasmid sample with a smaller molecular weight in Example 3 of this application. Lane 1 is the DNA marker, lane 2 is the anion exchange chromatography loading solution for plasmid 101, lane 3 is the anion exchange membrane elution solution for plasmid 101, and lane 4 is the hydrophobic chromatography elution solution for plasmid 101.

[0024] Figure 8 This is a 1% agarose electrophoresis pattern of the anion exchange membrane purification and chromatography process of the plasmid sample with a larger molecular weight in Example 4 of this application. Lane 1 is the DNA marker, lane 2 is the anion exchange chromatography loading solution for plasmid 103, lane 3 is the anion exchange membrane elution solution for plasmid 103, and lane 4 is the hydrophobic chromatography elution solution for plasmid 103.

[0025] Figure 9 Preferred steps for the purification method of plasmid DNA provided in this application. Detailed Implementation

[0026] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0027] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0028] Unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in the context of the document and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0030] When used in this article, the term "plasmid" refers to a DNA molecule that can accept exogenous nucleic acid fragments and replicate in prokaryotic or eukaryotic cells, and is often in the form of a circular double-stranded DNA molecule.

[0031] In this article, "chromatography" refers to a method that utilizes the active groups on the surface of the chromatographic medium to adsorb different solutes in the mobile phase, and separates them by utilizing the strength of their adsorption capacity for different solutes.

[0032] As used herein, "anion chromatography" refers to a chromatography technique that involves running an anion chromatography medium through a glass or metal tube, filling the tube to form a column; such a tubular column is called anion chromatography column. In some non-limiting embodiments of this application, column chromatography operations are relatively cumbersome, generally requiring a chromatography instrument to control the chromatography process, and in-situ cleaning of the equipment to ensure no cross-contamination; furthermore, the process of filling the empty column tube with the chromatography medium also requires considerable manpower and time.

[0033] As used herein, the terms "anion exchange membrane," "anion membrane," and similar expressions have the same meaning. Non-limiting examples include polymer membranes containing basic active groups that selectively bind and / or selectively adsorb anions. In some non-limiting embodiments of this application, the operation of filtration using anion membranes is very simple compared to traditional chromatography methods such as anion column chromatography, generally requiring only a peristaltic pump and a few tubing sections (and perhaps a pressure sensor).

[0034] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below.

[0035] This application provides a method for purifying plasmid DNA, the method comprising the following steps: sequentially concentrating and changing the buffer of the plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; and eluting the anion exchange membrane with an elution buffer to obtain purified plasmid DNA.

[0036] According to one embodiment of this application, a purified plasmid DNA is also provided, which is obtained using the purification method described in this application.

[0037] In some embodiments, the method includes the step of filtering the plasmid DNA sample to be purified through an anion exchange membrane. In some embodiments, the method does not include the step of using an anion exchange chromatography column.

[0038] This application employs anion exchange membrane filtration instead of traditional chromatography methods (such as anion exchange column chromatography), which can effectively remove impurities such as residual RNA, HCP, and endotoxins. Compared with traditional chromatography methods for purifying plasmid DNA, this method improves the recovery rate while meeting sample quality requirements; at the same time, it eliminates the need for fixed asset investment in chromatography equipment and columns, simplifying the operation process and shortening the processing time.

[0039] The anion exchange membrane can be of any suitable type. In some preferred embodiments of this application, anion exchange membranes are used. Q-series anion exchange membranes use stabilized reinforced cellulose as the membrane matrix and trimethylammonium-N... + (CH3)3 (trimethyl ammonium moiety) is used as the ion exchange ligand. Other suitable anion exchange membranes can be selected by those skilled in the art as needed. Furthermore, the thickness and area of ​​the anion exchange membrane can be selected according to the sample volume; the greater the membrane thickness and area, the larger the sample volume can be processed.

[0040] After filtering the plasmid DNA sample to be purified through an anion exchange membrane, washing and elution can be performed. In some embodiments, the method includes the following steps: filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; and eluting the anion exchange membrane with an elution buffer to obtain purified plasmid DNA. In some embodiments, after the step of filtering the plasmid DNA sample to be purified through the anion exchange membrane and before the step of eluting the anion exchange membrane with an elution buffer, a step of washing the anion exchange membrane with a washing solution is also included. By combining washing and elution steps with anion exchange membrane filtration, impurities such as residual RNA, HCP, and endotoxins can be removed more effectively, further optimizing the purification effect.

[0041] The purification method described in this application can employ various washing and elution schemes, and the washing and eluents can utilize different buffer systems and pH values. In some embodiments, the pH of the washing and eluents is 7.0–8.0; for example, any buffer system known in the art with a pH of 7.0–8.0 can be used as the washing and eluents. It is understood that the pH of the washing and eluents can be any value or range within the range of 7.0 to 8.0, such as 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, etc., or 7.0–7.5, 7.5–8.0, or other unlisted values ​​within the range of 7.0 to 8.0. In some embodiments, a Bis-Tris-HCl buffer system is used. In some embodiments, a Tris-HCl buffer system is used. In some embodiments, the washing and elution solutions contain bis(2-hydroxyethylamino)tris(hydroxymethyl)methane hydrochloride (Bis-Tris-HCl) or tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl). In some preferred embodiments of this application, a Tris-HCl buffer system is used, which can further improve the purification effect compared to other types of buffers.

[0042] In the purification method described in this application, two washing solutions can be used sequentially to wash the anion exchange membrane to further improve the purification effect. In some embodiments, washing the anion exchange membrane with the washing solutions includes washing the anion exchange membrane sequentially with a first washing solution and a second washing solution to remove impurities. In some preferred embodiments, the pH of the first washing solution, the second washing solution, and the eluent is 7.5. In some more preferred embodiments, the first washing solution contains 20 mM Tris-HCl, 10 mM EDTA, 400 mM NaCl, 0.05% Triton X-100, and 0.05% Triton X-114. In some embodiments, the washing volume of the first washing solution is greater than or equal to 100 volumes of the anion exchange membrane. In some embodiments, the washing process with the first washing solution is greater than or equal to 100 minutes. In some more preferred embodiments, the second washing solution contains 20 mM Tris-HCl, 10 mM EDTA, and 590 mM NaCl. In some embodiments, the washing volume of the second washing solution is greater than or equal to 50 volumes of the anion exchange membrane. In some more preferred embodiments, the eluent comprises 20 mM Tris-HCl, 1000 mM NaCl, and 10 mM EDTA. In some embodiments, the elution volume of the eluent is greater than or equal to 30 volumes of the anion exchange membranes.

[0043] Before filtering the plasmid DNA sample to be purified through an anion exchange membrane, RNA precipitation can be performed to remove a large amount of RNA impurities contained in the sample. In some embodiments, the step of treating the plasmid DNA sample to be purified with an RNA precipitant is included before filtering it through an anion exchange membrane. In some embodiments, the RNA precipitant includes at least one of calcium chloride or ammonium sulfate. In some preferred embodiments, the RNA precipitant includes calcium chloride, and the final concentration of the calcium chloride is 0.2 to 0.4 M. It is understood that the final concentration of the calcium chloride is any value or range within the range of 0.2 M to 0.4 M, such as 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, etc., or 0.2 M-0.3 M, 0.3 M-0.4 M, or other unlisted values ​​within the range of 0.2 M to 0.4 M. In some preferred embodiments, the RNA precipitant treatment time is greater than or equal to 30 minutes, and the RNA precipitant treatment temperature is 18 to 26 °C.

[0044] Before treatment with an RNA precipitant (e.g., calcium chloride), the plasmid DNA sample to be purified can be transferred to a neutral, low-salt buffer solution by concentration and buffer exchange, thereby enhancing the purification effect of the RNA precipitant; the concentration and buffer exchange step also removes some impurities from the sample. In some embodiments, before treating the plasmid DNA sample to be purified with the RNA precipitant, the process further includes a step of sequentially concentrating and buffering the plasmid DNA sample. In some preferred embodiments, the concentration and buffer exchange are performed using a tangential flow filtration membrane. In some preferred embodiments, the pore size of the tangential flow filtration membrane is 100 kDa. In some embodiments, after concentration, the concentration of plasmid DNA in the plasmid DNA sample to be purified is 0.5–1.5 mg / mL. It is understood that the concentration of plasmid DNA in the plasmid DNA sample to be purified is any value or range within the range of 0.5 mg / mL to 1.5 mg / mL, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, etc., or 0.5 mg / mL-1.0 mg / mL, 1.0 mg / mL-1.5 mg / mL, or other unlisted values ​​within the range of 0.5 mg / mL to 1.5 mg / mL. In some embodiments, the buffer exchange is an equal-volume exchange, where equal volume means that the volume of the sample solution before and after the exchange is equal. In some embodiments, the number of buffer exchanges is 6 to 8. In some embodiments, the buffer solution used in the buffer exchange contains Tris-HCl. In some embodiments, the pH of the buffer solution is 7.0 to 8.0. It is understood that the pH of the buffer solution is any value or range within the range of 7.0 to 8.0, such as 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, etc., or 7.0-7.5, 7.5-8.0, or other unlisted values ​​within the range of 7.0 to 8.0. In some preferred embodiments, the buffer solution contains 20 mM Tris-HCl and 10 mM EDTA. Based on filtration using an anion exchange membrane, the addition of a concentration and buffer exchange step and the application of an RNA precipitant before filtration can further remove impurities from the plasmid DNA sample, achieving better purification results.

[0045] When using an anion exchange membrane for filtration, the conductivity of the sample solution can be adjusted to further improve the purification effect. In some embodiments, the conductivity of the plasmid DNA sample to be purified is 29–31 mS / cm when filtered through the anion exchange membrane. In some embodiments, before the step of filtering the plasmid DNA sample through the anion exchange membrane and after the step of treating the plasmid DNA sample with an RNA precipitant, a step of diluting the plasmid DNA sample with pure water is included to make the conductivity of the plasmid DNA sample to be purified 29–31 mS / cm. It is understood that the conductivity of the plasmid DNA sample to be purified can be any value or range within the range of 29 mS / cm to 31 mS / cm, such as 29, 29.5, 30, 30.5, 31, etc., or 29 mS / cm-30 mS / cm, 30 mS / cm-31 mS / cm, or other unlisted values ​​within the range of 29 mS / cm to 31 mS / cm. Controlling the conductivity of the plasmid DNA sample to be purified within the above range can further improve the effect of the anion exchange membrane in capturing plasmid DNA, thereby further improving the purification effect.

[0046] In some embodiments, the method described in this application includes the following steps: obtaining a plasmid DNA sample to be purified; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; washing the anion exchange membrane with a washing solution to remove impurities; and eluting the anion exchange membrane with an elution solution to obtain purified plasmid DNA.

[0047] In some preferred embodiments, the method of this application includes the following steps: obtaining a plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; washing the anion exchange membrane with a washing solution to remove impurities; and eluting the anion exchange membrane with an elution solution to obtain purified plasmid DNA.

[0048] In some preferred embodiments, the method of this application includes the following steps: obtaining a plasmid DNA sample to be purified; sequentially concentrating and changing the buffer of the plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; washing the anion exchange membrane with a washing solution to remove impurities; and eluting the anion exchange membrane with an elution solution to obtain purified plasmid DNA.

[0049] In some more preferred embodiments, the method includes the following steps: obtaining a plasmid DNA sample to be purified; sequentially concentrating and changing the buffer of the plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; sequentially washing the anion exchange membrane with a first washing buffer and a second washing buffer to remove impurities; and eluting the anion exchange membrane with an elution buffer to obtain purified plasmid DNA.

[0050] In some more preferred embodiments, the method includes the following steps: obtaining a plasmid DNA sample to be purified; sequentially concentrating and changing the buffer in the plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; diluting the plasmid DNA sample to be purified to a conductivity of 29–31 mS / cm; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; and eluting the anion exchange membrane with an elution buffer to obtain purified plasmid DNA.

[0051] In some more preferred embodiments, the method includes the following steps: obtaining a plasmid DNA sample to be purified; sequentially concentrating and changing the buffer in the plasmid DNA sample to be purified; treating the plasmid DNA sample to be purified with an RNA precipitant; diluting the plasmid DNA sample to be purified so that the conductivity of the plasmid DNA sample to be purified is 29-31 mS / cm; filtering the plasmid DNA sample to be purified through an anion exchange membrane to capture the plasmid DNA; sequentially washing the anion exchange membrane with a first washing buffer and a second washing buffer to remove impurities; and eluting the anion exchange membrane with an elution buffer to obtain purified plasmid DNA.

[0052] In some embodiments, obtaining the plasmid DNA sample to be purified includes the following steps: harvesting cells containing plasmid DNA and / or cell culture supernatant to produce a harvest; and lysing the harvest to obtain the plasmid DNA sample to be purified. Methods for obtaining the desired plasmid DNA sample by culturing cells are known in the art and are not limited thereto. In some preferred embodiments of this application, *E. coli* is cultured to produce the plasmid DNA sample to be purified; during this process, *E. coli* needs to be lysed, and the lysis process should be uniform and gentle to minimize the generation of open circular plasmids.

[0053] Throughout the process, the composition, pH, conductivity, etc., can be finely adjusted according to the actual situation. In actual production, the process requirements shall prevail. As long as the deviation from the parameters of this application is not significant and no other functional requirements are introduced, they shall all be within the scope of protection of this application.

[0054] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0055] The exemplary embodiments of this application will now be described with reference to the accompanying drawings, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0056] Example

[0057] Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0058] Example 1: Obtaining plasmid DNA samples to be purified

[0059] The process of obtaining a plasmid DNA sample to be purified involves pretreatment of the sample, including the following steps: 1) bacterial resuscitation and culture; 2) bacterial lysis and clarification; 3) TFF concentration and medium replacement; and 4) RNA precipitation and clarification.

[0060] 1) Strain recovery and culture

[0061] Take one working seed of plasmid 102 (Beyotime Biotechnology, D2488) and thaw it at 30.0℃ for 5-10 minutes in a dry thermostat (Hangzhou Youning GA150-1) until the ice crystals melt.

[0062] Using a 200 μL pipette, pipette 100 μL of the working plasmid seed culture into a shaker tube containing 5 mL of LB medium. Tighten the cap and place the tube in a shaking incubator (Min Chuan MQT-60R) at 37.0 ± 2.0 °C and 230 ± 20 rpm for 6 hours. After incubation, start sampling to measure the OD600 of the seed culture, ensuring the OD600 value is between 2.0 and 4.0.

[0063] Use a 1000μL pipette to draw 500μL of seed culture and inoculate it into a conical cell culture flask containing TB medium. Tighten the cap and place the flask in a shaking incubator at 37.0±2.0℃ and 230±20rpm for 12 hours overnight. Take a sample to analyze the OD600 of the bacterial culture.

[0064] 2) Cell lysis and clarification

[0065] Centrifuge the bacterial suspension (medical centrifuge, Haier Medical LX-60T500-J) at 4000g for 15 minutes to collect the wet bacteria. Add the resuspension at a ratio of 1 (g) to 8 (mL) of wet bacterial weight to resuspension, and stir until a homogeneous suspension is formed. Observe for the presence of no lumpy or filamentous bacterial cells.

[0066] Add the alkaline lysis buffer at a 1:1 ratio, mix gently, and let stand for 4–5 minutes. Then add an equal volume of neutralizing solution and transfer to a 5L glass bottle.

[0067] The pyrolyzed sample was first filtered through a depth filter (Sartopure PP3, 0.65 μm) and then second filtered through a 0.22 μm Nylon filter to obtain a clear sample.

[0068] 3) TFF Concentration and Fluid Replacement

[0069] Disinfect the membrane pack with 0.5M NaOH for 30–60 minutes, then rinse the membrane pack with purified water (PALLCentramate). TM T-Series Cassette), with effluent and reflux conductivity <50 μS / cm.

[0070] The sample after secondary clarification was concentrated. The peristaltic pump speed was set (inlet flow rate of 300 LMH), the reflux flow rate was controlled, and the transmembrane pressure (TMP) was kept ≤2.0 Bar. The sample at the effluent end was collected and concentrated to a theoretical concentration of 0.5-1.5 mg / mL. The final concentration volume = amount of plasmid after secondary clarification (mg) / theoretical concentration (0.5-1.5 mg / mL).

[0071] After quantitatively concentrating the sample volume, add an equal volume of buffer solution for liquid exchange. The rotation speed and TMP are the same as in the concentration step, and the number of exchange cycles is 6–8. After the final exchange, concentrate to the volume obtained in the concentration step.

[0072] Close the outflow end, drain the membrane pack, wash the membrane pack with 100 mL of replacement buffer, and collect the wash buffer. Repeat the washing process once. Mix the wash buffer with the replaced liquid to form the TFF combined solution.

[0073] Sterilize the membrane pack with 0.5M NaOH for 30–60 minutes, and finally preserve the membrane pack with 0.1M NaOH.

[0074] 4) RNA precipitation and clarification

[0075] Add 3M CaCl2 stock solution to the TFF pooling solution to a final concentration of 0.3M, and let stand at room temperature (18–26°C) for >30 min. Then filter through a 0.22µm Nørgänger filter to obtain the plasmid DNA sample to be purified.

[0076] Example 2: Anion exchange membrane purification

[0077] The plasmid DNA sample to be purified obtained in Example 1 was filtered and purified using an anion exchange membrane. The specific steps are as follows:

[0078] Connect the tubing and adjust the flow rate of the peristaltic pump (Ripleykin KR1) to 1-2 mV / min.

[0079] The anion exchange membrane was equilibrated using an equilibration solution of 20 mM Tris-HCl, 10 mM EDTA, and 400 mM NaCl at pH 7.5, with an equilibration volume greater than 20 mV, until the pH and conductivity stabilized.

[0080] The clarified sample was diluted with purified water until the conductivity was below 30 mS / cm, and the pH was adjusted to 7.5.

[0081] The sample is passed through an anion exchange membrane (Sartorius) by a peristaltic pump. Q series).

[0082] The anion exchange membrane was rinsed with washing solution 1 (20 mM Tris-HCl, 10 mM EDTA, 400 mM NaCl, 0.05% Triton X-100, 0.05% Triton X-114, pH 7.5) for an equilibration volume greater than 100 mV and a time greater than 100 min.

[0083] The anion exchange membrane was washed with washing solution 2 (20 mM Tris-HCl, 10 mM EDTA, 590 mM NaCl, pH 7.5) with a washing volume greater than 50 mV.

[0084] The anion exchange membrane was eluted with an eluent (20 mM Tris-HCl, 10 mM EDTA, 1000 mM NaCl, pH 7.5), and the eluent was collected. The washing volume was greater than 30 mV.

[0085] Comparative Example 1: Purification by Chromatography

[0086] The plasmid DNA sample to be purified obtained in Example 1 was purified using chromatography as a comparative example in Example 2 of this application. The chromatography process included: 1) anion exchange chromatography (AEX); 2) hydrophobic chromatography (HIC).

[0087] 1) Anion chromatography

[0088] The anion exchange chromatography column (Seplife LXMS-30Q, 5 mL column volume) was washed with 0.5 M NaOH until the conductivity baseline stabilized, for at least 30 min; the packing material was then washed with purified water until the conductivity stabilized and <100 μS / cm. The chromatography system was a UniqueAutopre25D protein purification system.

[0089] The chromatography column was equilibrated using equilibration buffer (20 mM Tris-HCl, 10 mM EDTA, 400 mM NaCl, pH 7.5) with an equilibration volume greater than 5 CV until the pH and conductivity stabilized.

[0090] The clarified sample was diluted with purified water until the conductivity was below 30 mS / cm, and the pH was adjusted to 7.5. The sample was then loaded into an anion exchange column.

[0091] The chromatography column was equilibrated using equilibration buffer (20 mM Tris-HCl, 10 mM EDTA, 400 mM NaCl, pH 7.5) with an equilibration volume greater than 10 CV.

[0092] The column was washed with 30% eluent (20 mM Tris-HCl, 10 mM EDTA, 1000 mM NaCl, pH 7.5) for a wash volume greater than 5 CV.

[0093] The anion exchange chromatography column was eluted using eluent (20 mM Tris-HCl, 10 mM EDTA, 1000 mM NaCl, pH 7.5). The collection conditions were 100 mAu ≤ Peak (UV280) ≥ 100 mAu.

[0094] Wash the anion exchange chromatography column with 0.5M NaOH for at least 30 min. Then preserve the column with 0.1M NaOH, with a washing volume greater than 5 CV.

[0095] 2) Hydrophobic Chromatography

[0096] Wash the hydrophobic chromatography column (Capto Plasmid Select, 5 mL column volume) with 0.5 M NaOH until the conductivity baseline is stable, for at least 30 min. Then wash the packing material with purified water until the conductivity is stable and <100 μS / cm.

[0097] The chromatography column was equilibrated with equilibration buffer (20 mM Tris-HCl, 10 mM EDTA, 2 M (NH4)2SO4, pH 7.5) to a volume greater than 5 CV until the pH and conductivity stabilized.

[0098] Add 4M (NH4)2SO4 stock solution to the anion exchange chromatography eluent until the final concentration is greater than 2M (NH4)2SO4, and adjust the pH to 7.5. Load the sample treated by "1) anion exchange chromatography" into the hydrophobic column.

[0099] The chromatography column was equilibrated using equilibration buffer (20 mM Tris-HCl, 10 mM EDTA, 2 M (NH4)2SO4, pH 7.5) with an equilibration volume greater than 10 CV.

[0100] The chromatography column was linearly eluted using eluent (20 mM Tris-HCl, 10 mM EDTA, 0.7 M (NH4)2SO4, pH 7.5). The collection conditions were 100 mAu ≤ Peak (UV280) ≥ 100 mAu.

[0101] Wash the hydrophobic column with 0.5M NaOH for at least 30 minutes. Then preserve the column with 0.1M NaOH, with a washing volume greater than 5 CV.

[0102] Analysis of Results of Example 2 and Comparative Example 1

[0103] The anion chromatography pattern in Comparative Example 1 is as follows: Figure 1 As shown, the 1% agarose electrophoresis pattern of the sample during anion exchange chromatography is as follows: Figure 2 As shown. From Figure 2 It can be seen that the plasmid purity of the elution buffer (AEX-Elu) is normal, with virtually no RNA residue.

[0104] The hydrophobic chromatography pattern in Comparative Example 1 is as follows: Figure 3 As shown. The 1% Agarose electrophoresis patterns of the anion exchange membrane purification samples from Example 2 and the chromatography process of Comparative Example 1 are shown below. Figure 4 As shown. From Figure 4 It can be seen that the anion exchange membrane purified sample (lane 3) and the chromatography purified sample (lane 4) both have high purity. The proportion of supercoiled plasmids of the target component is very high, the proportion of open circular plasmids is very low, and there is basically no RNA residue.

[0105] The total plasmid content of the sample purified by anion exchange membrane was 3.18 mg, with a recovery rate of 67.66%; the total plasmid content of the sample purified by two-step chromatography (anion exchange chromatography and hydrophobic chromatography) was 2.47 mg, with a recovery rate of 52.55%. These results show that the recovery rate of the anion exchange membrane filtration purification process is significantly higher than that of the two-step chromatography purification process.

[0106] Meanwhile, the endotoxin level in the sample was <10 EU / mg. High-performance liquid chromatography (HPLC-AEX) analysis showed that the supercoil ratio of the anion exchange membrane purified sample was 92.90%, and the supercoil ratio of the two-step chromatography purified sample was 94.50%. The HPLC-AEX analysis results are as follows: Figure 5 (Anion exchange membrane eluent sample) Figure 6 (The hydrophobic chromatography eluent sample is shown.)

[0107] The results above show that, compared with traditional chromatography processes, the anion exchange membrane purification method provided in this application is simpler to operate, can greatly reduce production costs and process time, and does not require fixed asset investment such as chromatography equipment and chromatography columns; at the same time, it can achieve the same purity as traditional chromatography processes, and even the recovery rate is significantly higher than that of traditional chromatography processes.

[0108] Example 3: Purification of plasmids with small molecular weight

[0109] This comparative example purifies a plasmid with a relatively small molecular weight (101 plasmid, Beyotime Biotechnology, D2181). The sample pretreatment steps are the same as in Example 1, to obtain the plasmid DNA sample to be purified; then, a portion of the sample is purified by anion exchange membrane, the steps are the same as in Example 2; another portion of the sample is purified by anion exchange chromatography and hydrophobic chromatography as a comparative example, the steps are the same as in Comparative Example 1.

[0110] Anion exchange membrane purification and two-step chromatography purification of 1% agarose electrophoresis patterns are shown below. Figure 7 As shown. From Figure 7 It can be seen that the anion exchange membrane purified sample (lane 3) and the two-step chromatography purified sample (lane 4) both have high purity. The proportion of supercoiled plasmids in the target component is very high, the proportion of open circular plasmids is very low, and there is virtually no RNA residue.

[0111] The total amount of plasmid in the sample purified by the anion exchange membrane was 2.81 mg, with a recovery rate of 97.91%; the total amount of plasmid in the sample purified by the two-step chromatography process was 1.76 mg, with a recovery rate of 61.32%. These results indicate that when purifying plasmids with smaller molecular weights, the recovery rate of the anion exchange membrane purification process is higher than that of the two-step chromatography process.

[0112] Meanwhile, the endotoxin level in the sample was <10 EU / mg. HPLC-AEX analysis showed that the supercoil ratio of the anion exchange membrane purified sample was 97.78%, and the supercoil ratio of the two-step chromatography purified sample was 98.35%.

[0113] Example 4: Purification of plasmids with large molecular weight

[0114] This comparative example purifies a plasmid with a relatively large molecular weight (103 plasmid, Aono Gene, HG-VZH1643). The sample pretreatment steps are the same as in Example 1, to obtain the plasmid DNA sample to be purified; then, a portion of the sample is purified by anion exchange membrane, the steps are the same as in Example 2; another portion of the sample is purified by anion exchange chromatography and hydrophobic chromatography, the steps are the same as in Comparative Example 1.

[0115] The 1% agarose electrophoresis patterns of the samples purified by anion exchange membrane and two-step chromatography are shown below. Figure 8 As shown. From Figure 8 It can be seen that the samples purified by anion exchange membrane (lane 3) and the samples purified by two-step chromatography (lane 4) both have high purity. The proportion of supercoiled plasmids of the target component is very high, the proportion of open circular plasmids is very low, and there is basically no RNA residue.

[0116] The total amount of plasmid in the sample purified by the anion exchange membrane was 2.15 mg, with a recovery rate of 61.43%; the total amount of plasmid in the sample purified by the two-step chromatography process was 1.06 mg, with a recovery rate of 30.29%. These results indicate that when purifying plasmids with larger molecular weights, the recovery rate of the anion exchange membrane purification process is higher than that of the two-step chromatography process.

[0117] Meanwhile, the endotoxin level in the sample was <10 EU / mg. HPLC-AEX analysis showed that the supercoil ratio of the anion exchange membrane purified sample was 95.11%, and the supercoil ratio of the sample purified by the two-step chromatography process was 97.24%.

[0118] In summary, the results of Examples 1-2 and Comparative Example 1 demonstrate that the anion exchange membrane purification method used in this application, which does not rely on chromatography, achieves the clinical requirement of a supercoil ratio ≥90%, while the detection indicators such as RNA residue and endotoxins also meet clinical application standards. Furthermore, compared to the chromatography method used in Comparative Example 1, the anion exchange membrane purification method used in this application yields a higher final product yield.

[0119] In summary, as demonstrated in Examples 3-4 above, the anion exchange membrane purification method used in this application exhibits excellent purification effects for plasmids of different sizes. It can be applied to both large and small molecular weight plasmids, demonstrating its versatility.

[0120] In summary, the anion exchange membrane purification method provided in this application does not rely on traditional chromatography processes. It is not only simple to operate and industrially scalable, but also improves the overall recovery rate of plasmid purification and meets pharmaceutical standards. Furthermore, the separation and purification method described in this application offers good purification effect, strong reproducibility, and simple operation, meeting the requirements for industrial-scale purification production. It significantly reduces production costs and process time, and eliminates the need for fixed asset investment in chromatography equipment and columns, making it of significant application value in the industrial separation and purification of plasmid DNA.

[0121] It should be noted that the above are merely preferred embodiments of this application and are not intended to limit the application. Various modifications and variations are possible for those skilled in the art. Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or be unforeseeable to the applicant or other those skilled in the art. Therefore, the appended claims and any possible amendments to the claims are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Importantly, as technology evolves, many elements described herein can be replaced by equivalent elements appearing after this application.

Claims

1. A method for purifying plasmid DNA, characterized in that, The method includes the following steps: The plasmid DNA samples to be purified were concentrated and the solution was changed sequentially. The plasmid DNA sample to be purified was treated with an RNA precipitant. The plasmid DNA sample to be purified is filtered through an anion exchange membrane to capture the plasmid DNA; as well as The anion exchange membrane was eluted with an elution buffer to obtain purified plasmid DNA.

2. The method according to claim 1, characterized in that, The concentration and liquid exchange are performed through a tangential flow filtration membrane.

3. The method according to claim 2, characterized in that, The tangential flow filter membrane has a pore size of 100 kDa.

4. The method according to claim 1, characterized in that, After concentration, the concentration of plasmid DNA in the sample to be purified is 0.5–1.5 mg / mL.

5. The method according to claim 1, characterized in that, The fluid exchange is an equal-volume exchange, and the number of fluid exchanges is 6 to 8 times.

6. The method according to claim 1, characterized in that, The buffer solution used in the fluid exchange contains Tris-HCl.

7. The method according to claim 6, characterized in that, The buffer solution has a pH of 7.0 to 8.0 and contains 20 mM Tris-HCl and 10 mM EDTA.

8. The method according to any one of claims 1-7, characterized in that, The RNA precipitant includes at least one of calcium chloride or ammonium sulfate.

9. The method according to claim 8, characterized in that, The RNA precipitant is calcium chloride, and the final concentration of the calcium chloride is 0.2–0.4 M.

10. The method according to claim 9, characterized in that, in: The RNA precipitant treatment time is greater than or equal to 30 minutes; and / or The RNA precipitant treatment temperature is 18–26 °C.

11. The method according to any one of claims 1-10, characterized in that, When the plasmid DNA sample to be purified is filtered through an anion exchange membrane, the conductivity of the plasmid DNA sample to be purified is 29-31 mS / cm.

12. The method according to claim 11, characterized in that, Before the step of filtering the plasmid DNA sample to be purified through an anion exchange membrane and after the step of treating the plasmid DNA sample to be purified with an RNA precipitant, the method further includes a step of diluting the plasmid DNA sample to be purified so that the conductivity of the plasmid DNA sample to be purified is 29-31 mS / cm.

13. The method according to any one of claims 1-12, characterized in that, The method includes the following steps: Obtain the plasmid DNA sample to be purified; The plasmid DNA samples to be purified were concentrated and the solution was changed sequentially. The plasmid DNA sample to be purified was treated with an RNA precipitant. The plasmid DNA sample to be purified is diluted so that the conductivity of the plasmid DNA sample is 29-31 mS / cm; The plasmid DNA sample to be purified is filtered through an anion exchange membrane to capture the plasmid DNA; and The anion exchange membrane is eluted with an elution buffer to obtain purified plasmid DNA.

14. The method according to any one of claims 1-13, characterized in that, After filtering the plasmid DNA sample to be purified through an anion exchange membrane and before eluting the anion exchange membrane with an elution buffer, the process further includes washing the anion exchange membrane with a washing solution.

15. The method according to claim 14, characterized in that, The washing solution and the elution solution contain bis(2-hydroxyethylamino)tris(hydroxymethyl)methane hydrochloride (Bis-Tris-HCl) or tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl).

16. The method according to claim 14, characterized in that, The pH of the washing solution and the elution solution is 7.0 to 8.

0.

17. The method according to claim 14, characterized in that, Washing the anion exchange membrane with a washing solution includes washing the anion exchange membrane sequentially with a first washing solution and a second washing solution to remove impurities.

18. The method according to claim 17, characterized in that, in: The washing volume of the first washing solution is greater than or equal to the volume of 100 of the anion exchange membranes, and the washing process lasts for more than or equal to 100 minutes. The washing volume of the second washing solution is greater than or equal to the volume of 50 of the anion exchange membranes; and / or The elution volume of the eluent is greater than or equal to 30 volumes of the anion exchange membrane.

19. The method according to claim 17, characterized in that, The pH of the first washing solution, the second washing solution, and the eluent is 7.

5.

20. The method according to claim 17, characterized in that, in: The first washing solution contains 20 mM Tris-HCl, 10 mM EDTA, 400 mM NaCl, 0.05% Triton X-100, and 0.05% Triton X-114; The second washing solution contains 20 mM Tris-HCl, 10 mM EDTA, and 590 mM NaCl; and / or The eluent contains 20 mM Tris-HCl, 1000 mM NaCl, and 10 mM EDTA.

21. The method according to any one of claims 1-20, characterized in that, The method includes the following steps: Obtain the plasmid DNA sample to be purified; The plasmid DNA samples to be purified were concentrated and the solution was changed sequentially. The plasmid DNA sample to be purified was treated with an RNA precipitant. The plasmid DNA sample to be purified is diluted so that the conductivity of the plasmid DNA sample is 29-31 mS / cm; The plasmid DNA sample to be purified is filtered through an anion exchange membrane to capture the plasmid DNA; The anion exchange membrane is sequentially washed with a first washing solution and a second washing solution to remove impurities; and The anion exchange membrane is eluted with an elution buffer to obtain purified plasmid DNA.

22. The method according to claim 21, characterized in that, Obtaining the plasmid DNA sample to be purified includes the following steps: Harvesting cells and / or cell culture supernatants containing plasmid DNA to produce a harvest; and The harvested material is lysed to obtain the plasmid DNA sample to be purified.

23. A purified plasmid DNA, characterized in that, The plasmid DNA was obtained using the purification method described in any one of claims 1-22.

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