EA000785B1 - A method for large scale isolation and purification of plasmid dna - Google Patents

Abstract

1. A process for large scale isolation and purification of plasmid DNA from large scale microbial cell fermentations, comprising: a) harvesting microbial cells from a large scale fermentation; b) adding to the harvested microbial cells a sufficient amount of a lysis solution; optionally not comprising lysozyme; c) heating the microbial cells of step b) to a temperature between 70 degree C and 100 degree C in a flow-through heat exchanger to form a crude lysate; d) centrifuging the crude lysate and collecting supernatant e) filtering and diafiltering the supernatant of step d) providing a filtrate; f) contacting the filtrate of step e) with an anion exchange matrix; g) eluting and collecting plasmid DNA from the anion exchange matrix; h) contacting the plasmid DNA from step g) with a reversed phase high performance liquid chromatography matrix; and i) eluting and collecting the plasmid DNA of step g) from the matrix. 2. The process of claim 1 wherein optionally concentrating and/or diafiltering the product of step i) into a pharmaceutically acceptable carrier. 3. The process of claim 2 wherein optionally sterilizing the DNA product. 4. The process of claims 1-3 wherein the heating of step c) is to a temperature between 70 degree C and 77 degree C. 5. The process of claims 1-3 wherein the lysis solution of step b) contains a sub-microgram concentration of lysozyme. 6. The process of claims 1-3 optionally including RNAsa treatment at any step following step a). 7. The process of claims 1-3 wherein the lysis solution of step b) is modified STET buffer. 8. The process of claims 1-3 wherein the lysis solution of step b) is modified STET buffer containing a sub-microgram concentration of lysozyme.

Description

This application is a partial continuation of application US No. 08/446118, filed May 19, 1995

BACKGROUND OF THE INVENTION

Classical methods for isolating plasmid DNA from fermented microorganisms are suitable for the production of small quantities of plasmids or laboratory plasmid preparations. One of these methods involves alkaline lysis of microbial host cells containing the plasmid, followed by neutralization with acetate, which precipitates the genomic DNA of the host cell and proteins, which are then removed, for example, by centrifugation. The liquid phase contains plasmid DNA, which is precipitated with alcohol, and then subjected to isopycnic centrifugation using CsCl in the presence of ethidium bromide. Ethidium bromide is necessary for the isolation of plasmid DNA in three different forms, supercoiled (form I), annular with a gap (form II), and linearized (form III), while the desired plasmid form is collected. Then, butanol is extracted, which is necessary to remove residual ethidium bromide, after which the DNA is precipitated with alcohol. Additional purification steps are performed to remove host cell proteins. Removal of the host proteins is carried out by repeated extraction with phenol or a mixture of phenol and chloroform. Plasmid DNA is precipitated with alcohol, and the phenol residue is removed by extraction with isoamyl / chloroform. Finally, alcohol-precipitated plasmid DNA is dissolved in water or in a suitable buffer solution.

However, this method has many disadvantages and limitations:

a) this method requires the use of expensive and harmful chemical compounds (CsCl and EtBr, which are used in density gradient centrifugation; EtBr is a known mutagen and must be removed from the final products; in addition, it is also an intercalating agent and can break the plasmid) ;

b) the centrifugation stage in the density gradient is not easy to scale;

c) extraction with an organic solvent is necessary to remove residual EtBr;

g) to remove residual proteins and DNase, phenol is extracted, and therefore, when implementing the method, centrifugation is necessary to separate the phenol / water emulsion;

d) due to the implementation of a large number of repeated stages, this method is time-consuming and requires a lot of time (it takes several days to isolate);

f) significant difficulties are caused by the scalability of the chemical lysis stage, i.e., the lysozyme / alkali / KOAc stage is effective for small cell lysis, however, the increase in viscosity observed in this process is a serious obstacle to large-scale production; and

g) for enzymatic weakening of the bacterial cell wall before its lysis, it is necessary to use large quantities of lysozyme.

After that, the mixture is neutralized by adding acid, which leads to precipitation of high molecular weight chromosomal DNA. High molecular weight RNA and protein SDN complexes are precipitated by adding a high concentration of KOAc salt. After centrifugation, the plasmid product is in the clarified supernatant. The disadvantages of this method are that these procedures must be carried out quickly and under ice cooling in order to slow down the action of nucleases, which are not removed until extraction with phenol. The main impurity present in the supernatant with the product is RNA.

Another widely used method for the isolation and purification of plasmid DNA from bacterial cells is the rapid method, suitable for obtaining plasmid DNA only in very small quantities.

Holmes and Quigley (1981, Analytical Biochem. 114, pp. 193-197) describe a simple and fast method for producing plasmids in which bacteria are treated with lysozyme and then boiled in an appropriate buffer (STET) for 20-40 s at about 100 ° C, resulting in the formation of an insoluble clot of genomic DNA, protein and debris, leaving the plasmid in solution along with RNA as the main impurity. Obviously, for the implementation of this method, it is necessary to use lysozyme, and therefore an additional processing step, which is not very desirable for large-scale production of DNA used in medicine or veterinary medicine. However, the addition of lysozyme may enhance plasmid release during lysis. The advantage of this method is that heat treatment of cells also contributes to the denaturation of DNase.

However, this method cannot be used for bacterial fermentation in large volumes, that is, in volumes of 5 l or more.

Instead of isopycnic centrifugation using CsCl used for plasmid purification, alternative methods have been developed and published. These methods are applicable only for laboratory isolation of plasmids and provide for the implementation of:

a) size exclusion chromatography, which has mainly limited transmittance;

b) hydroxyapatite chromatography, which has the disadvantage that high concentrations of urea must be used for its effectiveness;

c) reverse phase chromatography; and

g) ion exchange chromatography.

Hence it is obvious that for large-scale isolation and purification of plasmid DNA from large volumes of fermented microorganisms, it is necessary to develop an improved method for producing plasmid preparations. The need to develop a large-scale method of plasmid DNA is dictated by the rapid development of many areas of molecular biology. In particular, recent advances in the manufacture of polynucleotide-based vaccines for administration to humans, as well as in the field of gene therapy, require the development of a method that would allow the production of highly purified polynucleotide vaccines in large quantities.

Therefore, for the development / implementation of a large-scale and technically feasible method of fermentation, isolation, purification and characterization of DNA for biopharmaceutical purposes, completely new technologies are needed.

SUMMARY OF THE INVENTION

The existing technology used for laboratory isolation and purification of plasmid DNA is a series of classical laboratory methods that cannot be used for industrial DNA production. For example, centrifugation in a density gradient is not scalable; and cleaning procedures that inevitably entail the use of harmful and expensive chemicals / solvents such as ethidium bromide (a known mutagen) are laborious and time consuming. Therefore, a scalable alternative method has been developed, which is disclosed in this application. In addition, an HPLC analysis was developed to track the production of the plasmid product during the corresponding steps and to identify various forms of plasmids. Bacterial cells, including a plasmid, are suspended and incubated (optionally) with lysozyme in a buffer containing a detergent, and then, to lyse the cells, these cells are heated using a flow-through heat exchanger and centrifuged. After centrifugation, the clarified lysate, which mainly contains RNA and a plasmid product, is filtered through a 0.45 μm filter and then diafiltered, after which the lysate is loaded onto an anion exchange column.

The plasmid product may, but is not necessarily, be treated with RNase before or after filtration, or at an earlier or later stage. A fraction of the anion exchange product containing the plasmid was loaded onto a reverse phase chromatography column and eluted with an appropriate buffer, whereby a highly purified plasmid DNA was obtained that could be introduced into humans.

Brief Description of the Drawings

In FIG. 1 schematically shows a suitable heat exchanger.

In FIG. Figure 2 shows a graph of the outlet temperature versus flow rate.

In FIG. 3 shows comparative chromatograms of the total plasmid fraction in the clarified supernatant obtained using 50 mM EDTA and 100 mM EDTA.

In FIG. 4 is a graph of the dependence of the output of a supercoiled plasmid on the exit temperature.

In FIG. 5 shows elution profiles obtained by passing through an anion exchange column treated with RNase (thick line) and untreated (thin line) clarified lysate.

In FIG. Figure 6 shows the elution profiles obtained by anion exchange chromatography of a clarified lysate, which was diafiltered before loading into a column, and a lysate, which was not diafiltered before loading into a column.

In FIG. 7 shows an elution profile of plasmid DNA obtained from a cell lysate.

In FIG. Figure 8 shows the results of the analysis of a DNA product obtained at various intermediate stages of purification, which was performed using agarose gel electrophoresis.

In FIG. Figure 9 shows a monitoring scheme for anion exchange HPLC analysis of a DNA product, confirming the purity of the specified product.

DETAILED DESCRIPTION OF THE INVENTION

The authors of this application have developed a new, scalable alternative method of lysing / removing debris for large-scale isolation and purification of plasmids, which involves the rapid heating of microbial cells in order to induce their lysis and precipitation of genomic DNA, proteins and the rest of the debris, with the plasmid retained in solution. This method is intended for large-scale isolation and purification of plasmid DNA. The authors of the present application, it was found that the suspension of microbial cells in a modified STET buffer (described below), followed by heating the suspension to about 70-1 00 ° C in a flow heat exchanger gives good lysis results. Continuous or periodic centrifugation of the lysate results in the formation of a precipitate that contains cell debris, protein, and most of the genomic DNA, while the plasmid remains in the supernatant. The method of the present invention has several advantages in obtaining higher levels of the product than in chemical lysis; in the inactivation of DNase; simplicity of implementation and the possibility of choosing the volume of products obtained.

The present invention relates to a method for large-scale isolation and purification of plasmid DNA from fermented microorganisms.

It is provided that the total volume of the fermentation medium with microorganisms is more than 5 l, or microorganisms are collected from a volume of medium of more than 5 l.

The proposed method allows to obtain plasmid DNA in highly purified form suitable for administration to humans.

Suitable for human introduction of DNA are, but are not limited to, polynucleotide vaccines and DNA intended for human genomic therapy. Polynucleotide vaccines are used for direct human injection Montgomery, D.L. et al., 1993, Cell Biol., 169, pp. 244-247; Ulmer, J.B. et al., 1993, Science, 259, pp. 1745-1749.

Also, the proposed method provides real-time monitoring for obtaining various forms of plasmid DNA in the process of stages of isolation and purification. As indicated above, such various forms of plasmid DNA that can be individually isolated by the claimed method are Form I (supercoiled plasmid), Form II (gap plasmid or relaxed plasmid), and Form III (linearized plasmid).

The method in accordance with the present invention is suitable, mainly for the isolation of plasmid DNA from fermented microorganisms. However, for any specialist it is obvious that in the implementation of the claimed method can be used in a variety of microbial cells, including, but not limited to, fungal cells such as yeast and bacterial cells. Preferred are fermented bacterial cells containing a plasmid that must be isolated and purified, in particular E. coli cells containing a plasmid intended for isolation and purification. At the same time, it is obvious to any specialist that when carrying out the claimed method instead of B. coli, any bacterial cells can be used. Fermentation of the cells can be carried out in any liquid medium suitable for culturing the bacteria used.

The plasmid isolated and purified by the method of the present invention may be any extrachromosomal DNA molecule. These plasmids can have a high or low copy number per cell. Moreover, they can be of virtually any size. It should be noted that virtually any plasmid present in microbial cells can be isolated by the method of the present invention.

Microbial cells containing the plasmid are harvested from the fermentation medium to obtain a cell paste or suspension. Any suitable method can be used to collect cells from a liquid medium, including, but not limited to centrifugation or microfiltration.

Isolation of plasmid DNA from harvested microbial cells by methods of modern laboratory technology consists mainly in the enzymatic treatment of microbial cells to weaken the cell wall, followed by lysis of these cells. Purification steps include repeated centrifugation using CsCl / EtBr followed by extraction with an organic solvent and precipitation to remove tRNA, residual proteins, EtBr and other impurities originating from host cells. These stages are not scalable, and therefore unsuitable for large-scale production. In contrast, preparative chromatography performed on an appropriate scale is a highly efficient purification method that achieves a high level of resolution and increased productivity in the purification of plasmid DNA products, and which is quite simple to implement. It was shown that the use of two different chromatography methods, namely, reverse phase and anion exchange chromatography, allows to achieve a high level of plasmid DNA purification that meets the requirements for preparations for administration to humans. Isolation by reverse phase chromatography is based on hydrophobic interactions, and isolation by anion exchange chromatography is based on electrostatic interaction. Through the implementation of these two orthogonal chromatographic steps, separation of the various forms of the plasmid is achieved (supercoiled, open, relaxed, linear and concatemer), and the removal of impurities originating from host cells such as LPS (endotoxin), RNA, DNA and residual proteins.

In the method of the present invention, the collected microbial cells are resuspended in a modified STET buffer containing about 50 mM Tris, about 50-100 mM EDTA, about 8% sucrose, about 2% Triton X-100, and optionally sub-microgram concentrations of lysozyme at pH 6 0-10.0. Concentrations of lysozyme, which may, but need not be, used in the method of the present invention, are significantly lower than the concentrations of lysozyme used in conventional methods. It will be apparent to those skilled in the art that the basic composition of the above buffer can be modified and adapted for use in the method of the present invention. It should be noted that modifications of the basic composition of the buffer should not adversely affect the productivity of the method of the present invention or alter the yield of a product that can be achieved using a method that is within the scope of the present invention. For best results, the pH range can be adjusted according to the particular bacterial strain used. A preferred pH range is about 8.0-8.5. Then the suspension is heated to about 70-100 ° C, and preferably to 70-77 ° C, in a flow heat exchanger. The lysate is centrifuged, resulting in a precipitate containing a large amount of cell debris, protein, and most of the genomic DNA.

In order to demonstrate the feasibility of flow-through thermal lysis of microbial cells containing a plasmid, an appropriate heat exchanger model was constructed. In particular, such a heat exchanger consists of a stainless steel tube (10 ft. X 0.25 in. (Outside diameter), 30.48 mm. X 6.35 mm) having a spiral shape. This spiral tube is completely immersed in a high constant temperature water bath. The amount of delay in the coil of the heat exchanger is about 50 ml. To measure the temperature at the inlet and outlet, and the temperature of the water bath, thermocouples and a thermometer are used, respectively. The product stream is fed into the heating coil using a Masterflex silicone tube peristaltic pump. The cell lysate exiting the coil is then centrifuged in a Beckman J-21 batch centrifuge to clarify. In FIG. 1 schematically shows a specific device, however, other types of heat exchangers may be used in the present invention, including, but not limited to, a shell-and-tube heat exchanger, which is preferred.

After centrifugation, the clarified lysate can be, but is not necessarily, treated with RNase, and the plasmid product can be filtered to further remove fine debris. In this method, various filtration methods can be used, including, but not limited to, filtering through a membrane having small pores. The preferred filtration method is filtration through a 0.45 micron filter.

For the subsequent removal of impurities of the DNA product, the resulting material can be diafiltered. In this method can be used standard commercially available materials for diafiltration, carried out in accordance with a known method.

A diafiltration method using an ultrafiltration membrane is preferred with a molecular weight cut-off ranging from 30,000 to 500,000, depending on the size of the plasmid. The DNA preparation described above is diafiltered using an ultrafiltration membrane (with a molecular weight cut-off of about 1 000 000) against a column buffer, and then loaded into an anion exchange column. Diafiltration prior to loading into the anion exchange column is preferred and can significantly increase the amount of lysate that can be loaded into the column.

A wide range of commercially available anion exchange chromatography matrices can be used for use in the present invention, including, but not limited to, POROS, Qiagen, Toso Haas, Sterogene, Spherodex, Nucleopac, and Pharmacia anion exchange resins. The column (Poros 11 P1 / M, 4.5 mm x 100) is first balanced with 20 mM Bis / Tris-propane (pH 7.5) and 0.7 M NaCl. A sample is then loaded and washed with the same buffer. Then an elution is performed with a gradient of 0.5-0.85 M NaCl in approximately 25 column volumes and the appropriate fractions are collected. Anion exchange chromatography is an ideal first stage of fine purification, as it provides excellent purification from RNA, genomic DNA and protein. In FIG. 5 (bold line) shows the elution profile of the filtered clarified cell lysate obtained by passing through an anion exchange column. Analysis by agarose gel showed that the second peak, which appears after passing through the through stream, consists of a plasmid product. An earlier broad peak refers to RNA. This was confirmed by incubating the clarified cell lysate with ribonuclease before loading into the column, resulting in the disappearance of a large peak, and instead, several smaller and faster elution peaks appeared due to the cleavage of the products as a result of hydrolysis by ribonuclease.

The product fraction obtained by anion exchange chromatography is loaded onto a reverse phase column. A wide range of commercially available 9 matrices can be used for use in the present invention, including, but not limited to, matrices from POROS, Polymer Labs, Toso Haas, Prarmacia, PQ Corp., Zorbax, BioSepra resins, BioSepra Hyper D resins, BioSepra resins QHyper D and Amicon. Matrices based on polymers or on the basis of silica gel may also be used. The reverse phase column (Poros P / H) was equilibrated with approximately 100 mM ammonium bicarbonate, pH = 8.5. A gradient of 0-11% isopropanol is used to elute the bound material. In this way, three forms of plasmids can be isolated, namely, forms I, II, III described above.

The eluted plasmid DNA is then concentrated and / or diafiltered to reduce the volume or to replace the buffer. In the case of DNA intended for administration to humans, the diafiltration of the DNA product can be carried out in a pharmaceutically acceptable carrier or buffer solution. Suitable pharmaceutically acceptable carriers or buffers are well known in the art and are described in the literature, for example, Remington s Pharmaceutical Sciences. For the purposes of the present invention, any suitable method for concentrating a DNA sample can be used. Such methods are diafiltration, precipitation with alcohol, lyophilization, etc., while diafiltration is the preferred method. After diafiltration, the final plasmid DNA product can be sterilized. In this case, any suitable method of sterilization of the DNA product can be used, provided that it will not adversely affect the properties of the DNA product; for example, sterilization of a DNA product can be carried out by passing it through a membrane having a sufficiently small pore size, for example, 0.2 microns or less.

Below are examples illustrating the implementation of the claimed method, however, these examples should not be construed as limiting the scope of the invention.

Example 1. Microbial cell growth, cell lysis and clarification.

One liter of frozen E. coli cell suspension was used to prepare 8 L of cell suspension in STET buffer (8% sucrose, 0.5% Triton, 50 mM Tris buffer at pH 8.5 and 50 mM EDTA). The optical density of the cell suspension at 600 nm was approximately 30. The measured viscosity of the cell suspension was approximately 1.94 centipoise at room temperature (24 ° C.). The resulting suspension was continuously stirred until homogeneous. Then, the cell suspension was pumped into the heat exchanger at 81 ml / min, which corresponds to a residence time of the cell solution in the heat exchanger of approximately 35 s. The temperature of the bath was maintained at 92 ° C. The measured temperature of the cell solution at the inlet and outlet of the heat exchanger was approximately 24 ° C and approximately 89 ° C (average value), respectively. About one liter of sample was passed through a heat exchanger. Clogging in the tube was not visually observed, although the lysate was slightly thicker than the starting material. After cooling the lysate to room temperature, its measured viscosity was approximately 40 cP. After that, the cell lysate was clarified by serial centrifugation at 9000 rpm for 50 min using Beckman J-21. Analysis of the supernatant confirmed the effectiveness of cell lysis and isolation of the product. The yield obtained by thermal lysis by the flow method is at least comparable to the yield obtained by the Quigley & Holms boiling method. However, the latter method is carried out in the laboratory to obtain small batches of the drug, and therefore it is not suitable for large-scale (5 L or higher) production. Since the method of heat exchange is flow-through, the upper limit of the volume of cell suspension that can be obtained does not exist. This method can be adapted for large-scale fermentation of bacteria to produce large quantities of highly purified plasmid DNA.

The clarified lysate was then filtered through a membrane having a pore size of 0.45 μm to remove fine debris. The resulting filtrate was diafiltered using a membrane with a molecular weight cut-off of about 10,000.

Example 2. Regulation and reproducibility of cell lysis using a heat exchanger.

The regulation of the flow rate at which the cell suspension is passed through a heat exchanger allows for direct control of the lysis temperature, i.e., the outlet temperature. A cell suspension solution was prepared as described in Example 1 and pumped into a heat exchanger at a flow rate of 160-850 ml / min. The corresponding outlet temperatures ranged from 93 to 65 ° C, respectively. In FIG. 2 shows the ratio of flow rate and temperature. The initial temperature of the cell suspension was 24 ° C, and the temperature of the bath was kept constant at 96 ° C.

In addition, a series of run cycles were carried out, where the outlet temperature was 80 ° C. The yield of 24 mg of circular DNA per liter of clarified supernatant illustrates the reproducibility of this method.

Example 3. Purification of plasmid DNA.

Microbial cells and lysates were obtained as described in examples 1 and 2, and the following analyzes were performed.

To illustrate that the addition of 100 mM EDTA compared to 50 mM EDTA increases the percentage of supercoiled DNA, and the following assays were performed to determine the permissible outlet temperature range (i.e., lysis temperature) for supercoiled DNA isolation. The supercoiled form of plasmid DNA is desirable because it is more stable than the relaxed ring form. Thus, the supercoiled form of DNA can be converted into an open circular form by single stranded rupture under the influence of DNase. It was found that the addition of 100 mM EDTA compared to 50 mM EDTA in STET buffer minimizes the formation of an open ring plasmid. In FIG. Figure 3 shows comparative chromatograms of the whole plasmid in the clarified supernatant with 50 mM EDTA compared to 100 mM EDTA. A cell suspension was obtained by the method described in example 1. The working flow rate for these series was approximately 186 ml / min. The inlet and outlet temperatures and the bath temperature were 24, 92, and 96 ° C, respectively.

Acceptable lysis temperature limits were determined by measuring the percentage of supercoiled plasmid generated for each batch. In FIG. 4 shows the concentration of a supercoiled plasmid as a function of outlet temperature. Acceptable temperature limits for lysis are 75 - 92 ° C. At temperatures below 75 ° C, a more relaxed ring plasmid was obtained, which is most likely due to an increase in DNase activity. At temperatures above 93 ° C, the levels of the supercoiled plasmid are likely to decrease, which is possibly due to thermal denaturation.

After continuous thermal lysis and centrifugation, 1 ml of clarified lysate was either incubated with 5 μg of RNase for 2 hours, or used without treatment. Then, untreated samples and RNase-treated samples were loaded onto an anion exchange column (Poros Q / M 4.6 x 100), which was previously equilibrated with a mixture (50:50) of solvents A and B [HPLC: solvent A: 20 mM Tris / bis-propane, pH 8.0; and solvent B 1M NaCl in 20 mM Tris / bis-propane, pH 8.0]. The column was eluted using a gradient of 50 to 85% B in 100 column volumes.

The open circular plasmid was eluted at approximately 68% B. and the supercoiled plasmid was eluted at approximately 72% B.

In FIG. 5 shows a comparison of the eluate on an anion exchange column from a clarified lysate treated with RNase (thin line) and not treated with RNase (thick line). After about 10 minutes, a peak was formed, which is plasmid DNA, and then a large peak was produced in an untreated sample, which is RNA. In the sample treated with RNase, a large peak of RNA is eliminated, and a clearer separation of the plasmid peak and the impurity peaks is ensured.

As described above, diafiltration before anion exchange chromatography leads to a significant increase in the amount of lysate that can be loaded onto the column. This is illustrated in FIG. 6, which shows a comparison of a clarified lysate that was diafiltered and a clarified lysate that was not diafiltered before anion exchange chromatography. Samples were obtained by the method described above, except that one sample was diafiltered before loading onto an anion exchange column and the other sample was not diafiltered. The column was washed and eluted as described above. In FIG. Figure 6 shows that the amount of impurities eluted from the column is significantly higher in the sample that was not diafiltered. A large number of impurities that bind the matrix on the anion exchange column can greatly reduce the maximum capacity of the column, which causes loss of the DNA product due to the inability of the matrix to bind to any other material. Therefore, diafiltration removes impurities and allows you to bind another DNA product with an anion exchange matrix, which, in turn, allows you to get a larger volume of clarified lysate loaded on the column.

Plasmid DNA eluted from the anion exchange column was separated into separate forms by reverse phase HPLC analysis. The separation of the supercoiled plasmid (Form 1) from the open circular plasmid (Form 2) is shown in FIG. 7. The two forms were easily separated, which allowed the isolation of individual plasmid forms.

Example 4. Highly purified plasmid DNA obtained by chromatographic method.

The cell paste for fermentation was resuspended in a modified STET buffer, and then subjected to thermal lysis in a batch device. Alternatively, the cell paste for fermentation was resuspended in a modified STET buffer, and then subjected to heat lysis by the flow method described above. The lysate was centrifuged as described above. 20 ml of the supernatant was filtered as described above, and then loaded onto an anion exchange column (Poros Q / M 4.6x100), which was pre-equilibrated with a mixture (50/50) of buffers A and B described above. A gradient of 50–85% B was run in 50 column volumes at a flow rate of 10 ml / min. Fractions (2.5 ml each) were collected from the column. Supercoiled plasmid DNA was eluted from the column at 72% B.

The product obtained by anion exchange chromatography was loaded onto a reverse phase chromatographic column (Poros R / H), which was preliminarily equilibrated with 100 mM ammonium bicarbonate at pH = 8.0, and a gradient of 0% 80% methanol was used to elute the bound material. Highly purified supercoiled plasmid DNA was eluted at 22% methanol. A photograph of an agarose gel with product fractions obtained at each of the main stages of the purification process is shown in FIG. 8. The final product shown in FIG. 9 is highly purified, as evidenced by agarose gel analysis, colorimetric analysis, and HPLC analysis as described in Example 3. The resulting product contained more than 90% supercoiled plasmid and less than 1 0% open loop plasmid. RNA levels were below the detection limits of the assay used. Levels of genomic DNA and protein impurities were below the detection limits of the analysis used. The total yield of the supercoiled plasmid in the final stage of the process was approximately 60% of the output of the supercoiled plasmid in the clarified lysate.

Example 5. Purification of plasmid DNA on a multi-gram scale.

4.5 L of a frozen E. coli cell suspension was used to produce 33.7 L of a cell suspension in STET buffer (8% sucrose, 2% Triton, 50 mM Tris buffer, 50 mM EDTA, pH 8.5) containing 2500 units / ml lysozyme. The calculated optical density of the suspension at 600 nm was about 30. The resulting suspension was stirred at room temperature for 1 to 5 minutes to ensure thorough mixing, and then incubated for 45 minutes with stirring at a temperature of 37 ° C. After incubation, stirring was continued at room temperature, and the cell suspension was pumped into the heat exchanger at a flow rate of 500 ml / min. The temperature of the bath was maintained at 10 ° C, and the measured temperatures at the inlet and outlet of the cell suspension were approximately 24 ° C and 70-77 ° C, respectively. The cell lysate exiting the heat exchanger was collected in Beckman centrifuge tubes (500 ml each), and the resulting material was immediately centrifuged in Beckman J-21 centrifuges for 50 min at 9000 rpm. After centrifugation, it was found that the supernatant contained 4-5 times more plasmid product than when incubation with lysozyme was not carried out. After centrifugation, the supernatant was immediately diafiltered against 3 volumes of TE buffer (25 mM

Tris-EDTA at pH 8.0), and then incubated with 20 x 10 ⁵ units of E. coli RNase for 2-4 hours at room temperature. After incubation was complete, the product solution was diafiltered against an additional 6 volumes of TE buffer using a membrane with a molecular weight cut-off of 1 00 kDa, and then filtered through a 0.45 μm filter to remove residual debris. The filtered lysate was diluted with 0.7 M NaCl in 20 mM bis / Tris-propane-NaCl buffer at pH = 7.5, and then, the diluted filtrate was loaded onto an anion exchange column. The anion exchange column (3.6 L POROS RI / M) was previously equilibrated with 20 mM bis / Tris-propane and 0.7 M sodium chloride. The filtered lysate was loaded onto a column. In this case, 5 g of a supercoiled plasmid was loaded onto an anion exchange column. After loading, the column was washed with 2-4 column volumes of 20 mM bistris-propane and 0.7 M NaCl. A gradient of 0.7 M NaCl, 2.0 M NaCl in 20 mM bis / Tris-propane in 10 column volumes was used to remove other E. coli proteins, RNA, and some endotoxins. The supercoiled plasmid fraction was eluted with 1.4-2.0 M sodium chloride. The supercoiled plasmid fraction obtained from the anion exchange column and containing 4 g supercoiled plasmid was then diluted 2-3 times with pyrogen-free water, adjusted to 1.2% IPA, and the pH adjusted to 8.5 by adding 1N. NaOH. The supercoiled plasmid fraction diluted on the anion exchange column was then loaded onto a 7 liter reverse phase column (POROS R2 / M), which was pre-equilibrated with 10 mM ammonium bicarbonate containing 1.2% IPA. In this case, 3.2 g of the supercoiled plasmid was loaded onto a reverse phase column, and then this column was washed with 6-10 column volumes of 1.2% IPA in 1 00 mM ammonium bicarbonate. This intensive washing was carried out to remove impurities. Elution was performed with a gradient of 1.2% IP A 11.2% IP A in 5 column volumes. The supercoiled plasmid fraction was eluted at approximately 4% IPA. Then, the supercoiled plasmid product fraction obtained from the reverse phase column was concentrated and diafiltered into normal saline using a membrane with 30 kDa molecular weight cutoff. The volume of the final product was filtered through a filter with a pore diameter of 0.22 μm. The above cleaning method was systematized in table 1, which shows data on the removal of impurities, as well as yields for each of the main stages of purification. The total yield of the product obtained in accordance with the claimed method amounted to more than 50% of the output of the supercoiled plasmid in the clarified cell lysate, as indicated by analysis carried out using anion exchange HPLC described in Example 3. The purity of the product was very high with less than 1% RNA and E. coli protein and less than 2.9% of the E. coli genomic DNA.

Table 1. Systematized method for purification and isolation on a multi-gram scale

Stage Plasmid product (mg) % Yield per stage Genomic DNA (mg / mg) Protein (mg / mg) RNA (mg / mg) Lal u / mg Clarified lysate 6750 one hundred 0.52 7.6 196 1.1 x 10 ⁷ Concentration / RNase / Diafiltration / Final Filtration 6500 93 0.50 1,6 2.21 3.4 x 10 ⁶ Anion exchange chromatography 4000 from 5000 80 0.41 0.3 0.1 1.2 x 10 ⁴ Reverse phase chromatography 2300 from 3200 77 0,029 <0.01 ++ <0.01 ++ 62 Concentration / diafiltration to the final buffer 2110 one hundred 0,029 <0.01 ++ <0.01 ++ 2,8 Final exit 54

lower detection limits of the analytical method

Claims (8)

1. The method of large-scale isolation and purification of plasmid DNA from large volumes of the fermentation medium of microbial cells, including the implementation of the following stages: a) the collection of microbial cells after their cultivation in large volumes; b) adding to the collected microbial cells the required amount of lysis buffer, optionally containing lysozyme; c) heating microbial cells with lysis buffer to a temperature of 70-100 ° C in a flow heat exchanger at a flow rate of 160-850 ml / min to obtain a crude lysate; d) centrifuging the crude lysate and collecting the supernatant; e) filtration and diafiltration of the supernatant collected in step d) to obtain a filtrate; e) contacting the filtrate obtained in step e) with an anion exchange matrix; g) elution and collection of plasmid DNA from the anion exchange matrix; h) contacting the plasmid DNA obtained in step g) with the matrix used for high-resolution reverse phase liquid chromatography, and i) elution and collection of plasmid DNA from the matrix indicated in step g). 2. The method according to claim 1, characterized in that the concentration and / or diafiltration of the plasmid DNA obtained in stage i) is additionally carried out in a pharmaceutically acceptable carrier. 3. The method according to claim 2, characterized in that it further sterilize the plasmid DNA. 4. The method according to any one of claims 1 to 3, characterized in that in step c) the heating is carried out to a temperature of 70-77 ° C. 5. The method according to any one of claims 1 to 3, characterized in that the lysing buffer used in stage b) contains a sub-microgram concentration of lysozyme. 6. The method according to any one of claims 1 to 3, characterized in that RNase is additionally treated with any product obtained after stage a). 7. The method according to any one of paragraphs. 1-3, characterized in that the lysis buffer used in stage b) is a modified STET buffer. 8. The method according to any one of paragraphs. 1-3, characterized in that the lysis buffer used in stage b) is a modified STET buffer containing a sub-microgram concentration of lysozyme.