CZ366197A3 - Large-scale isolation process of plasmid - Google Patents

Abstract

A process is disclosed for the large scale isolation and purification of plasmid DNA from large scale microbial fermentations. All three forms of plasmid DNA; supercoil (form I), nicked or relaxed circle (form II), and linearized (form III), are individually isolatable using the disclosed process. Highly purified DNA suitable for inclusion in a pharmaceutical composition is provided by the disclosed process.

Description

Technical field

The invention relates to the large-scale isolation of all three forms of plasmid DNA.

BACKGROUND OF THE INVENTION

Conventional methods for isolating plasmid DNA from bacterial cultures are suitable for the preparation of small or laboratory scale plasmids. One such procedure involves alkaline lysis of microbial host cells followed by neutralization with acetate to precipitate genomic DNA of the host cells and proteins. The precipitate is removed, for example, by centrifugation. The liquid phase contains a plasmid DNA that is precipitated with alcohol and then isolated by isopycnic centrifugation using CsCl in the presence of ethidium bromide. Ethidium bromide is required to split the total plasmid DNA into three distinct forms, namely the helix (Form I), the open circle (Form II) and the linearized form (Form III), and further to isolate the desired form of the plasmid. To remove residual ethidium bromide further extraction with butanol is required, followed by precipitation of the DNA with alcohol. Additional purification steps are used to remove proteins from the host cells, which include repeated phenol extractions or phenol / chloroform mixtures. The plasmid DNA is precipitated with alcohol and the residual phenol is removed by repeated extractions of a mixture of isopentyl and chloroform. After the final alcohol precipitation, the plasmid DNA is dissolved in water or in a suitable buffer solution.

The disadvantages and limitations of this procedure are as follows:

(a) this procedure requires the use of expensive and dangerous chemicals (CsCl and EtBr, which are used in density gradient centrifugation; EtBr is a known mutagen and must be removed from the product; it may also cause a break in the plasmid DNA because it is an can intercalate into DNA);

(b) the density gradient centrifugation is not easily adjustable;

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

d) phenol extraction is used to remove residual proteins and DNase, using centrifugation to separate the phenol and aqueous phases;

f) repetitive steps make the process laborious and time consuming by large application of viscosity skiing is difficult; and using a plurality of wall attenuation microbial cells prior to lysis.

(isolation requires a few days) the problem is changing the volume of the lysozyme / alkali / KOAc scale, isolation in small cells makes chemical lysis effective at increasing the large scale lysozyme to enzymatic

The mixture is then neutralized by the addition of an acid which results in the precipitation of the high molecular weight chromosomal DNA. The high molecular weight RNK and protein-SDS complexes are precipitated by the addition of a high concentration of KOAc salt. After centrifugation, the plasmid is found in the clarified supernatant. A limitation of this procedure is that it must proceed rapidly and the DNA must be kept on ice in order to dampen the nuclease activity, which was only removed by phenol extraction. The major contaminant, RNK, remains in the supernatant with the plasmid DNA.

Another commonly used method for isolating and purifying plasmid DNA from bacteria provides a rapid procedure suitable only for small-scale isolations.

Holmes and Quigley (1981, Analytical Biochem., 114, pp 193-197) describe a simple and rapid method of preparing a φ φ φ φ · · φ φ φ · φ φ φ · φ φ φ · The plasmids, where the bacteria are treated with lysozyme, are then boiled at a temperature of about 100 ° C in the appropriate buffer (STET) for 20 to 40 seconds. Plaz plaz φ φ φ φ φ φ φ , thereby forming an insoluble clot of genomic DNA, protein and waste, wherein the plasmid DNA together with the RNK as the main contaminant remains in solution. The degree of lysozyme digestion is necessary for the functionality of this method of isolation. This step is less useful for large-scale DNA isolation. Said DNA is then used for application to humans and animals. Addition of lysozyme may increase plasmid release during lysis. The advantage is that heat treatment of the cells also denature the enzyme DNase. However, this method is not suitable for isolation from large-scale microbial fermentations, it appears to be applicable to fermentations where the volume is less than five liters.

Other isopycnic centrifugation alternatives using CsCl suitable for plasmid isolation have been reported. These alternatives are only suitable for plasmid isolation on a laboratory scale and include:

(a) size exclusion chromatography based on inherently limited performance;

b) hydroxyapatite chromatography, which has the disadvantage of requiring high urea concentrations to be effective;

(c) reverse phase chromatography; and

d) ion-exchange chromatography.

Isolation and purification of large-scale plasmid DNA from large volume microbial fermentations therefore requires the development of a better plasmid isolation process. Developments in many areas of molecular biology require an isolation and purification procedure suitable for large-scale plasmid DNA production. In particular, the latest improved procedures in the field of human polynucleotide-based vaccines suitable for human gene therapy require the ability to produce large quantities of polynucleotide vaccines in highly pure form.

· · F f f f f f f f f f f f f f f f f f f f f f f f f f f f ·· · · ··

A completely new technology is required to develop and implement a commercially available process for fermentation, isolation, purification and characterization of DNA for pharmaceutical purposes.

SUMMARY OF THE INVENTION

The current laboratory method for isolating and purifying plasmid DNA includes a series of classical laboratory methods that are not suitable for use in the manufacturing process. For example, the density gradient centrifugation volume cannot be easily changed; the purification process necessitates the use of hazardous and expensive chemicals / solvents such as ethidium bromide, which is a known mutagen. The procedure is laborious and time consuming. For these reasons, an alternative procedure has been developed where the volume of insulation can be adjusted. This procedure is described herein. In addition, an assay was performed to monitor the plasmid product during the individual steps of the process, and can distinguish between different forms of the plasmid. The plasmid-bearing microbial cells were suspended and incubated with lysozyme in a buffer containing detergent. To lyse the cells, the suspension was heated by passing through a heat exchanger and then centrifuged. After centrifugation, the clarified lysate, which contains predominantly RNK and plasmid product, is filtered through a 0.45 micron filter and then subjected to diafiltration before being applied to the ion exchange column. The plasmid may be treated with the RNase enzyme before or after filtration or during previous or later steps. After the anion exchange, the plasmid-containing product fraction is applied to a reverse phase column and eluted with the appropriate buffer. A highly pure plasmid DNA suitable for human use is obtained.

A new, alternative, lysis procedure, and the removal of unwanted cellular debris, where the volume can be arbitrarily adjusted, are identified and suitable for large-scale plasmid isolation and purification. This procedure utilizes rapid heating to induce cell lysis of cells, and genomic DNA, proteins, and other unwanted cellular debris are precipitated while the plasmid remains in solution. This procedure can be used to isolate and purify large-scale plasmid DNA. It has been found that when microbial cells are suspended in a modified STET buffer (as described below) and then the suspension is heated to a temperature of about 70-100 ° C in a flow heat exchanger, excellent lysis occurs. Continuous flow or batch centrifugation of the lysate affects the pellet that contains cellular debris, proteins, and most of the genomic DNA, while the plasmid remains in the supernatant. The present invention offers a number of advantages such as high product yield compared to chemical lysis, furthermore DNase inactivation, simplicity and the possibility of arbitrarily adjusting insulation volume.

The invention relates to a process for the isolation and purification of large-scale plasmid DNA from microbial fermentations. The large-scale microbial fermentations used herein are whole cell fermentations with volumes greater than about five liters or cells obtained from fermentations greater than about five liters.

The invention further provides for obtaining a plasmid DNA in a highly pure form suitable for human application. Said DNA includes, for example, polynucleotide vaccines and DNA for human gene therapy. Polynucleotide vaccines are intended for direct administration to humans by injection (Montgomery, DL et al., 1993, Cell Biol., 169, 99, 244-247; Ulmer, JB et al., 1993, Sciences, 259, pp 1745-1749). .

The invention also describes a method for directly monitoring various forms of plasmid DNA during the individual isolation and purification steps. The various forms of plasmid DNA which are mentioned above and can be isolated individually by the process according to the invention are form I (supercoiled), form II (relaxed plasmid or β).

a single strand break DNA plasmid) and Form III (linearized plasmid).

The process of the invention is generally suitable for use in conjunction with microbial fermentation. Those skilled in the art will appreciate that various microbial cells may be used in the present invention, for example fungal cells including yeast and bacterial cells. A preferred microbial fermentation is bacterial fermentation of cells that contain the plasmid being digested. should insulate and clean. A preferred bacterial fermentation is the fermentation of E. coli containing a plasmid to be isolated and purified. The skilled artisan will appreciate that other types of fermentation other than E. coli fermentation can be used in the present invention. The microbial fermentation may take place in any liquid medium suitable for the growth of the bacteria used.

The plasmid to be isolated and purified by the process of the invention may be any extrachromosomal DNA molecule. Plasmids may be present in the cell in a high copy number or a low copy number. The size of the plasmids can be virtually arbitrary. Those skilled in the art will recognize that virtually any plasmid can be isolated by the process of the invention.

Cell paste or slurry was prepared from microbial cells containing plasmid and obtained from the fermentation medium. Any commercially available method, including, for example, centrifugation or microfiltration, is suitable to recover cells from the liquid medium.

Isolation of plasmid DNA from the obtained microbial cells using current laboratory scale procedures involves mainly the application of enzymes to microbial cells to attenuate the cell wall, followed by lysis of the cells. The purification steps then include repeated centrifugation using CsCl / EtBr followed by organic extractions

Solvents and precipitation to remove tRNK, residual proteins, EtBr or other contaminants from the host cells. These steps are not freely selectable and are therefore not suitable for use in large-scale processes. Conversely, the use of preparative chromatography in the process of purifying plasmid DNA products is very advantageous and provides high resolution, ease of use, and increased productivity. Two different types of chromatography are suitable for purifying the plasmid DNA to the very high level of purity required when administered to humans. These are anion exchange chromatography and reverse phase chromatography. Reverse phase-based separations are governed by hydrophobic interactions, while anion-exchange chromatography separations are based on electrostatic interactions. These two orthogonal chromatographic steps achieve the separation of different forms of the plasmid (n-admixture forms, open relaxed forms, linear forms and concateners) and the removal of host contaminants such as LPS (endotoxin), RNK, DNA and residual proteins.

According to the method of the invention, the obtained microbial cells were 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 a sub-microgram concentration of lysozyme, at a pH of 6, 0 to 10.0. The concentration of lysozyme used in the process of the present invention is substantially lower than those used in procedures known in the art. Those skilled in the art will appreciate that modifications to this basic buffer composition can be prepared and are suitable for use in the process of the invention. Modifications to the basic buffer composition that do not substantially affect or alter the result of the process are applied in the process of the invention. The pH range can be adjusted to a value that gives the best results for the strain used. A pH range of about 8.0 to 8.5 is preferred. The suspension is then heated to a temperature of about 70 ° C to 100 ° C, preferably a temperature of 70 ° C to 77 ° C, by passing through a heat exchanger. The lysate is centrifuged and cell debris, proteins, and most genomic DNA are pelleted.

To demonstrate that lysis of plasmid-containing microbial cells can be performed by passing the microbial suspension through a heat exchanger, a prototype of this heat exchanger was constructed. This exchanger consists of a stainless steel tube with external dimensions of 3.04 x 6, 35.10 ³ m coiled into a spiral. The tube was completely immersed in a constant temperature water bath. The volume of the coiled tube is approximately 50 ml. Thermocouples and a thermometer were used to measure exchanger inlet and outlet temperatures and water bath temperature. The product was pumped into the heating coil by a peristaltic Masterflex pump through a silicone line. After exiting the exchanger, the cell lysate was centrifuged for clarification in a Beckman J-21 batch centrifuge. Figure 1 shows a schematic of this apparatus, but other types of heat exchanger designs may be utilized in the described invention, including, for example, preferred jacket and tubular structures.

After centrifugation, RNase was applied to the clarified lysate and the plasmid product can be filtered to remove residual fine waste. Various types of filtration can be used in this process, for example membrane filtration with small pores. A preferred filtration method is filtration through a 0.45 micron filter.

Another way to remove contaminants from the DNA product is that the material may undergo diafiltration. Standard commercially available materials are suitable for use in the process of the invention. A preferred diafiltration method is the use of an ultrafiltration membrane having a molecular weight interface in the range of 30,000 to 500,000 depending on size. 999 99 99

From the plasmid. The isolated DNA was further diafiltered using an ultrafiltration membrane (molecular weight edge of approximately 100,000) against a buffer which is also used in subsequent anion exchange chromatography. It is preferable to include a diafiltration step over anion exchange chromatography, which allows the amount of lysate to be loaded onto the column to be increased.

A wide range of commercially available anion exchange matrices can be used in the present invention, including, for example, those available from POROS Anion Exchange Resins, Qiagen, Toso Haas, Sterogene, Spherodex, Nucleopac, and Pharmacia. The column (Poros II PI / M, 4.5mmx100) is initially equilibrated with 20mM Bis / TRIS propane at pH 7.5 and 0.7M NaCl. Samples were loaded onto the column and washed using the same initial buffer. An elution gradient of 0.5 M to 0.85 M NaCl in an amount of 25 column volumes is then applied to the column and the individual fractions collected. Anion exchange chromatography is the ideal first purification step because it removes RNK, genomic DNA and proteins. Figure 5 (thick line) shows the elution profile of a filtered clarified cell lysate sample from the anion exchange column. Analysis on an agarose gel showed that the second peak that appeared after passing through the column was a plasmid product. The previous large peak causes RNK. This finding was confirmed by incubating the clarified cell lysate with ribonuclease before loading onto the column. Then it turned out that the large peak had disappeared and was replaced by several smaller, faster eluted peaks, the fractions of these plots containing degraded ribonuclease digestion products.

Fractions from the anion exchange column are loaded onto a reversed phase column. Commercially available matrices, such as those available from POROS, Polymer Labs, Toso Haas, Pharmacia, PQ Corp., Zorbax,

4444 ··· ··· 4 · · * · · 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ····· · · ··· ·· · ·

BioSepra resins, BioSepra Hyper D resins, BioSepra Q-Hyper D resins, and Amicon. The matrices may consist of a polymer or silica. The reverse phase column (Poros R / H) is equilibrated with approximately 100 mM ammonium carbonate at pH 8.5. A gradient of 0 to 11% isopropanol is used to elute the bound material. In this way, the three plasmid forms I, II and III described above were separated.

The eluted plasmid DNA can then be concentrated and / or diafiltered to reduce buffer volume or swap. For the isolation of DNA for human use, it is recommended to use diafiltration of the DNA product into a pharmaceutically acceptable carrier or buffer. Pharmaceutically acceptable carriers or buffers are known in the art and include those described in a number of publications such as Remington's Pharmaceutical Sciences. Any method suitable for concentrating the DNA sample can be used in the present invention. Such methods include diafiltration, alcohol precipitation, lyophilization and the like. Diafiltration is preferred. After diafiltration, the final plasmid DNA product can be sterilized. Any method of sterilization that does not affect the use of the DNA product is suitable. Said sterilization methods include passage through a membrane having sufficiently small pores, for example 0.2 microns or less.

Overview of the drawings

Figure 1 shows a diagram of a suitable heat exchanger.

Figure 2 is a graphical representation of the relationship between outlet temperature and flow rate.

Figure 3 shows comparative chromatograms of all forms of plasmid in clarified supernatant with 50 mM EDTA and 100 mM EDTA.

• · ·

Figure 4 shows the yield of the superhelix form of the plasmid as a function of the outlet temperature.

Figure 5 shows the elution profiles of anion exchange columns on which clarified RNase enzyme lysates (thick line) or clarified RNase-free lysates (thin line) were applied.

Figure 6 shows the anion exchange chromatography elution profiles of the clarified lysate that has been subjected to diafiltration or not prior to column loading.

Figure 7 shows the elution profile of plasmid DNA from cell lysate.

Figure 8 shows an electrophoretic analysis on an agarose gel of a DNA product obtained from various intermediate purification steps.

Figure 9 shows anion-exchange HPLC analysis of the DNA product showing the purity of the product.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Cultivation of microbial cells, cell lysis and clarification.

A slurry of frozen E. coli cells was used to prepare eight liters of cell suspension in STET buffer (8% sucrose, 0.5% TRITON, 50 mM TRIS buffer, pH 8.5 and 50 mM EDTA). The absorbance value of the cell suspension at 600 nm was about 30. The suspension was stirred continuously to obtain homogeneity. The viscosity of the cell suspension was measured at room temperature (24 ° C) and was about 1.94 cp. The cell suspension was pumped through the exchanger at a rate of 81ml / min, meaning that the suspension remained in the heat exchanger for approximately 35 seconds. The temperature in the water tank was maintained at 92 ° C. The temperature of the inlet and outlet cell solution was about 24 ° C and 89 ° C (mean). Approximately 11 samples were passed through the exchanger. Unseen bonding of the lysate to the tube was observed, although the lysate was denser than the starting material. The lysate was cooled to room temperature and its viscosity reached approximately 40 cp. The cell lysate was clarified by batch centrifugation at 9,000 rpm. for 50 minutes using a Beckman J-21 centrifuge. Analysis of the supernatant confirmed efficient cell lysis and product recovery. The product yield was at least comparable to that obtained using the Quigley and Holmes cooking method. However, the latter method must be run in batch mode on a laboratory scale and is therefore unsuitable for large-scale processing (5 liters or more). Since the heat exchanger is flow-through, there is no limitation on the maximum volume of cell suspension that can be treated by this process. This process is therefore suitable for large-scale fermentations of bacteria in order to produce large quantities of highly pure plasmid DNA.

The clarified lysate was then filtered through a membrane having a pore size of 0.45 microns to remove residual waste. The filtrate was then diafiltered using a membrane having a molecular weight edge of about 100,000.

Example 2: Control and reproducibility of cell lysis using heat exchanger parameters.

Adjusting the flow rate (i.e., residence time) at which the cell slurry is pumped through the heat exchanger allows control of the ski temperature, which is the temperature of the suspension at the exit of the exchanger. The cell slurry solution was prepared as described in Example 1 and pumped through a heat exchanger. The flow rate was in the range of 160 to 850ml / min. The corresponding outlet temperatures were between 93 ° C and 65 ° C. Figure 2 illustrates the relationship between flow rate and temperature. Initial cell temperature • 44 • 4 * 4 • 4

the slurry was 24 ° C and the water bath temperature was kept constant at 96 ° C. The circulation stopped when the outlet temperature reached 80 ° C. The commonly achieved yield is 24mg of circular DNA per liter of clarified supernatant, demonstrating the reproducibility of the process.

Example 3: Purification of plasmid DNA.

Microbial cells and lysates were prepared as described in Examples 1 and 2, followed by the following analyzes.

The following analyzes were performed to show that the addition of 100mM EDTA, unlike 50mM EDTA, would increase the percentage of supercoiled DNA and in order to determine an acceptable exit temperature range (i.e., ski temperature) with respect to obtaining the supercoiled DNA. The supercoiled form of DNA is preferred because it is more stable than the relaxed ring form. One way to convert a supercoiled form into an open ring is to create a break using DNase. Addition of 100 mM EDTA in contrast to 50 mM EDTA in STET buffer was found to minimize the formation of the plasmid form of the open ring. Figure 3 shows comparative chromatographs of the whole plasmid in the clarified supernatant with 50 mM EDTA versus 100 mM EDTA. The cell suspension was prepared according to the procedure described in Example 1. The flow rate was approximately 186 ml / min. The inlet, outlet and water bath temperatures are 24 ° C, 92 ° C and 96 ° C.

The acceptable range of lysis temperatures was determined by measuring the percentage of supercoiled form of the plasmid formed at each cycle. Figure 4 shows the concentration of the supercoiled form of the plasmid as a function of the outlet temperature. An acceptable temperature range is between 75 ° C and 92 ° C. At a temperature below 75 ° C, more plasmid forms of the relaxed ring are formed, probably due to increased DNase activity. At a temperature above 93 ° C

14 4 • · · · · · · · • ··· · · · • · » • · · · · • · · · · · · · · · · · • · · · • · · · · · • · · • · · · amount supercoiled forms plasmid decreases, it probably happens denaturation heat. After continuous ski heat and centrifugation se lml

The clarified lysate was either incubated with 5μ9 RNase for 2 hours or used without RNase. Both samples were then loaded onto an anion exchange column (Poros Q / M 4.6x100) previously. equilibrated with a 50:50 mixture of solvent A and B (solvent A for HPLC having the composition: 20 mM Tris / Bis propane pH 8.0; and solvent B having the composition: 1M NaCl in 20 mM Tris / Bis propane pH 8.0). The column was eluted using a gradient of 50% to 85% solvent B by an amount equal to 100 column volumes. The open ring plasmid form is eluted into approximately 68% solution B and the supercoiled form of the plasmid is 72% solution B.

lysate, enzyme fraction,

Comparison of anion exchange eluates applied a line) to where (thick)

RNase (weak on the cleared column) and without application of Figure 5. A peak of approximately ten minutes which was untreated is followed by a large peak, which is RNK. For samples that produced plasmid DNA

By RNase, after the RNase-treated sample, the RNK peak was removed and a better separation of the plasmid peak from the contaminant peak was achieved.

As previously described, diafiltration, which is preceded by anion exchange chromatography, greatly increases the amount of lysate that can be loaded onto the column. This fact is demonstrated by comparing the clarified lysate, Figure 6, which shows which diafiltered and the clarified lysate which was not subjected to diafiltration prior to anion exchange chromatography. Samples were prepared as described above except that one sample was diafiltered prior to loading on the anion exchange column and the other not. The separation and elution of the column was the same as that described above. Picture No. 6 · · · · · · · · · · · · · · · · · · · · · · · · · · · · Shows that the amount of contaminants in the eluted material is greater in the case of a sample that has not been filtered. The large amount of contaminated material that binds to the anion exchange column matrix may completely exhaust the maximum column capacity, which may cause loss of the DNA product, as the matrix is unable to further bind any other material. Therefore, diafiltration, which removes contaminants, allows the binding of more DNA product to the matrix of the anion exchange column, thereby allowing a larger volume of clarified lysate to be applied to the column.

The individual forms of the eluted plasmid DNA were separated on reverse phase HPLC. The separation of the supercoiled form of the plasmid (form I) and the fracture ring (form II) is shown in Figure 7. These two forms were separated easily, allowing the isolation of the individual forms of the plasmid.

Example 4: High purity plasmid DNA isolated by a chromatography-based procedure.

The fermentation cell paste was resuspended in STET buffer and then thermally lysed in a batch process. Alternatively, the fermentation cell paste was resuspended in a modified STET buffer and then thermally lysed as described above. Twenty milliliters of supernatant was filtered as described above and then loaded onto an anion exchange column (Poros Q / M 4.6x100) which was previously equilibrated with a 50:50 mixture of buffers A and B as described above. A gradient of 50% to 85% of solution B was used to elute the column in an amount corresponding to 50 column volumes. The flow rate was 10ml / min. 2.5 ml of each fraction was collected from the column. The supercoiled form of the plasmid DNA was eluted from the column into 72% solution B.

The anion exchange column product was then loaded onto a reversed-phase chromatography column (Poros R / H) previously equilibrated with 100mM ammonium carbonate at pH 8.0 and using a gradient of 0% to 80% to elute the bound material. of methanol. The highly pure supercoiled form of the plasmid DNA was eluted in 22% methanol.

Figure 8 shows an agarose gel with fractions collected from each main stage of the purification process. The agarose gel analysis and HPLC analysis of the colorimetric assay described in Example 3 shows that the final product shown in Figure 9 is of high purity. The product contains more than 90% of the supercoiled form of the plasmid and less than 10% of the open ring form. The amount of RNK was below the detection limit of the assay used for this purpose. The amount of genomic DNA and proteins was also below the detection limit of the assay used for this purpose. The yield of the supercoiled form of the plasmid at the end of the process was approximately 60% of that form contained in the clarified lysate.

Example 5: Multi-gram isolation of plasmid DNA.

A 4.51 frozen slurry of E. coli cells in STET buffer (8% sucrose, 2% Triton, 50 mM Tris buffer, 50 mM EDTA, pH 8.5) containing 2500 units / ml lysozyme was used to prepare a 33.71 cell suspension. The absorbance of the suspension at 600nm was about 30. For complete mixing, the suspension was stirred at room temperature for 15 minutes and then incubated for 45 minutes with stirring at 37 ° C. After incubation, stirring was continued at room temperature and the cell suspension was passed through a heat exchanger at a flow rate of 500 ml / min. The batch temperature was held at 100 ° C and the inlet temperature of the cell suspension was approximately 24 ° C and the outlet temperature was between 70-77 ° C. The cell lysate exiting the heat exchanger was collected in Beckman centrifuge bottles (500ml) and then immediately centrifuged in Beckman J-21 centrifuges for a period of time. β · ο

50inin. at 9,000 rpm. The supernatant was found to contain 4 times more plasmid product after centrifugation than when no lysozyme incubation was used. After centrifugation, the supernatant was immediately diafiltered against three volumes of TE buffer (25 mM Tris-EDTA pH8.0) and then incubated with 20x10 ⁵ units of E. coli RNase for 2-4 hours at room temperature. At the end of incubation, the product solution was diafiltered with 6 volumes of TE buffer using a 100 kD MWCO membrane and then filtered through a 0.45 micron pore filter to remove residual waste. The filtered lysate was diluted with 20 mM Bis / Tris propane-NaCl pH7.5 to 0.7M NaCl. This buffer was prepared to dilute the filtrate, which was further loaded onto the anion exchange column. The anion exchange column (3.61 POROS PI / M) was previously equilibrated with 20 mM Bis / Tris propane buffer and 0.7 M NaCl. A filtered lysate corresponding to the column capacity was applied to the column. In this case, 5g of the supercoiled form of the plasmid was applied to the anion exchange column. After loading, the column was washed with 2-4 column volumes of 20 mM Bis / Tris propane buffer and 0.7 M NaCl. A gradient from 0.7 M NaCl to 2.0 M NaCl in 20 mM Bis / Tris propane in an amount corresponding to 10 column volumes was used to remove most of the proteins, RNKs and endotoxins contained in E.coli cells. The supercoiled fraction of the plasmid eluted at a concentration between 1.4M and 2.0M NaCl. The supercoiled fraction of the plasmid after passing through the anion exchange column was diluted 2-3 times with pyrogen-free water, adjusted to 1.2% IPA and pH8.5 with 1N NaOH. The diluted supercoiled fraction of the plasmid after passing through the anion exchange column was then loaded onto a 71-phase reversed-phase column (POROS R2 / M), which had previously been equilibrated with 100mM ammonium carbonate containing 1.2% IPA. In this case, a 3.2g supercoiled plasmid was loaded onto the inverted phase column and the column was

washed with 6-10 volumes of 1.2% IPA in 100mM ammonium carbonate. This washing was performed to remove impurities. A gradient of 1.2% IPA to 11.2% IPA in an amount corresponding to five column volumes was used for elution. The supercoiled fraction of the plasmid elutes at approximately 4% IPA. The supercoiled fraction of the product from the reversed phase column was then concentrated and diafiltered into normal saline using a 30 kD MWCO membrane. The final product volume was filtered through a 0.22 micron filter. Table 1 describes the removal of impurities and yields of each main process step. The yield of the process was more than 50% of the supercoiled form of the plasmid in the clarified cell lysate as determined by anion exchange HPLC as described in Example 3. The purity of the product was very high. The product contained less than 1% RNK and protein and less than 2.9% genomic DNA from E. coli cells.

Table 1: Multi-gram isolation and yield

Degree Plasmid Product (mg) G. Stupně yield grade genom. DNA (mg / mg) protein (mg / mg) RNK (mg / mg) LAL Eu / mg clarified iysate 6750 100 ALIGN! 0, 52 7.6 196 1,1x10 ' RNase concentration / diafiltration / dead end filtration 6500 93 0, 50 1, 6 2.21 3,4x10 ^b anion exchange 4000 z 5000 80 0, 41 0, 3 0.1 l, 2x10 ⁴ reverse 2300 z 3200 77 0, 029 <0.01 \ <0.01 \ 62 concentration of diafiltration into final buffer 2110 100 ALIGN! 0,029 <0, 0Γ ₊ <0, or ₊ 2, 8 final yield of the process 54

below the detection level of said method

Wf- #

Claims (11)

PATENT CLAIMS A method for the isolation and purification of plasmid DNA from large-scale microbial cell fermentations, characterized in that: (a) microbial cells are obtained from large-scale fermentation; b) adding a sufficient amount of lysing solution to the obtained microbial cells; c) heating the microbial cells of step b) to a temperature between 70 ° C and 100 ° C in a continuous heat exchanger to form a crude lysate; d) centrifuging the crude lysate; e) filtration and diafiltration of the supernatant of step d) to form a filtrate; f) contacting the filtrate of step e) with an anion exchange matrix; g) eluting the plasmid DNA from the anion exchange matrix and collecting it for further processing; h) contacting the plasmid DNA of step g) with a reverse-phase high performance liquid chromatography matrix; i) eluting the plasmid from the reverse phase high performance liquid chromatography matrix of step h) and collecting it for further processing; j) the product of step i) may be concentrated and / or diafiltered into a pharmaceutically acceptable carrier; and k) the DNA product can be sterilized. The method of claim 1, wherein the lysis solution of step b) is a modified STET buffer. • 9 The process according to claim 1, wherein the heating according to step c) is at a temperature between 70 ° C and 77 ° C. The method of claim 1, wherein the lysing solution of step b) comprises sub-programmatic concentrations of lysozyme. The method of claim 1, which may include, at any stage following step a), the application of RNase. The isolated and purified plasmid DNA obtained by the method of claim 1. The plasmid DNA of claim 6, wherein said plasmid is suitable for administration to humans. The plasmid DNA of claim 6, wherein said plasmid is suitable for administration to animals. The plasmid DNA of claim 6, wherein said plasmid is a polynucleotide vaccine. 10. Isolated and purified plasmid DNA suitable for human application. The plasmid DNA of claim 10, wherein said plasmid DNA is a polynucleotide vaccine.