WO2010046918A1

WO2010046918A1 - Purification of supercoiled plasmid dna

Info

Publication number: WO2010046918A1
Application number: PCT/IN2009/000558
Authority: WO
Inventors: Munishwar Nath Gupta; Pradeep K. Singh; Smita Raghava
Original assignee: VB Medicare Pvt Ltd
Current assignee: VB Medicare Pvt Ltd
Priority date: 2008-10-08
Filing date: 2009-10-08
Publication date: 2010-04-29
Anticipated expiration: 2011-04-08

Abstract

This invention comprises a process of purifying supercoiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in Gene Therapy or in uses where high purity supercoiled plasmid DNA is required, from a genetically transformed bacterium over-expressing the said supercoiled plasmid and the supercoiled plasmid DNA prepared by a process of this invention and products containing supercoiled DNA made by a process of this invention. The process of this invention comprises selective precipitation from a cell lyste of all DNA and endotoxin except supercoiled DNA by free metal ions. The said impurity comprising plasmid isoforms (linear, relaxed), genomic DNA, RNA, and an endotoxin from a host cell. The method is illustrated by using Cu²⁺ metal ions. The said Cu²⁺ metal ions were added as CuSO₄ to reach to a final concentration of at least 0.5 M. The removal of metal ions is achieved by dialysis.

Description

PURIFICATION OF SUPERCOILED PLASMID DNA.

TECHNICAL FIELD

The invention relates to isolation and purification of ScDNA (supercoiled plasmid DNA) for the purpose of use as a vaccine.

DESCRIPTION

DNA vaccines have come up as a very effective tool for immunization that has several advantages over conventional vaccines. The concept comprises introducing a gene of a pathogenic organism in a host in a way that facilitates expression of an antigen in the host organism that will trigger a response from the host organism for production of antibodies against that antigen and effectively against the organism from which the gene is derived.

This is achieved conveniently by constructing plasmids in which the desired pathogen gene is incorporated, transforming a microbe, such as Escherichia coli, large scale fermentative production of the transformed microbe, stimulating over expression of the plasmids containing the pathogen gene construct as overexpressed inclusion bodies in the form of super-coiled plasmids in the transformed microbe, recovering the super-coiled plasmids thus formed and using them as an active ingredient of a vaccine alone or in combination with other supercoiled plasmids.

Several advantages of such a vaccine include stability of this vaccine at room temperature, ability to make cocktail of several ScDNA species, ability of these vaccines to stimulate both antibody production and cytotoxic T-cell response and good safety profile as toxicity/immunogenicity is low.

However, such a vaccine can not afford to have various contaminants from other cell components as impurities, particularly plasmid isoforms (linear, relaxed), genomic DNA and RNA of the transformed cell, proteins and endotoxins from the host cell, particularly from E. coli.

SUMMARY OF INVENTION

This invention comprises a process of purifying supercoiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in Gene Therapy or in uses where high purity supercoiled plasmid DNA is required, from a boiling cell lysate or from a solution in water of a mixture comprising supercoiled plasmid DNA and an impurity, the said process comprising steps of treating the said solution by selective precipitation of all DNA and endotoxins except supercoiled DNA by free metal ions in a solution for a period of time, separating supernatant from the precipitate and removing metal ions from the said supernatant. The said impurity comprising plasmid isoforms (linear, relaxed), genomic DNA, RNA, and an endotoxin from a host cell. The method is illustrated by using Cu²⁺ metal ions. The said Cu²⁺ metal ions were added as CuSO₄ to reach to a final concentration of at least 0.5 M for a period of time of at least about one hour. The separation of supernatant from the precipitate may be achieved by centrifugation. The removal of metal ions is achieved by dialysis The dialysis is done, preferably, against a buffer containing EDTA.

This invention is illustrated by isolation of supercoiled plasmid DNA from a genetically transformed bacterium Escherichia coli.. However, the invention can be applied to supercoiled DNA isolation from water solution of over-expressed supercoiled DNA from any genetically modified micro-organism.

The purity and characteristics of supercoiled plasmid DNA prepared by process of this invention comprise freeness from any other plasmid isoforms as determined by agarose gel electrophoresis, freeness from genomic DNA and RNA as determined by agarose gel electrophoresis , freeness from any protein impurity that shall show Λ_26O//4₂₈o = 1.87, and freeness from endotoxins of the standard that by LAL assay shall show less than 0.025 Endotoxin Unit (EU) per μg plasmid.

In process of this invention, the said boiling cell lysate is prepared comprising steps of centrifuging culture broth to get a pellet of cells, suspending the said pellet in small quantity of buffer and adding Lysozyme to it, putting on boiling water bath for a short time.

A process of purifying super-coiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in a Gene Therapy or in uses where high purity supercoiled DNA is required, from a genetically engineered Escherichia coli containing over- expressed supercoiled DNA, the said process comprising steps of centrifuging the culture broth to get a pellet of cells, suspending the said pellet in small quantity of buffer, adding an enzyme to lyse the cells to the same, putting on boiling water bath for a short time to achieve lysis, centrifuging the lysed mixture to remove precipitate comprising proteins and DNA from a supernatant that contains native plasmid DNA and other impurities, adding sodium acetate and isopropanol to the said supenatant, centrifuging to get a pellet of DNA and separating supernatant containing impurities, washing the pellet with 70% ethanol accompanied by centrifugation and dissolving in sterile distilled water to get a solution containing all forms of DNA and endotoxin, adding CuSO₄ solution was added to reach a final concentration of 0.5 M, adding CuSO₄ solution was added to reach a final concentration of 0.5 M, incubating for 1 hour at 25° C, centrifuging to get a supernatant containing supercoiled DNA and a pellet comprising endotoxins, open circular plasmid DNA, linear plasmid DNA, RNA and residual denatured chromosomal (genomic) DNA.Removing the Cu²⁺ from supercoiled DNA by dialysis against Tris buffer containing EDTA. The enzyme sued in this invention is Lysozyme or may be any other enzyme or a step achieving lysis of the cells without adversely affecting the super-oiled plasmid DNA or its further isolation.

This invention also comprises a supercoiled plasmid DNA or a composition containing the same, wherein the said supercoiled plasmid DNA that is (a) free from any other plasmid isoforms as determined by agarose gel electrophoresis, (b) free from genomic DNA and RNA as determined by agarose gel electrophoresis and , (c) free from any protein impurity as indicated by Λ₂₆oΛ4_28O = 1.87, (d) free from endotoxins as indicated by LAL assay showing less than 0.025 Endotoxin Unit (EU) per μg plasmid when the said supercoiled plasmid DNA is made by a process comprising steps of treating the said solution by free metal ions for a period of time to achieve selective precipitation of all DNA except super-coiled DNA, separating supernatant from the precipitate and removing metal ions from the said supernatant.

DETAILED DESCRIPTION OF INVENTION

The present invention discloses a technology for obtaining supercoiled plasmids with the following characteristics/purity that meet the requirements of the regulatory agencies for the use of such preparations for DNA vaccines/gene therapy (Ferreira et al., 2000; Prather et al., 2003).

1) Supercoiled DNA (ScDNA) free of any other plasmid isoforms (linear, relaxed) as evaluated by agarose gel electrophoresis.

2) Free from genomic DNA and RNA by agarose gel electrophoresis.

3) Purity

⁼ 1-87 which shows absence of any protein impurity.

4) Endotoxins by LAL assay < 0.025 EU per μg plasmid

Prior art methods for recovering super coiled plasmid comprise column chromatography on silica gel, anion exchangers, hydrophobic interactions, chromatographic media. Partition methods have also been explored for large scale methods. Centrifugation in CsCl-ethidium bromide gradient is also used at lab scale.

Murphy et. al. (2003) have used immobilized metal-ion affinity chromatography (IMAC) for nucleic acid separations. IMAC, as the name applies, is a chromatographic technique which utilizes chelated metal ions (Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, Fe³⁺) and has been more extensively applied for protein purification (Porath et. al., 1975; Porath, 1992). Murphy et. al. (2003) have purified plasmid DNA from E. coli alkaline lysates ,wherein the IMAC matrixes adsorbed single-stranded nucleic acids through metal ion interactions with aromatic base nitrogen. It was reported that oligonucleotide duplexes, plasmid, and genomic DNA show low IMAC binding affinity, while RNA and single-stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose. It has been reported that RNA and damaged plasmid DNA are effectively removed without loss of the closed circular plasmid, presumably through interactions with exposed, single-stranded regions. This account suggests that their purified preparation by method of Murphy et al would have plasmid isoforms, as they, like any other oligonucleotide duplexes, shall have low affinity with IMAC matrix and will not be adsorbed Thus, IMAC matrices would not be useful to purify super-coiled DNA for the purpose of DNA vaccine.

More recently, Tan et. al. (2007) have talked of the differential interactions of plasmid DNA, RNA, and endotoxins with immobilized and free metal ions. Tan et. al., who worked with alkaline cell lysate on immobilized metal ion systems, however, never worked with a real system / cell lysates with free metal ions and it is not possible to simulate/extrapolate their results.

None of the methods available so far succeeded in making preparations of supercoiled plasmids with the purity that meet the requirements of the regulatory agencies, described above, for the use of such preparations for DNA vaccines/gene therapy by using free metal ions.

It was a surprising finding that a simple treatment of Cu²⁺ at a final CuSO₄ MNG concentration of 0.5 M resulted in clean separation of supercoiled DNA contained in a boiling lysate of bacterial cells from a precipitate of endotoxin, plasmid isoforms and RNA. The Cu²⁺ can be removed from the supercoiled DNA by dialysis against a buffer containing EDTA.

In one embodiment, the invention comprises use of free metal ions for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution.

In another embodiment, the invention comprises use of free metal ions for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from cell lysate of bacteria containing the said supercoiled DNA.

In yet another embodiment, the invention comprises use of free metal ions for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from boiling cell lysate of bacteria containing the said supercoiled DNA.

In a further embodiment, the invention comprises use of free Cu²⁺ for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution.

In another embodiment, the invention comprises use of free Cu²⁺ for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from cell lysate of bacteria containing the said supercoiled DNA. In yet another embodiment, the invention comprises use of free Cu²⁺ for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from boiling cell lysate of bacteria containing the said supercoiled DNA.

It is also an embodiment of the invention that a final concentration of 0.5 M with respect to CuSO₄ gives Cu²⁺ concentration that gives precipitation of endotoxin and plasmid isoforms and RNA in the embodiments of the invention mentioned above.

It is also an embodiment of the invention that treatment with Cu²⁺ in above embodiments is carried out for about one hour.

It is also an embodiment of the invention that treatment with Cu²⁺ in above embodiments is carried out for about one hour and at 25°C.

Other embodiments and any variations possible shall become evident to a person skilled in the art as the invention is illustrated by following description and examples and are also included within the scope and content of this specification.

In a further embodiment, this invention comprises a method of obtaining supercoiled DNA preparation useful for DNA Vaccines and gene therapy, and other uses where highly pure supercoiled DNA is required.

The vaccines and products produced from supercoiled DNA prepared by the method of this invention are also embodiments of this invention.

The following examples are only illustrative and the organisms used, chemicals used and conditions of treatments used may be varied within a limit that permits achievement of the intended results, and any such variations are included within this disclosure.

The invention deals with obtaining the pure supercoiled plasmids with above specifications by the following procedure: A. Cell lysis and primary isolation step. This followed the boiling lysis method as essentially described by Holmes and Quigley (1981). An alternative method called alkaline lysis method (Birnboim and DoIy, 1979) is also available for cell lysis (in case of bacteria) and in fact is often recommended (Brown, 1998; Alcamo, 1999) MNG for plasmid recovery. However, it was surprising that alkaline lysate failed to give purification of supercoiled DNA at 0.5 M concentration of CuSO₄ by the method of this invention; see Fig. 2 lane 4. It may be noted here that some contaminant DNA is lost at 0.5 M CuSO₄, and is an indication that if more CuSO₄ is used, it may lead to get purified supercoiled DNA. However, in practice, it is not possible to go beyond 0.5 M CuSO₄ concentration on account of precipitation of Cu(II) at this pH as the hydroxide and hence, on alkaline lysate the method of Tan et al did not work. (Ferreira et al., 2000). The steps for the boiling lysis method followed is shown in Fig 1.

B. Precipitation of nucleic acids is achieved by isopropanol treatment.

C. Purification from plasmid isoforms and endotoxins, which are present in this precipitate, is achieved in this invention by a simple precipitation with Cu²⁺ , and this constitutes an important embodiment of this invention. In this step, except supercoiled DNA that remains in solution, endotoxins and all other forms of DNA like open circular, linear plasmid DNA, RNA and remaining denatured chromosomal DNA (genomic DNA) get precipitated and can be removed by centrifugation.

D. Supercoiled DNA that remains in supernatant of above step is dialysed against Tris buffer containing EDTA to make them free from Cu²⁺ ions. Supercoiled DNA obtained in this way contains endotoxin level less than 0.5 EU mL ¹ . Steps followed in Figure 1 are in general as follows: Culture broth is centrifuged to get a pellet of cells that is suspended in small quantity of buffer. Lysozyme is added to it and the sample is put on boiling water bath for a short time. The lysed mixture is centrifuged to remove precipitate of proteins and DNA from a supernatant that contains native plasmid DNA and other impurities to which sodium acetate and isopropanol is added and centrifuged to get a pellet of DNA and impurities get separated in supernatant. The pellet is washed with 70% ethanol accompanied by centrifugation and dissolved in sterile distilled water. This solution contained all forms of DNA and endotoxin. To this, CuSO₄ solution was added to reach a final concentration of 0.5 M, incubated for 1 hour at 25° C and centrifuged to get a supernatant containing supercoiled DNA and pellet of endotoxins, open circular plasmid DNA, linear plasmid DNA, RNA and residual denatured chromosomal (genomic) DNA. The Cu²⁺ from supercoiled DNA could be separated by dialysis against Tris buffer containing EDTA. The supercoiled DNA obtained thus has endotoxin level of less than 0.5 EU mL^"1 which is within the limits required for a DNA vaccine.

The process of this invention as described above, is simple, scalable and applicable to isolation and purification of ScDNA in general that shall meet purity requirement of the regulatory agencies for ScDNA preparations for use, but not limited to, in DNA vaccines/ gene therapy. While final step, viz. precipitation step, was the crucial aspect of the present invention, equally crucial was the choice of boiling lysis method (rather than alkaline lysis method).

FIGURES AND THEIR DESCRIPTION

Figure 1 : protocol for isolation and purification of supercoiled DNA

Figure 2: 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5α harboring pGFPuv plasmid DNA by method described in this application and the one described by Tan et. al., 2007. Lane 1, total nucleic acid prepared by boiling lysis method; Lane 2, after metal precipitation (0.5M Cu2+) of boiling lysate; Lane 3, total nucleic acid prepared by alkaline lysis method (Tan et. al., 2007 ); Lane 4, after metal precipitation (0.5M Cu²⁺) of alkaline lysate.

Figure 3: 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5α harboring pGFPuv at different concentration of Cu²⁺. Lane 1, total nucleic acid prepared by boiling lysis method; Lane 2, at 0.1 M Cu²⁺; Lane 3, at 0.25M Cu²⁺; Lane 4, at 0.5M Cu²⁺; Lane 5, at 0.75M Cu²⁺; Lane 6, at 1.0M Cu²⁺.

Figure 4: 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5α harboring pTYB12 containing lipase gene. Lane 1, total nucleic acid (precipitated cell lysate by isopropanol/sodium acetate); Lane 2, precipitate containing RNA and other impurities; Lane 3, supercoiled plasmid DNA obtained by our method.

Figure 4a: CD spectra of purified supercoiled plasmid DNA, pTYB12 containing lipase gene (1) and total nucleic acid precipitated with isopropanol/ sodium acetate (2) in 10 mM Tris-HCl, ImM EDTA, pH 7.0, 25°C. Concentration of purified supercoiled plasmid DNA was 0.05 mg/mL and 0.06 mg/mL for total nucleic acid.

Figure 4b: Variation with temperature of UV absorption (245 nm) of purified supercoiled plasmid, pTYB12 containing lipase gene (0.06 mg/mL) in 10 mM Tris-HCl, ImM EDTA, and pH 7.0.

Figure 4c: Transmission electron micrograph of purified supercoiled plasmid DNA, pTYB12 containing lipase gene, at 50 nm resolution. Figure 4d: Comparison of yield of supercoiled plasmid DNA, pTYB12 containing lipase gene, purified by copper precipitation (grey) with Qiagen plasmid purification spin column (black) [QIAprep® Spin Miniprep Kit].

Figure 5: 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5α harboring pGEM^® T-Easy. Lane 1, total nucleic acid (precipitated cell lysate by isopropanol/sodium acetate); Lane 2, supercoiled plasmid DNA obtained by our method.

The invention is exemplified with three systems. EXAMPLE 1

PLASMID pGFPuv (CLONTECH LABORATORIES, PALO ALTO, CA)

Green fluorescent protein (GFP) has become well established as a marker of gene expression and protein targeting in intact cells and organisms (Tsien, 1998). The potential of GFP has also been recognized by gene therapy researchers (Wahlfors et. al., 2001). The plasmid pGFPuv was transformed into E. coli DH5α. A single colony was picked and inoculated into 5 mL Luria-Bertani (LB) medium containing 100 μgmL^"1 ampicillin. The tube was incubated at 37 ⁰C at 200 rpm for 14-16 h till an ^_6OO of approximately 4 was reached. The protocol described in Figure 1 was followed thereafter for getting a purified ScDNA preparation. Figure 3 shows the agarose gel for the purification of ScDNA at different concentration of Cu²⁺. It was observed that 0.5 M Cu²⁺ (lane 4, figure 3) was sufficient for getting ScDNA at a purity level in conformity with the criteria stated in the beginning of this application.

EXAMPLE 2 OF PLASMID pTYB12 (NEW ENGLAND BIOLABS, IPSWICH, MA) CONTAINING LIPASE GENE

The plasmid pTYB12 containing lipase gene was transformed into E. coli DH5α.. A single colony was picked and inoculated into 5 mL Luria-Bertani (LB) medium containing 100 μgmL ¹ ampicillin. The tube was incubated at 37 °C at 200 rpm for 14-16 h till an ^₆₀₀ of approximately 4 was reached. The protocol described in Fig 1 was followed thereafter for getting a purified ScDNA preparation. Figure 4 shows the agarose gel for the purification of ScDNA at a purity level in conformity with the criteria stated in the beginning of this application. Figure 4a and 4b (CD spectra and UV melt profile) strongly support that the preparation obtained was of ScDNA (Thumm et. al., 1988). Figure 4c shows the transmission electron micrograph of the final purified preparation. Figure 4d shows the comparison of yield of ScDNA purified by Cu²⁺ precipitation with Qiagen plasmid purification spin columns. It is shown that at large culture volumes, the yield obtained by Cu²⁺ precipitation was better than what was obtained by using the Qiagen kit.

EXAMPLE 3 OF PLASMID pGEM^®-T EASY (PROMEGA, MADISON, WI)

This was a commercially available vector, which is often used for cloning PCR products (http://www.promega.com/vectors/t vectors.htm). The plasmid pGEM^®-T Easy was transformed into E. coli DH5α. A single colony was picked and inoculated into 5 mL Luria-Bertani (LB) medium containing 100 μgmL^"1 ampicillin. The tube was incubated at 37 ⁰C at 200 rpm for 14-16 h till an ^₆₀₀ of approximately 4 was reached. The protocol described in box 1 was followed thereafter for getting a purified ScDNA preparation. Figure 5 shows the agarose gel for the purification of ScDNA at a purity level in conformity with the criteria stated in the beginning of this application.

REFERENCES

Alcamo I.E., DNA technology: the awesome skill. Second edition. Harcourt Academic Press, Farmingdale, NY: 1999.

Birnboim, H.C., DoIy, J., A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res. 7 (1979) 1513-1523. Brown T.A., Molecular biology lab fax I: recombinant DNA. Academic Press, San Diego, CA: 1998.

Dalai, S., Singh, P. K., Raghava, S., Rawat, S., Gupta, M.N. Purification and properties of alkaline lipase from Burkholderia cepacia ATCC 25609, Biotechnol. Appl. Biochem. (2008) doi: 10.1042/BA20070186.

Davranov, K., Microbial lipases in biotechnology, Appl. Biochem. Microbiol. 30 (1994) 527-534.

Ferreira, G.N.M., Monteiro, G.A., Prazeres, M.F., Cabral, J.M.S., Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications, TIBTECH 18 (2000) 380-387.

Holmes, D.S., Quigley, M., A rapid boiling method for the preparation of bacterial plasmids, Anal. Biochem. 114 (1981) 193-197.

Murphy, J.C., Jewell, D.L., White, K.I., Fox, G.E., Willson, R.C., Nucleic acid separations utilizing immobilized metal affinity chromatography, Biotechnol. Prog. 19 (2003) 982-986.

Porath, J., Carlsson, J., Olsson, I., Belfrage, G., Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258 (1975) 598-599.

Porath, J., Immobilized metal ion affinity chromatography, Protein Exp. Purif. 3 (1992) 263-281.

Prather, K.J., Sagar, S., Murphy, J., Chartrain, M., Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification, Enzyme Microb. Technol. 33 (2003) 865-883. Tan, L., Lai, W.-B., Lee, C.T., Kim, D.S., Choe, W.-S., Differential interactions of plasmid DNA, RNA and endotoxin with immobilized and free metal ions, J. Chromatogr. A 1141 (2007) 226-234.

Thumm, W., Seidl, A., Hinz, H.-J., Energy-structure correlations of plasmid DNA in different topological forms, Nucl. Acids Res. 16 (1988) 1 1737-11757.

Tsien, R.Y., The green fluorescent protein, Annu. Rev. Biochem. 67 (1998) 509-544.

Wahlfors, J., Loimas, S., Pasanen, T., Hakkarainen, T., Green fluorescent protein (GFP) fusion constructs in gene therapy research, Histochem. Cell Biol., 115 (2001) 59-65.

Claims

1. A process of purifying supercoiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in Gene Therapy or in uses where high purity supercoiled plasmid DNA is required, from a solution in water of a mixture comprising supercoiled plasmid DNA and an impurity, the said process comprising steps of: a. treating the said solution by selective precipitation of all DNA and endotoxin except supercoiled DNA by free metal ions in a solution for a period of time, b. separating supernatant from the precipitate, c. removing metal ions from the said supernatant.

2. A process of claim 1 comprising at least one of the following: a. the said impurity comprising plasmid isoforms (linear, relaxed), genomic DNA, RNA, and an endotoxin from a host cell, b. the said solution in water is derived from a boiling cell lysate of a microorganism, preferably a bacterium, c. wherein the said metal ion is Cu²⁺, d. the said period of time is at least about one hour, e. separating supernatant from the precipitate is achieved by centrifugation, f. the said removal of metal ions is achieved by dialysis.

3. A process of claim 2 wherein: a. the said micro-organism is a bacterium that may be genetically transformed to over-express a supercoiled plasmid DNA, b. the said Cu⁺² is added in the form of CuSO₄ to reach to a final concentration of at least 0.5 M, c. wherein the said dialysis is done against a buffer containing EDTA,

4. A process of claim 1 wherein the said purity and characteristics of supercoiled plasmid DNA comprise: d. freeness from any other plasmid isoforms, as determined by agarose gel electrophoresis, e. freeness from genomic DNA and RNA, as determined by agarose gel electrophoresis, f. freeness from any protein impurity that shall show Λ₂₆₀//4₂₈₀ = 1.87, and g. freeness from endotoxins of the standard that by LAL assay shall show less than 0.025 Endotoxin Unit (EU) per μg plasmid.

5. A process of claim 3 wherein the said boiling cell lysate is prepared comprising steps of; h. centrifuging culture broth to get a pellet of cells, i. suspending the said pellet in small quantity of buffer and adding Lysozyme to it, j. putting on boiling water bath for a short time

6. A process of purifying super-coiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in a Gene Therapy or in uses where high purity supercoiled DNA is required, from a genetically engineered Escherichia coli containing over-expressed supercoiled DNA, the said process comprising steps of: a. centrifuging the culture broth to get a pellet of cells, b. suspending the said pellet in small quantity of buffer, adding an enzyme to lyse the cells to the same, putting on boiling water bath for a short time to achieve lysis, c. centrifuging the lysed mixture to remove precipitate comprising proteins and DNA from a supernatant that contains native plasmid DNA and other impurities, d. adding sodium acetate and isopropanol to the said supenatant, centrifuging to get a pellet of DNA and separating supernatant containing impurities, e. washing the pellet with 70% ethanol accompanied by centrifugation and dissolving in sterile distilled water to get a solution containing all forms of DNA and endotoxin, f. adding CuSO₄ solution was added to reach a final concentration of 0.5 M, g. adding CuSO₄ solution was added to reach a final concentration of 0.5 M, h. incubating for 1 hour at 25° C, i. centrifuging to get a supernatant containing supercoiled DNA and a pellet comprising endotoxins, open circular plasmid DNA, linear plasmid DNA,

RNA and residual denatured chromosomal (genomic) DNA. Removing the Cu²⁺ from supercoiled DNA by dialysis against Tris buffer containing

EDTA.

7. A process of claim 6 wherein the said enzyme comprises Lysozyme.

8. A supercoiled plasmid DNA or a composition containing the same, wherein the said supercoiled plasmid DNA that is (a) free from any other plasmid isoforms that are detectable by agarose gel electrophoresis, (b) free from genomic DNA and RNA detectable by agarose gel electrophoresis and , (c) free from any protein impurity as indicated by Λ₂₆₀Λ4₂₈o = 1.87, (d) free from endotoxins as indicated by LAL assay showing less than 0.025 Endotoxin Unit (EU) per μg plasmid is made by a process comprising steps of: a. treating the said solution by free metal ions for a period of time to achieve selective precipitation of all DNA except super-coiled DNA, b. separating supernatant from the precipitate, c. removing metal ions from the said supernatant.

9. A supercoiled plasmid DNA or a composition of claim 16, wherein the said supercoiled DNA is made by a process of claim 1 or 6.