MX2007003214A - Stable liquid formulations of plasmid dna. - Google Patents

Abstract

This invention relates to plasmid DNA liquid formulations that are stable and stays un-degraded at +4 C to room temperature for long periods of time, and are thus useful for storage of plasmid DNA that are used research, plasmid-based therapy, such as DNA vaccine and gene therapy. The present invention also relates to a method of preserving plasmid DNA in a stable form over time at +4 C to room temperature. The present invention also relates to stable plasmid DNA liquid compositions for use in a method of treatment of the human or animal body by plasmid-based therapy, such as DNA vaccination or gene therapy.

Description

FORMU STABLE LIQUID LATIONS OF PLASMID DNA Field of the Invention The present invention relates to liquid formulations of Plasmid DNA which are stable and in which the plasmid remains without degrading at + 4 ° C to room temperature for long periods of time. Said formulations are thus useful for the storage of the plasmid DNA used in research or in plasmid-based therapies, such as DNA vaccines and gene therapy.

Background of the Invention Advances in molecular biology clearly suggest that plasmid-based therapy, in particular the fields of DNA vaccines and gene therapy, can be an effective means of treating diseases. A promising method for safely and efficiently administering a normal gene to human cells is by plasmid DNA. Plasmid DNA is a covalently closed (ccc) or supercoiled circular form of bacterial DNA in which a DNA sequence of interest can be inserted. Examples of DNA sequences of interest that can be introduced into mammalian cells include exogenous genes, functional genes or mutant genes, antisense sequences, RNAi or dsRNA sequences, ribozymes and those used, for example, in vaccines of DNA against viral infections or in the treatment of cardiovascular diseases, diseases related to angiogenesis or cancer. Once administered to the human cell, the plasmid DNA begins to replicate and to produce copies of the inserted DNA sequence. Thus, the use of plasmid DNA as active pharmaceutical ingredients (API) to treat different disease states can be considered, however, its storage has emerged as a significant obstacle for this technology. Indeed, if the plasmid DNA is stored under conditions that are not optimal, its structure is degraded and the supercoiled topology (ccc) of the molecule can be converted into inactive forms (open and linear circular) due to oxidative damage. Oxidizing agents, for example, hydrogen peroxide, superoxide and hydroxyl radicals, which are generated by Fenton-type reactions, are responsible for the oxidative degradations of DNA. Particularly, the routes of oxidation of free radicals and depurination and ß elimination represent the main sources of DNA degradation in the case of plasmid DNA highly purified in aqueous formulations, producing breaks or nicks in a strand of DNA and subsequent conversions of the DNA. Double coiled covalent circular DNA (ccc) supercoiled in a relaxed circle or in open or linear circular shapes. The plasmid DNA is usually formulated in aqueous solutions buffered with phosphate or Tris, in which the phosphate or Tris buffer are present in a concentration of about 10. mM. Generally, said compositions are, however, subjected to degradation processes that occur during storage in an aqueous solution. These plasmid DNA solutions have very low stability at both approximately + 4 ° C and + 25 ° C. In particular, its depuration rates are very high at + 4 ° C to room temperature (RT). The degradation processes are usually evaluated by determining the supercoiled, open and linear circular DNA content, as well as by the rate of depurination, that is, the accumulation of apurine sites and by oxidation, that is, formation of 8-hydroxyguanine in the weather. The long-term storage of a pharmaceutical product containing plasmid DNA results, therefore, in many degradation reactions that affect the stability of the DNA. To solve these problems the plasmid DNAs are usually freeze-dried to be stored at temperatures that go up to room temperature but require additional manipulation steps of reformulations and additional risks of contamination and / or degradation. Because any breakage of the strands that occurs in the plasmid DNA affects quality and behavior, it is critical to study the damage that occurs over time during storage and manipulations of the plasmid DNA and provide a storage composition that allows a stability of plasmid DNA in long-term storage and safe manipulations in wider temperature ranges of + 4 ° C to room temperature. The Applicants have thus discovered new liquid compositions for plasmid DNA which are stable and resistant over a wide range of temperatures, for example, to room temperature over a long period of time, thus facilitating storage, transport, manipulation and distribution of the drug based on DNA, DNA vaccines or gene therapy before safe administrations to subjects. In particular, said liquid formulations are useful for highly purified plasmid DNA which can be used for plasmid-based research and therapy, for example, in gene therapy and DNA vaccines.

Brief Description of the Invention A first object of the present invention is a composition for preserving plasmid DNA in a liquid formulation for long periods of time at temperatures up to + 25 ° C. The present invention thus relates to a liquid storage composition of plasmid DNA comprising a plasmid DNA and a buffer in a concentration of up to 5mM, up to 4mM, or up to 3mM, or also up to 2mM which is sufficient to maintain the pH of the composition of plasmid DNA between 6 and 9, thus preserving plasmid DNA with a supercoiled content of at least 80%, and a plasmid content subject to debridement and nicking of less than 20%. The present invention also relates to a stable liquid storage composition of plasmid DNA comprising a plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of said formulation or composition between 6.2 and 8 , 5, and / or approximately +/- 0.3 of one or both of these values, thus preserving plasmid DNA with shedding and nicking rates of less than 5% per year when stored at approximately + 4 ° C and less than 5% per month when stored at approximately + 25 ° C. The present invention also relates to a stable liquid storage composition of plasmid DNA comprising a plasmid DNA and a buffer solution in a concentration of up to 2mM sufficient to maintain the pH of said formulation or composition between 6.7 and 8, 0, and / or about +/- 0.3 of one or both of these values, thus preserving the plasmid DNA with shedding and nicking rates of less than 2% per year when stored at approximately + 4 ° C and less than 2% per month when stored at approximately + 25 ° C. The present invention further relates to a stable liquid storage composition of plasmid DNA comprising a plasmid DNA and a buffer solution in a concentration of up to 2 μM sufficient to maintain the pH of said formulation or composition between 7.0 and 7, 5, approximately +/- 0, 3, thus preserving the plasmid DNA with depuration speeds and nicking of less than 1% per year when stored at approximately + 4 ° C and less than 1% per month when stored at approximately + 25 ° C. Another object of the present invention is a method for preserving plasmid DNA in a stable form in a liquid storage composition, comprising (i) preparing a purified sample of plasmid DNA; (ii) combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6 and 9; and (iii) storing the plasmid DNA. The method according to the present invention allows to preserve high quality plasmid DNA with at least 80% supercoiled plasmid DNA.

The present invention relates to a method for preserving Plasmid DNA in a stable form in a liquid storage composition at temperatures of about + 4 ° C to + 25 ° C with debridement and nicking rates of less than 5% per month to less than 5% per year, comprising (i) ) prepare a purified sample of plasmid DNA; (I) combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6.2 and 8.5 and / or approximately +/- 0.3 of one or both of those values; and (iii) storing the plasmid DNA at the selected temperature. The present invention also relates to a method for preserving plasmid DNA in a stable form in a composition liquid storage at temperatures of about + 4 ° C to + 25 ° C with depurination and nicking rates of less than 2% per month to less than 2% per year, comprising (i) preparing a purified sample of plasmid DNA; (ii) combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6.7 and 8 or about +/- 0.3 of one or both of these values; and (iii) storing the plasmid DNA at the selected temperature. The present invention further relates to a method for preserving plasmid DNA in a stable form in a liquid composition at temperatures of about + 4 ° C to + 25 ° C with debridement and nicking rates of less than 1% per month less than 1% per year, comprising (i) preparing a purified sample of plasmid DNA; (ii) combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 7.0 and 7.5 and / or approximately +/- 0.3 of one or both of those values; and (iii) storing the plasmid DNA at the selected temperature. According to the plasmid DNA composition it comprises a buffer solution in a concentration of less than 5 mM, or less than 4 mM, or less than 3 mM. Preferably, the stable storage composition of plasmid DNA comprises a buffer solution at trace level or at a very high concentration. diluted up to 2mM, and more preferably between 1mM and 2mM. Most preferably, the buffer solution is present at a concentration of less than 1 mM, between 250 μM and 1 mM, or between 400 μM and 1 mM, to maintain the pH of said formulation or composition between 6 and 9, or between 6.2 and 8.5, preferably between 6.7 and 8, and most preferably between 7 and 7.5, and / or about +/- 0.3 of any one or more of those values. The stable composition according to the present invention is particularly useful for storing highly purified plasmid DNA, which has very low levels of chromosomal DNA, RNA, proteins and contaminating endotoxins. Said highly purified plasmid DNAs have less than about 0.01% RNA contaminating the host cell, and / or less than about 0.01% contaminating protein of the host cell, and / or less than about 0.01% of Genomic DNA contaminating the host cell. The preferred highly purified plasmid DNAs have less than about 0.001% RNA contaminating the host cell, and / or less than about 0.001% protein contaminating the host cell, and / or less than about 0.001% genomic DNA contaminating the host cell. host cell. The most preferred highly purified plasmid DNAs have less than about 0.0001% RNA contaminating the host cell, and / or less than about 0.0001% contaminating protein from the host cell, and / or less than about 0.0001 % DNA genomic contaminant of the host cell. Yet another object of the present invention is a method for preparing a stable liquid plasmid DNA composition for storage at a temperature of up to about 25 ° C, comprising (1) a lysis step of the cells comprising making the cells flow through (a) a turbulent flow to rapidly mix a cell suspension with a solution that lyses the cells; and (b) a laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, wherein the mixture formed in (a) flows from the turbulent flow to the laminar flow and optionally further comprises (c) adding a second solution that neutralizes the lysis solution, the mixture incubated in (b) of the laminar flow flowing to the second solution, to release the plasmid DNAs from the cells; (2) a chromatography step to purify the plasmid DNA released in this way; (3) combining said purified plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6 and 9, and (4) storing the plasmid DNA composition at a temperature of up to about 25 °. C. The present invention also relates to a method for preparing a stable liquid plasmid DNA formulation for storage at a temperature of up to about 25 ° C, comprising (1) a cell lysis step comprising making the cells flow through of (a) a medium for turbulent flow to rapidly mix a cell suspension with a solution that lyses the cells; and (b) a laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, wherein the mixture formed in (a) flows from the turbulent flow to the laminar flow and optionally further comprises (c) a medium to add a second solution that neutralizes the lysis solution, the mixture incubated in (b) flowing from the laminar flow medium to the medium to add a second solution, to release the plasmid DNAs from the cells; (2) performing a chromatography step to purify the plasmid DNA released in this manner; (3) performing a diafiltration and / or buffer exchange step; (4) combining said purified plasmid DNA with a buffer solution in a concentration of up to 2mM sufficient to maintain the pH of the resulting composition between 6 and 9; and (5) filling pathways with the liquid plasmid DNA composition and storing the plasmid DNA composition at a temperature of up to about 25 ° C. According to the method of the present invention, the buffer solution is added to the plasmid DNA composition in a concentration of less than 5mM, or less than 4mM, or less than 3mM. Preferably, the method comprises the addition of trace levels of the buffer solution or the addition of a buffer solution in a very dilute concentration of up to 2mM, and more preferably between 1mM and 2mM. Most preferably, the buffer solution is present in a concentration of less than 1 mM, between 250 μM and 1 mM, or between 400μM and 1mM, to maintain the pH of said formulation or composition between 6 and 9, or between 6.2 and 8.5, preferably between 6.7 and 8, and most preferably between 7 and 7.5, and / or approximately +/- 0.3 of any one or more of those values. A further object of the present invention is vials containing the stable liquid formulation of plasmid DNA as an active pharmaceutical ingredient for use in research or in plasmid-based therapy, such as gene therapy or DNA vaccine. Yet another object of the present invention is the vial containing a purified plasmid DNA is a plasmid called NV1 FGF which is a pCOR plasmid carrying an expression cassette encoding the FGF-1 gene, which is useful for the treatment of peripheral ischemia of the extremities, including peripheral arterial disease (PAOD or PAD) and critical ischemia of the extremities (CLI). The additional objects and advantages of the invention will be shown in part in the description that follows and in part will be obvious from the description or can be appreciated by practicing the invention. The objects and advantages of the invention will be carried out and will be achieved by the elements and combinations indicated particularly in the appended claims. It should be understood that both the foregoing general description and the following detailed description are only by way of example and explanation and that they are not restrictive of the invention, as vindicates The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments of the invention and together with the description serve to explain the principles of the invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic of the apparatus that can be used for the continuous cell lysis mode of the invention. Figure 2 is a schematic of the M 1 mixer in the continuous cell lysis apparatus. Figure 3 is a table comparing purification yields in terms of gDNA, RNA, protein, contaminating endotoxin using a single step anion exchange chromatography (AEC), or a two step method with an exchange chromatography step anionic in combination with triple helix affinity chromatography (THAC), and a three-step method comprising an anion exchange chromatography step, a triple helix affinity chromatography step and a hydrophobic interaction chromatography step (HIC) in combination. ND means not detected: low sensitivity analytical methods. Figure 4 is a table comparing various methods of separation and purification of plasmid DNA, such as ammonium exchange chromatography (AEC), hydroxyapatite chromatography (HAC), hydrophobic interaction chromatography (HIC), reverse phase chromatography (RPC), exclusion chromatography by size (SEC), tri-helix affinity chromatography (THAC) alone or in combination and the method according to the present invention. The results in terms of quality of the purified plasmid DNA are provided herein. ND, not detected (low sensitivity analytical methods). Figures 5A and 5B are graphs showing the rates of depurination and nicking (formation of the open circular plasmid form) of the plasmid DNA stored at + 25 ° C and + 5 ° C up to 90 days. Figures 6A and 6B are graphs showing the rates of depurination and nicking (formation of the open circular plasmid form) of the plasmid DNA stored at + 25 ° C and + 5 ° C up to 1 50 days. Definitions The formulation or composition of plasmid DNA means a composition comprising an effective amount of plasmid DNA or a formulation of a plasmid DNA present in an effective amount for use in research or in plasmid-based therapy, such as gene therapy or DNA vaccine. . The stable storage formulation of plasmid DNA means a formulation that can be used to store the plasmid DNA in a stable form for long periods of time before being used for research or plasmid-based therapy. The storage time can be as long as several months, 1 year, 5 years, 10 years, 1 5 years or up to 20 years at a temperature range from + 5 ° C to + 25 ° C (RT: room temperature). Generally, a stable formulation or composition of plasmid DNA means a plasmid DNA formulation having a double-stranded DNA ratio supercoiled at least 80%, the rest being in the form of open and / or linear circular plasmids. A stable formulation of plasmid DNA hereinafter means a composition comprising plasmid DNA having debridement and nicking rates (formation of the open circular plasmid form) of less than 5% per month when stored at + 25 ° C and less than 5% per year when stored at + 5 ° C. Preferably, a stable plasmid DNA formulation hereinafter means a composition comprising plasmid DNA having clearance and nicking rates (formation of the open circular plasmid form) of less than 2% per month when stored. at + 25 ° C and less than 2% per year when stored at + 5 ° C. More preferably a stable plasmid DNA formulation hereinafter means a composition comprising plasmid DNA having clearance and nicking rates (formation of the open circular plasmid form) of less than 1% per month when stored at + 25 ° C and less than 1% per year when stored at + 5 ° C.

Acid means that it refers to or that it contains an acid; which has a pH of less than 7. Alkali does not mean that it refers to or that it contains an alkali or base; which has a pH greater than 7. Conti nuo means uninterrupted, which is not interrupted. Genomic DNA (abbreviated as gDNA) means a DNA that comes from or exists on a chromosome. Laminar flow means the type of flow in an aqueous solution stream in which each particle moves in a direction parallel to all the particles. Lysate means the material produced by the cell lysis process. The term "lysate" refers to the action of breaking the cell wall and / or cell membrane of a cell that is in a buffered solution (ie, cell suspension) by chemical treatment using a solution containing a usable agent. Usage agents include, for example, alkali, detergents, organic solvents and enzymes. In a preferred embodiment, the lysis of the cells is performed to release intact plasmids from the host cells. It is neutralized to make (a solution) neutral or to neutralize (an acid or base / alkali). By this term we do not mean that something neutralizing a solution brings the pH of the solution to a pH between 5 and 7, and preferably about 7 or more preferably closer to 7 than it was at the beginning.

Newtonian fluid is a fluid in which the shear stress is proportional to the velocity gradient and perpendicular to the plane of shear. The constant of proportionality is known as viscosity. Examples of Newtonian fluids include liquids and gases. Non-Newtonian fluid is a fluid in which the shear stress is not proportional only to the velocity gradient and perpendicular to the plane of shear. Non-Newtonian fluids may not have a well-defined viscosity. Non-Newtonian fluids include plastic solids, fluids according to the law of potency, viscoelastic fluids (which have both viscous and elastic properties), and fluids with time-dependent viscosity. Plasmid DNA means a small cell inclusion consisting of a DNA ring that is not a chromosome, which may have the ability to have a non-endogenous DNA fragment inserted. As used herein, "plasmid DNA" may also be any form of plasmid DNA, such as a form of non-chromosomal DNA cut, processed or otherwise manipulated including, for example, any of, or any combination of, Circular nicked plasmid DNA, relaxed circular plasmid DNA, supercoiled plasmid DNA, cut plasmid DNA, linear or linear plasmid DNA and single-stranded plasmid DNA. The procedures for the construction of plasmids include those described in Maniatis et al. , Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press (1989). A protocol for a plasmid DNA mini-prep well known in the art (Birnboim and Doly, Nucleic Acids Research 7: 1513 (1979)), can be used to initially isolate plasmid DNA for further processing by some aspects of the invention and can contrast with the highly purified samples produced from the methods of the invention. Preferably, the plasmid DNA form is, or at least is after the preparation by the purification method of the invention, substantially a closed circular form of plasmid DNA or approximately 80%, 85%, 90%, 95%, or more of approximately 99% a closed circular form of plasmid DNA. Alternatively, a covalently closed supercoiled form of plasmid DNA (ccc) may be preferred in some therapeutic methods, where it may be more effective than open circular, linear or multimeric forms. Therefore, pharmaceutical grade plasmid DNA can be isolated from or separated from one or more plasmid forms and substantially comprises one or more desired forms. For the purposes of the present invention, the term "flow" refers to the action of passing a liquid at a particular flow rate (eg, liters per minute) through the mixer, usually by the action of a pump. It should be noted that it is believed that the flow rate through the mixer affects the efficiency of lysis, precipitation and mixing. The terms "nicked" and "relaxed" DNA mean DNA that does not It is supercoiled. "Supercoiled" DNA is a term known in the art to describe a particular, isolated form of plasmid DNA. Other forms of plasmid DNA are also known in the art. A "contaminating impurity" is any substance from which it is desired to separate or isolate the DNA. Contaminating impurities include, but are not limited to, host cell proteins, endotoxin, host cell DNA, such as chromosomal DNA or genomic DNA and / or RNA from the host cell. It is understood that what is or can be considered a contaminating impurity may depend on the context in which the methods of the invention are practiced. A "contaminating impurity" may or may not come from the host cell, that is, it may or may not be an impurity of the host cell. "Isolating" or "purifying" a first component (such as DNA) means enriching the first component with respect to other components with which the first component is initially located. The desired and / or obtainable degrees of purification are provided herein. The terms "essentially free and highly purified" are defined as approximately 95% and preferably more than 98., 99% pure or if n contaminants or that has less than 5%, and preferably less than 1 -2% of contaminants. Pharmaceutical grade DNA is defined herein as a DNA preparation containing no more than about 5%, and preferably not more than about 1-2%, of cellular components, such as cell membranes. Also disclosed is a method for producing and isolating highly purified plasmid DNA that essentially contains no contaminants and, therefore, is pharmaceutical grade DNA. The plasmid DNA produced and isolated by the method of the invention contains very low levels, that is, parts per million (ppm) of chromosomal DNA, RNA, proteins and contaminating endotoxins and mostly contains the closed circular form of plasmid DNA. The plasmid DNA produced according to the invention is of sufficient purity to be used in research and in plasmid-based therapy and optionally as a material for clinical trials in humans and gene therapy experiments in humans and in clinical trials. A "pharmaceutical grade plasmid DNA composition" of the invention is one that is produced by a method of the invention and / or is a composition having at least one of the purity levels defined below as a "Grade plasmid DNA". pharmacist". Preferably, a "pharmaceutical grade plasmid DNA composition" of the invention has a level of purity defined by at least two of those defined below as a "pharmaceutical grade plasmid DNA" for example, less than about 0.01% of Chromosomal or genomic DNA and less than about 0.01% contaminating proteins or for example less than about 0.01% chromosomal DNA or gene and less than about 0.1 EU / mg endotoxins. The pharmaceutical grade plasmid DNA preferably contains less than about 0.001% chromosomal or genomic DNA and less than about 0.001% contaminating proteins or for example less than about 0.001% chromosomal or genomic DNA and less than about 0.1 EU / mg of endotoxins. More preferably it contains less than about 0.0001% of chromosomal or genomic DNA and less than about 0.0001% of contaminating proteins or for example less than about 0.0001% of chromosomal or genomic DNA and less than about 0.1 EU / mg of endotoxins. The most preferred pharmaceutical grade plasmid DNA contains less than about 0.00008% chromosomal or genomic DNA and less than about 0.00005% contaminating proteins or for example less than about 0.00008% chromosomal or genomic DNA and less than approximately 0.1 EU / mg endotoxins. Other combinations of purity levels are included in the definition. Of course, the pharmaceutical grade plasmid DNA composition may also comprise or contain added components desired for any particular use, including use in combination treatments, compositions and therapies. The levels of chromosomal or genomic DNA, RNA, endotoxins or proteins refer to contaminants from the production of the plasmid based on cells or to another or other contaminants from the process of purification. Most preferably, "pharmaceutical grade plasmid DNA" is defined herein as a DNA preparation containing at the level of one part per million or ppm (< 0.0001%, ie < 0.0001 mg per 100 mg of plasmid DNA) or less of genomic DNA, RNA and / or contaminating proteins. Also, or more precisely, "pharmaceutical grade plasmid DNA" as used herein may mean a DNA preparation containing less than about 0.01%, or less than 0.001%, and preferably less than 0.0001%, or preferably less than 0.00008% (<0.00008%, ie <0.00008 mg per 100 mg of plasmid DNA) of chromosomal DNA or genomic DNA. "Pharmaceutical grade plasmid DNA" can also mean a DNA preparation containing less than about 0.01%, or less than 0.001%, and preferably less than 0.0001%, or preferably less than 0.00002% (< 0.00002%, ie <0.00002 mg per 100 mg of plasmid DNA) of contaminating RNA. "Pharmaceutical grade plasmid DNA" can also mean a DNA preparation containing less than about 0.0001%, and most preferably less than 0.00005% (< 0.00005%, ie <0.00005 mg per 100 mg of plasmid DNA) of contaminating proteins.

"Pharmaceutical grade plasmid DNA" can also mean a DNA preparation containing less than 0.1 EU / mg endotoxins. "Pharmaceutical grade" plasmid DNA means herein a DNA preparation that is preferably predominantly in a circular form and more precisely DNA containing more than 80%, 85%, 90%, 95%, or more than 99% of a closed circular form of plasmid DNA. T-tube refers to a T-shaped tube configuration, in which a T-shape is formed by a single tube piece created in that configuration or more than one piece of tube combined to create that configuration. The tube T has three arms and a central area in which the arms are joined. A tube T can be used to mix ingredients since two fluids can flow in each of the arms of the T, join in the central area and exit through the third arm. The mixing occurs when the fluids are combined. Turbulent flow means irregular random movement of fluid particles in directions transverse to the direction of primary flow, in which the velocity at a given point varies erratically in magnitude and direction. Viscoelastic refers to fluids that have both viscous and elastic properties.

Detailed Description Of The Invention The present invention relates to liquid formulations of Plasmid DNA which are stable and in which the plasmid DNA remains without degrading at room temperature for a long period of time. Said formulations or plasmid DNA compositions are thus useful for the storage of the plasmid DNA used in research, in plasmid-based therapies, such as gene therapy or DNA vaccines. According to the present invention, the stable liquid storage composition of plasmid DNA comprises a plasmid DNA and a buffer solution in a concentration of up to 5mM, or up to 4mM, or up to 3mM, or up to 2m which are sufficient to maintain the pH of said composition between 6 and 9, and the composition predominantly comprises the supercoiled form of plasmid DNA at temperatures of about 4 ° C to 25 ° C, for several months, 1 year, 2 years, 3 years, 4 years, 5 years and up to 10 years. A plasmid DNA composition having predominantly the supercoiled plasmid form comprises at least 80% supercoiled or closed circular plasmid DNA, or about 85%, and preferably about 90%, or about 95%. Most preferably, the stable plasmid DNA composition contains approximately 99% of the supercoiled or closed circular form of plasmid DNA. Alternatively, a stable composition for plasmid DNA storage provides depuration and nicking rates of less than 5% per month.

The stable liquid storage formulation of plasmid DNA according to the present invention therefore comprises a plasmid DNA and a very dilute buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the composition at least about 6, and at most at 9, or between 6.2 and 8.5, and preferably between 6.7 and 8, and more preferably between 7 and 7.5, and / or approximately +/- 0.3 of one or both of those values. The stable liquid composition of plasmid DNA comprises a buffer solution in a concentration of up to 2mM to maintain the pH of said formulation or composition between 6.2 and 8.5, and / or about +/- 0.3 of one or both of these values, thus allowing the storage of plasmid DNA with debridement and nicking speeds of less than 5% per year when stored at approximately + 4 ° C and less than 5% per month when stored at approximately + 25 ° C . Preferably, the stable liquid plasmid DNA composition comprises a buffer solution in a concentration of up to 2 mM to maintain the pH of said formulation or composition between 6.7 and 8, and / or about +/- 0.3 of one or both of these values, thus allowing the storage of plasmid DNA with rates of debridement and nicking of less than 2% per year when stored at approximately + 4 ° C and less than 2% per month when stored at approximately + 25 ° C .

More preferably, the stable liquid plasmid DNA composition comprises a buffer solution in a concentration of up to 2mM to maintain the pH of said formulation or composition between 7 and 7.5, and / or about +/- 0.3 of one or both of these values, thus allowing the storage of plasmid DNA with debridement and nicking speeds of less than 1% per year when stored at approximately + 4 ° C and less than 1% per month when stored at approximately + 25 ° C. The molar concentration of the buffer solution is determined so that the buffering effect is exerted within a limit and in a volume in which the pH value is stabilized between 6 and 9, or between 6.2 and 8.5, preferably between 6.7 and 8, and most preferably between 7 and 7.5, and / or about +/- 0.3 of any of those values. The buffer solution can therefore be added in a concentration of less than 5 mM. Preferably, the stable storage composition of plasmid DNA comprises traces of the buffer solution or a buffer solution in a very dilute concentration of up to 2mM, and more preferably between 1mM and 2mM. Most preferably, the buffer solution is present at a concentration of less than 1 mM, between 250 μM and 1 mM, or between 400 μM and 1 mM, to maintain the pH of said formulation or composition between 6 and 9, or between 6.2 and 8.5, preferably between 6.7 and 8, and most preferably between 7 and 7.5, and / or about +/- 0.3 of any one or more of those values. The buffer solution is present in a concentration of up to 2mM, or between 1 and 2mM. Preferably, the buffer solution is present in a concentration of less than 1 mM. Most preferably, the buffer solution is present as trace levels at a concentration as low as 250μM and up to 1mM. The trace levels of the buffer solution may be about 400μM, and just enough to maintain the pH at the ranges indicated hereinabove. The buffer solutions that can be used in the compositions of the present invention consist of an acid / base system comprising Tris [(tris (hydroxymethyl) -aminomethane], or lysine or an acid selected from a strong acid (hydrochloric acid, for example) and a weak acid (maleic acid, malic acid or acetic acid, for example), or an acid / base system comprising Hepes acid [2- (4- (2-hydroxyethylpiperazine) -1-yl) ethanesulfonic acid] and a strong base ( sodium hydroxide, for example), or phosphate buffers, such as sodium phosphate or potassium phosphate. The buffer solution may also comprise Tris / HCl, lysine / HCl, Tris / maleic acid, Tris / malic acid, Tris / acetic acid, or Hepes / sodium hydroxide. Preferably, the Tris buffer is used in the stable storage composition of plasmid DNA of the present invention. As shown in the Examples below, the plasmid DNA formulations according to the present invention they exhibit excellent stability at both 4 ° C and ambient temperature (RT), for example, 20 or 25 ° C. The composition of the present invention may further comprise a saline excipient. Saline excipients that can be used in the compositions of the present invention can comprise anions and cations selected from the group consisting of acetate, phosphate, carbonate, SO2"4, Cl", Br ", NO3" Mg2 +, Li +, Na +, K + , and NH +, and any other salt or form of a pharmaceutical compound available or previously used. The preferred saline excipient is NaCl at a concentration between 100 and 200 mM, and preferably at a concentration of approximately 150 mM. The stable compositions according to the present invention are particularly useful for storing highly purified plasmid DNA or pharmaceutical grade plasmid DNA having very low levels of chromosomal DNA, RNA, proteins and contaminating endotoxins. Said highly purified plasmid DNAs have less than about 0.01%; or 0.001%; or 0.0001% RNA contaminating the host cell, and / or less than about 0.01%; or 0.001%; or 0.0001% contaminating protein of the host cell, and / or less than about 0.01%; or 0.001%; or 0.0001% of genomic DNA contaminating the host cell. The compositions according to the present invention may further comprise an adjuvant, such as for example a polymer selected from polyethylene glycol, a pluronic or a sugar polysorbate or alcohol. According to another aspect, the present invention relates to a method for preserving plasmid DNA in a composition comprising a) preparing a purified sample of plasmid DNA and b) combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2mM which maintains the pH of the resulting composition between 6.2 and 9. Preferably, the pH is maintained between 6.5 and 8.5, preferably between 6.7 and 8, and most preferably between 7 and 7.5. , and more particularly to approximately 7.2. The present invention also relates to a method for preserving plasmid DNA in a composition comprising a) preparing a purified sample of plasmid DNA, b) combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2mM sufficient to maintain the pH of the resulting composition between 6 and 9, and c) to store the plasmid DNA. The method according to the present invention allows the plasmid DNA to be stored with at least 80% supercoiled plasmid DNA. The pH of the resulting composition can be maintained between 6.2 and 8.5 and approximately +/- 0.3 of one or both of these values, thus allowing to preserve the plasmid DNA at temperatures of approximately + 4 ° C to +25. ° C with debridement and nicking speeds of less than 5% per month to less than 5% per year. Preferably, the pH of the resulting composition can maintain between 6.7 and 8 and approximately +/- 0.3 thus allowing the plasmid DNA to be preserved at temperatures of approximately + 4 ° C to + 25 ° C with debridement and nicking speeds of less than 2% per month less of 2% per year. More preferably, the pH of the resulting composition can be maintained between 7 and 7.5 and approximately +/- 0.3 of one or more of these values, thus allowing the preservation of the plasmid DNA at temperatures of about + 4 ° C to +25. ° C with debridement and nicking speeds of less than 1% per month to less than 1% per year. According to the method of the present invention, the buffer solution is added to the plasmid DNA composition in a concentration of up to 2 mM, or between 1 and 2 mM. Preferably, the buffer solution is added to achieve a concentration of less than 1 mM. Most preferably, the buffer solution is present as trace levels at a concentration as low as 250μM and up to 1mM. The trace levels of the buffer solution may be about 400μM, and just enough to maintain the pH at the ranges indicated hereinabove. According to the present method, a saline excipient can also be added to the solution of plasmid DNA and buffer. These have been described hereinabove. The preferred saline excipient is NaCl at a concentration between 100 and 200 mM, and preferably about 150 mM.

The plasmid DNAs that are formulated in the compositions according to the present invention may be in an isolated form.

They can be isolated by lysis of bacterial cells and purified as described herein or synthesized by automated nucleic acid synthesis equipment. They may comprise a polynucleotide encoding a polypeptide, wherein the polynucleotide may be a transgene, such as a therapeutic gene, for example of mammalian origin, such as a rodent or human gene and is operably linked to a promoter sequence. The polynucleotide that is inserted into the plasmid DNA can have a genomic origin and therefore contain exons and introns as reflected in their genomic organization or can be obtained from complementary DNA. The polynucleotide can encode any of a variety of polypeptides, such as, without limitation, an immunogenic peptide or protein, an angiogenesis factor, erythropoietin, adenosine deaminase, Factor VII I, Factor IX, dystrophin, β-globin, LDL receptor, CFTR, insulin, an anti-angiogenesis factor, a growth hormone, a1-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, an interleukin, and an interferon. Preferably, the plasmid DNA comprises a polynucleotide encoding an angiogenesis factor, such as an FGF gene (FGF-1 to FGF-22), VEGF, HGF, or HI F-1. As an alternative to the use of a polynucleotide encoding a polypeptide, the polynucleotide can encode an siRNA, which can be used to inhibit the expression of a target gene, e.g. when the expression of the gene is undesirable (for example, the gene of a pathogen) or when the level of gene expression is undesirably high in a cell. Promoters suitable for use in various vertebrate systems are well known and include for example, RSV LTR, M PSV LTR, SV40, metallothionein promoter and CMV I EP which can be used advantageously. The plasmid DNA can include prokaryotic and eukaryotic vectors, and expression vectors, such as pBR322 and pUC vectors and their derivatives. They can incorporate multiple origins of replication, eg, prokaryotic replication origins, such as pM B1 and ColEl, or eukaryotic origins of replication, such as those that facilitate replication in yeast, fungal, insect and mammalian cells (e.g. , SV40 ori). The insert may include DNA from any organism, but will preferably have a mammalian origin and may include in addition to a gene encoding a therapeutic protein, regulatory sequences such as promoters, amplifiers, locus control regions, selectable genes, polylinkers for insertion of the transgene, leader peptide sequences, introns, polyadenylation signals or combinations thereof. The selection of vectors, origins and genetic elements will vary based on the requirements and is within the reach of those skilled in the art. Selectable markers can be, for example, antibiotic resistance gene, for example, SupPhe tRNA, the tetracycline resistance gene, the kanamycin resistance gene, puromycin resistance, neomycin resistance gene, hygromycin resistance gene, and thymidine kinase resistance. The central part of the plasmid advantageously allows inserts of mammalian, other eukaryotic, prokaryotic or viral DNA fragments and the resulting plasmid can be purified and used in plasmid-based therapy in vivo or ex vivo. Preferably, plasmid DNA with a conditional origin of replication is used, such as the pCOR plasmid which is described in US application publication 2003/1618445. The resulting high copy number greatly increases the ratio of plasmid DNA to chromosomal DNA, RNA, cellular proteins and co-factors, improves plasmid production and facilitates downstream purification. Accordingly, any plasmid DNA according to the invention can be used. Representative vectors include but are not limited to plasmid NV1 FGF. NV1 FGF is a plasmid encoding an acid Fibroblast Growth Factor or Fibroblast Growth Factor 1 (FGF-1), useful for treating patients with terminal arterial occlusive disease (PAOD) or peripheral arterial disease (PAD). Camerota et al. (J Vasc. Surg., 2002, 35, 5: 930-936) describe that in 51 patients with terminal non-reconstructible DBP, with pain at rest or tissue necrosis, they were injected intramuscularly single or repeated increasing doses of NV1 FGF in the thigh and ischemic calf muscles. Several parameters such as oxygen pressure were subsequently evaluated transcutaneous, brachial indexes in the ankles and toes, evaluation of pain and healing of ulcers. There was a significant increase in brachial indexes, pain reduction, resolution of the size of the ulcers and improved perfusion after administration of NV1 FGF. According to another aspect, the present invention provides a composition as defined herein above for use in a method of treating a human body or an animal body by therapy. Preferably, the composition according to the present invention contains a pCOR plasmid encoding an angiogenic gene of the FGF or VEGF family for the treatment of cardiovascular diseases such as peripheral ischemia, peripheral arterial diseases, eg, PAOD or PAD, critical ischemia of the extremities. (CLI), and intermittent claudication (IC). As another preferred use, the plasmid DNA comprises a polynucleotide that encodes an immunizing peptide and can be used as a DNA vaccine. The present invention thus provides a composition for the vaccination of humans or animals, thereby generating an effective immunity against infectious agents, including intracellular viruses, and also against tumor cells. Indeed, the stable composition of plasmid DNA can be used as a DNA vaccine to greatly increase the immunogenicity of certain viral proteins and cancer-specific antigens that they usually induce a weak immune response. They are useful for the induction of immunity induction of cytotoxic T cells against poorly immunogenic viral proteins of Herpes virus, non-A, non-B, and VI H. Plasmid DNA can encode polypeptides that confer immunity, which can act as endogenous immunogens to elicit a humoral or cellular response, or both, or even an antibody. In this regard, the term "antibody" encompasses complete immunoglobulins of any kind, chimeric antibodies and hybrid antibodies with dual or multiple epitope or antigen specificities and fragments such as F (ab) 2, Fab ', Fab and the like, including fragments hybrids Also referred to as "antibody" are conjugates of said fragments and so-called antigen-binding proteins (single-chain antibodies) as described, for example, in U.S. Patent 4,704,692 (the content of which is incorporated herein). memory by reference). Therefore, plasmid DNA comprising a polynucleotide encoding variable regions of an antibody can be used to produce antibody in situ. For an illustrative methodology for obtaining polynucleotides encoding antibodies, see Ward et al. Nature, 341: 544-546 (1989); Gillies et al., Biotechnol. 7: 799-804 (1989); and Nakatani et al. , loe. cit. , 805-810 (1989). For its part, the antibody will exert a therapeutic effect, for example, by binding to a surface antigen associated with a pathogen. Alternatively, the encoded antibodies can be anti-idiotypic antibodies (antibodies that bind to other antibodies) as described, for example, in Pat. of US No. 4,699,880. Such anti-idiotypic antibodies could bind to endogenous or foreign antibodies in a treated individual, to thereby improve or prevent pathological conditions associated with an immune response, for example, in the context of an autoimmune disease. The composition according to the present can thus be administered to humans or animals in vivo, allowing the plasmid DNA to reach several cells of the animal body, including muscle, skin, brain, lung, liver, spleen or blood cells. Administration of the polynucleotides directly in vivo is preferably performed to the muscle or skin cells. The injections can be performed for example in the muscle or the skin using an injection syringe or a vaccine to provide an effective immunization of the subject. In effect, the gene of an antigen that is introduced into the cells of the subject, the transfected cells, which now express the antigen, will be processed and presented to the immune system by normal cellular routes. Adjuvants or lymphokines can be coinjected to further increase immunization. For example, the stable composition of the plasmid DNA present can be used for vaccination against virus or as a DNA vaccine to treat latent viral infections such as, for example, Hepatitis B, VI H and members of the virus group of the virus.

Herpes, in which the virus remains intracellular in an inactive or partially active form. The plasmid DNA composition of the present invention can be further used for the treatment of malignant diseases, for increasing the cellular immune response against a specific protein of the malignant state, an oncogene, a fetal antigen or an activation marker. The plasmid DNAs (pDNAs) that are formulated for long-term storage according to the present invention are usually produced in bacterial cells that are lysed in order to release the cellular contents from which the pDNA is isolated. This process generally involves three stages comprising cell resuspension, cell lysis, neutralization and precipitation of host contaminants. The cell resuspension usually uses manual agitation or magnetic stirring and a homogenizer or blender to resuspend the cells in the resuspension buffer. Cell lysis can be performed by manual agitation or magnetic stirring in order to mix the resuspended cells with the lysis solution, which consists of dilute lysozyme or alkali (base), such as, for example, alkaline or potassium acetate (KOAc) and detergents; then maintain the mixture at room temperature (20-25 degrees Celsius) or on ice for a period of time, such as 5 minutes, to complete the lysis. Generally, RNase is also added to degrade the RNAs of the bacterial suspension.

The third stage is the neutralization and precipitation of host contaminants. The second stage lysate is usually mixed with a cold neutralization solution by gentle agitation or magnetic stirring to acidify the lysate before being placed on ice for 10-30 minutes to facilitate denaturation and precipitation of the high molecular weight chromosomal DNA. , host proteins and other host molecules. When cell lysis is performed using a lysozyme treatment, the bacteria are contacted with lysozyme and then boiled at approximately 1000 ° C in an appropriate buffer for 20 to 40 seconds forming an insoluble pool of genomic DNA, proteins. and cell debris leaving the plasmid in solution with RNA as the main contaminant. Then, a mixed solution of NaOH and sodium dodecyl sulfate (S DS) is added in order to dissolve the cytoplasmic membrane. NaOH partially denatures the DNA and partially degrades the RNA and S DS acts to dissolve the membrane and denature the proteins. Subsequently, the SDS-protein complex and the cell residues are precipitated by the addition of 5N potassium acetate (pH 4.8). At this time, the pH is important both to neutralize the NaOH used in said manipulation and to renature the plasmid. Subsequently, a centrifugation is applied to eliminate the precipitates, thus obtaining the plasmid DNAs desired in the supernatant. Alternatively, an alkaline lysis is performed which consists of mix a suspension of bacterial cells with an alkaline lysis solution. The alkaline lysis solution consists of a detergent, for example, sodium dodecyl sulfate (SDS), to lyse the bacterial cells and release the intracellular material and an alkali, for example, sodium hydroxide, to denature the proteins and nucleic acids of the cells ( particularly ADNg and RNA). When the cells are used and the DNA is denatured, the viscosity of the solution increases greatly. After denaturation, an acid solution, for example, potassium acetate (solution 3), is added to neutralize the sodium hydroxide by inducing renaturation of the nucleic acids. Long fragments of gDNA re-associate randomly and form networks that precipitate as flocs, trapping proteins, lipids and other nucleic acids. The potassium salt of dodecyl sulfate also precipitates by eliminating the proteins to which it is associated. The two strands of pDNA (plasmid DNA), intertwine with each other, reassociate normally to re-form the initial plasmid that remains in solution. These chemical stages may be suitable for lysing cells on a small scale or in small volumes of bacterial fermentations of less than five liters, but the increase in viscosity may hinder large-scale processing. The lysis technique can be carried out batchwise, that is, in which different solutions are mixed by the sequential addition of the solutions to containers or tanks. After the solution containing the cell suspension is has been mixed with the lysis solution, the non-viscoelastic alkali lysate is mixed with the neutralization solution. The continuous mixing of several cell lysis solutions using a series of static mixers can be used as an alternative to batch methods when large scale plasmid productions are intended. According to these methods, a dissolution of a cell suspension and a cell lysis solution are simultaneously added to a static mixer. The dissolution of lysed cells leaving the first static mixer and a precipitation dissolution are simultaneously added to a second static mixer. The solution leaving this second mixer contains the precipitated lysate and plasmids. Other continuous modes of lysing cells include the use of a direct-flow heat exchanger in which the suspended cells are heated to 70-100 ° C. After cell lysis in the heat exchanger, the output stream is subjected to continuous or discontinuous flow centrifugation during which the cell debris and genomic DNA precipitate, leaving the plasmid DNA in the supernatant. A preferred method for continuous alkaline lysis of bacterial cell suspension, particularly on a large scale, is described in International Patent Publication WO 05/026331, which is incorporated by reference. This preferred method deals with the problems caused by the viscoelastic properties of the fluids and the shear forces involved during the stages of mixed and provides an important advantage by limiting shear forces. Therefore, a high production of plasmid DNA can be prepared using the scalable continuous alkaline lysis method of the host cells which is further described herein. As a first step the host cells are inoculated, that is, they are transformed with a plasmid DNA into the cells in the exponential growth phase and plated in plates containing LB medium containing an antibiotic such as tetracycline. Separate colonies of the plate are inoculated each in 20 ml of LB medium supplemented with the appropriate antibiotic tetracycline in separate sterile plastic Erlenmeyer flasks and grown for 12-16 hours at 37 ° C in a shaking incubator. One of these cultures was used to inoculate 200 ml of sterile LB medium supplemented in a 2 L Erlenmeyer flask. This was grown at 37 ° C and 200 rpm in a shaking incubator and used to inoculate two 5 L Erlenmeyer flasks and they were grown at 30 ° C and 200 rpm in a shaking incubator and used to inoculate the fermentation vessel when they were in the middle of the exponential phase, after 5 hours and at an OD600 nm of 2 units. The cultures of the host cells and the inoculation are well known in the art. Generally, the host cells grow until they reach a large biomass and the cells are exponentially growing in order to have a large amount of plasmid DNA. Two different methods can be used, that is, discontinuous fermentation and discontinuous feeding. The discontinuous fermentation allows controlling the growth rate by manipulating the growth temperature and the carbon source used. As used herein, the term "discontinuous fermentation" is a cell culture process by which all the nutrients required for cell growth and for the production of the plasmid contained in the cultured cells are in the container in excess, such as, for example, up to a 10-fold excess of nutrient concentrations at the time of inoculation, thus avoiding the need for additions to the sterile container after post-sterilization additions and the need for complex models and feeding strategies. In particular, the amounts of yeast extract in the medium range are enriched from 5 g / l (as in the LB medium) to 20 g / l, thereby providing large amounts of growth factors and nucleic acid precursors. The medium is also supplemented with ammonium sulfate (5 g / l) which acts as a source of organic nitrogen. Another type of fermentation is fermentation of discontinuous feeding, in which the rate of cell growth is controlled by the addition of nutrients to the culture during cell growth. As used herein, "batch feed fermentation" refers to a cell culture process in which the rate of growth is controlled by carefully evaluated additions of metabolites to the culture during fermentation. The discontinuous feed fermentation according to the invention allows the cell culture to reach a higher biomass than the discontinuous fermentation. Examples of fermentation processes and exemplary rates of feed addition are described below for a 50 L preparation. However, other volumes may also be processed, eg, 10 L, 50 L, or more than 500 L, using the exemplary feed rates described below, depending on the scale of the equipment. Fermentations with highly enriched discontinuous medium and discontinuous feeding medium are suitable for the production of crops with high cell density to maximize the specific production of the plasmid and allow collection with a large biomass still in exponential growth. Batch feed fermentation uses glucose or glycerol as a carbon source. The fermentation is carried out in batch mode until the initial carbonized substrate (glucose) is exhausted. This point is noted by a rapid increase in OD and is confirmed by a glucose analysis in a sample taken immediately after this event. Then the pump of the previously primed feed medium is started. The speed of the pump is determined by a model obtained from Curless et al. (Bioeng 38: 1082-1090, 1991), which is incorporated herein by reference in its entirety. The model is designed to facilitate the control of the feeding phase of a process of discontinuous feeding. In the initial batch process, a non-inhibitory concentration of substrate is consumed by cells growing at their maximum specific growth rate, resulting in a rapid increase in biomass levels after inoculation. The culture can not grow at this rate indefinitely due to the accumulation of toxic metabolites (Fieschio et al., "Fermentation Technology Using Recombinant Microorganisms." In Biotechnology, eds. HJ Rhem and G. Reed, Weinheim: VCH Verlagsgesellschaft mbH 7b: 1 17-140, 1989). In order to allow continued logarithmic growth, the model calculates the time-based feed rate of the growth-limiting carbon substrate, without the need for retro-control, to result in a discontinuous feed growth phase adjusted by the operator . This is chosen at a level that does not produce the accumulation of inhibitory catabolites and is sufficient to yield a large biomass. Additions of precursors (organic nitrogen in the form of ammonium sulfate) during the feeding process in the batch feed fermentation are designed to prevent detrimental effects on the quality of the plasmid. Lysis methods well known in the art include, for example, heat lysis with direct flow of microbial cells containing the plasmid. This process is described among others in International Publication WO 96/02658. The particular heat exchanger consisted of a 305 cm x 0.64 stainless steel tube cm O. D. with serpentine form. The coil is completely submerged in a water bath with a constant high temperature. The retention volume of the coil is approximately 50 mL. Thermocouples and a thermometer were used to determine the inlet and outlet temperatures and the temperature of the water bath, respectively. The product stream is pumped into the heating coil using a Masterflex peristaltic pump with silicone tubes. The cell lysate exited the coil and was centrifuged in a discontinuous Beckman J-21 centrifuge to rinse it. After centrifugation, the plasmid DNA can be purified using the purification method according to the present invention. Alternatively, static mixers in series can be used for cell lysis. As described in WO97 / 23601 (incorporated herein by reference), a first static mixer can be used to lyse the cells through a first static mixer and to precipitate the cell lysate a second static mixer as an alternative method to lyse the cells before the method for purifying the plasmid DNA according to the present invention. A large volume of cells can be used online carefully and continuously using the static mixer and the other static mixers are brought online to achieve other functions such as dilution and precipitation. Suitable static mixers useful in the method of the present invention include any device direct flow referred to in the art as a static or non-moving mixer with a length sufficient to allow the processes of the present invention. For example, in order to lyse the cells, the static mixer will have to have a length that provides a sufficient contact time between the lysis solution and the cells to cause the lysis of the cells in question during their passage through the mixer . Suitable static mixers contain an internal helical structure that causes the two liquids to come into contact with each other in an opposite rotational flow causing the liquids to mix with each other in a turbulent flow. The most preferred method or device for cell lysis comprises (a) a turbulent flow medium for rapidly mixing a cell suspension (solution 1 in Figure 1) with a solution that lyses the cells (solution 2 in Figure 1); and (b) a means for laminar flow to allow incubation of the mixture formed in (a) without substantial agitation, in which the mixture formed in (a) flows from the turbulent flow medium to the laminar flow medium. Furthermore, this may comprise a means for adding a third solution which neutralizes the lysis solution (solution 3 in Figure 1), in which the mixture incubated in (b) flows from the laminar flow medium to the medium for the addition of a second solution. Thus, for example, this process can be used to isolate plasmid DNA from cells and comprises: (a) mixing the cells with an alkaline lysis solution in the medium for turbulent flow; and (b) neutralizing the alkaline lysis solution by the addition of an acid solution. This process uses T tubes to mix the cell suspension (solution 1) and the alkaline solution (solution 2) uniformly and very rapidly before the viscoelastic fluid appears, thereby providing a major advantage by limiting the shear forces. The tubes T generally have a small tube diameter, usually a diameter of less than 1 cm, preferably of about 2 and 8 mm, and more preferably of about 6 mm, in order to increase the contact time of the mixed fluids, but this method does not use induction of mixing by passing through the tube. Table 1 below shows the variation of parameters B1 a, B1 b, B2 of the medium for turbulent flow, laminar flow, and turbulent flow, respectively, and their corresponding flow velocities S 1, S2, and S3 as shown in Figure 1. Table 1 The process can use a mixer or injector with tubes instead of a T, which allows the dispersion of the cells in the lysis solution. Accordingly, the mechanical stress in the fluids passing through the tubes is greatly reduced compared to that of the fluids being agitated, for example, by shovels in tanks. The initial efficiency of the mixing results in an even greater efficiency in the following seconds, since this fluid still does not have the viscoelastic properties and the mixing performed by the small diameter tube is very effective. Conversely, when a T-tube is used for mixing, the initial mixing is only moderate as the fluid rapidly becomes viscoelastic, which results in considerable problems as it flows into the tube. This partial mixing results in the lysis of only a part of the cells and, therefore, can only release a part of the plasmids before neutralization. Lysis can be divided into two phases during lysis, Phase I and Phase I I. These two phases correspond to I) lysis of the cells and I I) denaturation of the nucleic acids, which produces a significant change in the rheological behavior that results in a viscoelastic fluid. The adjustment of the diameters of the tubes makes it possible to achieve the purpose of these two phases. In a tube with a small diameter (B1 a), mixing increases. This is the configuration used for Phase I. In a tube with a large diameter (B1 b), the mixing (and therefore the shear stress) is reduced. This is the configuration used for Phase I I. The preferred mixer used is the so-called M 1, as shown in Figure 2, but any T-shaped device to provide the dispersion of the cell suspension according to the present invention. One way to perform the lysis with this mixer is to inject solution 1 countercurrently into the alkaline lysis solution through one or more orifices with small diameter in order to obtain an effective dispersion. The diameters of these holes may be from about 0.5 mm to 2 mm, and preferably about 1 mm in the configuration shown. The mixture then leaves the mixer M 1 to pass through a tube with a small diameter (Figure 1) for a short period of time (about 2.5 sec). The combination of diameter and flow time can be easily calculated to maintain a turbulent flow. Examples of variations of these parameters are given in Table 1. All references to the diameter of the tube provide the inner diameter of the tube, not the outer diameter, which includes the thickness of the walls of the tube. This short residence time in the tube allows a very rapid homogenization of solutions 1 and 2. Assuming that solution 1 and solution 2 are still Newtonian fluids during Phase I, the flow mode is turbulent during the homogenization phase. At the outlet of this tube, solutions 1 and 2 are homogenized and the lysis of the cells in suspension begins. The homogenized mixture then passes through a second tube (B1 b) with a much larger diameter (Figure 1), in which the lysis of the cells and the formation of the fluid takes place. viscoelastic During this phase, mixing can be minimized and the solution allowed to "stand still" to limit turbulence as much as possible in order to minimize any shear stress that would fragment the gDNA. A contact time of about 1 to 3 min, about 2 min, and preferably 1 min 20 sec may be sufficient to complete the cell lysis and to denature the nucleic acids. During the denaturing phase, the flow mode can be laminar, which promotes the slow diffusion of SDS and sodium hydroxide to the cellular components. The lysate obtained in this way and the neutralization solution 3 can then be mixed with a mixer Y called M2. In one embodiment of the present invention, the internal diameter of the mixer Y is about 4 to 15 mm, or about 6 to 10 mm, and may be about 6mm or about 10mm. The tube with a small diameter (for example, a tube of approximately 6 mm) is placed in the outlet of the mixer Y to allow rapid (<1 sec) and efficient mixing of the lysate with solution 3. The neutralized solution is collected in a collection tank. During neutralization, the rapid decrease in pH induces flocculation (ie the formation of lumps or masses). On the other hand, the partially denatured plasmid is renatured very quickly and remains in solution. The flocs settle gradually in the collection tank, dragging most of the contant nantes The schematic drawing of Figure 1 shows an embodiment of the continuous lysis system (CL). Continuous lysis can be used alone or with additional processes. With this process any type of cell, ie, prokaryotic or eukaryotic, can be used for any purpose related to lysis, such as the release of the desired plasmid DNA from target cells to be further purified. This continuous alkaline lysis process can be carried out in cells collected from a fermentation that have grown to a cell biomass that has not reached the stationary phase and that are therefore in the exponential growth phase (2-1 0 g weight dry / liter). The continuous alkaline lysis step can also be performed on cells harvested from a fermentation that has grown to a large biomass of cells and which are not exponentially growing but have reached the stationary phase, with a cell concentration of about 10-100%. 200 g of dry weight per liter, and preferably 12-60 g of dry weight per liter. The plasmid DNAs can be purified using different methods before being formulated into the stable storage composition according to the present invention. Indeed, plasmid DNA preparations, which are produced from bacterial preparations frequently contain a mixture of relaxed and supercoiled plasmid DNA. Methods for purifying plasmid DNA are well known in the art. Generally, methods to isolate and purify DNA plasmid from bacterial fermentations consist in breaking the host bacterial cells containing the plasmid, as described above, and neutralizing the lysate with neutralization with acetate to produce the precipitation of the genomic DNA and proteins of the host cell, which are eliminated, for example, by centrifugation. The liquid phase contains the plasmid DNA that is precipitated with alcohol and is subjected to isopycnic centrifugation using CsCI in the presence of ethidium bromide to separate the different forms of plasmid DNA, that is, supercoiled, nicked circle, and linearized. Additional extraction with butanol is required to remove residual ethidium bromide followed by precipitation of the DNA using alcohol. Additional purification steps are used to remove proteins from the host cell. These methods are generally suitable for the preparation of plasmids at small or laboratory scales. Alternative methods include, for example, size exclusion chromatography, hydroxyapatite chromatography and different chromatographic methods based on reverse phase or anion exchange. These alternatives may be suitable for producing small amounts of the research material on a laboratory scale but may not be easily scalable to produce large amounts of plasmid DNA. For example, the methods available for separating plasmid DNA use ion exchange chromatography (Duarte et al., Journal of Chromatography A, 606 (1998), 31 -45) or size exclusion chromatography (Prazeres, DM, Biotechnology Techniques Vol. 1, No. 6, June 1997, p 417-420), coupled with the use of additives such as polyethylene glycol (PEG) ), detergents, and other components such as cobalt hexamine, spermidine, and polyvinylpyrrolidone (PVP). Alternative methods known for the separation of supercoiled and relaxed plasmid DNA forms utilize resins and solvents, such as acetonitrile, ethanol and other components such as triethylamine and tetrabutyl ammonium phosphate during processing. In the case where nucleic acids or plasmid DNA are introduced into humans or animals in a therapeutic context, a highly purified pharmaceutical-grade plasmid DNA is required, since said purified nucleic acid must comply with the drug quality standards for to safety, power and efficiency. The removal of contaminating endotoxins may be required particularly when the plasmid DNA has been purified from gram-negative bacterial sources having high levels of endotoxins. These endotoxins are generally lipopolysaccharides, or fragments thereof, which are components of the outer membrane of Gram-negative bacteria and which are present in the DNA preparation of the host cells and in the membranes or macromolecules of the host cells. They can produce inflammatory reactions, such as fever or sepsis in the host that receives the plasmid DNA. Therefore, the elimination of endotoxins can be a crucial and necessary step in the Purification of plasmid DNA for therapeutic or prophylactic use. The elimination of endotoxins from plasmid DNA solutions mainly utilizes the negatively charged structure of endotoxins. However, the plasmid DNA is also negatively charged and therefore the separation is usually achieved with anion exchange resins which bind both molecules and, under certain conditions, preferably elute plasmid DNA while binding endotoxins. In addition to preparing nucleic acids without contaminating endotoxins, which if administered to a patient could induce a toxic response, it may be desirable to produce highly pure nucleic acid that does not contain toxic chemical compounds, mutagens, organic solvents or other reagents that endanger safety or effectiveness of the resulting nucleic acid. Before formulating the plasmid DNA in a stable aqueous solution for long-term storage according to the present invention, the plasmid DNA is preferably purified by a combination of chromatographic steps, which make it possible to obtain a plasmid DNA preparation containing low levels, is say, parts per million (ppm) of chromosomal DNA, RNA, proteins and contaminating endotoxins and that mainly contains the closed circular form of plasmid DNA. More preferably, the purification methods described in the international publication WO95 / 026331 and in the international patent application No. PCT / EP2005 / 005213, are used for preparing plasmid DNA for research applications and in plasmid-based therapy, such as gene therapy and DNA vaccines. Purification methods comprise the use of triple helix affinity chromatography, which is preceded by or followed by at least one additional chromatographic technique, optionally or typically as the final purification steps or at least at or near the end of the scheme of purification of the plasmid. Triple-helical affinity chromatography is used in combination with one or more chromatographic steps, such as ion exchange chromatography, hydrophobic interaction chromatography, gel permeation or size exclusion chromatography, hydroxyapatite chromatography (type I and II), of reverse phase and affinity chromatography. Any available affinity chromatography protocol involving the separation of nucleic acids can be adapted to be used. Anion exchange chromatography or any of the other chromatographic steps or techniques used can use stationary phases, chromatographic displacement methods, simulated moving bed technology and / or columns or continuous bed systems. In addition, any one or more of the steps or techniques may utilize high performance chromatography techniques or systems. Therefore, the method preferably comprises purification steps that include triple helix affinity chromatography with an additional step of ion exchange chromatography and can also be hydrophobic interaction chromatography or gel permeation chromatography. The ion exchange step chromatography step can be ion exchange chromatography in fl uidized lepals and high resolution axial and / or radial anion exchange chromatography. The most preferred method includes a combination of steps of ion exchange chromatography, triplex affinity chromatography and hydrophobic interaction chromatography, occurring in that order. The first chromatographic step can be preceded by filtration of the lysate or other elimination of the floccule. Therefore, continuous lysis can be combined with the purification steps listed above and result in a product that contains pDNA. It can be combined, for example, with at least one of flocculin removal (such as lysate filtration, sedimentation or centrifugation), ion interpamil chromatography (such as cationic or anion exchange), triplex affinity chromatography and hydrophobic interaction chromatography. . In one embodiment, the continuous lysis is followed by anion exchange chromatography, triplex affinity chromatography and hydrophobic interaction chromatography, in that order. In another, continuous lysis is followed by lysate filtration, anion exchange chromatography, triplex affinity chromatography and hydrophobic interaction chromatography, in this order. That is, stages allow a process of plasmid manufacturing truly scalable that can produce large amounts of pDNA with unprecedented purity. The DNA and RNA as well as the host proteins are in the sub-ppm range. The method may also utilize additional steps of size exclusion chromatography (SEC), reverse phase chromatography, hydroxyapatite chromatography and / or other available chromatographic techniques, methods or systems, in combination with the steps described herein according to the present invention. request.

A flocculation can be used to provide a higher purity to the resulting pDNA product. This stage can be used to remove most of the precipitated material (floccule). A mechanism for performing the floccule removal is by a filtration step of the lysate, such as through a filter with a grid of 1 to 5 mm, and preferably 3.5 mm, followed by a depth filtration as a step of fine filtration. Other methods to achieve the elimination of the floccule are by centrifugation or sedimentation. Ion exchange chromatography can be used to provide a higher purity to the resulting pDNA product. The anion exchange can be selected depending on the properties of the contaminants and the pH of the solution. Anion exchange chromatography can be used to provide a higher purity to the resulting pDNA product. Anion exchange chromatography works by attaching negatively charged molecules (or acids) to a support that is charged positively The use of ion exchange chromatography allows to separate molecules based on their charge. Using this technique, families of molecules (acidic, basic and neutral) can easily be separated. Stepwise elution schemes can be used, eluting many contaminants in the first fractions and eluting the pDNA in the last fractions. Anion exchange is very effective in removing proteins and endotoxins from the preparation of pDNA. In the case of ion exchange chromatography, the packaging material and the method for preparing said material as well as the process for preparing, polymerizing and functionalizing anion exchange chromatography and for eluting and separating the plasmid DNA are well known in the art. present in this one. The compound to be used for the synthesis of the base materials that are used for the packaging material of the anion exchange chromatography can be any compound, if several hydrophobicity functional groups or several ion exchange groups can be introduced by a subsequent reaction to the synthesis of the base materials. Examples of monofunctional monomers include styrene, o-halomethylstyrene, m-halomethylstyrene, p-halomethylstyrene, o-haloalkylstyrene, m-haloalkylstyrene, p-haloalkylstyrene, α-methylstyrene, α-methyl-o-halomethylstyrene, α-methyl-m- halomethylstyrene, a-methyl-p-halomethylstyrene, a-methyl-o-haloalkylestyrene, a-methyl-m-haloalkylstyrene, a-methyl-p- Haloalkylstyrene, o-hydroxymethylstyrene, m-hydroxymethylstyrene, p-hydroxymethylstyrene, o -hydroxy to quil styrene, m -hydroxy to quil styrene, p -hydroxy to quil styrene, amyl I -o-hydroxymethylstyrene, a -methyl-m-hydroxy methyl styrene, a-methyl-p-hydroxymethyl I styrene, a-methyl-o-hydroxyalkylstyrene, a-methyl-m-hydroxyalkylstyrene, a-methyl-p-hydroxyalkylstyrene, glycidyl methacrylate, glycidyl acrylate, hydroxyethyl acrylate, hydroxymethacrylate, and vinyl acetate. The most preferred compounds are haloalkyl groups substituted on the aromatic ring, halogens such as Cl, Br, I and F and saturated straight-chain and / or branched hydrocarbons with 2 to 15 carbon atoms. Examples of polyfunctional monomers include divinylbenzene, trivinylbenzene, divinyl toluene, trivinyl toluene, vinylnaphthalene, trivinylnaphthalene, ethylene glycol dimethacrylate, ethylene glycol diacrylate, diethylene glycol dimethyl acrylate, diethylene glycol diacrylate, methylenebismethacrylamide, and methylenebisacrylamide. Several ion exchange groups can be introduced by the subsequent reaction. The preparation of the base material includes a first step in which the monofunctional monomer and the polyfunctional monomer are weighed in an appropriate proportion and the accurately weighed diluent or solvent which is used for the purpose of adjusting the pores of the formed particles is added. and the initiator of the heavy polymerization similarly in a precise manner, followed by good agitation. The mixture is then subjected to an oil-in-water type polymerization suspension in which the mixture is added in a solution The dissolved aqueous slurry of the stabilizer is accurately precipitated in advance and oil droplets of the desired size are formed by mixing with stirring and the polymerization is carried out by gradually heating the solution of the mixture. The ratio of the monofunctional monomer to the polyfunctional monomer is generally about 1 mole of monofunctional monomer and about 0.01 to 0.2 mole of the polyfunctional monomer to obtain soft particles of the base material. A polymerization initiator is not particularly restricted and commonly used azobis and / or peroxide type are used. Suspension stabilizers such as ionic surfactants, nonionic surfactants and polymers with amphipathic properties or mixtures thereof can also be used to prevent aggregation of the oil droplets with each other. The packaging material to be used for ion exchange chromatography to purify plasmid DNA preferably has a relatively large pore diameter, particularly in the range of 1,500 to 4,000 angstroms. The modification of the surface to introduce ion exchange groups to the base materials is well known in the art. Two types of eluents can be used for ion exchange chromatography. A first eluent containing a low salt concentration and a second eluent containing a high salt concentration can be used. The elution method consists of changing by stages from the first eluent to the second eluent and continuously changing the composition of the elution gradient of the first eluent to the second eluent. The buffers and salts that are generally used in these eluents can be used for ion exchange chromatography. For the first eluent containing a low salt concentration, an aqueous solution with a buffer concentration of 10 to 50 mM and a pH value of 6 to 9 is particularly preferable. For the second eluent which contains a high concentration of salt, it is particularly preferably an aqueous solution with 0.1 to 2M sodium salt added to the eluent C. Sodium chloride and sodium sulfate can be used for the sodium salts. In addition, a chelating agent for bivalent metal ions such as, for example, ethylenediaminetetraacetic acid, to inhibit the degradation of plasmids due to enzymes that degrade DNA in the Escherichia coli lysate. The concentration of chelating agent for bivalent metal ions is preferably 0.1 to 100 mM. A wide variety of commercially available anion exchange matrices are suitable for use in the present invention, including, but not limited to, those available from POROS Anion Exchange Resins, Qiagen, Toso Haas, Sterogene, Spherodex, Nucleopac, and Pharmacia. For example, the column (Poros I I Pl / M, 4.5 mm x 100) is initially equilibrated with 20 mM Bis / TRIS Propane at pH 7.5 and 0.7 M NaCl. The sample is loaded and washed with the same initial buffer. A gradient is applied from 0.5 M to 0.85 M NaCl in approximately 25 volumes of col umna and the fractions are collected. Preferred anion exchange chromatography includes Fractogel TMAE HiCap. Triple-helical affinity chromatography is described among others in the patents of US Pat. No. 6,319,672, 6,287,762 as well as in the international patent application published in WO02 / 77274 of the Applicant. Triple-stranded affinity chromatography is based on the specific hybridization of oligonucleotides and a target sequence in the double-stranded DNA. These oligonucleotides can contain the following bases: - ti midi na (T), which can form triplets with A.T doublets of double-stranded DNA (Rajagopal et al., Biochem 28 (1 989) 7859); - adenine (A), which can form triplets with A.T doublets of double-stranded DNA; - guanine (G), which can form triplets with doublets G. C of double-stranded DNA; - protonated cytosine (C +), which can form triplets with G. doublets of double-stranded DNA (Rajagopal et al., loc. cit.); - Uracil (U), which can form tri pletes with base pairs A. U or A.T. Preferably, the oligonucleotide used comprises a homopyridine sequence rich in cytosines and the specific sequence present in the DNA is a homopuri-na-homopyrimidine sequence. The presence of cytosines allows having a triple helix that is stable at acid pH in which the cytosines are protonated and destabilized at alkaline pH in which the cytosines are neutralized. The oligonucleotide and the specific sequence present in the DNA are preferably complementary to allow the formation of a triple helix. The best yields and the best selectivity can be obtained by using an oligonucleotide and a specific sequence that are totally complementary. For example, a poly (CTT) oligonucleotide and a poly (GAA) specific sequence. Preferred oligonucleotides have a sequence 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(GAGG (CTT) 7 (SEQ ID NO: 1), in which the GAGG bases do not form a triple helix but allow the oligonucleotide to be separated from the coupling arm; sequence (CTT) 7. These oligonucleotides are capable of forming a triple helix with a specific sequence containing complementary units (GAA) .The sequence in question may be, in particular, a region containing 7, 14 or 17 GAA units, such as described in the examples Another specific sequence of interest is the sequence 5'-AAGGGAGGGAGGAGAGGAA-3 '(SEQ ID NO: 2) This sequence forms a triple helix with the oligonucleotides 5'-AAGGAGAGGAGGGAGGGAA-3' (SEQ ID NO: 3) or 5'-TTGGTGTGGTGGGTGGGTT-3 '(SEQ ID NO: 4) In this case, the oligonucleotide binds in an antiparallel orientation to the polypurine chain These triple helices are stable only in the presence of Mg2 + (Vasquez et al. ., Biochemistry, 1995, 34 , 7243-7251; Beal and Dervan, Science, 1991, 251, 1360-1363). As indicated above, the specific sequence may be a sequence naturally present in the double-stranded DNA or a synthetic sequence artificially introduced into the latter. It is especially advantageous to use an oligonucleotide capable of forming a triple helix with a sequence that is naturally present in the double-stranded DNA, for example, in the origin of replication of a plasmid or in a marker gene. In this regard, it is known by sequence analysis that some regions of these DNAs, in particular at the origin of replication, could have homopurine-homopyrimidine regions. The synthesis of oligonucleotides capable of forming triple helices with these natural homopurine-homopyrimidine regions advantageously allows to apply the method of the invention to unmodified plasmids, in particular, to commercial plasmids of the pUC type, pBR322, pSV, and the like. Among the homopurin-homopyrimidine sequences naturally present in a double-stranded DNA, a sequence comprising all or part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3 '(SEQ ID NO: 5) present in the origin of replication of the plasmid Col E l of EJ coli. In this case, the oligonucleotide forming the triple helix has the sequence: 5'-GAAGGGCTTCCCTCTTTCC-3 '(SEQ ID NO: 6), and binds alternately to the two strands of the double helix, as described by Beal and Dervan (J. Am. Chem. Soc. 1992, 1 14, 4976-4982) and Jayasena and Johnston (Nucleic Acids Res. 1992, 20, 5279-5288). As well mention may be made of the sequence 5'-GAAAAAGGAAGAG-3 '(SEQ ID NO: 7) of the β-lactamase gene of plasmid pBR322 (Duval-Valentín et al., Proc. Nati. Acad. Sci. USA, 1992, 89, 504 -508). Appropriate target sequences that can form triplex structures with particular oligonucleotides have been identified in origins of replication of Col El plasmids as well as the pCOR plasmids. Plasmids pCOR are plasmids with a conditional origin of replication and are described among others in the USA 2004/142452 and the USA 2003/161844. ColE-derived plasmids contain a 12-mer homopurine sequence (5'-AGAAAAAAAGGA-3 ') (SEQ ID NO: 8) mapped 5' to the RNA-II transcript involved in the replication of the plasmid ( Lacatena et al., 1981, Nature, 294, 623). This sequence forms a stable triplex structure with the 12-mer complementary oligonucleotide 5'-TCTTTTTTTTCCT-3 '(SEQ I D NO: 9). The central part of pCOR contains a homopurine chain of 14 non-repeated bases (5'-AAGAAAAAAAAGAA-3 ') (SEQ ID NO: 10) located in the segment rich in A + T of the origin of replication and of pCOR (Levchenko et al. al., 1996, Nucleic Acids Res., 24, 1936). This sequence forms a stable triplex structure with the complementary oligonucleotide of 14-mer 5'-TTCTTTTTTTTCTT-3 '(SEQ ID NO: 11). The corresponding oligonucleotides 5'-TCTTTTTTTTCCT-3 '(SEQ ID NO: 8) and d'-TTCTTTTTTTTCTT-S "(SEQ ID NO: 1 1) efficiently and specifically locate their respective complementary sequences located at the origin of replication of Col E l ori pCOR (oriy). In fact, a single non-canonical triad (T * GC or C * AT) can result in the complete destabilization of the triplex structure. The use of an oligonucleotide capable of forming a triple helix with a sequence present in an origin of replication or in a marker gene is especially advantageous, since it makes possible, with the same oligonucleotide, the purification of any DNA containing said origin of replication or said marker gene. Therefore, it is not necessary to modify the plasmid or the double-stranded DNA in order to incorporate an artificial sequence into it. Although fully complementary sequences are preferred, it is understood, however, that some mismatches can be tolerated between the sequence of the oligonucleotide and the sequence present in the DNA, provided that they do not result in a large loss of affinity. The sequence 5'-AAAAAAGGGAATAAGGG-3 '(SEQ I D NO: 12) present in the β-lactamase gene of E ^ coli can be mentioned. In this case, the thymine that interrupts the polypurine sequence can be recognized by a guanine from the third strand, thus forming a G * TA triplet that is stable when flanked by two T * AT triplets (Kiessling et al., Biochemistry, 1992 , 31 2829-2834). According to a particular embodiment, the oligonucleotides of the invention comprise the sequence (CCT) n, the sequence (CT) n or the sequence (CTT) ", in which n is an integer between 1 and 15 inclusive. The use of sequences is especially advantageous type (CT) n or (CTT) ". The Applicant has shown, in effect, that the amount of C in the oligonucleotide influences the purification performance. In particular, as shown in Example 7, the purification performance increases when the oligonucleotide contains fewer cytosines. It is understood that the oligonucleotides of the invention can also combine units (CCT), (CT) or (CTT). The oligonucleotide used can be natural (composed of unmodified natural bases) or it can be chemically modified. In particular, the oligonucleotide can advantageously have certain chemical modifications that allow it to increase its resistance to or protection against nucleases or its affinity for the specific sequence. It is also understood that oligonucleotide means any succession of linked nucleosides that have undergone a modification of the backbone in order to make it more resistant to nucleases. Among the possible modifications, oligonucleotide phosphorothioates, which are capable of forming triple helices with DNA (Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), as well as oligonucleotides having formacetal or methylphosphonate backbones (Matteucci et al. al., J. Am. Chem. Soc, 1991, 1 13. 7767-7768). It is also possible to use oligonucleotides synthesized with nucleotide α-anomers, which also form triple helices with DNA (Le Doan et al., Nucleic Acids Res., 1987, 1_5, 7749-7760). Another modification of the skeleton is the phosphoramidate linkage. For example, mention may be made of the internucleotide linkage phosphoramidate N3 -P5 described by Gryaznov and Chen, which results in oligonucleotides forming triple helices with especially stable DNA (J. Am. Chem. Soc, 1994, 116. 3143-3144). Among other modifications of the skeleton, the use of ribonucleotides, of 2'-O-methylribose, phosphodiester, etc. can also be mentioned. (Sun and Héléne, Curr Opinion Struct. Biol., 116. 3143-3144). Finally, the phosphorus-based skeleton can be replaced by a polyamide backbone as in PNA (peptide nucleic acids), which can also form triple helices (Nielsen et al., Science, 1991, 254. 1497-1500; Kim et al. , J. Am. Chem. Soc, 1993, 115. 6477-6481), or by a guanidine-based skeleton, as in DNG (deoxyribonucleic guanidine, Proc Nati, Acad. Sci. USA, 1995, 92, 6097-6101) , or by polycationic DNA analogs, which also form triple helices. The thymine of the third strand can also be replaced by a 5-bromouracil, which increases the affinity of the oligonucleotide for the DNA (Povsic and Dervan, J. Am. Chem. Soc, 1989, 111. 3059-3061). The third strand may also contain non-natural bases, among which 7-deaza-2'-deoxyxantosine (Milligan et al., Nucleic Acids Res., 1993, 21_, 327-333), 1- (2-deoxy) can be mentioned. β-D-ribofuranosyl) -3-methyl-5-amino-1 H-pyrazolo [4,3-o '] pyrimidin-7-one (Koh and Dervan, J. Am. Chem. Soc, 1992, 114 1470-1478), 8-oxoadenine, 2-aminopurine, 2'-O-methylseudoisocitidine, or any other modification known to the person skilled in the art (for a review see Sun and Hélené, Curr Opinion Struct. Biol., 1993 , 3, 345-356). Another type of modification of the oligonucleotide has the purpose, more especially, to improve the interaction and / or affinity between the oligonucleotide and the specific sequence. In particular, a more advantageous modification according to the invention consists in methylating the cytosines of the oligonucleotide. The oligonucleotide methylated in this manner has the outstanding property of forming a stable triple helix with the specific sequence at pH ranges close to neutrality (> 5). Therefore, this makes it possible to work at higher pH values than the oligonucleotides of the prior art, that is, at pH values in which the risks of degradation of the plasmid DNA are much lower. The length of the oligonucleotide used in the method of the invention is between 5 and 30. An oligonucleotide with a length greater than 10 bases is advantageously used. The length can be adapted by one skilled in the art for each individual case to obtain the desired selectivity and stability of the interaction. The oligonucleotides according to the invention can be synthesized by any known technique. In particular, they can be prepared by nucleic acid synthesizers. Obviously any other method known to the person skilled in the art can be used. In order to allow its covalent coupling to the support, generally the oligonucleotide is functionalized. Thus, it can be modified by a thiol, amine or carboxyl terminal group in the 5 'or 3' position. In particular, the addition of a thiol, amine or group carboxyl allows, for example, coupling the oligonucleotide to a support having disulfide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions. These couplings are formed by the formation of disulfide, thioether, ester, amide or amine linkages between the oligonucleotide and the support. Any other method known to the person skilled in the art may be used, such as bifunctional coupling reagents, for example. Furthermore, in order to improve the hybridization with the coupled oligonucleotide, it may be advantageous if the oligonucleotide contains a base sequence "arm" and "spacer". The use of an arm allows, in fact, to join the oligonucleotide at a chosen distance from the support, allowing to improve its interaction conditions with the DNA. The arm advantageously consists of a linear carbon chain comprising 1 to 18 and preferably 6 or 12 groups (CH2), and an amine that allows the union to the column. The arm is linked to a phosphate of the oligonucleotide or a "spacer" composed of bases that do not interfere with hybridization. Thus, the "spacer" can comprise purine bases. As an example, the "spacer" may comprise the GAGG sequence. The arm is advantageously composed of a linear carbon chain comprising 6 or 12 carbon atoms. The affinity chromatography of triplex is very effective to eliminate RNA and genomic DNA. These can be functionalized chromatographic supports, in large quantity or pre-packaged in a column, functionalized plastic surfaces or latex beds, functionalized magnetic or others. Preferably, chromatographic supports are used. As an example, the chromatographic supports that can be used are agarose, acrylamide or dextran as well as their derivatives (such as Sephadex, Sepharose, Superose, etc.), polymers such as poly (styrene / divinylbenzene), or grafted or non-grafted silica, by example. The chromatography columns can operate in the diffusion or perfusion mode. To obtain better yields in the purification, it is especially advantageous to use, in the plasmid, a sequence containing several positions of hybridization with the oligonucleotide. The presence of several hybridization positions promotes, in effect, the interactions between said sequence and the oligonucleotide, which result in an improvement in the purification yields. Therefore, for an oligonucleotide containing n residue repeats (CCT), (CT) or (CTT), it is preferable to use a DNA sequence containing at least n complementary moieties and preferably n + 1 complementary moieties. A sequence having n + 1 complementary moieties provides two positions of hybridization with the oligonucleotide. Advantageously, the DNA sequence contains up to 1 1 hybridization positions, that is, n + 10 complementary residues. The method according to the present invention can be used to purify any type of double-stranded DNA. An example of this The latter is circular DNA, such as a plasmid, which generally has one or more genes of therapeutic importance. This plasmid can also have an origin of replication, a marker gene and the like. The method of the invention can be applied directly to a cell lysate. In this embodiment, the plasmid, amplified by transformation followed by cell culture, is purified directly after lysis of the cells. The method of the invention can also be applied to a clarified lysate, that is to say to the supernatant obtained after the neutralization and centrifugation of the cell lysate. Obviously it can also be applied to a prepurified solution by known methods. The method also makes it possible to purify linear or circular DNA having an important sequence from a mixture comprising DNA from different sequences. The method according to the invention can also be used to purify double-stranded DNA. The cell lysate may be a lysate of prokaryotic or eukaryotic cells. With respect to prokaryotic cells, E bacteria can be mentioned as examples. coli B. subtilis. S. typhi murium or Streptomyces. As regards eukaryotic cells, animal, yeast, fungal and similar cells can be mentioned and more specifically the Kluvveromyces or Saccharomvces yeasts or the COS, CHO, C127, N I H3T3 and similar cells. The method of the present invention that includes at least one The triplex affinity chromatography step can be used to provide a higher purity to the resulting pDNA product. In triplex affinity chromatography, an oligonucleotide is attached to a support, such as a chromatography resin or other matrix. The sample being purified is mixed with the bound oligonucleotide, for example by applying the sample to a chromatography column containing the oligonucleotide bound to a chromatography resin. The desired plasmid in the sample will bind to the oligonucleotide forming a triplex. The bonds between the oligonucleotide and the plasmid can be Hoogsteen bonds. This step can occur at a pH < 5, at a high salt concentration during a contact time of 20 minutes or more. A washing step can be used. Finally, the deprotonation of cytosine occurs in a neutral buffer, eluting the plasmid from the resin to which the oligonucleotide is bound. Hydrophobic interaction chromatography uses hydrophobic residues in a substrate to attract hydrophobic regions of the molecules in the sample for purification. It should be noted that these HIC supports work through a "clustering" effect; non-covalent or ionic bonds are formed or shared when these molecules are associated. Hydrophobic interaction chromatography is beneficial in that it very effectively removes open circular forms of plasmids and other contaminants, such as gDNA, RNA and endotoxins. The synthesis of the base materials for the chromatography of Hydrophobic interaction, as well as the processes for preparing, polymerizing and functionalizing hydrophobic interaction chromatography and eluting and separating plasmid DNA by this are well known in the art and are described among others in US Pat. No. 6,441,160 which is incorporated in the present memory by reference. The compound to be used for the synthesis of the base materials which are used for the packaging material of the hydrophobic interaction chromatography can be any compound, if various hydrophobicity functional groups or various ion exchange groups can be introduced by a Subsequent reaction to the synthesis of the base materials. Examples of monofunctional monomers include styrene, o-halomethylstyrene, m-halomethylstyrene, p-halomethylstyrene, o-haloalkylstyrene, m-haloalkylstyrene, p-haloalkylstyrene, α-methylstyrene, α-methyl-o-halomethylstyrene, α-methyl-m- halomethylstyrene, a-methyl-p-halomethylstyrene, a-methyl-o-haloalkylstyrene, a-methyl-m-haloalkylstyrene, a-methyl-p-haloalkylstyrene, o-hydroxymethylstyrene, m-hydroxymethylstyrene, p-hydroxymethylstyrene, o-hydroxyalkylstyrene, m-Hydroxyalkylstyrene, p-hydroxy to quil styrene, amyl I -o-hydroxymethylstyrene, a -methyl-m-hydroxymethylstyrene, a -methyl-p-hydroxymethylether, a-methyl- o-hid roxy to quil styrene, a-methym-hydroxy to quil styrene, a-methyl-p-hydroxyalkylstyrene, glycidyl methacrylate, glycidyl acrylate, hydroxyethyl acrylate, hydroxymethacrylate, and vinyl acetate. The most compounds Preferred are haloalkyl groups substituted on the aromatic ring, halogens such as Cl, Br, I and F and straight and / or branched chain saturated hydrocarbons with 2 to 15 carbon atoms. Examples of polyfunctional monomers include divinylbenzene, trivinylbenzene, divinyl toluene, trivinyl toluene, divinylnaphthalene, trivinylnaphthalene, ethylene glycol dimethacrylate, ethylene glycol diacrylate, diethylene glycol dimethacrylate, diethylene glycol diacrylate, methylenebismethacrylamide, and methylenebisacrylamide. Various functional hydrophobic groups or various ion exchange groups can be introduced by the subsequent reaction. In order to minimize the influence on the desired products that it is desired to separate due to the hydrophobicity that the base material presents itself, or to the increase or decrease in the volume of the base material by itself due to the change in salt concentration and Upon change of the pH value, the base material is preferably prepared using relatively hydrophilic monomers, such as glycidyl methacrylate, glycidyl acrylate, hydroxyethyl acrylate, hydroxymethacrylate and vinyl acetate. The preparation of the base material includes a first step in which the monofunctional monomer and the polyfunctional monomer are weighed in an appropriate proportion and the accurately weighed diluent or solvent that is used for the purpose of adjusting the pores of the particles is added. formed and the initiator of the polymerization weighed similarly in a precise manner, followed by good agitation. The mixture is then subjected to a polymerization suspension of oil-in-water type in which the mixture is added in an aqueous solution of dissolved suspension of the heavy stabilizer in a precise manner in advance and oil droplets of the desired size are formed by mixing with stirring and the polymerization is carried out by gradually heating the solution of mix. The ratio of the monofunctional monomer to the polyfunctional monomer is generally about 1 mole of monofunctional monomer and about 0.01 to 0.2 mole of the polyfunctional monomer to obtain soft particles of the base material. The proportion of the polyfunctional monomer can be increased to about 0.2 to 0.5 moles to obtain hard particles of base materials. The polyfunctional monomer can be used only to obtain even harder particles. A polymerization initiator is not particularly restricted and commonly used azobis and / or peroxide type are used. Suspension stabilizers such as ionic surfactants can also be used, nonionic surfactants and polymers with amphipathic properties or mixtures of these to prevent aggregation of the oil droplets with each other. The diameter of the particles formed is generally about 2 to 500 μm. The preferred diameter of the particles is between 2 and 30 μm, and more preferably about 2 to 10 μm. When large-scale purification of nucleic acids with high purity is sought, it is about 10 to 100 μm and when the desired product is separated from the crude stock solution it can be 100 to 500 μm, more preferably about 200 to 400 μm. To adjust the diameter of the particles, the rotational speed of the agitator can be adjusted during the polymerization. When particles with a small diameter are needed, the number of revolutions can be increased and, when large particles are desired, the number of revolutions can be lowered. Therefore, since the diluent to be used is used to adjust the pores of the particles formed, the selection of the diluent is particularly important. As a fundamental concept, for the solvent to be used for the polymerization, the adjustment is made by combining in a varied manner a solvent which is a poor solvent for the monomer with a solvent which is a good solvent for the monomer. The size of the pore diameter can be appropriately selected depending on the molecular size of the nucleic acids to be separated, although it is preferred that it be in a range of 500 to 4,000 angstroms for the packaging material for hydrophobic interaction chromatography and in a range of 1,500 to 4,000 angstroms for the packaging material for ion exchange chromatography. In hydrophobic interaction chromatography, in order to separate nucleic acids with different preferable hydrophobicity using packaging materials with different hydrophobicity, respectively, it is important to modify the surface of the base material. The hydrophobic groups can be selected from those of long or branched chain including saturated hydrocarbon groups or unsaturated hydrocarbon groups with 2 to 20 carbon atoms. The aromatic ring can also be contained in the hydrocarbon group. The hydrophobic groups may also be selected from compounds having the following formula: Mit rii wherein n = 0 to about 20 and the methylene group may have straight or branched chain, m = 0 to about 3 and the hydrocarbon group may have straight or branched chain, and A is a C = O group or an ether group, although the methylene group may be attached directly to the base material without A. The hydrophobic groups may further include an alkylene glycol ether group having 2 to 20 carbon atoms consisting of repeating units from 0 to 10, wherein the opposite end of the group functional group that reacts with the base material can be an OH group as such or it can be covered with an alkyl group with 1 to 4 carbon atoms. The hydrophobic groups described above can be used alone or as a mixture to modify the surface. Chains of alkyl groups with 6 to 20 are preferred carbon atoms for low hydrophobicity such as plasmids. Long chains of alkyl groups having 2 to 15 carbon atoms to separate compounds with high hydrophobicity such as RNA from Escherichia coli and RNA in the cells of humans and animals. Alkyl groups of 4 to 18 carbon atoms to separate compounds with a relatively low hydrophobicity such as DNA from Escherichia coli and DNA in the cells of humans and animals. After separating these compounds, the compounds may be appropriately selected to modify the surface without being confined to said exemplification. In effect, the degree of hydrophobicity of the packaging material varies depending on the concentration of salt in the medium or the concentration of salt in the eluent for adsorption. In addition, the degree of hydrophobicity of the packaging material differs depending on the amount of the group introduced into the base material. The pore diameter of the base material for hydrophobic interaction chromatography is particularly preferable to be 500 to 4,000 angstroms, but can be appropriately selected from said range depending on the molecular size of the nucleic acids to be separated. In general, because the retention of nucleic acids in the packaging material and the adsorption capacity (defined by the sample) differ depending on the pore diameter, it is preferable to use a base material with a large pore diameter for nucleic acids. with a large molecular size and a base material with a small pore diameter for nucleic acids with a small molecular size. For example, styrene base material can be reacted with hydrophobic group comprising long chain alkyl groups, using a halogen and / or carbonyl halide containing compound and a catalyst such as FeCl3, SnCl2 or AICI3, and using a Friedel reaction -Craft, it is possible to add it directly to the aromatic ring in the base material as a dehalogenated compound and / or acylated compound. In the case of the base material are particles containing a halogen group, for example, using compounds with OH contained in the functional group to be added, such as butanol, and using the Williamson reaction with an alkaline catalyst such as NaOH or KOH, it is possible to introduce the functional group via an ether link. In the case that the desired functional group to be added is an amino group-containing compound, such as hexylamine, addition using an alkaline catalyst such as NaOH or KOH and using a dehalogenic acid reaction is possible. In the case that the base material contains an OH group, on the other hand, if an epoxy group, halogen group or carbonyl halide group is introduced previously in the functional group that is to be added, it is possible to introduce the functional group via a link ether or ester. In the case where the base material contains an epoxy group, if it reacts with a compound with an OH group or amino group contained in the functional group that it is desired to add, it is possible to enter the functional group via an ether or amino link. Furthermore, in the case where the functional group that is desired to be added contains a halogen group, it is possible to add the functional group by means of an ether bond using an acid catalyst. Because the proportion of the functional group to be introduced into the base material is influenced by the hydrophobicity of the product to be separated, it can not be restricted, but, in general, the packaging material is suitable with about 0.05 to 4.0 mmoles of functional group added per 1 g of dry base material. With respect to the modification of the surface, a method for adding the functional group by a subsequent reaction to the formation of the base material or the particles is as described. The modification of the surface is carried out according to the same method, in which the base material is formed after the polymerization using monomers by adding said functional groups before the polymerization. The base material can also be porous silica gel. A method for making silica gel comprises coupling with silane using a compound such as alkyltrimethoxysilane directly in the particles manufactured according to the method described in "Latest High-Speed Liquid Chromatography", page 289 et seq., (Written by Toshio Nambara and Nobuo Ikegawa, published by Tokyo Hirokawa Bookstore in 1988). Before or after coupling the silane using a silane coupling agent containing an epoxy group, a group may be added functional according to the method mentioned above. A proportion of the functional group that is introduced from about 0.05 to 4.0 mmoles of functional group added per 1 g of dry base material is suitable. Eluents are used in the separation or purification step of hydrophobic interaction chromatography. Generally, two types of eluents are used. An eluent contains a high concentration of salt, while the second eluent contains a low salt concentration. The elution method comprises changing by stages of an eluent having a high salt concentration to an eluent having a low salt concentration and the elution gradient method which continuously changes the composition from one eluent to the other can be used. The buffers and salts generally used in hydrophobic interaction chromatography can be used. For the eluent that contains a high concentration of salt, an aqueous solution with a salt concentration of 1.0 to 4.5M and a pH value of 6 to 8 is particularly preferable. For the eluent which contains a low salt concentration, an aqueous solution with a concentration is particularly preferable salt of 0.01 to 0.5M and a pH value of 6 to 8. Generally, ammonium sulfate and sodium sulfate can be used as salts. The purification step of the plasmid DNA with hydrophobic interaction chromatography can be carried out by sequentially combining a packaging material in which introduced the functional group with a low hydrophobicity with a packaging material into which the functional group with high hydrophobicity has been introduced. Indeed, the cultivated Escherichia coli medium contains in large quantity several different components in hydrophobicity such as polysaccharides, genomic DNA, RNA, plasmid and Escherichia coli proteins. It is also known that there are differences in hydrophobicity even among nucleic acids. Proteins that are considered impurities have a higher hydrophobicity than plasmids. Many hydrophobic interaction chromatography resins are commercially available, such as Fractogel propyl, Toyopearl, Source isopropil, and any other resin having hydrophobic groups. The most preferred resins are Toyopearl polymeric medium. Toyopearl is a methacrylic polymer that incorporates a high mechanical and chemical stability. The resins are available as a series of non-functionalized resins "HW" and can be derivatized with surface chemistries for ion exchange chromatography or hydrophobic interactions. Four types of Toyopearl HIC resins that exhibit different surface chemistry and hydrophobicity levels can be used. The hydrophobicity of Toyopearl HIC resins is increased in the series: Ether, Phenyl, Butyl and Hexyl. The structures of the preferred Toyopearl HI C resins, ie, Toyopearl HW-65 with a pore diameter of 1000 angstroms are shown below: IJx oncjrl But? L G50. { H -! - 0- (~ H -CH - (. H ~ p i. Rox otunrl HH? L G50 I. - O- H-CH - (l l - i I - (l l- I.

The Toyopearl resins described above can have various particle size grades. Toyopearl 650C has a particle size of about 50 to 150 μm, preferably about 100 μm, while Toyopearl 650M has a particle size of about 40 to 90 μm, preferably about 65 μm and Toyopearl 650S has a particle size of about 20 to 50 μm , preferably about 35μm. It is well known that the size of the particles influences the resolution, that is, the resolution improves with a particle size degree from C to M to S, and therefore increases with smaller particle sizes. The most preferred Toyopearl resin used in the HIC chromatography step in the separation and purification process of the plasmid DNA according to the present invention is Toyopearl butyl-650S which is marketed by Tosoh Bioscience.

An additional diafiltration step can be performed. The commercially available standard diafiltration materials are suitable for use in this process, according to standard techniques known in the art. A preferred diafiltration method is diafiltration using an ultrafiltration membrane with a molecular weight cutoff in the range of 30,000 to 500,000, depending on the size of the plasmid. This diafiltration step allows the exchange of buffer and a concentration is performed. The eluate is concentrated 3 to 4 times by tangential flow filtration (membrane cut-off point, 30 kDa) to a target concentration of approximately 2.5 to 3.0 mg / mL and the concentrate buffer is exchanged by diafiltration to a constant volume with 10 volumes of saline solution and adjusted to the target plasmid concentration with saline. The concentration of plasmid DNA is calculated from the absorbance at 260 nm of samples of the concentrate. The plasmid DNA solution is filtered through a 0.2 μm capsule filter and divided into several aliquots, which are stored in containers in a cold room at 2-8 ° C until further processing. This yields a purified concentrate with a plasmid DNA concentration of supercoiled plasmid of about 70%, 75%, 80%, 85%, 90%, 95%, and preferably 99%. The global recovery of plasmid with this process is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 80%, with an average recovery of 60%.

Said diafiltration step is carried out according to the following conditions: buffer is used for stage a) and for stage b): i) a first diafiltration (stage a) against 12, 5 to 13, 0 volumes of 50 mM Tris / HCl, 1 50 mM NaCl, pH 7.4 (called buffer I), and ii) Perform a second diafiltration of the retentate from step a) above (step b) versus 3.0 to 3, 5 volumes of excipient did not (1 50 mM NaCl). This preferred diafiltration step according to the present invention effectively and extensively eliminates ammonium sulfate and EDTA. Also, after these diafiltration steps, an appropriate physiological concentration of NaCl (approximately 150mM) and a final concentration of Tris below 1mM (between 200μM and 1mM) are obtained. Preferably, the plasmid DNA composition that is used contains purified plasmid DNA which essentially does not contain contaminants or contains contaminants in the sub-ppm range and is therefore pharmaceutical grade DNA. The pharmaceutical grade plasmid DNA composition may comprise sub-ppm (< 0,0001%, ie, < 0.0001 mg per 1000 mg of plasmid DNA) of gDNA, RNA, &contaminating proteins The plasmid DNA composition The pharmaceutical grade can comprise less than about 0.01%, or less than 0.001%, and preferably less than 0.0001%, or preferably less than 0.00008% (<0.0008%, ie , <0.0008 mg per 100 mg of plasmid DNA) of chromosomal DNA or genomic DNA. The composition of pharmaceutical grade plasmid DNA may comprise less than about 0.01%, or less than 0.001%, and preferably less than 0.0001%, or preferably less than 0.00002% (<0.0002%, i.e., <0.0002 mg per 100 mg of plasmid DNA) of contaminating RNA. The pharmaceutical grade plasmid DNA composition may comprise a plasmid DNA preparation containing less than about 0.0001%, and most preferably less than 0.00005% (< 0.00005%, ie, <0, 00005 mg per 100 mg of plasmid DNA) of contaminating proteins of the host cell. The pharmaceutical grade plasmid DNA composition may also comprise a plasmid DNA preparation containing less than 0.1 EU / mg endotoxins. The pharmaceutical-grade plasmid DNA composition thus predominantly contains the circular form and more precisely contains more than 80%, 85%, 90%, 95%, or 99% of the closed circular form of the plasmid DNA. The pharmaceutical composition may have a detectable level of genomic DNA of the host cell of less than about 0.01% and less than about 0.001% of host cell RNA may be included in the invention. Most preferably, the pharmaceutical grade plasmid DNA composition can have less than about 0.00008% genomic DNA from the host cell and less than about 0.00002% host cell RNA and less than Approximately 0.00005% of proteins from the host cell. In fact, any combination of the purity levels indicated above can be used for any particular pharmaceutical grade plasmid DNA composition of the invention. The compositions may also comprise other pharmaceutically acceptable components, buffers, stabilizers, or compounds to improve gene transfer and particularly the transfer of plasmid DNA to a cell or organism. The plasmid DNA obtained in this manner can be formulated according to the present invention in NaCl as a saline excipient and an appropriate concentration of Tris buffer in order to maintain or control the pH value between 6.2 and 9, preferably between 6, 5 and 8, more preferably 7 and 7, 5. The plasmid DNA formulations according to the present application are particularly useful since their plasmid DNA can be surprisingly stored in a stable, non-degradable form under these conditions for a period of time. prolonged at 5 ° C and up to 25 ° C, that is, at room temperature. As indicated above, the purified plasmid DNA is present in a solution with less than or about 0.1 EU / mg endotoxin, less than or about 0.00005% contaminating proteins of the host cell, less than or about 0.00002% RNA contaminating the host cell, and less than or approximately 0.00008% of genomic DNA contaminating the host cell. A pharmaceutical grade plasmid DNA composition comprises sub-ppm (< 0.00001%) of gDNA, RNA, and contaminating proteins of the host cell. More precisely, the pharmaceutical grade plasmid DNA composition does not contain essentially detectable amounts of gDNA, RNA and contaminating proteins. In addition, the pharmaceutical grade plasmid DNA composition does not essentially contain chromosomal DNA of the detectable host bacteria and therefore comprises less than about 0.01%, or less than about 0.001%, or less than about 0.0001%, or preferably less than 0.00008% chromosomal DNA or genomic DNA. In addition, the pharmaceutical grade plasmid DNA composition contains essentially no detectable host cell RNA and more precisely comprises less than about 0.01%, or less than 0.001%, and preferably less than 0.0001%, or preferably less than 0.00002% RNA contaminants of the host cell. In addition, the pharmaceutical grade plasmid DNA composition contains substantially no detectable host cell contaminating proteins and more precisely less than about 0.0001%, and most preferably less than 0.00005% contaminating proteins of the host cell. Finally, the pharmaceutical grade plasmid DNA composition does not contain substantially contaminating endotoxins and more precisely less than 0.1 EU / mg of endotoxins The plasmid DNA is present in substantially supercoiled form and more precisely comprises approximately or more than 99% closed circular form of plasmid DNA. A sterilization step can be performed by filtration before filling the vials with the purified plasmid DNA. Purified plasmid DNA vials are also provided which can be obtained by these methods. The purification of any type of vector of different sizes can be carried out. The size range of the plasmid DNA that can be separated is from about 5 kb to about 50 kb, preferably 15 kb to 50 kb, DNA that includes a vector core of about 3 kb, a therapeutic gene and associated regulatory sequences. Therefore, a vector core useful in the invention may be capable of carrying inserts of about 10-50 kb or greater. The insert may include DNA from any organism, but preferably it will be of mammalian origin and may include, in addition to a gene encoding a therapeutic protein, regulatory sequences such as promoters, poly adenylation sequences, amplifiers, locus control regions, etc. The gene encoding a therapeutic protein may have a genomic origin and therefore contain exons and introns as reflected in its genomic organization or may come from complementary DNA. Such vectors may include, for example, a replicable vector core with a high number of replication. copies, which has a polylinker for the insertion of a therapeutic gene, a gene encoding a selectable marker, for example SupPhe tRNA, the tetracycline resistance gene and is physically small and stable. The vector core of the plasmid advantageously allows inserts of mammalian, other eukaryotic, prokaryotic or viral DNA fragments and the resulting plasmid can be purified and used in plasmid-based therapy in vivo or ex vivo. Vectors with a relatively high copy number, ie, in the range of 20-40 copies / cell to 1,000-2,000 copies / cell can be separated and purified by the method according to the present invention. For example, a vector including the origin of replication of pUC according to the method of the invention is preferred. The pUC origin of replication allows a more efficient replication of the plasmid DNA and results in a tenfold increase in the number of plasmid / cell copies, for example, to an origin of pBR322. Preferably, plasmid DNA can be separated with a conditional replication origin or pCOR as described in US 2003/1618445, by the process according to the present invention. The resulting high copy number greatly increases the ratio of plasmid DNA to chromosomal DNA, RNA, cellular proteins and co-factors, improves plasmid production and facilitates downstream purification. Accordingly, any vector (plasmid DNA) according to the invention can be used. Representative vectors include but are not limited to plasmidMk.

NV1 FGF. NV1 FGF is a plasmid encoding an acid Fibroblast Growth Factor or Fibroblast Growth Factor 1 (FGF-1), useful for treating patients with terminal arterial occlusive disease (PAOD) or peripheral arterial disease (PAD). Camerota et al. (J Vasc. Surg., 2002, 35, 5: 930-936) describe that in 51 patients with terminal non-reconstructible DBP, with pain at rest or tissue necrosis, they were injected intramuscularly single or repeated increasing doses of NV1 FGF in the thigh and ischemic calf. Several parameters were subsequently evaluated such as transcutaneous oxygen pressure, brachial indexes in the ankles and toes, pain assessment and ulcer healing. There was a significant increase in brachial indexes, pain reduction, resolution of the size of the ulcers and improved perfusion after administration of NV1 FGF. The plasmid DNA composition may further comprise at least one polymer to improve the transfer of plasmid DNA to a cell. The plasmid DNA composition may also comprise a pharmaceutically acceptable carrier or excipient. The plasmid DNA composition can be formulated for administration by injection, intravenous injection, intramuscular injection, intratumoral injection, bombardment of small particles or topical application in a tissue. The plasmid DNA of these compositions is substantially in the form of closed circular supercoiled DNA.

The host cells useful according to the invention can be any bacterial strain, ie, both Gram-positive and Gram-negative strains, such as E. coli and Salmonella Typhimurium or Bacillus which is capable of maintaining a high copy number of the plasmids described above; for example 20-200 copies. Host strains of E. coli according to the invention can be used and include HB1 O1, DH1, and DH5aF, XAC-1 and XAC-1 pir 1 16, TEX2, and TEX2pir42 (WO04 / 033664). Strains containing the plasmid F or derivatives of plasmid F (eg, JM 109) are not generally preferred because the plasmid F can be co-purified with the therapeutic plasmid product.

Examples General techniques of cloning and molecular biology Traditional methods of molecular biology, such as digestion with restriction enzymes, gel electrophoresis, transformation in E. coli. Precipitation of nucleic acids and the like are described in the literature (Maniatis et al., 1., EF Fritsch, and J. Sambrook, 1989. Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, Ausubel F. M., R. Brent, RE Kinston, DD Moore, JA Smith, JG Seidman and K. Struhl, 1987. Current protocols in molecular biology 1987-1988, John Willey and Sons, New York. ). The nucleotide sequences were determined by the chain termination method according to the previously published protocol (Ausubel et al., 1987). Restriction enzymes were obtained from New England Biolabs, Beverly, MA (Biolabs). To carry out the ligations, the DNA fragments are incubated in a buffer comprising 50 mM Tris-HCl pH 7.4, 10 mM MgCl2 l 10 mM DTT, 2 mM ATP in the presence of the phage T4 DNA ligase (Biolabs ). The oligonucleotides were synthesized using phosphoramidite chemistry with the phosphoramidites protected at the ß position by a cyanoethyl group (Sinha, ND, J. Biernat, J. McManus and H. Koster, 1984. Polymer support oligonucleotide synthesis, XVII I: Use of β -cyanoethyl-N, N-dialkylamino- / N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolate of the final product Nucí.Aids Res., 12, 4539-4557: Giles, JW 1985. Advances in Automated DNA synthesis, Am. Biotechnol., Nov. / Dec.) with an automatic Biosearch 8600 DNA synthesizer using the manufacturer's recommendations. The ligated DNAs or DNAs to be tested for their transformation efficiency are used to transform the following competent strain: E ^ coU DH5a. F / endAI. hsdR17, supE44. thi-1 recAl. ayrA96. re1A1. ? 1 acZYA-arqF) U 169. deoR. FdOdlac (lacZ? M 15)] (for any Col E1 plasmid); or E ^ coli XAC-pir (for any plasmid derived from pCor).

The mi or DNA preparations are made according to the protocol of Klein et al. , 1 980. The LB culture medium is used to grow E. coli strains (M aniatis et al., 1982). The strains are incubated at 37 ° C. Bacteria are plated on plates of LB medium supplemented with suitable anti biotics. Example 1 The adjustment of the diameters to the flow rates used is done according to the calculation of Reynolds numbers in coils of the continuous lysis system. Because the following analysis assumes that the behavior of the fluids is Newtonian, the figures shown below are only fully valid in B 1 a and partly in B2. The value of the Reynolds number allows the person skilled in the art to specify the type of behavior that occurs. Here, we will only deal with the fl ow of fluid in a tube (hydraulic engineering). 1) Non-Newtonian Fluid The two types of non-Newtonian fluids that appear most commonly in industry are Bingham and Ostwald de Waele. In this case, the Reynolds number (Re) is calculated as follows: ReN is the generalized Reynolds number ReN = (1 / (2 n-3)) x (n / 3n + 1) nx ((px Dn xw 2 -n) / m) (1) D: internal diameter of the cross section (m) p: volumetric mass of the fluid (kg / m3) w: spatial velocity of the fluid (m / s) n: index of the behavior of the fluid (without dimension) m: coefficient of the Fluid consistency (dyn. s "/ cm2) And n and m are empirically determined (study of rheological behavior) 2) Newtonian Fluid As in the first section, in Equation (1) we have: Re = f (internal diameter) , μ, p, yu) because n and m are functions of μ Re = (ux D xp) / μ (2) p: Volumetric flow rate of the fluid (kg / m3) μ: Viscosity of the fluid (Pa.s, and 1 mPa. s = 1 cP) D: Internal diameter of the cross section (m) u: average spatial velocity of the fluid (m / s) Equation (1), for n = 1, is reduced to Equation (2) With Q = flow velocity (m3 / h) and S = surface area of the cross section (m2) and if μ is given in cP, then: Re = (4 x (Q / 3,600) xp) / ( (μ / 1 .000) xpx D) (3) In a circular duct, the flow is lam Inar for a number of Reynolds below 2,500, and it is hydraulically soft turbulent fl ow for a Reynolds number between 2,000 and 500,000.

The limit is deliberately vague between 2,000 and 2,500, when both types of behavior are used to determine what can be occur and the choice is made a posteriori. 3) Calculations Since n and m are generally not known, the following approximations have been used to estimate trends: Newtonian fluid (in all cross sections) p = 1,000 kg / m3 (for all fluids) μ = 5 cP in B1 and 40 cP in B1 b (our data) 2.5 cP in B2 (our data) The following calculations were made using Equation (3) for two standard tube configurations tested called configuration 1 and configuration 2 (without tube B1 b): Table 2 In these two configurations, the flows are laminar in all stages and the solutions do not mix properly in each other. For other tube configurations (without tube B1 b), we have: Table 3 Similar calculations were performed using Equation (3) for several tube configurations with tubes B1 a and B1 b present: Table 4 Clearly, predefined Reynolds values can be obtained by adjusting the diameters of the tubes and the flow rates. One skilled in the art can envisage many combinations of diameters and lengths for B2 or for the two sections of B1 (B1 a and B1 b). For example, the first section of B1 can be reduced from 6 mm to 3 mm in order to reduce the length and increase the agitation. Additionally, n and m can be determined from the study of the rheological behavior of the fluids and used to determine the correct characteristics of the tubes. In addition to the effectiveness of agitation, also the duration of the stirring can be considered, which in some embodiments of the present invention is obtained by adjusting the length of the coils. The diameter of the tubes or the velocity of the fluid do not seem to dominate in Equation (1) for a non-Newtonian fluid (data not shown). In other words, it does not seem to be more effective to alter the diameter than to alter the flow velocity if equation (1) is used for the calculations in B1 b and in B2. When high flow rates are desired, the diameter can be varied along with the flow rate. These principles can be used as a basis to limit agitation as much as possible in B1 b and B2 in order to avoid fragmentation of gDNA. During lysis, agitation can be quite vigorous as long as the gDNA is not denatured. The diameter reduction at the beginning of B1 it makes possible the increase of the agitation (Re increased) in order to sufficiently mix the solution 2 with the cells. On the other hand, when the cells are lysed, the agitation and frictional forces against the wall can be reduced to avoid fragmentation of the nucleic acids. The increase in diameter makes it possible to reduce agitation (decreased Re) and friction (lower speed). M 1: mixed fluids. B1 a: precise tuning of the mixture at the beginning of the lysis: convection phenomenon (macromixing). B1 b: let denaturation occur plus diffusion phenomenon (micromixing). It is assumed that the generalized Reynolds number has the same meaning for a non-Newtonian fluid that the classical Reynolds number has for a Newtonian fluid. In particular, it is assumed that the limit for the laminar regime in a duct with a circular cross-section is ReN < 2,300. The neutralization is done in B2. High flow rates tend to increase the fragmentation of genomic DNA by producing too vigorous agitation and wall friction forces (mechanical stress). The use of a tube with a large diameter makes it possible to reduce the agitation (Re) and the friction forces (speed). We put here a tube with a small diameter (6 mm) to avoid insufficient agitation. Our observations show that it is better to have only a tube with a small diameter for B2, in order to agitate "violently and quickly" the neutralized lysate. Example 2 The CL system can be divided into 5 stages. In a particular embodiment, the configuration is as follows: 1) Mixing: cells (in solution 1) + solution 2 (M 1 + 3 m of 6 mm tube). Start of cell lysis with S DS, there is no risk of DNA fragmentation as long as it does not denature. 2) End of lgGNA lysis and denaturation (1 3 m of 16 mm tube). 3) Mixing: Lisado + dissolution 3 (M2 + 3 m of 6 mm tube). 4) Collection of the neutralized lysate at 4 ° C. 5) Sedimentation of flocs and large fragments of gDNA overnight at 4 ° C. The following conditions can be used to carry out continuous lysis: Solution 1: 10 mM EDTA, glucose (Glc) 9 g / l and 25 mM Tris HCl, pH 7.2. Solution 2: SDS 1% and NaOH 0.2 N. Dissolution 3: Acetic acid 2 M and potassium acetate 3M. Flow rate 60 l / h: Dissolution 1 and dissolution 2 Flow rate 90 l / h: Dissolution 3 - Cells adjusted to 38.5 g / l with solution 1.

The cells in solution 1 pass through 3 nozzles that disperse them in solution 2, which arrives from the opposite direction. The mixer M 1 has a geometry that makes it possible to optimize the mixing of the two fluids (see Figure 2, schematic drawing of the mixer). The first section of the tube after the mixer M 1 is B1 a and the next section is B1 b. B1 a: 3 m in length, 6 mm in diameter, 2.5 sec in residence time B1 b: 13 m in length, 16 mm in diameter, 77 sec in residence time The process of the present invention provides an advantage in terms of effectiveness, summarized as: dispersion, brief violent mixing, and soft mixing by diffusion. Using the process of the present invention, the number of lysed cells is increased and therefore the amount of plasmid DNA recovered is increased. The idea of diffusion is especially important because of the difficulty of mixing these fluids due to their properties, in particular the viscoelasticity. The process of the present invention makes it possible to limit the shear stress and therefore limit the fragmentation of the gDNA, which facilitates its elimination during the subsequent chromatographic purification.

The problem is mixing with solution 3, which can be cooled down to 4 ° C. In one embodiment, the process of the invention uses: M2 mixer, which is a Y with an internal diameter of approximately 10 mm The section of the tube B2 located after the mixer M2. B2: 2 m of 6 mm tube; residence time: 1 sec Table 5 below provides the results obtained in comparative tests to show the advantages of our continuous lysis process compared to discontinuous lysis. Table 5 Proportion Amount of plasmid gDNA / pDNA in the extracted one per g cell lysate (mg / g) Discontinuous lysis 16.9 1, 4 Continuous lysis with 1, 6 1, 9 CL system described in example 1 Example 3 The column used is a 1 ml HiTrap column activated with N HS (N-hydroxysuccinimide, Pharmacia) connected to a peristaltic pump (<1 ml / min.exit.The specific oligonucleotide used has an NH2 group at the 5 end. and its sequence is as follows: 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(S EQ ID NO: 1) The buffers used in this example are as follows: Coupling buffer: 0.2 M NaHCO3 l 0.5 M NaCl, pH 8, 3.

Buffer A: 0.5 M ethanolamine, 0.5 M NaCl, pH 8, 3. Buffer B: 0.1 M acetate, 0.5 M NaCl, pH 4. The column is washed with 6 ml of 1 mM HCl, and the oligonucleotide diluted in the coupling buffer (50 nmol in 1 ml) is applied to the column and left for 30 minutes at room temperature. The column is washed three times successively with 6 ml of buffer A and then with 6 ml of buffer B. The oligonucleotide is thus bound covalently to the cell via a CONH connection. The column is stored at 4 ° C in PBS, 0.1% NaN3, and can be used at least four times. The following two oligonucleotides were synthesized: oligonucleotide 4817: 5'-GATCCGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGA AGAA GAAGAAGG-3 '(SEQ ID NO: 1 3) and oligonucleotide 481 8 5'-AATTCCTTCTT CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCG-3"(SEQ ID NO: 14) These oligonucleotides, when hybridized and cloned into a plasmid, introduce a homopuri sequence na-homopyrimidine (GAA) 1 7 (S EQ ID NO: 15) into the corresponding plasmid, as has been described above The sequence corresponding to these two hybridized oligonucleotides was cloned into the multiple cloning site of the plasmid pBKS + (Stratagene Cloning System, La Jolla CA), which carries a resistance gene to ampicillin. For this purpose, the oligonucleotides are hybridized in the following manner: one μg of these two oligonucleotides are put together in 40 ml of a final buffer comprising 50 mM Tris-HCl pH 7.4, 10 mM MgCl2. This mixture is heated to 95 ° C and set at room temperature so that the temperature decreases slowly. Ten ng of the mixture of hybridized oligonucleotides are ligated with 200 ng of plasmid pBKS + (Stratagene Cloning System, La Jolla CA) digested with BamHl and EcoRI in 30 μl final. After ligation, an aliquot is transformed into DH5a. The transformation mixtures are plated in medium L supplemented with ampicillin (50 mg / l) and X-gal (20 mg / l). The recombinant clones must show an absence of blue coloration in this medium, unlike the parental plasmid (pBKS +) that allows the complementation of the fragment? of β-galactosidase from E. coli. After minipreparation of the plasmid DNA from 6 clones, all showed the disappearance of the Pstl site located between the EcoRI and BamHl sites of pBKS +, and an increase in the molecular weight of the Pvull band of 448-bp containing the site of multiple cloning A clone is selected and the corresponding plasmid is designated? XL2563. The cloned sequence is verified by sequencing using primer -20 (5'-TGACCGGCAGCAAAATG-3 ') (SEQ ID NO: 16)) (Viera J. and J. Messing, 1982. The pUC plasmids, an M 13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers Gene, 19, 259-268) for the plasmid pBKS + (Stratagene Cloni ng System, La Jolla CA). Plasmid pXL2563 is purified according to the Wizard M egaprep kit (Promega Corp. Madison, Wl) according to the supplier's recommendations. This preparation of plasmid DNA is used in the examples described below. Plasmid pXL2563 is purified in the HiTrap col umn coupled to the oligonucleotide, described in 1 .1. , from a solution that also contains the plasmid pBKS +. The buffers used in this purification are as follows: Buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5 to 5. Buffer E: 1 M Tris-HCl, pH 9, 0.5 mM EDTA. The column is washed with 6 ml of buffer F, and the plasmids (20 μg of pXL2563 and 20 μg of pBKS + in 400 μl of buffer F) are applied to the col umn and incubated for 2 hours at room temperature. The column is washed with 10 ml of buffer F and the elution is carried out with buffer E. The plasmids are detected after electrophoresis in 1% agarose gel and staining with ethidium bromide. The proportion of the plasmids in the solution is estimated by determining their transforming activity in E. coli. Starting with a mixture containing 30% pXL2563 and 70% of pBKS +, a solution containing 1 00% of pXL2563 is recovered at the exit of the column. The purity, estimated by the DO ratio at 260 and 280 nm, increases from 1.9 to 2.5, which indicates that contaminating proteins have been eliminated by this method.

Example 4 Coupling of the oligonucleotide (5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(SEQ ID NO: 1)) to the column is performed as described in Example 3. For coupling, the oligonucleotide is modified at the 5'-end with an ami group not attached to the phosphate of the spacer by an arm containing 6 carbon atoms (Modified oligonucleotide Eurogentec SA, Belgium). Plasmid pXL2563 is purified using the Wizard Megaprep kit (Promega Corp. Madison, Wl) according to the supplier's recommendations. The buffers used in this example are as follows: Buffer F: 0-2 M NaCl, 0.2 M acetate, pH 4.5 to 5. Buffer E: 1 M Tris-HCl pH 9, 0.5 mM EDTA. The column is washed with 6 ml of buffer F, and 1 00 μg of plasmid pXL2563 diluted in 400 μl of buffer F are applied to the column and incubated for 2 hours at room temperature. The column is washed with 10 ml of buffer F and the solution is carried out with buffer E. The plasmid is quantified by determining the optical density at 260 nm. In this example, the union is carried out in a buffer whose molarity with respect to NaCl varies from 0 to 2 M (buffer F). The purification performance decreases as the molarity of NaCl decreases. The pH of the binding buffer can vary from 4.5 to 5, with the purification yield greater than 4.5. It is also possible to use another basic pH elution buffer: the elution is carried out then with a buffer comprising 50 mM borate, pH 9, 0.5 mM EDTA. The coupling of the oligonucleotide (5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(SEQ ID NO: 1)) to the column is performed as described in Example 3. The plasmid pXL2563 is purified using the Wizard Megaprep kit (Promega Corp. Madison, Wl ) according to the supplier's recommendations. The buffers used in this example are as follows: Buffer F: 0.1 M NaCl, 0.2 M acetate, pH 5. Buffer E: 1 M Tris-HCl pH 9, 0.5 mM EDTA. The column is washed with 6 ml of buffer F, and 100 μg of plasmid pXL2563 diluted in 400 μl of buffer F are applied to the column and incubated for one hour at room temperature. The column is washed with 10 ml of buffer F and the elution is carried out with buffer E. The content of genomic or chromosomal DNA of E coli present in the plasmid samples before and after passing through the column with the oligonucleotide. This genomic DNA is quantified by PCR using primers in the qalK gene of E. coli. According to the following protocol: The sequence of these primers is described by Debouck et al. (Nucleic Acids Res. 1985, 13 841-1853): 5'-CCG AAT TCT GGG GAC CA AGC AGT TTC-3 '(SEQI DNO: 17) and 5'-CCA AGC TTC ACT GTT CAC GAC GGG TGT-3' (SEQI DNO: 18). The reaction mixture comprises, in 25 μl of PCR buffer (Promega France, Charbonnieres): 1.5 mM MgCl 2; 0.2 mM dXTP (Pharmacia, Orsay); 0.5 μM of primer; 20 U / ml Taq polymerase (Promega). The reaction is carried out according to the sequence: - 5 min at 95 ° C - 30 cycles of 10 sec at 95 ° C 30 sec at 60 ° C 1 min at 78 ° C - 10 min at 78 ° C. The amplified DNA fragment 124 base pairs in length is separated by 3% agarose gel electrophoresis in the presence of SybrGreen I (Molecular Probes, Eugene, USA), and quantified by reference with a series of Ultrapur genomic DNA. of strain B of E. coli (Sigma, ref D4889). Example 5 This example describes the purification of plasmid DNA from a lysed rinse of a bacterial culture, in the so-called "miniprep" scale: 1.5 ml of an overnight culture of DH5a strains containing the plasmid pXL2563 are centrifuged. and the pellet is resuspended in 100 μl of 50 mM glucose, 25 mM Tris-HCl, pH 8, 10 mM EDTA. Add 200 μl of 0.2 M NaOH, 1% SDS invert the tubes to mix, add 150 μl of 3 M potassium acetate pH 5 and the tubes are inverted to mix. After centrifugation, the supernatant is recovered and loaded onto the column of the obtained oligonucleotide as described in Example 1. The binding, washing and elution are identical to those described in Example 3. About 1 μg of plasmid is recovered from 1, 5 ml of culture. The plasmid obtained, analyzed by agarose gel electrophoresis and staining with ethidium bromide, forms a single band of "supercoiled" circular DNA. No trace of high molecular weight (chromosomal) DNA or RNA is detected in the purified plasmid by this method. Example ß This example describes a piasmidic DNA purification experiment performed under the same conditions as in Example 5, starting from 20 ml of bacterial culture of DH5a strains containing the plasmid pXL2563. The cell pellet is resuspended in 1.5 ml of 50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA. The lysis is performed with 2 ml of 0.2 M NaOH, 1% SDS, and neutralization with 1.5 ml of 3 M potassium acetate, pH 5. The DNA is precipitated with 3 ml of 2-propanol, and the The pellet is resuspended in 0.5 ml of 0.2 M sodium acetate, pH 5.0, 0.1 M NaCl and loaded onto the oligonucleotide column obtained as described in the previous Example. The binding, washing of the column and elution are carried out as described in the previous Example, except for the washing buffer whose molarity with respect to NaCl is 0.1 M. The plasmid obtained, analyzed by agarose gel electrophoresis and staining with ethidium bromide, forms a single band of "supercoiled" circular DNA. No trace of high molecular weight (chromosomal) DNA or RNA is detected in the purified plasmid. The digestion of the plasmid with a restriction enzyme provides a single band at the expected molecular weight of 3 kilobases. He The plasmid contains a cassette containing the cytomegalovirus promoter, the gene encoding the luciferase and the homopurine-homopyrimidine (GAA) sequence 17 (SEQ ID NO: 15) which originates from the plasmid pXL2563. The DH1 strain (Maniatis et al., 1989) containing this plasmid is cultured in a 7 liter fermenter. A clarified lysate is prepared from 200 grams of cells: the cell pellet is resuspended in 2 liters of 25 mM Tris, pH 6.8, 50 mM glucose, 10 mM EDTA, to which 2 liters of 0.2 are added. M NaOH, 1% SDS. The lysate is neutralized by the addition of one liter of 3M potassium acetate. After diafiltration, 4 ml of this lysate is applied to a 5 ml HiTrap-NHS column coupled to the sequence oligonucleotide 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(SEQ ID NO: 1), according to the method described in Example 3. Washing and elution are carried out as described in the previous Example. Example 7 This example describes the use of an oligonucleotide having methylated cytosines. The sequence of the oligonucleotide used is as follows: 5'-GAGGMßCTTMeCT ™ CTTM? CTTM? CCTM? CTTMeCTT-3 '(SEQ ID NO: 19) This oligonucleotide has an NH2 group at the 5' terminus. M? C = 5-methylcytosine. This oligonucleotide makes it possible to purify the plasmid pXL2563 under the conditions of Example 1 with a binding buffer of pH 5 (in this way the risk of degradation of the plasmid). Example 8 In the above examples, the oligonucleotide used is modified at the 5'-terminal end with an amino group linked to the phosphate by an arm containing 6 carbon atoms: NH2- (CH2) 6. In this example, the amino group is attached to the terminal 5 'end phosphate by an arm containing 12 carbon atoms: NH2- (CH2) 12. The coupling of the oligonucleotide and the passage through the column are carried out as described in Example 3 with a buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5. This oligonucleotide allows obtaining better purification yields: a yield of 53% is obtained while with the oligonucleotide containing 6 carbon atoms, this yield is of the order of 45% under the same conditions. Example 9 Following the cloning strategy described in Example 3, two other plasmids carrying homopurine-homopyrimidine sequences are constructed: the plasmid pXL2725 containing the sequence (GGA)? 6, (SEQ ID NO: 20) and the plasmid pXL2726 which contains the sequence (GA) 25 (SEQ ID NO: 21). Plasmids pXL2725 and pXL2726, analogous to plasmid pXL2563, are constructed according to the cloning strategy described in Example 3, using the following oligonucleotide pairs: 5986: S'-GATCCyGA sGGG-S1 (SEQ ID NO: 22) 5987: d '-AATTCCCíTC sG-S' (SEQ ID NO: 23) 5981: 5'-GATCC (GGA) 17GG-3 '(SEQ ID NO: 24) 5982: AATTíCCT ^ d'-CCG-S1 (SEQ ID NO: 25) The oligonucleotide pair 5986 and 5987 is used to construct plasmid pXL2726 by cloning the oligonucleotides at BamHI and EcoRI sites of pBKS + (Stratagene Cloning System, La Jolla CA), while the oligonucleotides 5981 and 5982 are used for pXL2725 plasmid construction. The same experimental conditions are used as for the construction of plasmid pXL2563, and only pairs of oligonucleotides are changed. Similarly, the cloned sequences are verified by sequencing on the plasmids. This allows to observe that the plasmid pXL2725 has a modification with respect to the expected sequence: Instead of presenting a 17-fold repetition of the GGA sequence, it presents GGAGA (GGA)) 5 (SEQ ID NO: 26). Example 10 Oligonucleotides forming triple helices with these homopurine sequences are coupled to the HiTrap columns according to the technique described in Example 1 .1. the oligonucleotide of sequence 5'-AATGCCTCCTCCTCCTCCTCCTCCT-3 '(SEQ ID NO: 27) for plasmid purification pXL2725, and the oligonucleotide of sequence 5'-AGTGCTCTCTCTCTCTCTCTCTCTCT-3' is used (SEQ ID NO: 28) is used for purification of plasmid pXL2726. The two columns obtained in this way allow to purify the corresponding plasmids according to the technique described in Example 2, with the following buffers: Buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5. Buffer E: 1 M Tris-HCl, pH 9, 0.5 mM EDTA. The yields obtained are 23% and 31% for pXL2725 and pXL2726, respectively. Example 11 This example illustrates the influence of the length of the specific sequence present in the plasmid in the purification yields. The reporter gene used in these experiments to demonstrate the activity of the compositions of the invention is the gene encoding luciferase (Luc). Plasmid pXL2621 contains a cassette containing the promoter 661 -PB cytomegalovirus (CMV) cloned 5 'to the gene coding for luciferase, at the sites M l ul and Hindl II in pGL basic Vector vector (Promega Corp., Madíson, Wl). This plasmid is constructed using standard molecular biology techniques. Plasmids pXL2727-1 and pXL2727-2 are constructed in the following manner: Two micrograms of plasmid pXL2621 were linearized with BamHl; the enzyme is inactivated by treatment for 10 min at 65 ° C; simultaneously, oligonucleotides 6006 and 6008 hybridize as described for the construction of plasmid pXL2563. 6006: S'-GATCTÍGAA ^ CTGCAGATCT-S '(SEQ ID NO: 29) 6008: 5'-GATCAGATCTGCAG (TTC)? 7A-3 '(SEQ I D NO: 30). This hybridization mixture is cloned at the BamHI ends of plasmid pXL2621 and, after transformation into DH5a, recombinant clones are identified by restriction enzyme analysis with PstI because the oligonucleotides introduce a PstI site. Two clones were selected and the sequence of the cloned fragment nucleófidos is verified using the primer (6282, 5'-ACAGTCATAAGTGCGGCGACG-S '(SEQ ID NO: 31)) as primer sequencing reaction (Viera J. and J. Messing , 1982). The pUC plasmids an M 13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. (Gene 19: 259-268). The first clone (pXL2727-1) contains the sequence GAA repeated 10 times. The second (pXL2727-2) contains the sequence 5'-GAAGAAGAG (GAA) 7GGAAGAGAA-3 '(SEQ ID NO: 32). A column such as that described in Example 3 is used and is coupled to the oligonucleotide 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(SEQ ID NO: 1). Plasmid pXL2727-1 has 14 repeats of the GAA sequence. The oligonucleotide described above, which contains only 7 repeats of the corresponding CTT hybridization sequence, can thus hybridize to the plasmid in 8 different positions. The plasmid pXL2727-2, on the other hand, has a hybridization sequence (GAA) 7 (SEQ ID NO: 36) of the same length as that of the oligonucleotide bound to the column. This oligonucleotide can thus hybridize only in one position in pXL2727-2.

The experiment is identical to that described in Example 4, with the following buffers: Buffer F: 2 M NaCl, 0.2 M acetate, pH 4, 5. Buffer E: 1 M Tris-HCl, pH 9, 5 mM E DTA. The yield of the purification is 29% with the plasmid pXL2727-1 and 19% with pXL2727-2. The cells used are NI H 3T3 cells, inoculated the day before the experiment in 24-well culture plates with 50,000 cells / well. The plasmid is diluted in 1 50 mM NaCl and mixed with the lipofectant RPR1 1 5335. A ratio of positive charges of lipofectant / DNA negative charges equal to 6. The mixture is stirred, left for 10 minutes at Room temperature is diluted in medium if n fetal calf serum and added to the cells in the proportion of 1 μg of DNA per culture well. After two hours at 37 ° C, 1 0% volume / volume of fetal calf serum is added and the cells are incubated for 48 hours at 37 ° C in the presence of 5% CO2. The cells are washed twice with PBS and the luciferase activity is determined according to the protocol described (Promega kit, Promega Corp. Madison, Wl) in a Lumat LB9501 luminometer (EG and G Berthold, Evry). Plasmid pXL2727-1, purified as described in Example 8.2, yields transfection twice as high as those obtained with the same purified plasmid using the Wizard Megaprep kit (Promega Corp. Madison, Wl). Example 12 The following example demonstrates the purification of pCOR-derived plasmids using triple-helical affinity chromatography. It has been shown that this technology eliminates contaminating nucleic acids (particularly genomic DNA and RNA from the host cell) to levels that have not been achieved with conventional chromatographic methods. A triplex affinity gel is synthesized with Sephacryl S-1000 SF (Amersham-Pharmacia Biotech) as the chromatographic matrix. Sephacryl S-1000 is first activated with sodium m-periodate (3 mM, room temperature, 1 h) in 0.2 M sodium acetate (pH 4.7). Then, the oligonucleotide is coupled via its terminal 5'-NH2 moiety to aldehyde groups of the activated matrix by reductive amination in the presence of ascorbic acid (5 mM) as previously described for protein coupling (Hornsey et al., J Immunol. Methods, 1986, 93, 83-88). The homopyrimidine oligonucleotide used in these experiments (from Eurogentec, purified by HPLC) had a sequence that is complementary to a short homopurine sequence of 14-mer (5'-AAGAAAAAAAAGAA-3 ') (SEQ ID NO: 10) present in the origin of replication (oriy) of the pCOR plasmid (Soubrier et al., Gene Therapy, 1999, 6, 1482-1488). As discussed above, the sequence of the homopyrimidine oligonucleotide is 5'-TTCTTTTTTTTCTT-31 (SEQ ID NO: 11). The following plasmids are chromatographed: pXL3296 (pCOR without transgene, 2.0 kbp), pXL3179 (pCOR-FGF, 2.4 kbp), pXL3579 (pCOR-VEGFB, 2.5 kbp), pXL3678 (pCOR-AFP, 3.7 kbp), pXL3227 (pCOR-lacZ 5.4 kbp) and pXL3397 (pCOR-Bdeleted FVI I I, 6.6 kbp). All these plasmids are purified by two steps of anion exchange chromatography from clarified lysates obtained as described in Example 4. Plasmid pBKS + (pBluescript II KS + from Stratagene), a plasmid derived from ColEl, purified by ultracentrifugation in CsCI. All the plasmids used are in their topological state or supercoiled form (> 95%). In each plasmid DNA purification experiment, 300 μg of plasmid DNA is loaded in 6 ml of 2 M NaCl, 0.2 M potassium acetate (pH 5.0) with a flow rate of 30 cm / h in a column of affinity containing the aforementioned oligonucleotide 5'-TTCTTTTTTTTCTT-3 '(SEQ ID NO: 11). After washing the column with 5 volumes of the same buffer, the bound plasmid is eluted with 1 M Tris / HCl, 0.5 mM EDTA (pH 9.0) and quantified by UV (260 nm) and ion exchange chromatography with a Millipore Gen-Pak column (Marquet et al., BioPharm, 1995, 8, 26-37). The recoveries of the plasmids in the collected fraction are 207 μg for pXL3296, 196 μg for pXL3179, 192 μg for pXL3579, 139 μg for pXL3678, 97 μg for pXL3227, and 79 μg for pXL3397. No plasmid binding (< 3 μg) was detected when pBKS was chromatographed on this column. This indicates that the oligonucleotide d'-TTCTTTTTTTTCTT-S1 (SEQ I D NO: 1 1) forms stable triplex structures with the complementary sequence of 14-mer 5'- AAGAAAAAAAAGAA-3 '(SEQ ID NO: 10) present in pCOR (ori?), but not with the closely related sequence 5'-AGAAAAAAAGGA-3' (SEQ ID. NO: 8) present in pBKS. This indicates that the introduction of a single non-canonical triad (T * GC in this case) results in a complete destabilization of the triplex structure. As a control, no plasmid binding (< 1 μg) was observed when pXL3179 was chromatographed on a white column synthesized under strictly similar conditions but without oligonucleotide. By performing this affinity purification column under the conditions set forth herein, the level of contamination by the host genomic DNA was reduced from 2.6% to 0.07% for a preparation of pXL3296. Similarly, the level of contamination by the host DNA is reduced from 0.5% to 0.008% for a preparation of pXL3179 when the sample is chromatographed by the same affinity column. Example 13 The following example demonstrates the purification of Col E derived plasmids using triple helical affinity chromatography. It has been shown that this technology eliminates contaminating nucleic acids (particularly genomic DNA and RNA from the host cell) to levels that have not been achieved with conventional chromatographic methods. A triplex affinity gel is synthesized by coupling a oligonucleotide having the sequence 5'-TCTTTTTTTTTCCT-3 '(SEQ I D NO: 9) in Sephacryl S-1000 SF oxidized with periodate as described in the previous Example. The plasmids pXL3296 (pCOR without trene) and pBKS, a plasmid derived from Col El, are chromatographed on a 1 ml column containing the oligonucleotide 5'-TCTTTTTTTTCCT-3 '(SEQ ID NO: 9) under the conditions described in Example 9. The recoveries of the plasmids in the fraction collected are 175 μg for pBKS and < 1 μg for pXL3296. This indicates that the oligonucleotide 5'-TCTTTTTTTTCCT-3 '(SEQ ID NO: 9) forms stable triplex structures with the complementary sequence of 12-mer (5'-AGAAAAAAAGGA-3') (SEQ ID NO: 8) present in pBKS , but not with the closely related sequence of 12-mer (5'-AGAAAAAAAAGA-3 ') (SEQ ID NO: 34) present in pCOR. This indicates that the introduction of a single non-canonical triad (C * AT in this case) may result in a complete destabilization of the triplex structure. Example 14 A seed culture is produced in an Erlenmeyer flask without deflectors by the following method. The cell workbench is inoculated in an Erlenmeyer flask containing M9modG5 medium, at a seed rate of 0.2% v / v. The strain is cultivated at 220 rpm on a rotary shaker at 37 ° ± 1 ° C for approximately 18 ± 2 hours until the glucose disappears. This results in a 200 ml seed culture. It is expected that the optical density of the culture is A600 approximately 2-3.

A pre-culture is created in a first fermenter. The seed culture is trerred aseptically to a pre-fermentor containing M9modG5 medium to ensure a planting rate of 0.2% (v / v) and is cultivated with aeration and agitation. The pO2 remains above 40% saturation. The culture is collected when the glucose is consumed after 16 hours. This results in approximately 30 liters of pre-culture. It is expected that the optical density of the culture is A600 approximately 2-3. A main culture is created in a second fermenter. 30 liters of preculture are trerred aseptically to a fermentor with 270 liters of sterilized FmodG2 medium to ensure a seed ratio of approximately 10% (v / v). The culture starts in a discontinuous way to obtain biomass. The glucose feed starts once the initial sugar has been consumed after approximately 4 hours. Aeration, agitation, pO2 (40%), pH (6.9 ± 0.1), temperature (37 ± 1 ° C) and glucose feed are controlled in order to maintain a specific growth rate close to 0, 09h'1. The culture is finished after 35 hours of feeding. This results in approximately 400 liters of culture. It is expected that the optical density of the culture is A600 approximately 100. A first separation stage is carried out, which is called cellular collection. The biomass is collected with a disk centrifuge. The medium is concentrated 3 to 4 times to remove the remaining culture medium and resuspended continuously in 400 liters of buffer S 1 sterile. This results in approximately 500 liters of preconditioned biomass. DCW = 25 ± 5 g / L. A second separation step is carried out, which is called the concentration stage. After resuspension / homogenization in buffer S 1, the cells are processed again with the separator to yield a concentrated solids suspension. This results in approximately 60-80 liters of solids suspension washed and concentrated. DCW = 150 + 30 g / L; Plasmid DNA = 300 ± 60 mg / L. Then, a freezing step is performed. The suspension of solids is aseptically distributed in 20 L Flexboy ™ bags (filled to 50% capacity) and subsequently frozen at -20 ° ± 5 ° C before further processing. This results in a frozen biomass. PDNA = 300 ± 60 mg / L; supercoiled form > 95% Then, a cell thawing step is performed. The frozen bags are heated to 20 ° C and the cell suspension is diluted to 40 g / L, pH 8.0 with 100 mM Tris hydrochloride, 10 mM EDTA, 20 mM glucose and the suspension is left at 20 ± 2 ° C during 1 h with shaking before cell lysis. This results in a suspension of defrosted biomass solids. pH = 8.0 ± 0.2. Temperatures of approximately 20 ° C can be used during this stage. Then, an alkaline lysis step is performed. The cell lysis stage is comprised of the pumping of the diluted cell suspension through an in-line mixer with a 0.2 N solution. of NaOH-35 mM SDS (solution S2), followed by a step of continuous contact in a coiled tube. The continuous contact stage is to ensure complete cell lysis and denaturation of genomic DNA and proteins. The solution of the lysed cells is mixed in line with the ice-cold solution 3 (S3) of 3 M potassium acetate-2 N acetic acid, before collecting it in a stirred ice-cold vessel. The addition of solution S3 results in the precipitation of a genomic DNA, RNA, proteins and KDS. Next, a filtration of the lysate is performed. Neutralized lysate is incubated at 5 ± 3 ° C for 2 to 24 hours without agitation and filtered through a 3.5 mm mesh filter to remove most of the precipitated material (flocculation phase) followed by filtration of depth as fine filtration stage. This results in a lysed rinse, with a supercoiled plasmid concentration of more than 90%. Then, anion exchange chromatography is performed. The solution of the rinsed lysate is diluted with purified water to a target conductivity value of 50 mS / cm, filtered through a double-layer filter (3 μm-0.8 μm) and loaded onto a chromatography column. anion exchange A 300 mm column packed with 11.0 L of Fractogel® TMAE HiCap (M) resin (Merck; # 1.10316.5000) is used. The clarified lysate is loaded onto the column and elution is performed using a gradient NaCl step. Most of the contaminants bound to the column are elute with a NaCl solution at approximately 61 mS / cm, and the plasmid DNA is eluted with a NaCl solution at approximately 72 mS / cm. This results in an ion exchange chromatography eluate which has a high concentration of plasmid DNA. Then, triplex affinity chromatography is performed. The eluate of the anion exchange chromatography column is diluted with about 0.5 volumes of a 500 mM sodium acetate solution (pH 4.2) containing 4.8 M NaCl and pumped through a column of affinity chromatography of triplex equilibrated with 50 mM sodium acetate (pH 4.5) containing 2 M NaCl. The column has a diameter of 300 mm and contains 10.0 L of THAC Sephacryl ™ S-1000 gel (Amersham Biosciences, Piscataway, NJ). The column is washed with a 50 mM sodium acetate solution (pH 4.5) containing 1 M NaCl and NV1 FGF is eluted with 100 mM Tris (pH 9.0) containing 0.5 mM EDTA. This results in a triplex affinity chromatography eluate that has a high concentration of plasmid. Then, a step of hydrophobic interaction chromatography is carried out. The eluate of the affinity chromatography column is diluted with 3.6 volumes of 3.8 M ammonium sulfate in Tris (pH 8.0). After filtering through a 0.45 μm filter, the filtrate is loaded at 60 cm / h in a hydrophobic interaction column (diameter 300 mm) packed with 9.0 L of Toyopearl® Butyl-650S resin (TosoH corp). ., Grove City, OH). The column is Wash with a solution of ammonium sulfate at about 240 mS / cm and NV1 FGF is eluted with ammonium sulfate at 220 mS / cm. This results in an HIC eluate without relaxed forms. According to a preferred embodiment, an additional diafiltration step is carried out. The commercially available standard diafiltration materials are suitable for use in this process, according to standard techniques known in the art. A preferred diafiltration method is diafiltration using an ultrafiltration membrane with a molecular weight cutoff in the range of 30,000 to 500,000, depending on the size of the plasmid. This diafiltration step allows the exchange of buffer and a concentration is performed. The eluate from step 12 is concentrated 3 to 4 times by tangential flow filtration (membrane cut-off point, 30 kDa) to a target concentration of approximately 2.5 to 3.0 mg / mL and the buffer is exchanged. Concentrated by diafiltration at a constant volume with 10 volumes of saline and adjusted to the concentration of target plasmid with saline. The concentration of NV1 FGF is calculated from the absorbance at 260 nm of samples of the concentrate. The NV1 FGF solution is filtered through a 0.2 μm capsule filter and stored in containers in a cold room at 2-8 ° C until further processing. This yields a purified concentrate with a plasmid DNA concentration of supercoiled plasmid of about 70%, 75%, 80%, 85%, 90%, 95%, and preferably 99%. The global recovery of Plasmid with this process is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 80%, with an average recovery of 60%. Example 15 The method of the above Example comprising an ion exchange chromatography (AEC) step, a triple helix chromatography (THAC) step, and a hydrophobic chromatography (HIC) step results in a preparation of plasmid DNA plus purified compared to previously known methods. This new method has been compared to previously known methods and has resulted in preparations of plasmid DNA having much smaller amounts of genomic DNA, RNA, proteins and endotoxins. This is reflected in Figure 3. These experiments show that AEC, THAC and HIC provide a surprisingly high purification performance compared to some 2-step combinations for efficient removal of all contaminants. The combination of these steps provides a clear synergy in terms of efficiency in the separation of the plasmid DNA from other biological materials and contaminants, such as proteins and endotoxins, RNA and genomic DNA, as well as open circular plasmid. In addition, the synergistic combination of the steps, ie, AEC / THAC / HIC according to the present invention allows not only the obtaining of highly purified pharmaceutical-grade plasmid DNA but also of highly pure and fully supercoiled plasmid DNA compositions of more than 80 %, 85%, 90%, 95% and more than 99%.

Example 16 The method of the above Example comprising an ion exchange chromatography step, a triple helix affinity chromatography step, and a hydrophobic chromatography step for the preparation of a highly purified plasmid DNA preparation is compared to the known methods previously.

As shown in Figure 4, the method according to the present invention surprisingly results in preparations of pDNA having much smaller amounts of genomic DNA, RNA, proteins and endotoxins in the sub-ppm range. In addition, as shown in Figure 4, the process of the present invention shows a product quality obtained up to 10g. Example 17 The diafiltration step as described in Example 14 is carried out according to the following conditions: the buffers for step a and for step b were used to determine the best conditions for: iii) a first diafiltration (step a) versus 12.5 to 13.0 volumes of 50 mM Tris / HCl, 150 mM NaCl, pH 7.4 (called buffer I), and iv) Perform a second diafiltration of the retentate from step a) above (step b) against 3.0 to 3.5 volumes of saline excipient (150 mM NaCl). This alternative diafiltration step according to the present invention effectively and extensively eliminates ammonium sulfate and the EDTA. Also, subsequent to these diafiltration steps, an appropriate target NaCl concentration of approximately 150mM and a final concentration of Tris between 400μM and 1mM is obtained. Examples of plasmid DNA formulations compositions are given in Table 6 below, and Table 6 Example 18 A technical lot of plasmid DNA NV1 FGF API (active pharmaceutical ingredients) named LS06 according to Examples 13 and 17 was manufactured with the diafiltration process step described in Example 17. The eluate is first diafiltrated at approximately 2 mg. of API / mL versus approximately 1 3 volumes of buffer I and the resulting retentate was diafiltered against approximately 3 volumes of saline excipient. The final retentate was then filtered through a 0.2 μm filter and adjusted to 1 mg / mL. The final API (pH 7.24) was stored in a glass bottle at + 5 ° C until DP production. A stability study was performed on samples of LS06 stored in Duran glass bottles (API) as well as in 8 mL vials used for the manufacture of the Pharmaceutical Product. After 90 days at + 5 ° C the degree of both depurination and open circularization in all the samples was difficult to detect (<0.3%). After 90 days at + 25 ° C, the debridement and open circularization rates of the LS06 samples were also quite low. The debridement and open circularization rates calculated from this study were < 1% per month (Fig 8). This study demonstrates that the stability profile of plasmid DNA NV1 FGF is very stable in the formulation of the present invention in which the pH values are maintained at about 7.0 to 7.5. This clearly demonstrates that the plasmid DNA remains stable in a non-degraded form with low rates of depurination and plasmid nicking for a long period of time at + 25 ° C. Example 19 Batches of plasmid DNA NV1 FGF API (active pharmaceutical ingredients) designated LS04, LS04, LS06, LS07, and LT05 were manufactured according to Example 13 with the step of the diafiltration process described in Example 17. The eluate was first diafiltered. Place at approximately 2 mg API / mL against approximately 13 volumes of buffer I and the resulting retentate was diafiltered against approximately 3 volumes of saline excipient. The final retentate was filtered through a 0.2 μm filter and adjusted to 1 mg / ml to be stored in 8 ml vials. Plasmid DNAs having a NaCl concentration of about 150 mM and a final concentration of Tris between 1 mM and 2 mM are obtained. A stability study was carried out on all the aforementioned samples stored in 8 mL vials used for the manufacture of a Pharmaceutical Product. During 150 days at + 25 ° C, the pH of the plasmid DNA compositions did not change detectably, as shown in Figure 6A. The pH of LS04 dropped significantly to 6.54 (-0.27 units) after 203 days. For all lots except LS04, the rates of deburring and deboning at + 25 ° C were approximately 1.0% per month and were linearly time dependent for 140 days. The clearance rate of LS04 was significantly higher (2.7% per month) due to the significantly lower pH of this batch of API (> 0.4 units at T0). The nicking speed of LS04 was significantly lower than its clearance rate (2.4% per month). At + 5 ° C, the pH of all solutions remained stable over time and the degree of debridement and nicking was very low (below 0.5% after 200 days, Fig. 6B). This study demonstrates that the stability profile of plasmid DNA NV1 FGF is very stable over time at + 5 ° C and at + 25 ° C in the formulations of the present invention with low debridement and nicking rates. The specification must be understood in light of the indications of the references cited in the specification. The embodiments in the specification provide an illustration of the embodiments of the invention and should not be construed as limiting the scope of the invention. The person skilled in the art readily recognizes that the invention encompasses many other embodiments. All publications and patents cited in this description are incorporated by reference in their entirety. When the material incorporated by reference contradicts or is inconsistent with this specification, the specification shall be imposed on said material. The citation of any reference herein is not an admission that such references are prior art to the present invention.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so on, used in the specification, including the claims, should be understood in all cases that are modified by the term "approximately. " Accordingly, unless otherwise indicated, the numerical parameters are approximations and may vary depending on the desired desired properties desired by the present invention. Finally, and without intending to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter must be interpreted in light of the approximations of the number of significant digits and ordinary rounding. Unless otherwise indicated, the term "at least" precedes a series of elements should be understood that refers to each element of the series. Those skilled in the art will recognize, or be able to discover using only routine experimentation, many equivalents of the specific embodiments of the invention described herein. It is intended that said equivalents be encompassed by the following claims. One skilled in the art can rely on the contents of any of the references or documents referred to herein and each reference or document is incorporated herein by reference in its entirety. However, none of the references or documents referred to herein should change the meaning of any term or concept specifically defined in this document. The references and documents as well as the knowledge available to the person skilled in the art will allow changes and variations in the specific embodiments described herein. The specific examples and embodiments set forth herein should not be taken as limiting the scope or scope of the invention.

Claims (1)

1. CLAIMS 1. A stable liquid storage composition of plasmid DNA comprising a plasmid DNA and a buffer solution, wherein the buffer is present at a concentration of less than 2mM to maintain the pH of said composition between 6 and 9, and the composition comprises predominantly the supercoiled form of plasmid DNA. 2. The composition according to claim 1, wherein the plasmid DNA is stable at temperatures of about 4 ° C to 25 ° C. 3. The composition according to any one of the preceding claims, wherein the plasmid DNA is stable for several months, 1 year, 2 years, 3 years, 4 years, 5 years and up to 10 years. 4. The composition according to any one of the preceding claims, wherein the plasmid DNA is stable at about 4 ° C for several months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years and up to 20 years The composition according to any one of the preceding claims, comprising at least 80% supercoiled or closed circular plasmid DNA. The composition according to any one of the preceding claims, comprising about 80%, about 85%, about 90%, about 95%, and about or more than 99% of supercoiled form or closed circular plasmid DNA. The composition according to any one of the preceding claims, wherein the rates of depurination and nicking are less than 5% per month. The composition according to any one of the preceding claims, wherein the buffer is present in a concentration of up to 2mM. 9. The composition according to any one of the preceding claims, wherein the buffer is present in a concentration between 2 mM and 1 mM. The composition according to any one of the preceding claims, wherein the buffer is present in a concentration of less than 1 μM. eleven . The composition according to any one of the preceding claims, wherein the buffer is present in a concentration between 250 μM and 1 μM. 12. The composition according to any one of the preceding claims, wherein the buffer is present in a concentration of about 400 μM. 13. The composition according to any one of the preceding claims, wherein the buffer solution is present in a concentration such as to maintain the pH of said formulation or composition between 6.2 and 8.5 or about +/- 0, 3 of one or both of these values. 14. The composition according to any one of the previous claims, wherein the buffer solution is present in a concentration such as to maintain the pH of said formulation or composition between 6.2 and 8.5, or approximately +/- 0.3 of one or both of these values, and the plasmid DNA has debridement and nicking rates of less than 5% per year when stored at approximately + 4 ° C and less than 5% per month when stored at approximately + 25 ° C. 15. The composition according to any one of the preceding claims, wherein the buffer solution is present in a concentration such as to maintain the pH of said formulation or composition between 6.7 and 8.0 or about +/- 0.3. of one or both of these values. The composition according to any one of the preceding claims, wherein the buffer solution is present in a concentration such as to maintain the pH of said formulation or composition between 6.7 and 8.0, or approximately +/- 0, 3 of one or both of these values, and the plasmid DNA has debridement and nicking rates of less than 2% per year when stored at approximately + 4 ° C and less than 2% per month when stored at approximately + 25 ° C. The composition according to any one of the preceding claims, wherein the buffer solution is present in a concentration such as to maintain the pH of said formulation or composition between 7.0 and 7.5 or about +/- 0.3 of one or both of these values. 18. The composition according to any one of the preceding claims, wherein the buffer solution is present in a concentration such as to maintain the pH of said formulation or composition between 7.0 and 7.5, or about +/- 0.3 of one or both of these values, and the plasmid DNA has debridement and nicking rates of less than 1% per year when stored at approximately + 4 ° C and less than 1% per month when stored at approximately + 25 ° C. The composition according to any one of the preceding claims, wherein the buffer solution comprises: (a) Tris or lysine and an acid selected from a strong acid or a weak acid; (b) Hepes and a strong base; or (c) phosphate buffer. The composition according to any one of the preceding claims, wherein the buffer solution comprises Tris / HCl, lysine / HCl, Tris / maleic acid, Tris / malic acid, Tris / acetic acid, or Hepes / sodium hydroxide. twenty-one . The composition according to any one of the preceding claims, wherein the buffer is Tris. 22. The composition according to any one of the preceding claims, further comprising a saline excipient. 23. The composition according to claim 22, wherein the saline excipient is NaCl. The composition according to claim 23, wherein the NaCl is present in a concentration between 100 and 200mM, and preferably about 150mM. 25. The composition according to any one of the preceding claims, wherein the plasmid DNA is highly purified or is a pharmaceutical grade plasmid DNA. 26. A stable plasmid DNA composition comprising a plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration sufficient to preserve the plasmid DNA in a stable form at temperatures of about + 4 ° C to + 25 ° C. 27. A stable plasmid DNA composition comprising a plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration sufficient to preserve the plasmid DNA with at least 80% supercoiled plasmid DNA at a temperature of about + 4 ° C until at least approximately 4 years. 28. A stable plasmid DNA composition comprising a plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration sufficient to preserve the plasmid DNA at debridement and nicking rates of less than 5% by year up to 5% per month when stored at approximately + 4 ° C to + 25 ° C. 29. A stable salt composition of plasmid DNA comprising a plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration sufficient to preserve the plasmid DNA in a stable form with the minus 80% supercoiled plasmid DNA at 4 ° C to 25 ° C for a prolonged period of time. 30. A stable plasmid DNA composition comprising a plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration sufficient to preserve the plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA at 4 ° C to 25 ° C for up to 20 months. 31. The composition according to any one of claims 26 to 30, further comprising a saline excipient. 32. The composition according to claim 31, wherein the saline excipient is NaCl that is present in a concentration between 100 and 200mM, and preferably about 150mM. 33. The composition according to any one of claims 26 to 32, wherein the plasmid DNA is highly purified or is a pharmaceutical grade plasmid DNA. 34. A method for preserving plasmid DNA in a stable form in a composition comprising: preparing a purified sample of plasmid DNA; combining said purified sample of plasmid DNA and a buffer solution in a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6 and 9; and store the plasmid DNA. 35. The method according to claim 34, wherein the plasmid DNA contains at least 80% plasmid DNA Supercoiled 36. The method according to any of the foregoing claims 34 and 35, wherein the buffer solution is present in a concentration such as to maintain the pH of said composition between 6.2 and 8.5 or approximately +/- 0. , 3 of one or both of these values. 37. The method according to claim 36, wherein the plasmid DNA is preserved at temperatures of about + 4 ° C to + 25 ° C with debridement and nicking rates of less than 5% per month to less than 5% per year. 38. The method according to any of the preceding claims 34 to 37, wherein the buffer solution is present in a concentration such as to maintain the pH of said composition between 6.7 and 8.0 or about +/- 0.3. of one or both of these values. 39. The method of claim 38, wherein the plasmid DNA is preserved at temperatures of about + 4 ° C to + 25 ° C with clearance and nicking rates of less than 2% per month to less than 2%. by year. 40. The method according to any one of the foregoing claims 34 to 39, wherein the buffer solution is present in a concentration such as to maintain the pH of said composition between 7.0 and 7.5 or approximately +/-. 0, 3. 41. The method according to claim 40, wherein the plasmid DNA is preserved at temperatures of approx. + 4 ° C to + 25 ° C with clearance and de-icing speeds of less than 1% per month to less than 1% per year. 42. The method according to any one of the preceding claims 34 to 41, wherein the buffer is added in a concentration of up to 2mM. 43. The method according to claim 42, wherein the buffer is added in a concentration of between 2mM and 1mM. 44. The method according to claim 43, wherein the buffer is added in a concentration of less than 1 mM. 45. The method according to claim 43, wherein the buffer is added in a concentration between 250μM and 1mM. 46. The method according to claim 45, wherein the buffer is added at a concentration of approximately 400μM. 47. The method according to previous claims 34 to 46, wherein a saline excipient is further added to the plasmid DNA and buffer solution. 48. The method of claim 47, wherein the saline excipient is NaCl. 49. The method of claim 48, wherein the NaCl is added in a concentration between 100 and 200mM, and preferably about 150mM. 50. The method according to any one of the preceding claims 34 to 49, wherein highly purified plasmid DNA or a pharmaceutical grade plasmid DNA is combined with the buffer solution. 51 A method for preserving plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA in a liquid composition at a temperature of up to about 25 ° C for several months, comprising: preparing a purified sample of plasmid DNA; combine the purified sample of plasmid DNA and a buffer solution in which the buffer solution is present at a concentration of less than 2 μM; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 52. A method for preserving plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA in a liquid composition at a temperature of up to about 25 ° C for several months, comprising: preparing a purified sample of plasmid DNA; combining the purified sample of plasmid DNA and a buffer solution in which the buffer solution is present in a concentration between 1 and 2mM; and by mak- ing the plasmid DNA composition at a temperature of up to about 25 ° C. 53. A method for preserving plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA in a liquid composition at a temperature of up to about 25 ° C for several months, comprising: preparing a purified sample of plasmid DNA; combining the purified sample of plasmid DNA and a buffer solution in which the buffer solution is present at a concentration of up to 1 mM; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 54. A method for preserving plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA in a liquid composition at a temperature of up to about 25 ° C for several months, comprising: preparing a purified sample of plasmid DNA; combining the purified sample of plasmid DNA and a buffer solution in which the buffer solution is present at a concentration between about 250 μM and 1 mM; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 55. The method according to any one of claims 51 to 54, wherein the plasmid DNA is preserved at temperatures of about + 4 ° C to about + 25 ° C with debridement and nicking rates of less than 5% per month. at less than 5% per year. 56. The method according to any one of claims 51 to 55, wherein a saline excipient is further added to the plasmid DNA composition. 57. The method according to claim 56, wherein the saline excipient is NaCl. 58. The method according to claim 57, wherein the NaCl is present in a concentration between 100 and 200mM, and preferably about 150mM. 59. A stable plasmid DNA composition obtained by the method defined in any one of claims 34 to 58. 60. The stable plasmid DNA composition according to claim 59, wherein the plasmid DNA is highly purified or has a pharmaceutical grade. 61 A method for preparing a stable composition of Plasmid DNA at a temperature of up to about 25 ° C, comprising: a cell lysis step comprising flowing the cells through (a) turbulent flow medium to rapidly mix a cell suspension with a smooth solution the cells; and (b) a means for laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, wherein the mixture formed in (a) flows from the turbulent flow medium to the laminar flow medium and further comprises optionally (c) a means for adding a second solution that neutralizes the lysis solution, the mixture incubated in (b) flowing from the laminar flow medium to the medium to add a second solution, to release the plasmid DNAs from the cells; a chromatography step to purify the plasmid DNA released in this manner; combining said purified plasmid DNA and a buffer solution in a concentration of up to 2 μM sufficient to maintain the pH of the resulting composition between 6 and 9, and store the plasmid DNA composition at a temperature of up to about 25 ° C. 62. A method for preparing a stable plasmid DNA composition at a temperature of up to about 25 ° C, comprising: a cell lysis step comprising fl owing the cells through (a) a medium for turbulent flow to quickly mix a cell suspension with a solution that lyses the cells; and (b) a means for laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, wherein the mixture formed in (a) flows from the turbulent flow medium to the laminar flow medium and further comprises optionally (c) a means for adding a second solution that neutralizes the lysis solution, the mixture incubated in (b) flowing from the laminar flow medium to the medium to add a second solution, to release the plasmid DNA from the cells; - a chromatography step to purify the plasmid DNA released in this way; combining said purified plasmid DNA and a buffer solution in a concentration of up to 2mM sufficient to maintain the pH of the resulting composition between 6.2 and 8.5 or about +/- 0.3 of one or both of these values; Y store the plasmid DNA composition at a temperature of up to approximately 25 ° C. 63. A method for preparing a stable plasmid DNA composition at a temperature of up to about 25 ° C, comprising: a cell lysis step comprising flowing the cells through (a) a medium for flow turbulent to quickly mix a cell suspension with a solution that lyses the cells; and (b) a means for laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, in which the mixture formed in (a) flows from the turbulent flow medium to the medium for laminar flow and optionally further comprising (c) a means for adding a second solution neutralizing the lysis solution, fluting the mixture incubated in (b) from the laminar flow medium to the medium to add a second solution, to free the Plasmid DNA of the cells; a chromatography step to purify the plasmid DNA released in this manner; combining said purified plasmid DNA and a buffer solution in a concentration of up to 2mM sufficient to maintain the pH of the resulting composition between 6.7 and 8.0 or about +/- 0.3 of one or both of these values; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 64. A method for preparing a stable composition of Plasmid DNA at a temperature of up to about 25 ° C, comprising: a cell lysis step comprising fl owing the cells through (a) a means for turbulent fl ow to rapidly mix a cell suspension with a dissolution that smooths the cells; and (b) a means for laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, in which the mixture formed in (a) flows from the turbulent fl uid medium to the laminar flow medium and optionally further comprising (c) a means for adding a second solution that neutralizes the lysis solution, the mixture incubated in (b) flowing from the medium for laminar flow to the medium to add a second solution, to release the plasmid DNAs of the cells; a chromatography step to purify the plasmid DNA released in this manner; combining said purified plasmid DNA and a buffer solution in a concentration of up to 2 mM which maintains the pH of the resulting composition between 7.0 and 7.5 or approximately +/- 0.3 of one or both of these values, and - store the plasmid DNA composition at a temperature of up to about 25 ° C. 65. The method according to any one of claims 61 to 64, wherein the buffer solution is present in a concentration of less than 2mM. 66. The method according to any one of the claims 61 to 65, wherein the buffer solution is present in a concentration of about 1 to 2 mM. 67. The method according to any one of claims 61 to 66, wherein the buffer solution is added to reach a concentration of about 250 μM to less than 1 mM in the plasmid DNA composition. 68. A method for preparing a stable plasmid DNA composition at a temperature of up to about 25 ° C, comprising: - a cell lysis step comprising making the cells flow through (a) a turbulent flow medium to rapidly mix a cell suspension with a solution that lyses the cells; and (b) a means for laminar flow to allow incubation of a mixture formed in (a) without substantial agitation, wherein the mixture formed in (a) flows from the turbulent flow medium to the laminar flow medium and further comprises optionally (c) a means for adding a second solution that neutralizes the lysis solution, the mixture incubated in (b) flowing from the laminar flow medium to the medium to add a second solution, to release the plasmid DNAs from the cells; a chromatography step to purify the plasmid DNA released in this manner; combining said purified plasmid DNA and a buffer solution in which the buffer solution is present at a concentration of less than 2mM, or less than 1mM, or between 250μM and 1mM, and preferably 400μM; and by storing the plasmidic DNA composition at a temperature of up to about 25 ° C. 69. The method according to any one of claims 61 to 68, wherein an excipient is further added to the plasmid DNA composition. 70. The method according to claim 69, wherein the salt excipient is NaCl. 71 The method according to claim 70, wherein the NaCl is present at a concentration between 100 and 200mM, and preferably about 50mM. 72. The method according to any one of claims 61 to 71, wherein the lysis solution is a solution containing a lysis agent selected from the group consisting of an alkali, a detergent, an organic solvent and an enzyme. or a mixture of these. 73. The method according to any one of Claims 61 to 72, wherein the plasmid DNA is purified by at least one step of chromatography including anionic exchange chromatography, triplex affinity chromatography or hydrophobic interaction chromatography. 74. The method of claim 73, wherein the anion exchange chromatography step is combined with a triple helix chromatography step for the purification of idiodic plasmid DNA. 75. The method of claim 74 further comprising a step of hydrophobic interaction chromatography. 76. The method according to any one of claims 61 to 75, wherein the plasmid DNA is purified by a 3-step chromatographic process comprising anion exchange chromatography, triplex affinity chromatography and hydrophobic interaction chromatography, in this order . 77. The method according to any one of claims 61 to 76, wherein the first chromatography performed is preceded by a filtration of the lysate. 78. The method according to any one of the claims 61 to 77, in which the first chromatography performed is preceded by the elimination of the floccule. 79. The method according to any one of claims 61 to 78, wherein the last chromatographic step is followed by a diafiltration and / or buffer exchange step. 80. The method according to any one of claims 61 to 79, wherein the previous step of removing the floccule is carried out by passing the solution through a grid filter and by a depth filtration. 81 The method according to any one of the claims 61 to 80, wherein the diafiltration step to reach appropriate target values of salt, buffer and pH. 82. The method according to any one of claims 61 to 81, wherein the diafiltration step comprises the following steps: collecting the solution of the last chromatographic step; perform a first diafiltration step against Tris / NaCl buffer; performing a second diafiltration step against saline under conditions suitable for controlling the final concentration of the buffer and for stabilizing the pH of the final plasmid DNA formulation. 83. The method according to any one of claims 61 to 82, further comprising a stage of sterilization by filtration, formulation and filling of vials with the purified plasmid DNA. 84. A vial of highly purified plasmid DNA obtained by the method of claim 83. The vial according to claim 84, wherein the purified plasmid DNA is a plasmid designated NV1 FGF which is a pCOR plasmid having a cassette of expression that encodes the FGF-1 gene. 86. The vial according to claim 85 for use in the treatment of peripheral ischemia of the extremities, including peripheral arterial disease (PAOD or PAD) and critical ischemia of the extremities (CLI). 87. The method according to any one of claims 61 to 83, wherein the chromatographic steps allow the removal of impurities such as proteins, denatured genomic DNA, RNA, proteins, oligoribonucleotides, oligodeoxyribonucleotides, Denatured plasmid DNA and lipopolysaccharides. 88. The method according to any one of claims 61 to 83, wherein the chromatographic steps are carried out in solid support which is any organic, inorganic or composite, porous, superporous or non-porous material, suitable for chromatographic separations, which is derivatized with poly (alkene glycols), alkanes, alkenes, alkynes, locks or other molecules that confer a hydrophobic character on the support. 89. The composition according to any one of claims 1 to 33, wherein the plasmid DNA comprises a therapeutic and / or immunogenic coding sequence. 90. The composition according to claim 89, wherein the therapeutic gene is a mammalian gene. 91 The composition according to claim 89 as a DNA vaccine. 92. The composition according to any of claims 89 or 90 as plasmid-based therapy, such as gene therapy. 93. The composition according to any one of claims 1 to 33 for use in a method of treating the human or animal body by therapy.