EA011554B1 - 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

Field of Invention

The present invention relates to stable liquid plasmid DNA compositions, wherein the plasmid remains non-degraded at a temperature of from 4 ° C to room temperature for extended periods of time. Thus, such compositions are suitable for storing plasmid DNA used in research or plasmid-based therapy, such as, for example, DNA vaccines or gene therapy agents.

BACKGROUND OF THE INVENTION

Discoveries in molecular biology clearly show that plasmid-based therapy, specifically the field of DNA vaccines and gene therapy, can provide effective treatments for diseases. One promising way to safely and efficiently deliver a normal gene to human cells is through plasmid DNA delivery. Plasmid DNA is a covalently closed ring (ccc) or a supercoiled form of bacterial DNA into 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, mRNA or dsRNA sequences, ribozymes, and sequences for use in, for example, DNA vaccines against viral infection or in the treatment of cardiac -vascular diseases associated with the angiogenesis of diseases or malignant tumors. Once delivered to a human cell, plasmid DNA begins to replicate and synthesize copies of the DNA sequence of interest. Thus, the prospect of using plasmid DNA as an active pharmaceutical ingredient (ΑΡΙ) is sufficient for the treatment of a number of disease states, but storage is a significant obstacle to this method. In fact, if plasmid DNA is stored under adverse conditions, its structure degrades, and the supercoiled (ccc) topology / structure of the molecule can turn into inactive forms (open ring and linear) due to oxidative damage. Oxidizers, such as hydrogen peroxide, superoxide and hydroxyl radicals, formed as a result of reactions such as the Fenton reaction, are responsible for the oxidative degradation of DNA. Specifically, free radical oxidation pathways and β-cleavage are the main sources of DNA degradation for highly purified plasmid DNA in aqueous preparations, causing single-stranded breaks or nick-breaks of DNA and subsequent conversion of covalently closed ring (scc) double-stranded supercoiled DNA into a relaxed ring or open ring and linear shapes.

Plasmid DNA is usually formulated in phosphate or Tm8 buffer aqueous solutions, where the phosphate or Tmsc buffer solution is present at a concentration of about 10 mM. However, such compositions, as a rule, are subject to degradation processes that occur during storage in an aqueous solution. These plasmid DNA solutions have very low stability at approximately 4 and 25 ° C. Specifically, their depurinization ratios are very high from 4 ° C to room temperature (CT). Depurinization processes are usually controlled by measuring the content of supercoiled, open circular and linear DNA, as well as the depurinization ratio, i.e. accumulation of upurized sites, and oxidation, i.e. the formation of 8-hydroxyguanine over time.

Thus, long-term storage of a pharmaceutical product of plasmid DNA leads to many degradation reactions affecting DNA stability. To overcome these problems, plasmid DNA is usually lyophilized for storage at temperatures up to room temperature, but, therefore, requiring additional manipulation steps for processing the formulations and, in addition, creating an additional threat of contamination and / or degradation.

Since any breakage of a chain occurring in plasmid DNA affects its quality and functioning, it is necessary to consider damage occurring during long-term storage and manipulation of plasmid DNA and provide a storage composition that ensures the stability of plasmid DNA during long-term storage and safe manipulation at temperatures at range from 4 ° C to room temperature.

Thus, the authors found new liquid compositions for plasmid DNA that are stable and resistant over a long period of time to a wide temperature range, for example up to room temperature, thereby facilitating the storage, transportation, manipulation and distribution of DNA-based drug, DNA- vaccines or gene therapy agents prior to safe administration to subjects. Specifically, such liquid compositions are suitable for highly purified plasmid DNA, which can be used for research and plasmid-based therapy, for example in gene therapy and DNA vaccines.

SUMMARY OF THE INVENTION

The first object of the present invention is a composition that stores plasmid DNA in a liquid preparation for long periods of time at temperatures up to 25 ° C.

Thus, the present invention relates to a liquid composition that maintains stable plasmid DNA containing plasmid DNA and a buffer solution at a concentration of up to 5, 4, or up to 3, or even up to 2 mM, sufficient to maintain the pH of the plasmid DNA composition between 6 and 9, such

- 1 011554 thus allowing the storage of plasmid DNA with a supercoiled content of at least 80% and a content of plasmids susceptible to depurinization and single-strand breaks of less than 20%.

The present invention also relates to a liquid composition that keeps plasmid DNA stable, containing plasmid DNA and a buffer solution at a concentration of up to 2 mM, sufficient to maintain the pH of said drug or composition between 6.2 and 8.5 and / or approximately +/- 0, 3 from one or both of these values, thereby preserving plasmid DNA with ratios of depurinization and single strand breaks of less than 5% per year when stored at about 4 ° C and less than 5% per month when stored at about 25 ° C.

The present invention also relates to a liquid composition that maintains stable plasmid DNA containing plasmid DNA and a buffer solution at a concentration of up to 2 mm, sufficient to maintain the pH of the specified drug or composition between 6.7 and 8.0 and / or approximately +/- 0, 3 from one or both of these values, thereby preserving plasmid DNA with ratios of depurinization and single strand breaks of less than 2% per year when stored at about 4 ° C and less than 2% per month when stored at about 25 ° C.

In addition, the present invention relates to a liquid composition that maintains stable plasmid DNA containing plasmid DNA and a buffer solution at a concentration of up to 2 mm, sufficient to maintain the pH of the specified drug or composition between 7.0 and 7.5 approximately +/- 0.3 thereby preserving plasmid DNA with ratios of depurinization and single strand breaks of less than 1% per year when stored at about 4 ° C and less than 1% per month when stored at about 25 ° C.

Another object of the present invention is a method for storing plasmid DNA in a stable form in a liquid composition for storage, including:

(ί) obtaining a purified plasmid DNA sample;

(ίί) mixing said purified sample of plasmid DNA and a buffer solution at a concentration of up to 2 mM necessary to maintain the pH of the resulting composition between 6 and 9; and (ίίί) storage of plasmid DNA.

The method of the present invention provides a method for storing high quality plasmid DNA with at least 80% supercoiled plasmid DNA.

The present invention relates to a method for storing plasmid DNA in a stable form in a liquid composition for storage at temperatures from about 4 to 25 ° C with ratios of depurinization and single strand breaks of less than 5% per month and less than 5% per year, including:

(ί) obtaining a purified plasmid DNA sample;

(ίί) mixing said purified sample of plasmid DNA and a buffer solution at a concentration of up to 2 mM necessary to maintain the pH of the resulting composition between 6.2 and 8.5 and / or approximately +/- 0.3 from one or both of these values; and (ίίί) storage of plasmid DNA at a selected temperature.

The present invention also relates to a method for storing plasmid DNA in a stable form in a liquid composition for storage at temperatures from about 4 to 25 ° C with ratios of depurinization and single strand breaks of less than 2% per month and less than 2% per year, including:

(ί) obtaining a purified plasmid DNA sample;

(ίί) mixing said purified sample of plasmid DNA and a buffer solution at a concentration of up to 2 mM necessary to maintain the pH of the resulting composition between 6.7 and 8, or approximately +/- 0.3 from one or both of these values; and (ίίί) storage of plasmid DNA at a selected temperature.

In addition, the present invention relates to a method for storing plasmid DNA in a stable form in a liquid composition at temperatures from about 4 to 25 ° C with ratios of depurinization and single strand breaks of less than 1% per month and less than 1% per year, including:

(ί) obtaining a purified plasmid DNA sample;

(ίί) mixing said purified sample of plasmid DNA and a buffer solution at a concentration of up to 2 mM necessary to maintain the pH of the resulting composition between 7.0 and 7.5 and / or approximately +/- 0.3 from one or both of these values; and (ίίί) storage of plasmid DNA at a selected temperature.

Accordingly, the plasmid DNA composition contains a buffer solution in a concentration of less than 5, or less than 4, or less than 3 mm. Preferably, the composition for storing stable plasmid DNA contains a buffer solution in a predetermined amount or in a very low concentration of up to 2 mM, and more preferably between 1 and 2 mM. Most preferably, the buffer solution is present in 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 drug 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 these values.

The stable composition of the present invention is particularly suitable for storing highly purified plasmid DNA with very low amounts of contaminating chromosomal DNA, RNA, protein, and

- 2 011554 endotoxins. Such highly purified plasmid DNAs have less than about 0.01% of the host cell contaminant RNA and / or less than about 0.01% of the host cell genomic DNA contaminant. Preferred highly purified plasmid DNAs have less than about 0.001% of the host cell contaminating RNA, and / or less than about 0.001% of the host cell contaminating protein, and / or less than about 0.001% of the host cell contaminating DNA. Most preferred highly purified plasmid DNAs have less than about 0.0001% of the host cell contaminating RNA, and / or less than about 0.0001% of the host cell contaminating protein, and / or less than about 0.0001% of the host cell contaminating DNA.

Another object of the present invention is a method for producing a stable plasmid DNA liquid composition for storage at temperatures up to about 25 ° C, including:

(1) a cell lysis step consisting of passing cells through (a) a turbulent stream to quickly mix the cell suspension with a cell lysing solution; and (b) a laminar stream to allow incubation of the resulting mixture (a) without significant activation, where the formed mixture (a) passes from the turbulent stream to the laminar stream and, in addition, optionally includes (c) adding a neutralizing lysis solution of the second solution, incubated in (b) the laminar stream in the second solution of the mixture to isolate plasmid MDs K from cells;

(2) one chromatography step to purify plasmid DNA thus isolated;

(3) mixing the specified plasmid DNA and the buffer solution at 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 producing a liquid preparation for storing stable plasmid DNA at a temperature of up to about 25 ° C, including:

(1) a cell lysis step consisting of passing cells through (a) a turbulent flow device for rapidly mixing the cell suspension with a cell lysing solution; and (b) a laminar flow to allow incubation of the resulting mixture (a) without significant mixing, where formed in ( a) the mixture passes from the turbulent flow to the laminar flow and, in addition, optionally includes (c) a device for adding a neutralizing solution that neutralizes the lysing solution; the mixture incubated in (b) flows from the laminating device Nogo flow into the means for adding a second solution to isolate plasmid DNA from the cells;

(2) performing a chromatography step to purify plasmid DNA thus isolated;

(3) performing the step of diafiltration and / or buffer replacement;

(4) mixing said purified plasmid DNA and buffer solution at a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6 and 9; and (5) filling the vials with a liquid plasmid DNA composition and storing the plasmid DNA composition at a temperature of up to about 25 ° C.

By the method of the present invention, a buffer solution is added to the plasmid DNA composition at a concentration of less than 5, or less than 4, or less than 3 mM. Preferably, the method includes adding the prescribed amounts of the buffer solution or adding the buffer solution in a very low concentration of up to 2 mM and more preferably between 1 and 2 mM. Most preferably, the buffer solution is present in 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 drug 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 these values.

An additional object of the present invention are bottles (ampoules) containing, as an active pharmaceutical ingredient, a stable plasmid DNA liquid preparation for use in research or in plasmid-based therapy, such as, for example, gene therapy or a DNA vaccine.

Another object of the present invention is an ampoule containing purified plasmid DNA, which is a plasmid designated LU1ECE, which is a pCOK plasmid carrying an expression cluster encoding an ECE-1 gene suitable for treating peripheral limb ischemia, including peripheral artery disease ( ΡΆΟΌ or ΡΆΌ) and critical limb ischemia (CI1).

Additional objects and advantages of the invention are indicated in the following section of the description and partially become apparent from the description or can be studied by applying the invention. The objects and advantages of the invention are understood and realized by means of the elements and combinations detailed in the attached claims.

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It should be understood that in the framework of the above general description and the following detailed description are only illustrative and explanatory and do not limit the scope of the invention. The complementary drawings included and forming part of this description illustrate some embodiments of the invention and, together with the description, help explain the principles of the invention.

Brief Description of the Drawings

FIG. 1 is a diagram of a device that can be used to continuously lyse cells according to the invention.

FIG. 2 is a diagram of an M1 mixer in a continuous cell lysis device.

FIG. 3 is a table comparing the purification yields depending on the contaminating gDNA, RNA, proteins, endotoxin using one step of anion exchange chromatography (AEC) or a two-step method with anion exchange chromatography in combination with three-helix affinity chromatography (TNAC) and a three-step method, including an anion exchange chromatography step, a three-helix affinity chromatography step, and a hydrophobic interaction chromatography step (H1C); BUT - means "not detected" (low sensitivity of analytical methods).

FIG. 4 is a table comparing various methods for separating and purifying plasmid DNA, such as anion exchange chromatography (AEC), hydroxyapatite chromatography (HAC), hydrophobic interaction chromatography (H1C), reverse phase chromatography (HRC), size exclusion chromatography (8EC) ), triple helix affinity chromatography (TNAC), alone or in combination, and the method of the present invention. Here are the results, depending on the quality of the purified plasmid DNA; BUT - means not detected (low sensitivity analytical methods).

FIG. 5A and 5B are graphs showing the relationships of depurinization and single-strand breaks (formation of a plasmid form of a broken ring) stored at 25 and 5 ° C for up to 90 days of plasmid DNA.

FIG. 6A and 6B are graphs showing the relationships of depurinization and single-strand breaks (formation of the plasmid form of a broken ring) stored at 25 and 5 ° C for up to 150 days of plasmid DNA.

Definitions “A plasmid DNA preparation or composition” means a composition containing the required amount of plasmid DNA or a plasmid DNA preparation in which the necessary amount is present for use in research or plasmid-based therapy, such as, for example, gene therapy or a DNA vaccine.

“Preparation for stable storage of plasmid DNA” means a preparation that can be used to store plasmid DNA in a stable form for long periods of time before use, as, for example, in studies or plasmid-based therapy. Shelf life can be several months, 1 year, 5, 10, 15, or up to 20 years at a temperature range of 5 to 25 ° C (BT - room temperature).

Generally, “stable plasmid DNA preparation or composition” means a plasmid DNA preparation with a relative content of supercoiled double-stranded DNA of at least 80%, which is in the form of open circular and / or linear plasmids.

"Stable plasmid DNA preparation" hereinafter means a composition containing plasmid DNA with a relationship of depurinization and single stranded breaks (formation of a plasmid open ring 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 preparation hereinafter means a composition containing plasmid DNA with ratios of depurinization and single stranded breaks (formation of a plasmid open ring 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 preparation hereinafter means a composition containing plasmid DNA with a ratio of depurinization and single strand breaks (formation of a plasmid open ring form) of less than 1% per month when stored at 25 ° C. and less than 1% per year when stored at 5 ° C. .

"Acidic" means bound to or containing acid; having a pH of less than 7.

"Alkaline" means bound to alkali or containing an alkali metal or base; having a pH of more than 7.

“Continuous” means uninterrupted, has no interruptions.

"Genomic DNA (abbreviated gDNA)" means derived from a chromosome or found in a chromosome DNA.

"Laminar flow" means the type of current in the flow of a solution of water in which each particle moves in a parallel direction to each particle.

“Lysate” means material obtained during cell lysis. The term "lysis" refers to the process of rupture of the cell wall and / or cell membrane of a cell in buffer

- 4 011554 solution (i.e. cell suspension), by chemical treatment using a solution containing a lysing agent. Lysing agents include, for example, alkalis, detergents, organic solvents and enzymes. In a preferred embodiment, cell lysis is performed to isolate intact plasmids from host cells.

“Neutralizes” to make (solution) neutral, or is the cause (acid or base / alkali) of neutralization. By this term, the authors mean that something that neutralizes the 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 before.

A “Newtonian fluid” is a fluid in which the shear stress is proportional to the velocity gradient and perpendicular to the shear plane. The constant of proportionality is known as viscosity. Examples of Newtonian fluids include liquids and gases.

A “non-Newtonian fluid” is a fluid in which the shear stress is not proportional only to the velocity gradient and perpendicular to the shear plane. Non-Newtonian fluids may not have a pronounced viscosity. Non-Newtonian fluids include plastic solids, power fluids, viscoelastic fluids (with viscous and elastic properties) and non-stationary viscous fluids.

"Plasmid DNA" means a small cellular inclusion, consisting of a non-chromosome DNA ring, which may have the ability to receive inserted into it a fragment of non-endogenous DNA. As used herein, plasmid DNA can also be any form of plasmid DNA, such as, for example, a stripped-down, transformed, or other driven form of non-chromosomal DNA, including, for example, any of the forms, or any combination of single-stranded ring plasmid DNA forms, relaxed circular plasmid DNA, supercoiled plasmid DNA, truncated plasmid DNA, linearized or linear plasmid DNA, and single-stranded plasmid DNA. Methods for the construction of plasmids include those described in Mushasha c1 a1., Mo1esi1ag S1oshpd, A aogaFgu Mapia1, 26, Co 16 Brppd Natot aogaFgu Rgs55 (1989). For the initial isolation of plasmid DNA for later processing by means of some aspects of the invention, a well-known protocol for isolating plasmid DNA with “miniprep” (BlnpO1, ap6 Oo1y, Lyslc Ac16§ Kskeats 7: 1513 (1979)) can be used and can be contrasted with highly purified samples, obtained by the methods of the invention. Preferably, the plasmid DNA form is, or at least, after preparation by the purification method of the invention, a substantially closed circular form of plasmid DNA, or approximately 80, 85, 90, 95 or approximately more than 99% closed circular form of plasmid DNA. Alternatively, in some therapeutic methods, a supercoiled covalently closed form of plasmid DNA (ccc) may be preferred, where it may be more effective than an open circular, linear or multimeric form. Consequently, pharmaceutical grade plasmid DNA may be a plasmid isolated or separated from one or more forms and generally contain one or more desired forms.

For the purposes of the present invention, the term flow refers to the passage of fluid through a mixer at a specific flow rate (e.g., liters per minute), typically by 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 “single stranded DNA” and “relaxed” DNA mean non-coiled DNA.

“Supercoiled” DNA is a term well understood in the art in the description of a particular, separate form of plasmid DNA. Other forms of plasmid DNA are also known in the art.

A “contaminant” is any substance from which it is desirable to separate or isolate DNA. Contaminants include, but are not limited to, host cell proteins, endotoxin, host cell DNA, such as chromosomal DNA or genomic DNA and / or RNA of the host cell. It is understood that what is or may be considered as a contaminant may depend on the context in which the methods of the invention are applied. A “contaminant” may or may not be derived from a host cell, i.e. the impurity may or may not be an impurity of the host cell.

"Isolation" or "purification" of the original component (such as DNA) means the enrichment of the original component from other components with which the first component was originally detected. The volumes of the desired and / or obtained purification are presented here.

The terms “substantially free and highly purified” mean approximately 95%, and preferably more than 98.99% pure or free of pollutants, or containing less than 5%, and preferably less than 1-2% of pollutants.

The pharmaceutical category of DNA is here referred to as a DNA preparation containing not more than about 5%, and more preferably not more than about 1-2% of cellular components, such as cell membranes.

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Also described is a method for producing and isolating highly purified plasmid DNA, which is practically free of contaminants and thus represents a pharmaceutical category of DNA. Using the method of the invention, plasmid DNA containing very low amounts is obtained and isolated. millionths (ppm) of polluting chromosomal DNA, RNA, protein and endotoxins, and contains mainly a closed circular form of plasmid DNA. The plasmid DNA obtained according to the invention is of sufficient purity for use in research and plasmid-based therapy, and optionally for data on human clinical trials and experiments on human gene therapy and clinical studies.

A “pharmaceutical grade plasmid DNA composition” according to the invention is a plasmid DNA composition and / or composition of at least one of the purity levels indicated below as “pharmaceutical grade plasmid DNA” obtained by the method of the invention.

Preferably, the “pharmaceutical grade plasmid DNA composition” of the invention has a purity indicated by at least two of the purity grades defined below as “pharmaceutical grade plasmid DNA,” for example, less than about 0.01% chromosomal or genomic DNA and less than about 0 , 01% contaminating protein, or, for example, less than about 0.01% chromosomal or genomic DNA and less than about 0.1 IU / mg of endotoxins. Pharmaceutical grade plasmid DNA preferably contains less than about 0.001% chromosomal or genomic DNA and less than about 0.001% contaminating protein or, for example, less than about 0.001% chromosomal or genomic DNA and less than about 0.1 IU / mg of endotoxins. More preferably, it contains less than about 0.0001% chromosomal or genomic DNA and less than about 0.0001% contaminating protein or, for example, less than about 0.0001% chromosomal or genomic DNA and less than about 0.1 IU / 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 protein or, for example, less than about 0.00008% chromosomal or genomic DNA and less than about 0.1 IU / mg of endotoxins . Other combinations of degrees of purity are included in the definition. Of course, a pharmaceutical grade plasmid DNA composition, in addition, may include or contain additional components desired for any particular application, including use in combination treatments, compositions and therapies. Amounts of chromosomal or genomic DNA, RNA, endotoxins, or protein refer to contaminants obtained by cell-based plasmid production or other contaminants obtained during purification.

Most preferably, “pharmaceutical grade plasmid DNA” refers here to a DNA preparation containing genomic DNA, RNA and / or contaminating proteins at the level of one ppm or ppm. (<0.0001%, i.e. <0.0001 mg per 100 mg of plasmid DNA) or less.

Also or more precisely, “pharmaceutical grade plasmid DNA” here 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%, t ie <0.00008 mg per 100 mg of plasmid DNA) 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%, i.e. < 0.00002 mg per 100 mg of plasmid DNA) contaminating RNA.

“Pharmaceutical grade plasmid DNA” may also mean a DNA preparation containing less than about 0.0001%, and most preferably less than 0.00005% (<0.00005%, i.e. <0.00005 mg per 100 mg of plasmid DNA) contaminating proteins.

"Pharmaceutical grade plasmid DNA" may also mean a DNA preparation containing less than 0.1 IU / mg of endotoxins.

Pharmaceutical-grade plasmid DNA refers here to a DNA preparation, preferably located mainly in a circular form, and more specifically, a DNA containing more than 80, 85, 90, 95, or more than 99% of the closed circular form of plasmid DNA.

A T-tube refers to a T-shaped tube configuration, where the T shape is formed using a solid tube produced in such a configuration or several tubes connected to form such a configuration. The T-tube has three sleeves and a central area for connecting the sleeves. The T-tube can be used to mix the ingredients, since two liquids can flow, each from one of the T arms, connecting in the central region, and exit the third sleeve. Mixing takes place as a combination of liquids.

"Turbulent flow" means the random chaotic motion of fluid particles in directions transverse to the direction of the main flow, in which the speed at a given point randomly changes in magnitude and direction.

"Viscoelastic" refers to liquids with viscous and elastic properties.

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DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to stable liquid preparations of plasmid DNA, where the plasmid DNA remains not degraded at room temperature for a long period of time. Thus, such plasmid DNA preserving plasmid DNA preparations or compositions are suitable for research based on therapeutic plasmids, such as, for example, gene therapy or a DNA vaccine.

According to the present invention, a stable plasmid DNA liquid composition contains plasmid DNA and a buffer solution at a concentration of up to 5, or up to 4, or up to 3, or up to 2 mM, sufficient to maintain the pH of said composition between 6 and 9, and the composition contains a basic supercoiled form of plasmid DNA at temperatures from about 4 to 25 ° C for several months, 1 year, 2, 3, 4, 5 and up to 10 years.

A plasmid DNA composition with a predominantly supercoiled plasmid form contains 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% supercoiled or closed circular plasmid DNA. Alternatively, a stable plasmid DNA storage composition gives a depurinization to single strand break ratio of less than 5% per month.

Thus, the liquid plasmid DNA-stable preparation of the present invention contains plasmid DNA and a highly diluted buffer solution, a buffer solution at a concentration of up to 2 mM, sufficient to maintain a pH of the composition of at least 6 and not more than 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 from one or both of these values.

The stable plasmid DNA liquid composition contains a buffer solution at a concentration of up to 2 mM to maintain the pH of the indicated preparation or composition between 6.2 and 8.5 and / or approximately +/- 0.3 from one or both of these values, thus ensuring storage plasmid DNA with ratios of depurinization and single strand breaks 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 plasmid DNA liquid composition contains a buffer solution at a concentration of up to 2 mM to maintain the pH of said drug or composition between 6.7 and 8 and / or approximately +/- 0.3 from one or both of these values, thereby ensuring plasmid storage DNA with ratios of depurinization and single strand breaks 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 plasmid DNA liquid composition contains a buffer solution at a concentration of up to 2 mM to maintain the pH of said drug or composition between 7 and 7.5 and / or approximately +/- 0.3 from one or both of these values, thereby providing storage plasmid DNA with ratios of depurinization and single strand breaks 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 buffer effect appears in the range of values and in volume to stabilize the pH 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 from any of these values. Thus, a buffer solution can be added at a concentration of less than 5 mM. Preferably, the stable plasmid DNA composition contains traces of a buffer solution or buffer solution in a highly diluted concentration of up to 2 mM, and more preferably between 1 and 2 mM. Most preferably, the buffer solution is present in 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 drug 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 these values.

The buffer solution is present at a concentration of up to 2 mm or between 1 and 2 mm. Preferably, the buffer solution is present in a concentration of less than 1 mM. Most preferably, the buffer solution is present in trace amounts at a concentration as low as, for example, 250 μM and up to 1 mM. Trace amounts of the buffer solution can be approximately 400 μM and are precisely sufficient to maintain the pH in the ranges indicated above.

Suitable buffers for use in the compositions of the present invention consist of either an acid-base system comprising Tpc [tris- (hydroxymethyl) aminomethane] or lysine and an acid selected from strong acid (hydrochloric acid, for example) or weak acid (maleic , malic or acetic acid, for example) or from the acid-base system, including Nerek [2- (4- (2-hydroxyethylpiperazine) -1-yl) ethanesulfonic acid] and a strong base (sodium hydroxide, for example) or phosphate buffers such as sodium phosphate oia or potassium phosphate. The buffer solution may also contain Tpc / HCT lysine / HCl, Tpc / maleic acid, Tpc / malic acid, Tg / acetic acid or Nerek / sodium hydroxide. Preferably, a Tpc buffer solution is used in the stable plasmid DNA storage composition of the present invention.

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As shown in the examples below, the plasmid DNA preparations of the present invention exhibit exceptional stability at 4 ° C and at room temperature (CT), for example 20 or 25 ° C.

The composition of the present invention may further comprise a salt excipient. Suitable for use in the compositions of the present invention saline excipient may comprise anions and cations selected from the group consisting of acetate, phosphate, carbonate, 8o 4 ^2-, C1 ^-, Br ^-, ΝΟ3 ^-, Mg ^2+, L1 ^+, Να '. K ⁺ and ΝΗ ⁴⁺ and any other salt or form of an existing or previously used pharmaceutical compound.

A preferred salt excipient is ΝαΟΊ at a concentration between 100 and 200 mM, and preferably at a concentration of about 150 mM.

The stable compositions of the present invention are particularly suitable for storing highly purified plasmid DNA or the pharmaceutical category of plasmid DNA with very low amounts of contaminating chromosomal DNA, RNA, protein, and endotoxins. Such highly purified plasmid DNAs have less than about 0.01, or 0.001, or 0.0001% of the host cell contaminating RNA, and / or less than about 0.01, or 0.001, or 0.0001% of the host cell contaminating protein, and / or less approximately 0.01, or 0.001, or 0.0001% of the host cell contaminating genomic DNA.

The compositions of the present invention may further include an adjuvant, such as, for example, a polymer selected from polyethylene glycol, pluronic or polysorbate sugar or alcohol.

In yet another aspect, the present invention relates to a method for storing plasmid DNA in a composition, comprising:

a) obtaining a purified sample of plasmid DNA and

b) the connection of the specified purified sample of plasmid DNA and a buffer solution in a concentration of up to 2 mm, maintaining 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 about 7.2.

The present invention also relates to a method for storing plasmid DNA in a composition comprising:

a) obtaining a purified sample of plasmid DNA;

b) the connection of the specified purified sample of plasmid DNA and a buffer solution at a concentration of up to 2 mm, sufficient to maintain the pH of the resulting composition between 6 and 9; and

c) storage of plasmid DNA.

The method of the present invention allows the storage of plasmid DNA 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 from one or both of these values, thus allowing the storage of plasmid DNA at temperatures from about 4 to 25 ° C with ratios of depurinization and single strand breaks from less than 5% per month to less than 5% per year.

Preferably, the pH of the resulting composition can be maintained between 6.7 and 8, approximately +/- 0.3, thus allowing the storage of plasmid DNA at temperatures from about 4 to 25 ° C with ratios of depurinization and single-strand breaks from less than 2% per month to less than 2 % in year.

Most preferably, the pH of the resulting composition can be maintained between 7 and 7.5 and approximately +/- 0.3 from one or both of these values, thus allowing the storage of plasmid DNA at temperatures from about 4 to 25 ° C. with ratios of depurinization and single strand breaks from less than 1% per month to less than 1% per year.

By the method of the present invention, a buffer solution is added to the plasmid DNA composition at a concentration of up to 2 mM or between 1 and 2 mM. Preferably, the buffer solution is added until a concentration of less than 1 mM is reached. Most preferably, the buffer solution is present in small amounts at a concentration as low as 250 μM and up to 1 mM. Minor amounts of the buffer solution may be approximately 400 μM and are precisely sufficient to maintain the pH in the ranges indicated above.

In the present method, a salt excipient can be added to the plasmid DNA and the buffer solution.

Salt excipients are described hereinabove. A preferred salt excipient is No. 1C1 at a concentration between 100 and 200 mM, and preferably about 150 mM.

The plasmid DNAs presented in the compositions of the present invention may be in isolated form. They can be isolated by lysis of bacterial cells and purification, as described here, or synthesized by an automatic device for the synthesis of nucleic acids.

They can contain a polynucleotide encoding a polypeptide, where the polynucleotide can be a transgene, such as, for example, a therapeutic gene, for example, from a mammal, such as a rodent, or a human gene, and operably linked to a promoter sequence. A polynucleotide inserted into plasmid DNA may be a genomic pro

- 8 011554 origin and, therefore, contain exons and introns, as reflected in its genomic structure or can be obtained from complementary DNA. The polynucleotide can encode any of a number of polypeptides, as non-limiting examples, which are an immunogenic peptide or protein, angiogenic factor, erythropoietin, adenosine deaminase, factor VIII, factor IX, dystrophin, β-globin, LOB receptor, NETWORKS, insulin, antiangiogenic factor, hormone growth, αΐ-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, interleukin and interferon. Preferably, the plasmid DNA contains a polynucleotide encoding an angiogenic factor, such as, for example, the ECE gene (from ECE-1 to ECE-22), VΕCE, HCE or SHE-1. As an alternative to the use of a polynucleotide-encoding polypeptide, the polynucleotide can encode interfering RNA that can be used to inhibit the expression of a target gene, for example, where gene expression is undesirable (e.g., a pathogen gene) or where the level of gene expression in the cell is undesirably high. Suitable promoters for use in various vertebrate systems are well known and include, for example, Κ.δν LТК, ΜΡδν LТК, 8ν40, the metallothionein promoter, and ΟΜν SR can be successfully used.

Plasmid DNA may include prokaryotic and eukaryotic vectors and expression vectors, such as, for example, pBC.322 and pIS vectors and their derivatives. They can include various origin of replication, for example, prokaryotic origin of replication, such as pMB1 and Co1E1, or eukaryotic origin of replication, such as, for example, replicating origin of replication of yeast, fungus, insect and mammalian cells (e.g. δν40 op).

The insert may include DNA from any organism, but preferably the source of origin is a mammal and, in addition to the gene encoding the therapeutic protein, may include regulatory sequences, such as, for example, promoters, enhancers, locus controlling the region, selectable genes, polylinkers for insertion transgene, leader peptide sequences, introns, polyadenylation signals, or combinations thereof. The selection of vectors, sources, and genetic elements varies depending on needs and is well known to those skilled in the art. Selectable markers may be, for example, an antibiotic resistance gene, for example 8irRye tRNA, a tetracycline resistance gene, a kanamycin resistance gene, a puromycin resistance gene, a neomycin resistance gene, a hygromycin resistance gene, and a thymidine kinase resistance gene. The basis of the plasmid mainly allows the insertion of DNA fragments of mammals, other eukaryotic, prokaryotic or viral DNA, and the resulting plasmid can be cleaned and used применятьη νίνο or ex νίνο in plasmid-based therapy.

A plasmid DNA with a conditional replicator is preferably used, such as, for example, the plasmid pCOI described in published application US 2003/1618445. The resulting high copy number greatly increases the ratio of plasmid DNA to chromosomal DNA, RNA, cell proteins and cofactors, improves plasmid yield and facilitates easier subsequent purification. Thus, according to the invention, any plasmid DNA can be used. By way of non-limiting examples, representative vectors include the plasmid XU1ECE. ХУ1ЕСЕ is an encoding acidic fibroblast growth factor or type 1 fibroblast growth factor (ECE-1) plasmid suitable for treating patients with end-stage peripheral arterial occlusion disease (ΡΆΟΌ) or with peripheral artery disease (ΡΆΌ). Sachego1a e1 a1. (I. ν; · ι ^. 8igd., 2002, 35, 5: 930936) described that 51 patients with restless terminal stage ΡΆΌ with pain at rest or tissue necrosis in the ischemic thigh and lower leg were injected intramuscularly with XU1ESE in an increasing single or repeated doses. Then, various parameters were determined, such as transdermal oxygen pressure, ankle and finger-shoulder indices, pain analysis and ulcer healing. After administration of HU1ECE, a significant increase in shoulder indices, a decrease in pain, a clear size of ulcers, and an improvement in perfusion were observed.

In yet another aspect, the present invention relates, as indicated hereinabove, to a composition for use in a method of treating a human or animal organism through therapy. Preferably, the composition of the present invention contains a plasmid pCOI encoding an angiogenic gene of the ECE or VECE family for the treatment of cardiovascular diseases such as peripheral ischemia, peripheral arterial diseases, such as ΡΆΟΌ or ΡΆΌ, critical limb ischemia (CI), and intermittent claudication DS).

As another preferred use, plasmid DNA contains a polynucleotide encoding an immunizing peptide and can be used as a DNA vaccine. Thus, the present invention relates to compositions for vaccinating humans or animals, thereby inducing effective immunity against infectious agents including intracellular viruses, as well as against tumor cells. In fact, a stable plasmid DNA composition can be used as DNA vaccines to significantly enhance the immunogenicity of specific viral proteins and tumor-specific antigens that usually cause a weak immune response. They are useful for inducing cytotoxic T-cell-mediated immunity against weakly immunogenic herpes virus virus, ni-A, ni-B hepatitis and NS.

- 9 011554

Plasmid DNA can encode immunogenic polypeptides that can act as endogenous immunogens to elicit a humoral or cellular, or both, or, in addition, an antibody. In this sense, the term "antibody" includes complete immunoglobulin of any class, chimeric antibodies and hybrid antibodies with double or multiple specificity for an antigen or epitope, and fragments, such as E (aB) ₂ , EaB ', EaB, etc. p., including hybrid fragments. Also included in the term “antibody” are conjugates of such fragments and the so-called antigen binding proteins (single chain antibodies), as described, for example, in US Pat. No. 4,704,692 (the contents of which are incorporated herein by reference). Thus, plasmid DNA containing the polynucleotide encoding the variable regions of the antibody can be used to produce the ίη δίΐιι antibody. For an illustrative method related to the production of antibody-encoding polynucleotides, see \ Uay e! a1. Laiige, 341: 544-546 (1989); С1Ше8 е! A1., Vu1esipo1. 7: 799-804 (1989) and Yaaka1ap1 e! A1., 1os. p. 11, 805-810 (1989). In turn, the antibody may exhibit a therapeutic effect, for example, by binding a surface antigen associated with a pathogen. Alternatively, the encoded antibodies can be anti-idiotypic antibodies (antibodies that bind other antibodies), as described, for example, in US Pat. No. 4,699,880. Such anti-idiotypic antibodies can bind endogenous or foreign antibodies to the person being treated, thereby improving or preventing associated pathological conditions, for example, in the context of an autoimmune disease, with an immune response.

Thus, the composition of the present invention can be administered to humans or animals ίη νίνο, delivering plasmid DNA to various cells of the animal’s body, including muscle cells, skin, brain, lung, liver, spleen, or blood cells. The delivery of polynucleotides directly to ίη νίνο is preferred for muscle cells or skin. In order to ensure effective immunization of the subject, injections can be made, for example, into muscle or skin using a medical syringe or a syringe for vaccines. In fact, the antigen gene introduced into the subject’s cells, now transfected cells expressing the antigen, will be processed and presented to the immune system using conventional cellular mechanisms. To further enhance immunization, adjuvants or lymphokines can be administered simultaneously if possible.

For example, a stable composition of the present plasmid DNA can be used for vaccination against viruses or as a DNA vaccine for the treatment of latent viral infections, such as, for example, hepatitis B, HIV and caused by a group of herpes viruses, where the virus is stored in the cell in an inactive or partially active form . In addition, the plasmid DNA composition of the present invention can be used to treat a cancer to enhance the cellular immune response to the cancer-specific protein, oncogen, embryonic antigen or activation marker.

Plasmid DNAs (pDNAs) prepared for long-term storage of the present invention are typically obtained in bacterial cells, which are then lysed to release the contents of cells from which pDNA is isolated.

Typically, this process involves three stages, including cell resuspension: cell lysis, neutralization and precipitation of host cell contaminants. When resuspending cells, manual mixing or a magnetic stirring device and a homogenizer or impeller stirrer are usually used to resuspend the cells in resuspending buffer.

Cell lysis can be performed by spinning with hands or magnetic stirring to mix the resuspended cells with a lyse solution consisting of lysozyme or diluted alkali (base), such as, for example, alkaline or potassium acetate (KOAc) and detergents; then, to complete the lysis, the mixture was kept at room temperature (20-25 ° C) or on ice for a time interval, such as, for example, 5 min. RNase is also usually added to degrade the RNA of the bacterial suspension. The third stage is the neutralization and precipitation of the pollutants of the host cells. The lysate from the second stage is usually mixed with a cold solution to neutralize by gentle stirring or magnetic stirring to acidify the lysate before placing it on ice for 10-30 minutes to facilitate denaturation and precipitation of high molecular weight chromosomal DNA, host proteins and other host molecules.

When performing lysis using lysozyme treatment, bacteria interact with lysozyme and then they are boiled at approximately 100 ° C in the appropriate buffer for 20 to 40 s to form an insoluble clot from genomic DNA, protein and debris, leaving the plasmid in solution with RNA in as the main pollutant. After this, a mixed solution of IAOH and sodium dodecyl sulfate (δΌδ) is added to dissolve the cytoplasmic membrane. IaOH causes partial DNA denaturation and partial RNA degradation, while δΌδ acts to dissolve the membrane and denature proteins. Next, the δΌδ-protein complex and cell debris are precipitated by the addition of 5Ν potassium acetate (pH 4.8). At this stage, the pH is important to neutralize the NO used in the indicated operation and to renature the plasmid. After that, centrifuges are performed.

- 10 011554 to remove precipitates, thus obtaining the target plasmid DNA in the supernatant.

Alternatively, alkaline lysis is performed, consisting of mixing the bacterial cells with an alkaline lysis solution. An alkaline lysing solution consists of a detergent, for example, sodium dodecyl sulfate (8-8) for the lysis of bacterial cells and the release of intracellular matter, and an alkali, for example, sodium hydroxide to denature cell proteins and nucleic acids (specifically, gDNA and RNA). Since the cells are lysed and DNA is denatured, the viscosity of the solution increases dramatically. After denaturation, an acidic solution, such as potassium acetate (solution 3), is added to neutralize sodium hydroxide, stimulating the renaturation of nucleic acids. Long fragments of gDNA combine randomly and form flocculent networks, trapping proteins, lipids, and other nucleic acids. The potassium salt of dodecyl sulfate also settles, removing the proteins with which it is bound. Two interlinked strands of pDNA (plasmid DNA) are usually reassociated to restore the original plasmid remaining in the solution.

These chemical steps may be suitable for the lysis of cells in small numbers or small volumes of bacterial cultures less than 5 L, but an increase in viscosity may make it difficult to handle large quantities.

The lysis technique can be performed in serial production, i.e., where various solutions are mixed by the sequential addition of solutions to vessels and containers.

As an alternative to the batch method, especially when a large volume of the plasmid is to be obtained, continuous mixing of various solutions for cell lysis using a number of static mixers can be used. In these methods, a cell suspension solution and a cell lysis solution are simultaneously added to a static mixer. The lysed cell solution leaving the first static mixer and the precipitation solution are then simultaneously added to the second static mixer. The solution emerging from this second mixer contains precipitated lysate and plasmids. Other continuous methods of cell lysis include the use of a stream passing through a heat exchanger, where the suspended cells are heated to 70-100 ° C. After cell lysis in a heat exchanger, the effluent is centrifuged in a continuous fraction or in parts, in which cell debris and genomic DNA are precipitated, leaving plasmid DNA in the supernatant.

A preferred method for continuous alkaline lysis of a suspension of bacterial cells, especially on a large scale, is described in international patent publication XVO 05/02 6331, incorporated herein by reference. This preferred method addresses the problems caused by the viscoelastic properties of liquids and the frictional forces involved in the mixing stages, and provides a major advantage in limiting frictional forces. Therefore, a high yield of plasmid DNA can be obtained using the method of zooming in on a continuous alkaline lysis of host cells, described hereinafter.

In the first stage, host cells are infected, i.e. transform plasmid DNA at the exponential phase of cell growth and seeded in tablets containing LB medium, which includes an antibiotic, such as tetracycline. Then, each individual colony from the tablet is introduced into 20 ml of LB medium containing the corresponding antibiotic tetracycline into separate sterile Erlenmeyer plastic bottles and grown for 12-16 hours at 37 ° C in a shaking incubator. Then one of these cultures is used to infect 200 ml of sterile supplemented LB medium in 2 L Erlenmeyer bottles. They are grown at 37 ° C and 200 rpm in a shaking incubator and used to infect two 5 L Erlenmeyer bottles, and they are also grown at 30 ° C and 200 rpm in a shaking incubator and used to infect a fermenter vessel in the middle exponential phase after 5 hours and at ΟΌ600 nm, equal to 2 units.

Host cell cultures and infection are well known in the art. Typically, cell hosts grow until they reach high biomass, and the cells are in an exponential growth phase then to have a large amount of plasmid DNA. Two different methods can be used, i.e. cultivation with a single charge and cultivation with periodic feeding.

Single loading cultivation involves a growth rate controlled by controlling the growth temperature and the carbon source used. As used here, the term “one-shot culture” is a cell cultivation method in which all the nutrients necessary for cell growth and plasmid synthesis are contained in a large excess of cell culture vessel, such as, for example, up to 10-fold excess concentrations nutrients during infection, thus eliminating the need to make supplements in a sterile vessel after supplements after sterilization and the need for sophisticated feeding models and methods. In particular, the loading medium is supplemented with an excess of yeast extract from 5 (as in LB) to 20 g / l, thus providing huge amounts of growth factors and nucleic acid precursors. The medium is also supplemented with ammonium sulfate (5 g / l), acting as a source of organic nitrogen.

- 11 011554

Another type of cultivation is a culture with periodic feeding, in which the cell growth rate is controlled by the addition of nutrients to the culture during cell growth. As used herein, “batch-fed cultivation" refers to a cell culture method in which the growth rate is controlled by carefully measured culture metabolite additives during cultivation. The batch-fed culture of the invention allows a cell culture to achieve a higher biomass than a one-shot culture. Examples of the cultivation method and illustrative amounts of food addition are described below for 50 L of the composition. However, using the illustrative amounts of additives described below, it is also possible to process other volumes, for example 10, 50 or more than 500 L, depending on the size of the equipment. The highly enriched one-time loading medium and a batch-fed culture medium are designed to produce a culture with a high cell density to maximize the yield of a particular plasmid and to provide high biomass of cells still in the exponential growth phase. When cultivated with periodic feeding, glucose or glycerin is used as a carbon source. Cultivation is controlled in a batch mode until the starting carbon substrate (glucose) is depleted. This moment is noted by a rapid increase in ΌΟ and is confirmed by the analysis of glucose of a sample taken immediately after this event. After that, a pre-charged pump is switched on. The injection rate is determined using the obtained Cig1e88 e (a1. (Ref. 38: 1082-1090, 1991) model, which is fully incorporated by reference. The model is designed to facilitate control of the feeding stage in a batch fed method. In the initial serial production, growing with the maximum specific growth rate, the cells consume a non-inhibitory concentration of the substrate, providing a rapid increase in biomass after infection.Culture cannot grow at such a rate indefinitely due to the accumulation of toxic metabolites (Ex. gtep (a (yup Tesipoduod Iztd Kesotshap! Mkgogudashztz. ”1p Vyu (espoduodu, ebz. H.1. K1et and O. Keeb. ShetNepp: USN Ug1ad8de8e118syay tn 7b: 117-140, 1989). the models calculate the time-controlled feed rate of the growth-limiting carbon substrate without the need for a feedback control system, providing an operator-defined growth phase with periodic replenishment, which is chosen at a concentration that does not lead to the accumulation of inhibitory catabolites and is sufficient to obtain sokoy biomass. Additives of precursors (organic nitrogen in the form of ammonium sulfate) during the nutritional stage during cultivation with periodic feeding are designed to prevent harmful effects on the quality of the plasmid.

Lysis methods well known in the art can be used, including, for example, lysate-containing plasmid microbial cells by heating the stream. This method, among other things, is described in international patent publication SHO 96/02658. A particular heat exchanger consists of 10 ftx 0.25 inches in outer diameter of a spiral stainless steel pipe. The spiral is completely immersed in a water bath with constant high temperature. The enclosing volume of the spiral is approximately 50 ml. To measure the inlet and outlet temperatures and the temperature of the water bath, thermocouples and a thermometer were used, respectively. The product stream is pumped into a heated spiral using a peristaltic pump with a Ma8 silicone tube (exDex. After passing the spiral, the cell lysate is centrifuged in a pulse centrifuge to clean the Vesktap 1-21. After centrifugation, plasmid DNA can be purified using the purification method of the present invention.

Alternatively, cell lysis can be performed using sequentially arranged static mixers. As described in SHO 97/23601 (incorporated herein by reference), the first static mixer for lysing cells with a single static mixer and for precipitating a cell lysate, however, as an alternative cell lysis method to the plasmid DNA purification method of the present invention a second static mixer may be used. Using a sequentially located mixer, large volumes of cells can be carefully and continuously lysed, and other static mixers are arranged in series to perform other tasks, such as dilution and sedimentation. Acceptable static mixers suitable for the method of the present invention include any continuous flow device referred to in this area as a static or fixed mixer with sufficient length to allow the methods of the present invention. For example, for lysis of cells, a static mixer must have up to 5 sections, providing sufficient interaction time between the lysing solution and the cells, causing lysis of the exposed cells when passing through the mixer. Acceptable 5 static mixers contain an internal spiral structure, causing the two fluids to interact with each other in an oppositely rotating flow, causing the mixing of fluids in a turbulent flow.

The most preferred method or device for cell lysis includes:

(a) a turbulent flow device for rapidly mixing a cell suspension (see FIG. 1) with a cell lysing solution (solution 2 in FIG. 1) and

- 12 011554 (b) a device for laminar flow to allow incubation of the mixture formed in (a) without substantial mixing, where the mixture formed in (a) flows from the device for turbulent flow into the device for laminar flow.

In addition, it may comprise a device for adding a third solution neutralizing the lyse solution (solution 3 in FIG. 1), where the mixture incubated in (b) flows from the laminar flow device to the device for adding a second solution. Thus, for example, to isolate plasmid DNA from cells, this method can be applied, including:

(a) mixing the cells with an alkaline lysing solution in a turbulent flow device; and (b) neutralizing the alkaline lysing solution by adding an acidic solution.

In this method, T-shaped tubes are used until a viscoelastic fluid appears to continuously and very quickly mix the cell suspension (solution 1) and the alkaline solution (solution 2), thus providing a great advantage with respect to frictional forces. Typically, T-shaped tubes have a small tube diameter, usually with a diameter of less than cm, preferably about 2 and 8 mm, and more preferably about 6 mm, in order to increase the reaction time of the mixed liquids, but this method does not use stirring through the tube. Further in the table. 1 shows options for parameters B1a, B1b, B2 of a device for turbulent flow, laminar flow and turbulent flow, respectively, and their respective flow rates 81, 82, and 83, as shown in FIG. one.

Table 1

B1a (60 l / h) B1b (60 l / h) B2 (90 l / h) Flow rates Diameter Length Diameter Length Diameter Length 31, 32 and 33 Range from 5 Up to 7 mm 2-6 m from 12.5 Up to 19 mm from 13 to 23 m from 5 to 8 mm from 2 to 4 m 60/60/9 0 l / h ± 20%

The method can use a mixer or an injector with tubes instead of a T-shaped tube, which ensures the destruction of cells in a lysing solution. Accordingly, the mechanical stress of the fluids passing through the tubes is significantly reduced compared to when the fluids in the tanks are mixed, for example, with stirrers with blades. The initial mixing efficiency leads to even greater efficiency in seconds, from which it follows that this liquid does not yet have viscoelastic properties and the mixing carried out in a tube with a small diameter is very effective. On the contrary, when a T-shaped tube is used for mixing, the initial mixing only softens, and the liquid quickly becomes viscoelastic, leading to significant difficulties in flowing through the pipe. This partial mixing leads to the lysis of only part of the cells and therefore can only release part of the plasmids before neutralization. Lysis can be divided into two phases during lysis: phase I and phase II. These two phases correspond to I) lysis of cells and II) denaturation of nucleic acids, causing a significant change in rheological properties, leading to the formation of a viscoelastic fluid. Standardization of tube diameters can meet the needs of these two phases. Mixing is improved with a small tube diameter (B1a). This is a condition applicable to phase I. With a large tube diameter (B1b), mixing (and thus the friction force) is reduced. This is a condition applicable to phase II.

A preferred mixer used is a mixer designated as M1, as shown in FIG. 2, but any T-shaped device can also be used to ensure destruction of the cell suspension of the present invention. To obtain effective disruption, one of the ways to perform lysis with this mixer is to inject solution 1 into the back flowing alkaline lysis solution through one or more small diameter holes. The diameters of such holes can be from about 0.5 to 2 mm, and preferably about 1 mm, with this configuration. The mixture then passes mixer M1 to pass through the tube with a small diameter (Fig. 1) for a short period of time (approximately 2.5 s). To obtain a turbulent flow, a combination of diameter and current time can be easily calculated. Examples of options for these parameters are presented in table. 1. All references to the diameter of the tube are given for the inner diameter of the tube, not the outer diameter, which includes directly the wall thickness of the tube. Such a short lifetime in the tube provides a very fast homogenization of solutions 1 and 2. Provided that during phase I, solution 1 and solution 2 are still Newtonian fluids, in the homogenization phase the flow regime is turbulent. At the exit from this tube, solutions 1 and 2 are homogenized, and cell lysis begins in suspension.

Then the homogenized mixture passes through a second tube (B1b) of a significantly larger diameter (Fig. 1), in which the cells are lysed and a viscoelastic fluid is formed. During this phase, mixing can be minimized, and the solution should be “rested” to limit maximum turbulence in order to minimize any friction force that can fragment gDNA. Time

- 13 011554 interactions from about 1 to 3 minutes, about 2 minutes, and preferably 1 minute 20 seconds, may be sufficient to complete cell lysis and denaturation of nucleic acids. During the denaturation phase, the fluid flow regime may be laminar, causing slow diffusion of 8–8 and sodium hydroxide to the cellular components.

Thus, then the resulting lysate and solution 3 can be mixed to neutralize in mixer Υ designated as M2. In one embodiment, the internal diameter of the mixer Υ is from about 4 to 15 mm, or from about 6 to 10 mm, and may be about 6 mm or about 10 mm. A tube with a small diameter (for example, approximately 6 mm tube) is installed at the outlet of the mixer Υ to ensure quick (<1 s) and effective mixing of the lysate with solution 3. After that, the neutralized solution is collected in a collecting container.

When neutralized, a rapid decrease in pH causes the formation of flakes (i.e., the formation of lumps or clusters). On the other hand, the partially denatured plasmid renatures very quickly and remains in solution. Flakes settle gradually in a collecting tank, capturing most of the pollutants. In the schematic drawing in FIG. 1 shows one embodiment of a continuous lysis method (Cb). Continuous lysis can be used alone or with additional steps.

Any type of cell, i.e. prokaryotic or eukaryotic, can be lysed using this method for any lysis-related purposes, such as, for example, the release of desired, then purified plasmid DNA from target cells.

This method of the stage of continuous alkaline lysis can be performed on cells harvested as a result of cultivation, which was carried out to obtain high biomass of cells that have not yet reached the stationary phase and, thus, are in the phase of exponential growth (2-10 g dry weight / l). The step of continuous alkaline lysis can also be performed on cells harvested as a result of cultivation, which was carried out before obtaining a high biomass of the cells, and the cells are no longer in the exponential growth phase, but have reached a stationary phase with a cell concentration of about 10-200 g dry weight / l, preferably 12-60 g dry weight / l.

Plasmid DNAs can be purified using various methods before they are formulated in the stable storage composition of the present invention. In fact, plasmid DNA preparations derived from bacterial preparations often contain a mixture of relaxed and supercoiled plasmid DNA. Methods for purifying plasmid DNA are well known in the art.

Typically, methods for isolating and purifying plasmid DNA after culturing the bacteria consist of rupturing the plasmid-containing bacterial host cells as described above and neutralizing the lysate by neutralizing with acetate to precipitate genomic DNA and host cell proteins, which are then removed, for example by centrifugation. The liquid phase contains alcohol-precipitated plasmid DNA, which is then subjected to zone centrifugation using CgCl in the presence of ethidium bromide to separate various forms of plasmid DNA, i.e. super-twisted, annular with a single-stranded gap and linearized. After DNA precipitation with alcohol, additional extraction with butanol is necessary to remove the residual amount of ethidium bromide. The following are additional purification steps to remove host cell proteins. Typically, these methods are acceptable for small or laboratory scale plasmid preparations.

Alternative methods include, for example, size exclusion chromatography, hydroxyapatite chromatography, and various reverse phase or anion exchange chromatographic methods. These alternatives may be adequate to produce small quantities of the test substance on a laboratory scale, but cannot be easily expanded to obtain large quantities of plasmid DNA. For example, ion-exchange chromatography (EiPe s1 a1., 1oGi1o £ CHrothaudgarNu A, 606 (1998), 31-45) or size exclusion chromatography (Prhegeh., S., B.Ves11podu Tes11pk, etc.) are used as available methods for separating plasmid DNA. UO1. 1, No. 6, Liche 1997, p. 417-420) associated with the use of additives, such as polyethylene glycol (PEC), detergents and other components, such as cobalt hexamine, spermidine and polyvinylpyrrolidone (RUR). In known alternative processes, resins and solvents, such as acetonitrile, ethanol and other components such as triethylamine and ammonium tetrabutyl phosphate, are used in the treatment to separate supercoiled and relaxed forms of plasmid DNA.

In the event that nucleic acids or plasmid DNA is administered to humans or animals for therapeutic purposes, a highly purified pharmaceutical grade plasmid DNA is required, as such, purified nucleic acid must meet drug quality standards in terms of safety, activity and efficacy. Removal of contaminating endotoxins may be necessary, especially when plasmid DNA is purified from gram-negative bacterial sources with high levels of endotoxins. Typically, these endotoxins are lipopolysaccharides or fragments thereof, which are components of the outer membrane of gram-negative bacteria, and are present in the DNA preparation of host cells and cell membranes

- 14 011554 hosts or macromolecules. In the host receiving the plasmid DNA, they can cause inflammatory reactions, such as, for example, fever or sepsis. Therefore, for therapeutic or prophylactic use, endotoxin removal may be a key and necessary step in the purification of plasmid DNA. When removing endotoxin from plasmid DNA solutions, the negatively charged structure of endotoxins is mainly used. However, plasmid DNA is also negatively charged and, therefore, separation is usually achieved using anion exchange resins that bind both of these molecules and, under certain conditions, preferably plasmid DNA elutes upon binding of endotoxins. In addition, to obtain endotoxin-free nucleic acid contaminants that, when administered to a patient, can cause a toxic reaction, it may be desirable to obtain a highly purified nucleic acid free of toxic chemicals, mutagens, organic solvents or other reagents that could compromise the safety or effectiveness of the resulting nucleic acid. acids.

Prior to preparing plasmid DNA in a stable aqueous solution for long-term storage of the present invention, the plasmid DNA is preferably purified by a combination of chromatography steps to obtain a plasmid DNA preparation containing low amounts, i.e. parts per million (ppm) of contaminating chromosomal DNA, RNA, protein and endotoxins, and containing a predominantly closed circular form of plasmid DNA. More preferably, the purification methods described in international patent publication XVO 95/026331 and in international patent publication PCT / EP2005 / 005213 are used to obtain plasmid DNA for research applications and plasmid-based therapies, such as, for example, gene therapy and a DNA vaccine.

Purification methods include the use of triple helix affinity chromatography, which is preceded or followed by at least one additional chromatography method, optionally or usually as final purification steps, or at least at the final stage or before the final stage of the plasmid purification method. Three-helix affinity chromatography is used in combination with one or more chromatography steps, such as ion exchange chromatography, hydrophobic interaction chromatography, size exclusion or size exclusion chromatography, hydroxyapatite chromatography (type I and II), reverse phase chromatography and affinity chromatography. Any available affinity chromatography protocol involving nucleic acid separation can be adapted for use. Anion exchange chromatography or any one or more of the other chromatographic steps or methods used can be used, stationary phases, size exclusion chromatography methods, a fluidized bed method and / or continuous columns or systems. In addition, you can apply methods or systems of high performance chromatography, any one or more stages or methods.

Thus, the method preferably comprises purification steps including triple helix affinity chromatography with an additional step of ion exchange chromatography and, furthermore, may include hydrophobic interaction chromatography or gel permeation chromatography. The step of ion exchange chromatography may be moving bed ion exchange chromatography and high resolution axial and / or radial anion exchange chromatography. The most preferred method involves combining the steps of ion exchange chromatography, tri-helix affinity chromatography, and hydrophobic interaction chromatography. The first chromatographic step may be preceded by lysate filtration or another precipitate removal method.

Thus, continuous lysis can be combined with the above purification steps and result in a high purity product containing pDNA. It can be combined, for example, with at least one of the methods of sediment removal (such as, for example, lysate filtration, settling or centrifugation), ion-exchange chromatography (such as cationic or anion-exchange), three-spiral affinity chromatography and hydrophobic interaction chromatography. In one embodiment, continuous lysis in this order is followed by anion exchange chromatography, three-helix affinity chromatography, and hydrophobic interaction chromatography. Another continuous lysis in this order is followed by lysate filtration, anion exchange chromatography, three-helix affinity chromatography and hydrophobic interaction chromatography. These steps provide a truly large-scale method for plasmid production, with which large amounts of pDNA can be synthesized with unprecedented purity. DNA and RNA, as well as host proteins, are present in the range of less than ppm.

The method may also employ additional size exclusion chromatography steps (8EC), reverse phase chromatography, hydroxyapatite chromatography and / or other available chromatographic techniques, methods or systems in combination with the steps of the present invention described herein.

To obtain a higher purity of the resulting pDNA product, the precipitate can be removed. This step can be used to remove the precipitated substance (precipitate). One way to perform the removal of sediment is the stage of filtration of the lysate, such as, for example, through a strainer from 1 to

- 15 011554 mm and preferably 3.5 mm, followed by a deep filtration as the final filtration step. Other methods for performing sludge removal are centrifugation or sedimentation.

To obtain a higher purity of the resulting pDNA product, ion exchange chromatography can be performed. Anion exchange can be selected depending on the properties of pollutants and the pH of the solution.

To obtain higher purity of the resulting pDNA product, anion exchange chromatography can be performed. The principle of the operation of anion exchange chromatography is the binding of negatively charged (or acidic) molecules to a positively charged carrier. Thus, the use of ion exchange chromatography allows us to separate the molecules according to their charge. By this method, families of molecules (acidic, basic and neutral) can be easily separated. Stepwise elution methods can be used with many contaminants eluting in the early fractions and eluting in the later pDNA fractions. Anion exchange is very effective for removing protein and endotoxin from the pDNA preparation.

A sealant and a method for producing such a substance, as well as a method for producing, polymerizing and operating anion exchange chromatography and eluting and separating plasmid DNA for ion exchange chromatography are well known to those skilled in the art.

The compound used for the synthesis of basic substances that serve as a sealant for anion exchange chromatography can be any compound if various hydrophobic functional groups or various ion exchange groups can be introduced by subsequent interaction after synthesis of the basic substances.

Examples of monofunctional monomers include styrene, o-halogenmethyl styrene, m-halogenmethyl styrene, p-halogenmethyl styrene, o-haloalkyl styrene, m-haloalkyl styrene, p-haloalkyl styrene, α-methyl styrene, α-methyl-o-m-hal halogenmethylstyrene, α-methyl-p-halogenomethylstyrene, α-methyl-o-haloalkylstyrene, α-methyl-m-haloalkylstyrene, α-methyl-p-haloalkylstyrene, o-hydroxymethylstyrene, m-hydroxymethylstyrene, p-hydroxymethylstyrene, p-hydroxymethylstyrene m-hydroxyalkylstyrene, p-hydroxylalkylstyrene, α-methyl-o-hyd roxymethyl styrene, α-methyl-m-hydroxymethyl styrene, α-methyl-p-hydroxymethyl styrene, α-methyl-o-hydroxyalkyl styrene, α-methyl-m-hydroxyalkyl styrene, α-methyl-p-hydroxyalkyl styrene, glycidyl methacrylate, glycidyl acrylate, hydroxyethyl vinyl acetate.

Most preferred compounds are aromatic-substituted haloalkyl groups, halogens, such as C1, Bg, I and P, and straight and / or branched saturated hydrocarbons with carbon atoms from 2 to 15. Examples of polyfunctional monomers include divinylbenzene, trivinylbenzene, divinyltoluene, trivinyltoluene, divinylnaphthalene, trivinylnaphthalene, ethylene glycol dimethacrylate, ethylene glycol diacrylate, diethylene glycol dimethacrylate, diethylene glycol bisacrylate, methylene etacrylamide and methylene bisacrylamide.

Various ion exchange groups can be introduced through subsequent interaction. The preparation of the basic substance includes the first stage, where the monofunctional monomer and the multifunctional monomer are weighed in an appropriate ratio and a precisely weighed diluent or solvent used to standardize the pores in the formed particles is added in a similar way to a precisely weighed polymerization initiator, followed by thorough mixing. The mixture is then subjected to oil-in-water suspension polymerization, where the mixture is added to an aqueous solution of a pre-weighed dissolved suspension stabilizer and oil droplets with a target size are formed by stirring with a stirrer, and the mixture is polymerized by gradually heating the mixed solution. To obtain soft particles of the basic substance, the ratio of monofunctional monomer to polyfunctional monomer, as a rule, is approximately 1 mol of monofunctional monomer and from about 0.01 to 0.2 mol of multifunctional monomer. The polymerization initiator is also not particularly limited and the azo-bis type and / or peroxide type initiators used are commonly used.

Suspension stabilizers, such as, for example, ionic surfactants, nonionic surfactants and amphipathic polymers or mixtures thereof, can also be used to prevent aggregation among the oil droplets themselves.

The sealant used for ion exchange chromatography for purification of plasmid DNA preferably has a relatively large pore diameter, specifically within the range of 1500 to 4000 A. Surface modification is well known in the art for introducing ion exchange groups into basic substances in this field.

Two types of eluents can be used for ion exchange chromatography. A first eluent containing a low salt concentration and a second salt containing a high salt concentration can be used. The elution method consists in stepwise switching from the first eluent to the second eluent and the gradient elution method, continuously changing the composition from the first eluent to the second

- 16 011554 eluent. Buffer solutions and salts commonly used in these eluents for ion exchange chromatography can be used. For a low concentration salt of the first eluent, an aqueous solution with a buffer concentration of 10 to 50 mM and a pH value of 6 to 9 are particularly preferred. For a high concentration of salt of the second eluent, an aqueous solution with an added from 0.1 to 2 M sodium salt in eluent C. With respect to sodium salts, sodium chloride and sodium sulfate can be used.

In addition, a chelating agent, such as ethylenediaminetetraacetic acid, can be used to inhibit the degradation of plasmids due to DNA-degrading enzymes in the ExExepysoy lysate for a divalent metal ion. The concentration of the chelating agent for the divalent metal ion is preferably from 0.1 to 100 mm.

A wide range of commercially available anion exchange matrices are acceptable for use in the present invention as non-limiting examples, including matrices available from POK. O8 Άηίοη Exxayide Ks81I8, Οίαβοη. Toyu Naak, 8th year, 8th year, Yiskoras and Ryagtaaa. For example, a column (Roy II P1 / M, 4.5 mm x 100) is initially balanced with 20 mM B18 / TP8 Proriae at pH 7.5 and 0.7 M No. 1C1. The sample is applied to the column and washed with the same stock buffer solution. Then an elution gradient of 0.5 to 0.85 M No. 101 was applied in approximately 25 column volumes and fractions were collected. Preferred anion-exchange chromatography includes Egas! Ode1 TMAE H1Car.

Three-helix affinity chromatography is described, inter alia, in US Pat.

Three-helix affinity chromatography is based on the specific hybridization of oligonucleotides and the target sequence in double-stranded DNA. These oligonucleotides may contain the following bases:

thymidine (T) capable of forming triplets with A.T doublets of double-stranded DNA (Ka) adora1 e! A1., Vusyesh 28 (1989), 7859);

adenine (A) capable of forming triplets with A. T doublets of double-stranded DNA; guanine (O), capable of forming triplets with doublets of O. C double-stranded DNA; protonated cytosine (C ⁺ ), capable of forming triplets with O doublets. With double-stranded DNA (Ka) adora1 e! A1., 1os. Sy.);

uracil (I), capable of forming triplets with base pairs A. and A.T.

Preferably, the oligonucleotide used contains a cytosine-rich homopyrimidine sequence, and the specific sequence represented in the DNA is a homopurin-homopyrimidine sequence. The presence of cytosines makes it possible to obtain a triple helix stable at acidic pH, where cytosines are protonated, and destabilized at alkaline pH, where cytosines are neutralized.

The oligonucleotide and the specific sequence presented 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 a fully complementary oligonucleotide and a specific sequence. For example, a poly oligonucleotide (CTT) and a specific poly sequence (OAA). Preferred oligonucleotides have the sequence 5'-OAOOSTTSTTSTSTSTTSTSTTSTT-3 '(6L60 (CTT)) (8EO GO No. 1), in which the bases of the OAOO do not form a triple helix, but allow the oligonucleotide to be separated from the connecting bond; sequence (CTT) 7. These oligonucleotides are capable of forming a triple helix with a specific sequence containing complementary units (OAAs). The sequence in question may, in particular, be a region containing 7, 14 or 17 OAA units, as described in the examples.

Another sequence of particular interest is the sequence 5'-AAOOOAOOOOAOOAOAOOAA-3 '(8EO W No. 2).

This sequence forms a triple helix with oligonucleotides 5'-AAOOAOAOOAOOOAOOOAA-3 '(8ЕО Ш No. 3) or 5'-ТТУТОТОООООООООТТ-3' (8ЕО Ш No. 4). In this case, the oligonucleotide binds in an antiparallel orientation to the polypurine chain. These triple helixes are stable only in the presence of Mg ^ (Uaktsiek e! A1., Vusyetzkyu, 1995, 34, 72437251; Bea1 aib Oeguai, 8c1eise, 1991, 251, 1360-1363).

As indicated above, the specific sequence may be a naturally occurring sequence in double-stranded DNA or a synthetic sequence artificially inserted into the latter. It is particularly preferable to use an oligonucleotide capable of forming a triple helix with a naturally occurring sequence in double-stranded DNA, for example, at the origin of plasmid replication or in the marker gene. In this regard, it is known from sequence analysis that some regions of these DNA, especially at the origin of replication, may have homopurin-homopyrimidine regions. The synthesis of capable of forming triple helices with these natural homopurin-homopyrimidine regions of oligonucleotides successfully allows the method of the invention to be applied to unmodified plasmids, in particular commercial plasmids pIS, pBK.322, p8U, etc. type. Among those found in nature in double-sleeping

- 17 011554 DNA of homopurine-homopyrimidine sequences, you can specify a sequence containing all or part of the 5'-STTSSSSAASSSSAAAASS-3 'sequence present at the E.co11 replication start point of the Co1E1 plasmid (Betz GO No. 5). In this case, the triple helix forming oligonucleotide has the sequence 5'-CAASSSTSTSSSTSTTTSS-3 '(Betz GO No. 6) and alternatively binds to two double helix chains, as described in Bea1 apb Oeguap (I. At. Bos. 1992, 114, 4976-4982) and Dauakeya apb 1o11pk1op (Yis1e1s Ashbk Kek. 1992, 20, 5279-5288). You can also specify the sequence of the β-lactamase gene 5'-CAAAAAACCAASAC-3 '(Betz GO No. 7) of the plasmid pBK322 (Pyua1-Va1epyi!

Appropriate target sequences capable of forming triple structures with specific oligonucleotides have been identified at the replication start points of Co1E1 plasmids and pCOK plasmids. The pCOK plasmids are plasmids with a conditional origin of replication and, among other things, are described in IB 2004/142452 and IB 2003/161844. The plasmids obtained from Co1E1 contain a 12-membered homopurine sequence (5'-ACAAAAAAASSA-3 ') (BetZ GO No. 8) mapped before the RNA-11 transcript involved in the replication of the plasmid (Eacaepa e! A1., 1981, Ba1ige, 294, 623). This sequence forms a stable triple structure with a 12-membered complementary oligonucleotide 5'-TSTTTTTTTTSST-B '(Betz GO No. 9). The pCOK chain contains a homopurine region of 14 non-repeating bases (5'-AA6AAAAAAAA6AA-3 ') (Betz GO No. 10) located in the A + T-rich segment of the pCOK replication origin γ point (Euseuyepko e! A1., 1996, Lyshs Ac16k Kek ., 24, 1936). This sequence forms a stable triple structure with a 14-membered complementary oligonucleotide 5'-TTSTTTTTTTTTSTT-3 '(BETZ GO No. 11). The corresponding oligonucleotides 5'-TSTTTTTTTSTS-3 '(BER GO No. 8) and 5'-TTTTTTTTTTTTT-3' (BES) GO No. 11) efficiently and specifically label their corresponding Co1E1 op or pCOK (op · /) replication origin complementary sequences. In fact, a separate noncanonical triad (T * SS or C * AT) can lead to complete destabilization of the triple structure.

The use of an oligonucleotide capable of forming a triple helix with the sequence shown at the origin of the replication or marker gene is particularly advantageous since it allows the purification of any DNA containing the indicated origin of replication or the indicated marker gene with the same oligonucleotide. Therefore, there is no need to modify a plasmid or double-stranded DNA in order to introduce a synthetic specific sequence into it.

Although fully complementary sequences are preferred, it is nevertheless understood that some inconsistencies may be tolerated between the oligonucleotide sequence and the sequence represented in the DNA, provided that they do not result in too much loss of affinity. You can specify the sequence 5'-AAAAAA666AATAA666-3 'presented in the β-lactamase E.coH gene (Betz GO No. 12). In this case, the thymine breaking the polypurine sequence can recognize the third chain guanine, thereby forming a C * TA triplet, stable in cases when it is flanked by two T * AT triplets (K1ekk1td e! A1., VusyetShgu, 1992, 31, 28292834).

In a particular embodiment, the oligonucleotides of the invention comprise a sequence (CCT) p, a sequence (CT) p, or a sequence (CTT) p, where p is an integer between 1 and 15 inclusive.

It is especially advantageous to use sequences of the type (CT) _p or (CTT) _p . In fact, the inventors showed that the yield after purification depends on the amount of C in the oligonucleotide. Specifically, as shown in Example 7, the yield after purification is increased if the oligonucleotide contains less cytosines. It should be understood that the oligonucleotides of the invention can also combine units (CCT), (CT) or (CTT).

The oligonucleotide used may be natural (consisting of unmodified natural bases) or chemically modified. Specifically, the oligonucleotide may preferably have certain chemical modifications that provide it with resistance or protection against nucleases or increase its affinity for a specific sequence. It is also understood that an oligonucleotide means any linked nucleoside sequence susceptible to modification of the structure to make it more resistant to nucleases. Among the possible modifications, oligonucleotide phosphorothioates capable of forming triple helices with DNA (Khobo e! A1., Hysshs Ac16k Kek., 1994, 22, 3322-3330), as well as oligonucleotides containing skeletons with formacetal or methylphosphonate (MaEiss! E) can be mentioned. ! a1., I. At. Syet. Bos, 1991, 113, 7767-7768). It is also possible to use oligonucleotides synthesized with α-anomers of nucleotides, which also form triple helices with DNA (Be Eoap e! A1., Lyshs Ashbk Kek., 1987, 15, 7749-7760). Another modification of the skeleton is a phosphoramidate bond. For example, you can specify the internucleotide phosphoramidate bond Ν ³ -Ρ ⁵ described in Schuahpow apb Syep, which gives oligonucleotides that form especially stable triple helices with DNA (I. At. Syet. Bos., 1994, 116, 3143-3144). Among other skeleton modifications, the use of 2'-O-methylribose ribonucleotides, phosphodiester, etc. can also be indicated. (Beep apb Ne1epe, Sigg. Orshyup B1gis1. Vu1., 116, 3143-3144). Finally, the skeleton on wasps

- 18 011554 new phosphorus can be replaced by a polyamide backbone, as in PNA (peptide nucleic acids), also capable of forming triple helices (Mekep s1 a1., 8s1epse, 1991, 254, 1497-1500; K1t e1 a1., 1. At. Szet. Zos., 1993, 115, 6477-6481), or a skeleton based on guanidine, as in DNG (deoxyribonucleic guanidine, Proc. No. 111. Asab. 8c1. I8A, 1995, 92, 6097-6101), or also forming triple helices by polycationic DNA analogues.

It is also possible to replace thymine of the third chain with 5-bromouracil, which increases the affinity of the oligonucleotide for DNA (Roch81c apb Eeguap. 1. At. Syet. 8os., 1989, 111, 3059-3061). The third chain may also contain non-natural bases, among which 7-dease-2'-deoxyxanthosine can be mentioned (MbIdap e1 a1., No. 1s1e1s As1bk Kek., 1993, 21, 327-333), 1- (2-deoxy-3 E-ribofuranosyl) -3-methyl-5-amino-1-pyrazole [4,3-b] pyrimidin-7-one (Coy apb Eeguap, 1. At. Syet. 8os, 1992, 114, 1470-1478), 8 -oxoadenine, 2-aminopurine, 2'-O-methylpsevdoisocytidine, or any other modification known to a person skilled in the art (for information see 8ip apb Ne1pe, Art. Orlshop 81gis1. Vyu., 1993, 3, 345-356).

Another type of modification of the oligonucleotide is to more specifically enhance the interaction and / or affinity between the oligonucleotide and the specific sequence. Specifically, the most advantageous modification of the invention is the methylation of cytosines of an oligonucleotide. Thus, the methylated oligonucleotide demonstrates a noteworthy property of the formation of a triple helix with a specific sequence in close to neutral (> 5) pH ranges. Therefore, this allows working at higher pH values than for oligonucleotides of the previous region, i.e., at pH values, where the risks of plasmid DNA degradation are much lower.

The length of the oligonucleotide used in the method according to the invention is between 5 and 30. It is preferable to use an oligonucleotide with a length of more than 10 bases. To obtain the desired selectivity and stability of interaction, a person skilled in the art can adapt the length for each particular case.

The oligonucleotides of the invention can be synthesized using any known method. Specifically, they can be obtained using a nucleic acid synthesizer. It is obvious that you can apply any other method known to the person skilled in the art.

To ensure covalent binding to the carrier, the oligonucleotide is typically functionalized. Thus, it can be modified with a thiol, amine or carboxyl end group at the 5 ′ or 3 ′ position. Specifically, the addition of a thiol, amine or carboxyl group makes it possible, for example, to bind an oligonucleotide to a carrier bearing a disulfide, maleimide, amine, carboxyl, ester, epoxy, bromine or aldehyde functional groups. Such crosslinking is formed by introducing disulfide, thioether, ester, amide or amine bonds between the oligonucleotide and the carrier. Any other method known to those skilled in the art can be used, such as, for example, bifunctional binding reagents.

In addition, to enhance hybridization with the linked oligonucleotide, the content of the “shoulder” and “spacer” base sequences may be advantageous for the oligonucleotide. The use of the shoulder actually allows you to bind the oligonucleotide at a selected distance from the carrier, helping to improve the conditions for its interaction with DNA. Preferably, the arm consists of an unbranched carbon chain containing from 1 to 18 and preferably 6 or 12 groups (CH ₂ ), and provides binding to the amine column. The shoulder is connected to the phosphate of the oligonucleotide or “spacer” consisting of non-hybridizing bases. Thus, the “spacer” may contain purine bases. As an example, a spacer may comprise a CASS sequence. The arm preferably consists of 6 or 12 carbon atoms of an unbranched carbon chain.

Triplex affinity chromatography is very effective for removing RNA and genomic DNA. They can be functionalized, non-packaged chromatographic media, or prefilled in a column, functionalized plastic surfaces, or functionalized latex spheres, magnetic, or others. Chromatographic media are preferably used. Suitable chromatographic supports as an example are agarose, acrylamide or dextran, as well as their derivatives (such as Zeryabeh, Zeryagoke, Ziregoke and others), polymers such as, for example, poly (styrene divinylbenzene) or grafted or ungrafted silica . Chromatographic columns can be used in diffusion or perfusion mode.

To obtain higher yields after purification, it is especially advantageous to use a plasmid sequence containing several hybridization points with an oligonucleotide. In fact, the presence of several hybridization points activates the interactions between the indicated sequence and the oligonucleotide, which leads to an increase in the yields after purification. Thus, for an oligonucleotide containing c repeating fragments (CCT), (CT), or (CTT), it is preferable to use a DNA sequence containing at least p complementary motifs, and preferably n + 1 complementary motif. Thus, the sequence carrying the n + 1 complementary motif represents two points of hybridization with the oligonucleotide. Preferably after

- 19 011554 DNA contains up to 11 points of hybridization, i.e. n + 10 complementary motives.

The method of the present invention can be used to purify any type of double-stranded DNA. An example of the latter is ring DNA, such as a plasmid, typically including one or more genes of therapeutic value. This plasmid may also include an origin of replication, a marker gene, and the like. The method according to the invention can be performed directly in the cell lysate. In this embodiment, the amplified transformation followed by culturing the cells, the plasmid is purified immediately after cell lysis. Also, the method according to the invention can be performed in a purified lysate, i.e. in the obtained after neutralization and centrifugation of the cell lysate of the supernatant. It is quite obvious that it can also be performed in a solution previously purified by known methods. This method also allows you to clean including an important sequence of linear or circular DNA from a mixture containing DNA with different sequences. The method of the invention can also be used to purify double-stranded DNA.

The cell lysate may be a lysate of prokaryotic or eukaryotic cells.

As for prokaryotic cells, bacteria Ε.οοίί can be mentioned as examples. B. kib1Ϊ1Ϊ8, 8.1uritit1it or 81herotus§. Regarding eukaryotic cells, one can indicate cells of animals, yeast, fungi, etc. , and mainly the yeast K1tsueguotus§ or Zassyagotusek, or cells of CO8, CHO, C127, ΝΙΗ3Τ3, etc.

To obtain a higher degree of purification of the final pDNA product, the method of the present invention, comprising at least a step of triplex affinity chromatography, can be used. In triplex affinity chromatography, an oligonucleotide is coupled to a carrier, such as a chromatographic resin or other matrix. Thereafter, the sample to be purified is mixed with the bound oligonucleotide, such as, for example, by depositing the sample on a chromatographic column containing an oligonucleotide bound to a chromatographic resin. The desired plasmid in the sample binds to the oligonucleotide to form a triplex. The bonds between the oligonucleotide and the plasmid can be Hoogsteen interactions. Such a transition can occur at pH <5, at high salt concentration, with a reaction time of 20 minutes or more. You can apply the stage of washing. Finally, in a neutral buffer solution, deprotonation of cytosine occurs with elution of the plasmid from the oligonucleotide-bound resin.

In hydrophobic interaction chromatography, hydrophobic substances on a substrate are used to attract hydrophobic sites in the sample molecules for purification. It should be noted that these H1C carriers work using the “clustering” effect; in the case of binding of these molecules, non-covalent or ionic bonds are formed or distributed.

Chromatography of the hydrophobic interaction is useful because it very effectively removes open circular plasmid forms and other contaminants, such as gDNA, RNA, and endotoxin.

The synthesis of basic substances for hydrophobic interaction chromatography, as well as the method for producing, polymerizing and functionalizing hydrophobic interaction chromatography and the elution and separation of plasmid DNA through it, are well known in the art and, inter alia, are described in US Pat. No. 6,441,160, incorporated herein by reference.

Compounds used for the synthesis of basic substances, which serve as a sealant in hydrophobic interaction chromatography, can be any compounds if various hydrophobic functional groups or various ion-exchange groups can be introduced by subsequent interaction after the synthesis of basic substances.

Examples of monofunctional monomers include styrene, o-halogenmethyl styrene, m-halogenmethyl styrene, p-halogenmethyl styrene, o-haloalkyl styrene, m-haloalkyl styrene, p-haloalkyl styrene, α-methyl styrene, α-methyl-o-m-hal halogenmethylstyrene, α-methyl-p-halogenomethylstyrene, α-methyl-o-haloalkylstyrene, α-methyl-m-haloalkylstyrene, α-methyl-p-haloalkylstyrene, o-hydroxymethylstyrene, m-hydroxymethylstyrene, p-hydroxymethylstyrene, p-hydroxymethylstyrene m-hydroxyalkylstyrene, p-hydroxylalkylstyrene, α-methyl ogide oksimetilstirol, α-methyl-m-gidroksimetilstirol, α-methyl-p-gidroksimetilstirol, α-methyl-ogidroksialkilstirol, α-methyl-m-gidroksialkilstirol, α-methyl-p-gidroksialkilstirol, glycidyl methacrylate, glycidyl acrylate, hydroxyethyl acrylate, gidroksimetakrilat and vinyl acetate.

The most preferred compounds are aromatic substituted halogenated alkyl groups, halogens, such as C1, Br, I and P, and straight and / or branched saturated hydrocarbons with carbon atoms from 2 to 15.

Examples of polyfunctional monomers include divinylbenzene, trivinylbenzene, divinyltoluene, trivinyltoluene, divinylnaphthalene, trivinylnaphthalene, ethylene glycol dimethacrylate, ethylene glycol diacrylate, di-diamethylene bis-dimethacrylate, dimethacrylate bimethyl

Different hydrophobic functional groups or different ion exchange groups can

- 20 011554 enter through subsequent interaction. To minimize the effect on the desired products for separation due to the hydrophobicity exhibited by the basic substance itself, or an increase or decrease in the basic substance due to changes in salt concentration and pH, the basic substance is preferably prepared using relatively hydrophilic monomers such as glycidyl methacrylate, glycidyl acrylate hydroxyethyl acrylate, hydroxymethyl acrylate and vinyl acetate. The preparation of the basic substance includes the first stage, where the monofunctional and multifunctional monomers are weighed in an appropriate ratio and a precisely weighed diluent or solvent is used to standardize the pores in the formed particles, and a precisely weighed polymerization initiator is added in the same way, followed by thorough mixing. The mixture is then subjected to oil-in-water suspension polymerization, where the mixture is added to an aqueous solution of a pre-weighed dissolved suspension stabilizer and oil droplets with a target size are formed by stirring with a stirrer, and the mixture is polymerized by gradually heating the mixed solution.

To obtain soft particles of the basic substance, the ratio of monofunctional monomer to polyfunctional monomer, as a rule, is approximately 1 mol of monofunctional monomer and from about 0.01 to 0.2 mol of multifunctional monomer. To obtain hard particles of basic substances, the ratio of the multifunctional monomer can be increased from about 0.2 to 0.5 mol. To obtain even stiffer particles, only a multifunctional monomer can be used.

The polymerization initiator is also not particularly limited and the azo-bis type and / or peroxide type initiators used are commonly used.

Suspension stabilizers, such as, for example, ionic surfactants, nonionic surfactants and amphipathic polymers or mixtures thereof, can also be used to prevent aggregation among the oil droplets themselves.

The diameter of the formed particles, as a rule, is approximately from 2 to 500 microns. A preferred particle diameter is from 2 to 30 microns and more preferably from about 2 to 10 microns. If it is necessary to purify a large number of nucleic acids with a high degree of purity, the particle diameter is from about 10 to 100 microns, and when the target product is separated from the crude mother liquor, it can be from 100 to 500 microns, more preferably from about 200 to 400 microns. To standardize the particle diameter, you can select the speed of rotation of the mixer during polymerization. If particles with a small diameter are necessary, the number of rotations can be increased, and if large particles are desired, the number of rotations can be reduced. In this work, the choice of solvent is especially important, since the solvent used is used to standardize the pores in the formed particles. As a basic concept for the solvent used for the polymerization, the adjustment is carried out by various combinations of a weak solvent for the monomer and a good solvent for the monomer. The pore diameter size can be selected, respectively, depending on the size of the nucleic acids to be separated, but it is preferably in the range from 500 to 4000 A for the sealant for hydrophobic interaction chromatography and in the range from 1500 to 4000 A for the sealant for ion exchange chromatography.

In hydrophobic interaction chromatography, for the separation of nucleic acids with different hydrophobicity, it is preferable to use seals with different hydrophobicity, respectively, modification of the surface of the basic substance is important.

Hydrophobic groups can be selected from long chain or branched groups, including saturated hydrocarbon groups or unsaturated hydrocarbon groups with carbon atoms from 2 to 20. The hydrocarbon group may also contain an aromatic nucleus.

Hydrophobic groups can also be selected from compounds with the following formula:

Main substances

where η is from 0 to about 20 and the methylene group may be a straight or branched chain;

m is from 0 to about 3, and the hydrocarbon group may be a straight or branched chain;

A represents a C = O group or an ether group, but the methylene group may be bound to the basic substance without A.

Hydrophobic groups may also include an ether group of alkylene glycol with a carbon number of 2 to 20, consisting of from 0 to 10 repeating units, where the opposite end of the functional group interacting with the basic substance may be an OH group leaving this way or may overlap with an alkyl group with the number of carbon atoms from 1 to 4.

- 21 011554

To modify the surface, the hydrophobic groups described above can be used alone or in a mixture.

A chain of alkyl groups with a carbon number of 6 to 20 carbon atoms is preferred for plasmids with low hydrophobicity; a long chain of alkyl groups with a number of carbon atoms from 2 to 15 - for the isolation of compounds with high hydrophobicity, such as, for example, that obtained from EcenePea co11 RNA and RNA in human and animal cells; alkyl groups with the number of carbon atoms from 4 to 18 - for the isolation of compounds with relatively low hydrophobicity, such as, for example, DNA and DNA obtained from ExNepsyse in human and animal cells.

When separating these compounds, compounds for surface modification can be selected appropriately, not limited to this example. In fact, the degree of hydrophobicity of the sealant 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 sealant differs depending on the number of groups introduced into the basic substance.

The pore diameter of the base material for hydrophobic interaction chromatography is particularly preferably 500 to 4000 A, but it can be appropriately selected from the indicated range depending on the size of the molecules desired for nucleic acid separation. Usually, since the retention of nucleic acids on the sealant and the adsorption capacity (sample yield) differ depending on the pore diameter, for nucleic acids with a large molecular size it is preferable to use a basic substance with a large pore diameter and for nucleic acids with a small molecular size - the main substance with small pore diameter.

For example, a styrene base material may interact with a hydrophobic group containing a long chain of alkyl groups using a halogen-containing compound and / or a carbonyl halide and a catalyst, such as, for example, HerC1 ₃ , 8iC1 ₂ or A1C1 ₃ , and using the Friedel-Crafts reaction, directly into the aromatic nucleus into a basic substance, such as, for example, a dehalogenated compound and / or an acylated compound. The functional group can be introduced via an ether bond in the case of the basic substance, which is a fraction containing a halogen group, for example, using compounds containing OH, such as butanol, and using a Williamson reaction with an alkaline catalyst, such as ΝαΟΗ or KOH. If a functional group is desired to be introduced, which is a compound containing an amino group, such as, for example, hexylamine, it is possible to add using an alkaline catalyst such as No. 1OH or KOH and using an acid dehalogenation reaction. In the case of a basic substance containing an OH group, on the contrary, by introducing an epoxy group, a halogen group or a carbonyl halide group, the functional group can be introduced into the functional group desired for introduction via an ether or ester bond. A functional group can be introduced via an ether or amine bond in the case of an epoxy-containing basic substance, by reaction with a compound with an OH group or an amino group contained in a functional group desired for administration. In addition, in the case where a functional group containing a halogen group is desired for introduction, the functional group can be introduced via an ether bond using an acid catalyst. Since a part of the functional group introduced into the basic substance is influenced by the hydrophobicity of the desired product for separation, it cannot be reduced, but a binder material with approximately 0.05 to 4.0 mmol added functional group per gram of dry basic substance is usually suitable.

With respect to surface modification, a method of adding a functional group by subsequent reaction after the formation of a basic substance or particles is the described method. Surface modification is carried out by the same method, where the main substance is formed after polymerization using monomers with the indicated functional groups added before polymerization.

The base material may also be porous silica gel. A method for producing silica gel involves the addition of a silane using a compound, such as, for example, alkyltrimethoxysilane directly to the particles obtained by the method described in "La1e81 H1dy-8rea ί-ίςshy Sygota Fdgarju", p. 289 ίί. (\\ TTS1ep LU Tokyo nypaga apy Νοόιιο 1 keda ^ a, riNKkey Ly Tokuo Npoka \\ 'a VookkFge ίη 1988). Before or after the addition of a silane using a binder containing a silane epoxy group, a functional group can be added according to the above method. Acceptable is the part of the functional group, introduced from about 0.05 to 4.0 mmol of the functional group added per 1 g of dry basic substance.

Eluents are used in the separation by hydrophobic interaction chromatography or in the purification step. As a rule, two types of eluents are used. One eluent contains a high concentration of salt, while the second eluent contains a low concentration of salt. The elution method includes a stepwise switch from an eluent with a high salt concentration to an eluent with a low salt concentration, and a gradient elution method can be applied that continuously switches the composition from one eluent to another. Buffer solutions and salts can be used.

22 011554 for hydrophobic interaction chromatography. For an eluent containing a high concentration of salt, an aqueous solution with a salt concentration of 1.0 to 4.5 M and a pH value of 6 to 8 is particularly preferred. For an eluent containing a low concentration of salt, particularly preferred salts are an aqueous solution with a salt concentration of 0.01 up to 0.5 M and a pH value of 6 to 8. As a rule, ammonium sulfate and sodium sulfate can be used as salts.

The purification step of chromatography of the hydrophobic interaction of plasmid DNA can be carried out by sequentially combining a binder material with an introduced functional group with weak hydrophobicity with a binder material with an introduced functional group with strong hydrophobicity. In fact, the culture medium of Escherichia coli contains in large quantities various components differing in hydrophobicity, such as polysaccharides, Eczepsysoi gDNA, plasmid RNAs and proteins. It is also known that there are differences in hydrophobicity even among the nucleic acids themselves. Proteins that become contaminants have a higher hydrophobicity compared to plasmids.

Many hydrophobic interaction chromatography resins, such as 1tac1ore1 ogoru1, Touoreag1, 8oigse 1orgoru1, or any other hydrophobic group resins, are commercially available. The most preferred resins are bulky polymer substances Touoreag1. Touoreag1 is a methacrylic polymer with high mechanical and chemical stability. Resins are suitable as non-functionalized 11 \ C series resins, and derivatives can be obtained using surface chemistry for ion exchange chromatography or hydrophobic interaction chromatography. Four types of Touoreag 1 AL resins with different surface chemistry and levels of hydrophobicity can be used. The hydrophobicity of Touoreag1 AL resins increases in the series: E1eeg, Riepu1, Vi1u1 and Nehu1. Structures of the preferred resins Touoreag 1 GC, i.e. Touoreag1 11 \\ '- 65, with a pore diameter of 1000 A are shown below:

Touoreag1 resins described above may have a different particle size class. Touoreag1 650C has a particle size of about 50 to 150 microns, preferably about 100 microns, while Touoreag1 650M has a particle size of about 40 to 90 microns, preferably about 65 microns, and Touoreag1 6508 has a particle size of about 20 to 50 microns, preferably approximately 35 microns. It is well known that particle size affects the release, i.e. the release improves from C to M and to particle size class 8 and thus increases with decreasing particle size. The most preferred Touoreag1 resin used in the GC chromatography step in the process of separating and purifying the plasmid DNA of the present invention is Touoreag1 Liu1G6508, launched into serial production by Toza Wojs1epse.

An additional diafiltration step may be performed. Conventional, commercially available diafiltration agents suitable for use in this method by standard techniques are known in the art. A preferred diafiltration method is diafiltration using an ultrafiltration membrane with a molecular weight exclusion limit in the range of 30,000 to 50,000 depending on the size of the plasmid. This diafiltration step involves changing the buffer and then determining the concentration. The eluate is 3-4 times concentrated by tangential flow filtration (membrane exclusion limit of 30 kDa) to a target concentration of approximately 2.5 to 3.0 mg / ml, and the concentrate is a buffer replaced by diafiltration with a constant volume of 10 volumes of saline and selected for the target plasmid concentration with saline.

The concentration of plasmid DNA is calculated based on the absorption at 260 nm of samples of the concentrate. The plasmid DNA solution is filtered through a 0.2 μm membrane filter and divided into several aliquots, which are stored in containers in a cold room at 2-8 ° C until further processing. This gives a purified plasmid DNA concentrate, the concentration of supercoiled plasmid is approximately 70, 75, 80, 85, 90, 95, and preferably 99%. Limit plasmid production using

- 23 011554 of this method is at least 35, 40, 45, 50, 55, 60, 65, 70 and 80% with an average yield of 60%.

A similar diafiltration step is carried out in accordance with the following conditions: a buffer is used for step (a) and for step (b):

ί) first diafiltration (stage (a)) versus 12.5–13.0 volumes of 50 mM Tg / HO. 150 mM NaCl, pH 7.4 (marked with buffer Ι) and ίί) performing a second diafiltration of the concentrate from the above stage (a) (stage (b)) against 3.0-3.5 volumes of saline carrier (150 mM NaCl).

This is the preferred diafiltration step of the present invention efficiently and thoroughly removes ammonium sulfate and thoroughly EDTA. In addition, after these diafiltration steps, an appropriate physiological concentration of No. 1C1 (approximately 150 mM) and a final concentration of Mn8 below 1 mM (between 200 μM and 1 mM) are obtained.

Preferably, the plasmid DNA composition used contains purified, substantially free of contaminants or with a contaminant content in the range of less than ppm. plasmid DNA and thus represents the pharmaceutical category of DNA. A pharmaceutical grade plasmid DNA composition may contain less than ppm. (<0.0001%, i.e. <0.0001 mg per 100 mg of plasmid DNA) gDNA, RNA and protein contaminants.

A pharmaceutical grade plasmid DNA composition may contain less than about 0.01% or less than 0.001%, and preferably less than 0.0001% or preferably less than 0.00008% (<0.0008%, i.e. <0.0008 mg per 100 mg plasmid DNA) chromosomal DNA or genomic DNA.

A pharmaceutical grade plasmid DNA composition may contain 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 plasmid DNA) contaminating RNA.

A pharmaceutical grade plasmid DNA composition may contain a plasmid DNA preparation containing less than about 0.0001%, and most preferably less than 0.00005% (<0.00005%, i.e. <0.00005 mg per 100 mg of plasmid DNA) contaminating host cell proteins.

The composition may also contain a plasmid DNA preparation containing less than 0.1 IU / mg of endotoxins.

Thus, a pharmaceutical grade plasmid DNA composition advantageously contains a ring shape, and more specifically contains more than 80, 85, 90, 95, or 99% of a closed circular form of plasmid DNA.

The pharmaceutical composition included in the invention may have a detectable level of genomic DNA of the host cells of less than about 0.01% and less than about 0.001% of the host cell RNA. Most preferably, a pharmaceutical grade plasmid DNA composition may have less than about 0.00008% of the genomic DNA of the host cells, and less than about 0.00002% of the RNA of the host cells, and less than about 0.00005% of the protein of the host cells. In fact, any combination of the above purification levels can be used for any particular pharmaceutical grade plasmid DNA composition of the invention. The compositions may also contain other pharmaceutically acceptable components, buffers, stabilizers or compounds to improve gene transfer and specifically transfer of plasmid DNA to a cell or organism.

The plasmid DNA thus obtained can then be prepared as a composition of the present invention in NaCl as a salt filler and an appropriate concentration of Mn8 buffer solution to maintain or control the pH between 6.2 and 9, preferably between 6.5 and 8, more preferably between 7 and 7.5. The plasmid DNA preparations of the present invention are particularly effective since they can surprisingly maintain plasmid DNA in a stable, non-degraded form under these conditions for a long time interval at 5 ° C and up to 25 ° C, which is room temperature.

As indicated above, purified plasmid DNA is present in a solution with an endotoxin amount of less than or about 0.1 IU / mg, less than or about 0.00005% of the host cell contaminating protein, less than or about 0.00002% of the host cell contaminating RNA, or less or approximately 0.00008% contaminating the genomic DNA of the host cells. The pharmaceutical grade plasmid DNA composition contains less than ppm. (<0.00001%) gDNA of host cells, RNA and contaminating proteins. More precisely, a pharmaceutical grade plasmid DNA composition is substantially free of detectable contaminating gDNA, RNA, and protein. In addition, the pharmaceutical grade plasmid DNA composition is substantially free of the detectable chromosomal DNA of the bacterial host, and thus contains 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 is substantially free of detectable host cell RNA and more specifically contains less than about 0.01% or less than 0.001% and preferably less than 0.0001% or preferably less than 0.00002% of the cell-contaminating RNA - the hosts. In addition, the pharmaceutical grade plasmid DNA composition is substantially free of detectable host cell contaminating proteins and more accurately contains less than about 0.0001% and most preferably less than 0.00005% contaminating

- 24 011554 proteins of the host cells. Finally, a pharmaceutical grade plasmid DNA composition is substantially free of measurable contaminating endotoxins and more precisely contains less than 0.1 IU / mg of endotoxins. Plasmid DNA is present predominantly in supercoiled form and more precisely contains approximately or more than 99% of the closed circular form of plasmid DNA.

A sterilizing filtration step may be performed before filling the containers with purified plasmid DNA. A container of purified plasmid DNA obtained by these methods is also provided.

You can clean any type of vectors with different sizes. A suitable size range for plasmid DNA separation is from about 5 to about 50 kb, preferably from 15 to 50 kb, this DNA includes a vector backbone from about 3 kb, therapeutic gene and related regulatory sequences. Thus, a vector skeleton acceptable according to the invention can carry inserts of from about 10-50 or more, etc. The insert may include DNA from any organism, but preferably the source is a mammal, and may include, in addition to a gene encoding a therapeutic protein, regulatory sequences such as promoters, polyadenylation sequences, enhancers, locus control regions, etc. A gene encoding a therapeutic protein can be of genomic origin and, therefore, contain exons and introns, as reflected in its genomic structure, or can be obtained from complementary DNA. Such vectors may include, for example, a vector sequence that replicates with a high replication copy number, with a polylinker for inserting a therapeutic gene encoding a selected marker gene, e.g., ZirREe tRNA, a tetracycline and kanamycin resistance gene, and is physically small and stable. The vector sequence of the plasmid effectively provides insertion of DNA fragments of mammals, other eukaryotes, prokaryotes or viruses, and the resulting plasmid can be cleaned and used in plasmid-based therapy ίη νίνο or ex νίνο. By the method of the present invention, vectors with a relatively high copy number, i.e. in the range of 20-40 to 10002000 copies / cell. For example, a vector comprising an origin of replication of RIS is preferred by the method of the invention. The pIS origin of replication provides more efficient plasmid DNA replication and yields a ten-fold increase in the number of copies of the plasmid / cell relative to, for example, the pBK322 origin. Using the method of the present invention, it is possible to separate preferably plasmid DNA with a conditional replication point or pCOK, as described in I8 2003/1618445. The resulting high number of copies dramatically increases the ratio of plasmid DNA to chromosomal DNA, RNA, cell proteins and cofactors, increases the plasmid yield and facilitates easier further purification. Thus, any vector (plasmid DNA) of the invention can be used. Representative vectors, by way of non-limiting examples, include plasmid No. U1ECE.

KU1ECE is an encoding acidic fibroblast growth factor or type 1 fibroblast growth factor (ECE-1) plasmid suitable for the treatment of patients with end-stage peripheral arterial occlusion (RAE) or with peripheral artery disease (RAO). This e! a1. (L. Wax. 8igd., 2002, 35, 5: 930-936) described that 51 patients with non-recoverable terminal stage of RAW with resting pain or tissue necrosis in the ischemic thigh and lower leg were intramuscularly injected with NU1ECE in increasing single or repeated doses . Then, various parameters were determined, such as transdermal oxygen pressure, ankle and finger-shoulder indices, pain analysis and ulcer healing. After the introduction of NU1ECE, a significant increase in shoulder indices, a decrease in pain, a clear size of ulcers and an improvement in perfusion were observed.

The plasmid DNA composition may further comprise at least one polymer to improve the transfer of plasmid DNA into the cell. The plasmid DNA composition may also contain a pharmaceutically acceptable base or carrier. A plasmid DNA composition can be formulated for administration by injection, intravenous injection, intramuscular injection, intratumoral injection, particle bombardment, or topical application to tissue. In these compositions, plasmid DNA is generally in the form of a supercoiled closed circular DNA.

Suitable host cells of the invention may be any bacterial strain, i.e. Gram-positive and Gram-negative strains, such as E.co11 and Zaegene Turytigst or VasShik, capable of maintaining a high number of copies of the plasmids described above; for example 20-200 copies. According to the invention, E. co11 host strains can be used, which include HB101, OH1 and OH5oE, XAC-1 and XAS-1rp116, TECH2 and TECH2rp42 (No. O04 / 033664). Strains containing plasmid E or derivatives of plasmid E (for example, LM 109) are generally not preferred since plasmid E can be purified along with the therapeutic plasmid product.

- 25 011554

Examples

General methods for cloning and molecular biology.

Conventional molecular biology methods, such as restriction enzyme digestion, gel electrophoresis, E. soy transformation, nucleic acid isolation, are described in the literature (Mashayk e! A1., T., E.R. Rtyksy, aib 1. 8atgook, 1989. Moobsi1ag s1shid: aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ... KA. 8ιηί11τ 1.0. 8e1btai aib K. 8ΐπι1ι1. 1987. Stew! rgo! os1k тоη to1esi1ag юu1odu 1987-1988. Nucleotide sequences were determined by the method of chain termination in accordance with the already published protocol (AiklE1 e! A1., 1987).

Restriction enzymes were obtained from Νο \ ν Eid1aib Vu1ab, Veuu1u, MA (Vu1abk).

During ligation, DNA fragments were incubated in a buffer solution containing 50 mM Tpc-HCl pH 7.4, 10 mM MdC1 ₂ , 10 mM S, 2 mM ATP in the presence of T4 phage DNA ligase (B1o1bk).

Oligonucleotides were synthesized on a Vukateats 8600 automatic DNA synthesizer using the manufacturer's recommendations using phosphoramidite chemistry using β-position protecting cyanoethyl phosphoramidites (801113, Ν.Ό., 1. В1ета !, 1. МсМайик аиб N. Кбк! Ег, 1984. Rooter kirroy o11dois1eobbe kuschekNeck XVIII: Ike o! Β-suaηοеΐyu1-N, N-b ^ a1k1at ^ uo- / N-to ^ ryu1 ^ u pHokrNogapybёy!! aib 1ko1a1yui o! (Not ya1 rgobis !. No. p1. As1bk Kek., 12, 4539-4557: Oyek,! \ ¥. 1985. Abuisek 1i ai! ota! eb ^ NA kugINek . Am. Vyu1esio1., №u. / Ees.).

Ligated DNA or tested for DNA transformation efficiency was used to transform the following eligible strains:

E.soy EN5a [R / eibA1, NkbK17, kirE44, 1I-1, gesA1, arcA96, ge1A1, D (1ac2UA-aschR) I169, beoK, F80b1ac (1ac2DM15)] (for any Co1 E1 plasmid) or

E.soy XAS-rp (for any plasmid derived from pCog).

Minimap preparations of plasmid DNA were obtained in accordance with the protocol K1esh e! A1., 1980.

For the growth of E. coli strains, LB culture medium was used (Mashayk e! A1., 1982). Strains were incubated at 37 ° C. Bacteria were plated on plates supplemented with acceptable antibiotic LB medium.

Example 1

The selection of diameters for the applied flow rates follows from the calculation of the Reynolds coefficients in the spirals of the continuous lysis system. Since the following analysis assumes that the fluid properties are Newtonian, the figures presented below are fully valid only in B1a and, to some extent, in B2.

The value of the Reynolds coefficient allows a person skilled in the art to indicate the type of motion detected. In this work, the authors considered only the fluid flow through the pipe (hydraulics).

1) Non-Newtonian fluid.

The two types of non-Newtonian fluids most commonly found in industry are Bingham and Ostwald de Weil fluids.

In this case, the Reynolds coefficient (Ke) was calculated as follows:

Kek = (1 / (2 ' ³ )) x (η / 3η + 1) ^η χ ((ρχϋ ^η χν ² ^) / т) (1) where Kе _N is the generalized Reynolds coefficient;

Ό is the inner diameter of the cross section (m);

ρ is the bulk density of the liquid (kg / m ³ );

ν is the velocity of the liquid in space (m / s);

and - flow rate (dimensionless);

t is an indicator of the degree of density of the liquid (dyne-s ^and / cm ² );

and and m were determined empirically (study of rheological properties).

2) Newtonian fluid.

As in the first section, in formula (1), the authors received:

Ke =! (Inner diameter, μ, ρ and u), since u and t are functions of μ

Ke = (ihRxr) / p (2) ρ is the bulk mass of the liquid (kg / m ³ );

μ is the viscosity of the fluid (Pa-s, 1 MPa-s = 1 sR);

Ό is the inner diameter of the cross section (m);

and - the average velocity of the liquid in space (m / s).

Formula (1) with and = 1 reduces to formula (2).

С О = flow velocity (m ³ / h) and 8 = cross-sectional surface area (m ² ) and if μ is given in сР, therefore, (4х (2/3600) хр) / ((μ / ООО) хпхС) ( 3)

- 26 011554

In a round pipe, the flow is laminar for a Reynolds coefficient below 2500 and is a hydraulically even turbulent flow with a Reynolds coefficient between 2000 and 500000. The boundary value is not specifically defined and is between 2000 and 2500, where both types of properties are used to determine what can then happen and make empirical choices.

3) Calculations.

Since n and m are, as a rule, not known, the following approximations were used to estimate the motions.

Newtonian fluid (in all cross sections): ρ = 1000 kg / m ³ (for all fluids);

μ = 5 cP in B1a and 40 cP in B1b (authors 'data) and 2.5 cP in B2 (authors' data).

For two standard test configurations, designated configuration 1 and configuration 2 (without B1b pipe), using the formula (3), the following calculations were performed.

table 2

Spiral Config B1a walkie talkie 1 AT 2 Config B1a walkie talkie 2 AT 2 Viscosity* (cp) 5 2,5 5 2,5 Diameter (mm) 12.7 9.5 6 6 Flow rate (l / h) 60 105 12 21 Reynolds Ratio Movement 334 1564 141 495 laminar laminar laminar laminar

In these two configurations, the flows are laminar at all stages and the solutions do not mix sufficiently.

For other pipe configurations (without B1b pipe), the authors obtained the following.

Table 3

Spiral High speed / standard High speed / V.D. 16 mm High speed / V.D. 6 mm diameter B1a AT 2 B1a AT 2 B1a AT 2 Viscosity * (cp) 5 2,5 5 2,5 5 2,5 Diameter (mm) 12 10 sixteen sixteen 6 6 Flow rate (l / h) 120 210 120 210 120 210 Coeff. Reynolds 707 2971 531 1857 1415 4951 Traffic Laminar Turbulen Laminar Laminar Laminar Turbulen

Similar calculations were performed using formula (3) for various pipe configurations with the presented pipes B1a and B1b .:

Table 4

Spiral High speed High speed / maximum agitation B1a B1b AT 2 B1a B1a B1a Viscosity* (cp) 5 5 2, 5 5 5 5 Diameter 6 sixteen 6 3 2 3 Flow rate (l / h) 120 120 210 120 120 160 Reynolds coefficient 1415 531 4951 2829 4244 3773 Traffic Laminar Laminar Turbulen Turbulen Turbulen Turbulen

Obviously, predefined Reynolds coefficients can be obtained by selecting pipe diameters and flow rates.

A person skilled in the art can imagine many combinations of diameters and lengths for B2 or for two sections B1 (B1a and B1b). For example, in order to reduce length and increase mixing, the first section B1 can be cut from 6 to 3 mm. In addition, from the study of the rheological properties of liquids, p and t can be determined and used to determine the correct characteristics of the pipes.

In addition to the mixing efficiency, it is also possible to consider the duration of mixing, which in some embodiments of the present invention is obtained by selecting the length

- 27 011554 spirals.

The pipe diameter or fluid velocity is not dominant in the formula (1) for non-Newtonian fluid (data not shown). In other words, changing the diameter is no more effective than changing the flow rate if formula (1) is used for calculations in B1b and B2. The diameter can be changed together with the flow rate in those cases where high flow rates are desired.

These principles can be used as the basis for the maximum possible restriction of mixing in B1b and B2 in order to avoid fragmentation of gDNA.

During lysis, stirring can be quite intense until the gDNA is denatured. Reducing the diameter at the beginning of B1 allows you to increase mixing (increased Be) for sufficient mixing of solution 2 with cells. On the other hand, when lysing cells, the mixing and friction forces at the wall can be reduced to avoid nucleic acid fragmentation. Increasing the diameter reduces mixing (reduced Be) and friction (reduced speed).

M1: mixing liquids.

B1a: fine-tuning the mixing at the beginning of the lysis: convection effect (macro-mixing).

B1b: denaturation occurs plus diffusion (micro-mixing).

It is believed that the generalized Reynolds coefficient has the same value for a non-Newtonian fluid as the classical Reynolds coefficient has for a Newtonian fluid. Specifically, it is believed that the boundary value for the laminar regime in a pipe with a circular cross-section is Be \ <2300.

In B2, neutralization is performed. High flow rates tend to increase the fragmentation of genomic DNA due to too strong mixing and as a result of friction forces at the wall (mechanical stresses). The use of a large diameter pipe reduces mixing (Be) and friction (speed). In this work, the authors installed a pipe with a small diameter (6 mm) in order to avoid insufficient mixing. The observations of the authors show that a pipe with a small diameter is best suited for B2 in order to mix the neutralized lysate “sharply and quickly”.

Example 2

The authors propose to break down the cell lysis system into 5 stages. In one specific embodiment, the configuration is as follows:

1) Mixing: cells (in solution 1) + solution 2 (M1 + 3 m, 6 mm, tube). Start cell lysis by 8–8, without the risk of DNA fragmentation until the DNA is denatured.

2) End of lysis and denaturation of gDNA (13 m, 16 mm, tube).

3) Mixing: lysate + solution 3 (M2 + 3 m, 6 mm, pipe).

4) Collection of neutralized lysate at 4 ° C

5) Precipitation of flakes and large fragments of gDNA all night at 4 ° C. For the implementation of continuous lysis, you can use the following conditions:

Solution 1: EDTA 10 mm, glucose (C1c) 9 g / L and Tn5-HC1 25 mM, pH 7.2.

Solution 2: 8Ό8 1% and ΝαΟΗ 0.2n.

Solution 3: acetic acid 2 M and potassium acetate 3 M.

Solution 1 and solution 2: flow rate 60 l / h.

Solution 3: flow rate 90 l / h.

Cells were adjusted to 38.5 g / L with solution 1.

Cells in solution 1 pass through 3 holes in order to spray them into solution 2 going in the opposite direction.

The mixer M1 is shaped so as to ensure optimal mixing of the two liquids (see FIG. 2, schematic illustration of the mixer).

The first part of the pipe after mixer M1 is B1a, and the next part is B1b.

B1a: pipe 3 m length, diameter 6 mm, residence time 2.5 s.

B1b: 13 m length, diameter 16 mm, residence time 77 s.

The method of the present invention with respect to efficiency provides an advantage in total representing dispersion, short-term sharp mixing and soft mixing by diffusion. Using the method of the present invention, the number of lysed cells increases and, consequently, the amount of isolated plasmid DNA increases. The idea of diffusion is especially important because of the difficulty of mixing these fluids due to their properties, especially viscoelasticity.

The method of the present invention allows to limit the shear stress and therefore to limit the fragmentation of gDNA, contributing to its removal during subsequent purification by chromatography.

- 28 011554

The difficulty is the subsequent mixing with solution 3, which can be cooled to 4 ° C. In one embodiment, the process includes an M2 mixer with an Υ-shape and an inner diameter of about 10 mm; part of the pipe B2, placed after the mixer M2;

B2: pipe 2 m, 6 mm; stay time: 1 s.

In the table. 5, the results obtained in comparative tests are presented to demonstrate the advantages of the continuous lysis method developed by the authors in comparison with a one-shot lysis.

Table 5

The ratio of gDNA / pDNA in the lysate The number of selected plasmids per g of cells (mg / g) One-time Lysis 16, 9 1.4 Continuous lysis in the system Cb described in example 1 1,6 1.9

Example 3

The column used is a 1 ml H1Tgar column activated with N118 (β-hydroxysuccinimide, P1agtac1a) connected to a peristaltic pump (speed <1 ml / min). The particular oligonucleotide used had an NH2 group at the 5'-end, its sequence is as follows: 5'-OAOOSTTSTSTSTSTSTSTTSTTSTT-3 '(8Er GO No. 1).

The buffer solutions used in this example were the following solutions: Binding buffer: 0.2 M ΝηΙ 1CO ₃ , 0.5 M No. C1, pH 8.3.

Buffer A: 0.5 M ethanolamine, 0.5 M No. C1, pH 8.3.

Buffer B: 0.1 M acetate, 0.5 M No. C1, pH 4.

The column was washed with 6 ml of 1 mM HCl and the oligonucleotide (50 nmol in 1 ml) dissolved in binding buffer was then applied to the column and left for 30 min at room temperature. The column was sequentially washed three times with 6 ml of buffer A and then 6 ml of buffer B. Thus, the oligonucleotide was covalently bound to the column via a CΟNH bond. The column was stored at 4 ° C in PBS, 0.1% ΝηΝ ₃ and could be used at least four times.

The following two oligonucleotides were synthesized:

oligonucleotide 4817: 5'-OATSSOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOAAOO-3 '(GO 8Er № 13) and oligonucleotide 4818: 5'-AATTSSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSTTSO-3' (GO 8Er № 14)

After hybridization and cloning of these oligonucleotides into the plasmid, the homopurin-homopyrimidine sequence (OAA) _p (8E () GO No. 15) was introduced into the corresponding plasmid, as described above.

The sequence corresponding to these two hybridized oligonucleotides was cloned into the multiple cloning site of the plasmid pBK8 + (81g1adepe C1op1pd 8uz1et, Ba 1-11a CA) containing the ampicillin resistance gene. For this, the oligonucleotides were hybridized under the following conditions: 1 μg of these two oligonucleotides were placed together in 40 ml of the final buffer containing 50 mM TP8-HCl pH 7.4, 10 mM MdCl ₂ . This mixture was heated to 95 ° C and then left at room temperature so that the temperature slowly dropped. 10 ng of the mixture of hybridized oligonucleotides were ligated with 200 ng of the plasmid pBK8 + (81G1Adepe S1bsp8S1et, Ba 1-11A CA), digested with BATH1 and EcoK1 in 30 μl of the final buffer. After ligation, an aliquot was transformed into OH5a. The transformation mixtures were isolated on medium supplemented with ampicillin B (50 mg / L) and X-da1 (20 mg / L). Recombinant clones should show the absence of blue color on this medium, in contrast to their maternal plasmid (pBK8 +), which provides α-complementation from E.coH β-galactosidase fragment. After obtaining plasmid DNA from 6 clones, all showed a loss of the P11 region located between the EcoK1 and BatH1 regions of pBK8 + and an increase in molecular weight of 448 kb. RuiP strip containing the site for multiple cloning. One clone was selected and the corresponding plasmid rHB2563 was designated. The cloned sequence was checked by sequencing using primer -20 (5'-TOACCOOAAAAAAATO-3 '(8EP GO No. 16)) (U1ega B and B Mezstd. 1982. T1e riC rBztkB, ap M13tr7-bebueb 8U81et ог tzegyop tiedepededep. νίΐ1ι zupFeys ishueg8a1 rpteg. Oepe, 19, 259-268) for plasmid pBK8 + (81g1adepe C1otpd 8uz1et, Ba 1o11a CA). Plasmid rHB2563 was purified using \\ l / ags. 1 Medarger ky (Rgoteda Comp. MaFzop, Sh1) in accordance with the manufacturer's recommendations. This plasmid DNA preparation was used in the examples described below.

- 29 011554

Plasmid pXL2563 was purified on an H1Tgar column linked to the oligonucleotide described in 1.1 from a solution also containing plasmid pBK8 +. The buffers used in this purification were buffer E: 2 Μ NaCl, 0.2 Μ acetate, pH 4.5 to 5;

buffer E: 1 Μ TY8-HC1, pH 9, 0.5 mM EDTA.

The column was washed with 6 ml of buffer E, applied to a plasmid column (20 μg pXB2563 and 20 μg pBK8 + in 400 μl buffer E) and incubated for 2 hours at room temperature. The column was washed with 10 ml of buffer E, and then elution was performed with buffer E. Plasmids were detected after electrophoresis on a 1% agarose gel and stained with ethidium bromide. Part of the plasmids in solution was evaluated by measuring their transforming activity on E.co11.

Based on a mixture containing 30% pXL2563 and 70% rBK8 +, a solution containing 100% pXL2563 was collected at the column outlet. Estimated by the ratio of OI at 260 to 280 nm, the purity increased from 1.9 to 2.5, which indicates the removal of contaminating proteins by this method.

Example 4

The binding of the oligonucleotide (5'-SASSSTTSTSTSTTSTSTTSTTSTT-3 '(8EO GO No. 1)) to the column was performed as described in example 3. To bind the oligonucleotide was modified at the 5'-end of the amine group bound to the spacer phosphate via a shoulder containing 6 carbon atoms (Άοά ^ Γ1еά о1щогсс1еоббы Еигодеесес 8А, Ве1дшт). Plasmid pXL2563 was purified using νί / agb Μ edar ^ er ΕίΙ (Tgoteda Sogr., Μаά ^ 8οη, VI) in accordance with the manufacturer's recommendations. The buffers used in this example were buffer E: 0-2 Μ NaCl, 0.2 Μ acetate, pH 4.5 to 5;

buffer E: 1 Μ TY8-HC1, pH 9, 0.5 mM EDTA.

The column was washed with 6 ml of buffer E and 100 μg of plasmid pXb2563 was dissolved in 400 μl of buffer E, and then applied to the column and incubated for 2 hours at room temperature. The column was washed with 10 ml of buffer E, and then elution was performed with buffer E. The amount of plasmid was determined by measuring the optical density at 260 nm.

In this example, the binding was performed in a buffer, the molarity of which in relation to No. 1C1 ranged from 0 to 2 M (buffer E). The yield after purification decreased if the molarity of NaCl decreased. The pH of the binding buffer can vary from 4.5 to 5, the yield after purification was obtained above at 4.5. You can also use another elution buffer of basic pH: while the elution is performed with a buffer containing 50 mm borate, pH 9, 0.5 mm EDTA.

The binding of the oligonucleotide (5'-SASSSTTSTSTSTSTSTSTSTSTTST-3 '(8EO GO No. 1)) to the column was performed as described in Example 3. The plasmid pXL2563 was purified using νί / agb Kedar ^ er ΕίΙ (Tgoteda Comp., Μaά ^ 8ο in accordance with the manufacturer's recommendations. The buffers used in this example were buffer E: 0.1 Μ NaCl, 0.2 M acetate, pH 5;

buffer E: 1 Μ TY8-HC1, pH 9, 0.5 mM EDTA.

The column was washed with 6 ml of buffer E and 100 μg of plasmid pXb2563 was dissolved in 400 μl of buffer E, and then applied to the column and incubated for 1 h at room temperature. The column was washed with 10 ml of buffer E, and then elution was performed with buffer E. The content of E.co11 genomic or chromosomal DNA present in the plasmid samples was measured before and after passing through the column with the oligonucleotide. This genomic DNA was calculated by Ρί.Έ using primers for the E.co11 da1K gene. Followed the protocol: the sequence of these primers is described in IEb e! a1. (Heaths As1b8 Ie8. 1985, 13, 1841-1853):

5'-СССААТТСТССССССАССААССАСТАТТС-3 '(8Ер ГО No. 17); and

5'-SSAASSTTSASTSTTSASSASSSSSTST-3 '(8EO GO No. 18).

The reaction medium contained in 25 μl of PCR buffer (Ggoteda Egapse, Skaopn1e8):

1.5 mM ΜβΕΕ;

0.2 mM PXTT (Natasha, Og8au);

0.5 μM primer;

U / ml Tad polymerase (Ggoted).

The reaction was carried out in the following sequence:

min at 95 ° C;

cycles of 10 s at 95 ° C, 30 s at 60 ° C, 1 min at 78 ° C;

min at 78 ° C.

An amplified 124 kb DNA fragment were separated by electrophoresis in a 3% agarose gel in the presence of 8bbCrGeep I (Gnocecum aaaaa, Eidepe, I8A), and then the amount was determined by the set of control genomic DNAs of Iqargz strain B E.co11 (81 gta, hen Ό 4889).

- 30 011554

Example 5

This example describes the purification of plasmid DNA from a purified bacterial culture lysate, on the so-called “miniprep” scale: 1.5 ml of an overnight culture of plasmid pXL2563 ΌΗ5α strains were centrifuged and the pellet was resuspended in 100 μl of 50 mM glucose, 25 mM TrzNS1, pH 8, 10 mM EDTA. 200 μl of 0.2 Μ ΝαΟΗ, 1% 8Ό8 was added, the tubes were mixed by inversion, then 150 μl of 3 Μ potassium acetate, pH 5 was added and the tubes were mixed by inversion. After centrifugation, the supernatant was recovered and applied to an oligonucleotide column prepared as described in Example 1. Binding, washing and elution were identical to the procedures described in Example 3. About 1 μg of plasmid was isolated from 1.5 ml of culture. The resulting plasmid was analyzed by agarose gel electrophoresis and staining with ethidium bromide, taking the form of a single strip for "supercoiled" circular DNA. In the plasmid purified by this method, the absence of traces of high molecular weight (chromosomal) DNA or RNA was observed.

Example 6

This example describes an experiment on the purification of plasmid DNA, carried out under the same conditions as in example 5, based on 20 ml of plasmid containing pXL2563 strains of bacterial culture ΌΗ5α. The cell pellet is placed in 1.5 ml of 50 mm glucose, 25 mm TP8-HC1, pH 8, 10 mm EDTA. Lysis was performed with 2 ml of 0.2 Μ ΜαΝ, 1% 8Ό8, and neutralization with 1.5 ml of 3 Μ potassium acetate, pH 5. After this, 3 ml of 2-propanol was precipitated with DNA and the residue was placed in 0.5 ml of 0, 2 Μ sodium acetate, pH 5, 0.1 Μ No. 101 and applied to a column with an oligonucleotide obtained as described above in the example. Binding, column washing and elution were performed as described above in the example, with the exception of the wash buffer, the molarity of which with respect to Να01 was 0.1 Μ. The resulting plasmid was analyzed by agarose gel electrophoresis and staining with ethidium bromide, taking the form of a single strip for "supercoiled" circular DNA. In a purified plasmid, the absence of traces of high molecular weight (chromosomal) DNA or RNA was observed. Cleavage of the plasmid with restriction enzymes gave a separate band with an expected molecular weight of 3 kb. The plasmid contained a plasmid comprising the cytomegalovirus promoter encoding the luciferase gene and the homopurin-homopyrimidine sequence (OAA) ₁₇ (8ED # 15), originating from the plasmid pXb2563. Strain эту1 containing this plasmid (ΜαηίαΙίδ c1 a1., 1989) was cultured in a 7-liter fermenter. A purified lysate was obtained from 200 g of cells: the cell pellet was placed in 2 L of 25 mM Tp8, pH 6.8, 50 mM glucose, 10 mm EDTA, to which 2 L 0.2 Μ ΝαΟΗ, 1% 8Ό8 was added. The lysate was neutralized by adding 1 l of 3 Μ potassium acetate. After diafiltration, 4 ml of this lysate was loaded onto a 5 ml H1Tgar-YH8 column, coupled to an oligonucleotide with the sequence 5'-OA6 OSTTSTSTSTSTSTTSTSTTST-3 '(8EO No. 1) in accordance with the method described in Example 3. Washing and elution were performed as described above in the example.

Example 7

This example describes the use of an oligonucleotide carrying methylated cytosines. The sequence of the oligonucleotide used was

5'-6A66 ^Μe STT ^Μe STT ^Μe STT ^Μe STT ^{Μ STF Μe} STT ^Μe STT-3 '(8ЕО ΙΌ No. 19).

This oligonucleotide had an XH ₂ group at the 5'-end; ^Me C = 5-methylcytosine. This oligonucleotide purifies the plasmid pXb2563 under the conditions of Example 1 with a binding buffer of pH 5 (thereby reducing the risk of plasmid degradation).

Example 8

In the examples described above, the oligonucleotide used was modified at the 5'-end with an amine group bound to a phosphate via a 6-carbon shoulder arm: OH ₂ - (CH ₂ ) ₆ . In this example, it was associated with phosphate of the 5 'end by means of a shoulder containing 12 carbon atoms: XH ₂ - (CH ₂ ) 1 ₂ . The binding of the oligonucleotide and passage through the column was carried out, as described in example 3, with buffer P: 2 Μ BaCl, 0.2 Μ acetate, pH 4.5. This oligonucleotide allows a better yield after purification: a yield of 53% was obtained, whereas under the same conditions with an oligonucleotide containing 6 carbon atoms, this yield was approximately 45%.

Example 9

After the cloning procedure described in Example 3, two more plasmids were constructed that carried homopurin-homopyrimidine sequences:

containing the sequence (OAA) 1 ₆ plasmid pXL2725 (8ED ΙΌ No. 20) and containing the sequence (OA) ₂₅ plasmid pXL2726 (8ED ΙΌ No. 21).

Plasmids pXB2725 and pXB2726, similar to plasmid pXB2563, were constructed according to the cloning procedure described in Example 3 using the following oligonucleotide pairs:

5986: 5'-OATSS (OA) ₂₅ 666-3 '(8EO ΙΌ No. 22);

5987: 5'-AATTSSS (TS) ₂₅ O-3 '(8EO ΙΌ No. 23);

5981: 5'-OATSS (66A) ₁₇ 6O-3 '(8EO ΙΌ No. 24);

5982: 5'-AATT (CCT) ₁₇ CCO-3 '(8EO ΙΌ No. 25).

- 31 011554

The oligonucleotide pair 5986 and 5987 were used to construct plasmid pXB2726 by cloning oligonucleotides in the BHH1 and EcoK1 regions of pBK8 + (81a1adepe C1ocsp8 8b1aet, ba ba11a CA), while oligonucleotides 5981 and 5982 were used to construct plasmids25. The same experimental conditions were used as for the construction of plasmid pXL2563, and only oligonucleotide pairs were changed. Similarly, cloned sequences were verified by plasmid sequencing. This allows us to show that the plasmid pXL2725 has a modification with respect to the expected sequence: instead of the OCA sequence repeated 17 times, there is CCACA (CCA) 1 ₅ (8EC ΙΌ No. 26).

Example 10

The oligonucleotides forming triple helices with these homopurine sequences were linked to H1Tgar columns according to the method described in Example 1.1. An oligonucleotide with the sequence 5'-AATSSSTSTSTSTSTSTSTST-3 '(8EC No. 27) was used to clean plasmid pXB2725, and an oligonucleotide with the sequence 5'-ATSTSTSTSTSTSTSTSTSTSTST-3' (8E0 ΙΌ No. 28) was used to clean plasmids26.

Thus, two columns were obtained that provided the purification of the corresponding plasmids as described in Example 2 with the following buffers:

buffer E: 2 M No. C1, 0.2 M acetate, pH 4.5;

buffer E: 1 M TY8-HC1, pH 9, 0.5 mM EDTA.

The yields obtained were 23 and 31% for pXB2 725 and pXB2726, respectively.

Example 11

This example illustrates the effect of the length of the specific sequence shown in the plasmid on the yields after purification.

The reporter gene used in these experiments to demonstrate the activity of the compositions of the invention is the luciferase (Luc) coding gene.

Plasmid pXb2621 contains a cluster including 661-bp. the cytomegalovirus promoter (SMU), cloned to the luciferase-encoding gene in the M1i1 and ΗίηάΙΙΙ regions into the pCb Lazyu Ueslog vector (Rgoteda Comp., Mablzop, XVI). This plasmid was constructed using traditional molecular biology methods.

Plasmids pXB2727-1 and pXB2727-2 were constructed under the following conditions.

μ plasmid pXB2621 linearized BATH1; the enzyme was inactivated by treatment for 10 min at 65 ° C; at the same time, the oligonucleotides 6006 and 6008 were hybridized as described for the construction of plasmid pXL2563:

6006: 5'-SATST (CAA) ₁₇ STSSASATST-3 '(8EO ΙΌ No. 29);

6008: 5'-SATSASATSTSSAS (TTS) 1 ₇ A-3 '(8EO ΙΌ No. 30).

This hybridization mixture was cloned at the WatCh ends of the plasmid pX262621 and, after transformation into β5α, recombinant clones were determined by PZP restriction enzyme analysis, since the oligonucleotides included the PZB site. Two clones were selected and, using a primer (6282: 5'-ACASTSATAAASTSSCCCoAcC-3 '(8EO ΙΌ No. 31)), the nucleotide sequence of the cloned fragment was determined, such as, for example, by sequencing the reaction primer (U1ega. App. М. Mezztd, 1982) using the resulting system from plasmids RIS M13tr7 for insertion mutagenesis and sequencing with synthetic universal primers (CEPE 19: 259-268).

The first clone (pXB2727-1) contained a 10-fold CAA sequence. The second (pXB2727-2) contained the sequence 5 -CAACAACACA (CAA) ₇ CCAACACAA-3 '(8EO ΙΌ No. 32).

A column was used as, for example, described in Example 3 and linked to the oligonucleotide 5'-SASSSTTSTSTSTSTSTSTSTSTTST-3 '(8EE ΙΌ No. 1).

Plasmid pXb2727-1 carried 14 repeats of the CAA sequence. The oligonucleotide described above containing only 7 repeats of the corresponding CTT hybridizable sequence can thus be hybridized with the plasmid at 8 different positions. In contrast, the plasmid pXb2727-2 had a hybridization sequence (CAA) ₇ (8EO No. 36) of the same length as the oligonucleotide bound to the column. Thus, this oligonucleotide can only be hybridized at one position per pXb2727-2. The experiment is similar to the experiment described in example 4 with the following buffers:

buffer E: 2 M No. C1, 0.2 M acetate, pH 4.5;

buffer E: 1 M TY8-HC1, pH 9, 0.5 mM EDTA.

The yield after purification with plasmid pXB2727-1 is 29 and 19% with pXB2727-2. The cells used are ΝΙΗ 3T3 cells, which were introduced into the 24-well culture plates at the rate of 50,000 cells / well the day before the experiment. The plasmid was dissolved in 150 mm No. 1 C1 and mixed with lipopectant KRK115335. A positively charged lipofectant / negatively charged DNA ratio of 6 was used. The mixture was shaken, left for 10 min at room temperature, diluted in medium without fetal calf serum, and then added to the cells in the ratio of 1 μg of DNA per culture well. After 2 hours at 37 ° C, 10% (v / v) fetal calf serum was added and the cells were incubated for 48 hours at 37 ° C in the presence of 5% CO ₂ . Cells from

- 32 011554 was washed twice with the FSB and the luciferase activity was measured according to the described protocol (Rgoteda K11. Rgoteda Comp. MaFzop, XVI) on a Beat luminometer! BV9501 (EU apb. With VePyo, Eughu). Purified as described in Example 8.2, the plasmid pXb2727-1 yielded twice the yield after transfection than the yield after transfection obtained with the same plasmid purified using ΧνίζηΓά Medarger k11 (Rgoteda Comp. MabEop, VI).

Example 12

The following example demonstrates the purification of rCOK derived plasmids using three-helix affinity chromatography. It was shown that using this method, contaminating nucleic acids (specifically, genomic DNA and host RNA) can be removed to levels not achievable with conventional chromatography methods.

A triplex-affinity gel was synthesized as a chromatographic matrix with Speryasku8-1-1000 8B (Integra-Mpyagtas1a Vu1esy). At first, 8eryasgu1 8-1000 was activated with sodium m-periodate (3 mM, room temperature, 1 h) in 0.2 M sodium acetate (pH 4.7). The oligonucleotide was then linked through its 5'-ΝΗ ₂ terminal part to the aldehyde groups of the activated matrix by reductive amination in the presence of ascorbic acid (5 mM), as described above for protein binding (Notzeu e1 a1., 1. Ittiо1. МеШобз, 1986, 93, 83-88). The homopyrimidine oligonucleotide used for these experiments (from Etcogepepc, HPCc-ccpEeb) had a sequence complementary to the short 14-membered homopurine sequence 5'-AACAAAAAAAACAA-3 '(8EC GO no. 10), present at the point of replication start (cpu cpu) e1 a1., Sepe Tiegaru, 1999, 6, 1482-1488). As previously discussed, the sequence of the homopyrimidine oligonucleotide is 5'-TTSTTTTTTTTSTT-3 '(8ES GO No. 11).

After that, the plasmids were subjected to chromatography:

pXX3296 (pCOK without transgene, 2.0 kb), pXB3179 (pCOK-ECE, 2.4 kb), pXB3579 (pCOK-UESEV, 2.5 kb) , pXB3678 (pCOK-AEP, 3.7 kb), pXB3227 (pCOK-1ac2 5.4 kb) and pXB3397 (pCOK-Bb1e! eb EUSH, 6.6 kb. m.).

All these plasmids were purified by two stages of anion exchange chromatography from the purified lysates obtained as described in Example 4. The plasmid pBK8 + purified by ultracentrifugation in CzC1 (pB1iezspr1 II K8 + Egot 81a1adepe) and the plasmid obtained from Co1E1 were also examined. All plasmids used were in a supercoiled (> 95%) topological state or form.

In each plasmid DNA purification experiment, 300 μg of plasmid DNA in 6 ml of 2 M No. C1, 0.2 M potassium acetate was applied to an affinity column containing 5'-TTSTTTTTTTTTSTT-3 'containing the above oligonucleotide (8ES GO No. 11). 0) at a flow rate of 30 cm / h. After washing the column with 5 volumes of the same buffer, the bound plasmid was eluted with 1 M TI8 / HC1, 0.5 mM EDTA (pH 9.0), the amount in IU (260 nm) was determined and ion-exchange chromatography was performed on a MPNroge Sep-Can column (Mashchets! e1 a1., VyuRyagt, 1995, 8, 26-37). The resulting plasmids in the collected fractions were:

207 mcg for pXB3296,

196 mcg for pXB3179,

192 mcg for pXb3579,

139 μg for pXB3678, μg for pXB 3227 and μg for pXB 3397.

The absence of bound plasmid (<3 μg) was determined by chromatography on this pBK8 column. This indicates that the oligonucleotide 5'-TTSTTTTTTTTTSTT-3 '(8ES GO No. 11) forms stable three-helix structures with a 14-member complementary 14-membered sequence 5'-AACAAAAAAAACAA-3' (8ES GO No. 10), but not with the 5'-ASAAAAAAAASSA-3 'tightly bound sequence present in rVK8 (8ES GO No. 8). This indicates that the introduction of one non-canonical triad (in this case, T * SS) leads to a complete destabilization of the structure of the triplex.

As a control, binding was observed without plasmid (<1 μg) if pXL3179 chromatography on an empty column synthesized under strictly similar conditions but without an oligonucleotide.

When performing this affinity purification on a column under the conditions indicated here, the level of contamination of the host genomic DNA decreased from 2.6 to 0.07% for the preparation pXb3296. Similarly, the level of contamination of the host DNA decreased from 0.5 to 0.008% for the preparation pXb3179, if chromatography with the same affinity column was used for the sample.

- 33 011554

Example 13

The following example demonstrates the purification of plasmids derived from Co1E1 using three-helix affinity chromatography. It was shown that using this method, contaminating nucleic acids (specifically, genomic DNA and host RNA) can be removed to levels not achievable with conventional chromatography methods.

The triplex affinity gel was synthesized by linking the oligonucleotide with the sequence 5'-TSTTTTTTTSST-3 '(ZEO No. 9) on periodically oxidized Zeryasgu1 3-1000 ZE, as described above in the example.

With plasmids pXB3296 (pCOK without transgene) and plasmid pBK3 obtained from Co1E1, chromatography was performed on a 1 ml column containing the 5'-TSTTTTTTTSTSST-3 'oligonucleotide (ZEO No. 9) under the conditions described in Example 9. The resulting plasmids in the collected fractions were 175 μg for pVHC and <1 μg for pXB3296. This indicates that the 5'-TSTTTTTTTTSST-3 'oligonucleotide (ZEO Ш No. 9) forms stable three-helix structures with the complementary 12-membered 5'-АСОАААААССА-3' sequence (ЗОО ΙΌ No. 8) present in rHEC, but not with present in pCOK very tightly bound 12-membered sequence 5'-ASAAAAAAAASA-3 ') (ZEO No. 34). This indicates that the introduction of one noncanonical triad (in this case, C * AT) leads to a complete destabilization of the structure of the triplex.

Example 14

Sowing culture was performed in an unopened Erlenmeyer bottle in the following way. A working bank of cells was introduced into an Erlenmeyer bottle containing M9tobC5 medium with a culture ratio of 0.2% (v / v). The strain was cultured at 220 rpm in a rotary shaker at 37 ± 1 ° C for approximately 18 ± 2 hours until glucose was depleted. The result was 200 ml of seeded culture. The estimated optical density of the A600 culture was approximately 2-3.

Then in the first fermenter received preculture. The seeded culture was sterilely transferred to a pre-fermenter with M9tobC5 medium to ensure a culture ratio of 0.2% (v / v) and cultured with aeration and stirring. pO _{2 was} maintained above 40% saturation. The culture was harvested when glucose ended after 16 hours. The result was approximately 30 l of preculture. The estimated optical density of the A600 culture was approximately 2-3.

Then in the second fermenter received the main culture. To ensure an inoculation ratio of approximately 10% (v / v), 30 l of preculture are sterilized transferred into 270 l of sterile EtobC2 fermenter medium. The culture is launched on a periodic mode for the formation of some biomass. Glucose was added once, the initially added sugar was depleted after about 4 hours. To maintain a specific growth rate close to 0.09 h ^-1 , aeration, mixing, pO ₂ (40%), pH (6.9 ± 0.1) were controlled temperature (37 ± 1 ° C) and the addition of glucose. The cultivation was stopped after approximately 35 hours of feeding. This results in approximately 400 L of culture. The estimated optical density of the A ₆₀₀ culture was approximately 100.

After collecting these cells, the first isolation step was performed. Biomass was collected in a centrifuge with conical baffles in the rotor. To eliminate the used culture medium, the medium was 3-4 times concentrated and continuously resuspended in 400 L of sterile buffer Z1. This results in approximately 500 L of the above biomass. ES \ Y = 25 ± 5 g / l.

After this concentration step, a second isolation step was performed. After resuspension / homogenization in buffer Z1, the cells were again centrifuged until a concentrated thick suspension was obtained. This results in approximately 60-80 L of washed and concentrated thick suspension, ES \ Y = E50 ± 30 g / l; plasmid DNA = 300 ± 60 mg / L.

Then performed the stage of freezing. The thick suspension was sterile transferred into 20-liter E1exbou ™ bags (filled up to 50% of their volume), and then frozen at -20 ± 5 ° С until further sequential treatment. This results in frozen biomass, pDNA = 300 ± 60 mg / l; supercoiled shape> 95%.

Then performed the stage of thawing cells. Frozen bags were warmed to 20 ° C and a thick cell suspension was diluted in 40 g / l, pH 8.0 with 100 mM TPC hydrochloride, 10 mM EDTA, 20 mM glucose, and the suspension was left at 20 ± 2 ° C per 1 cell lysis h with stirring. This leads to a thawed thick suspension of biomass, pH 8.0 ± 0.2.

At this stage, temperatures of approximately 20 ° C can be used.

Then, the alkaline lysis step was performed. The stage of cell lysis included pumping the diluted cell suspension through an in-line mixer with a solution of 0.2 N NaOH-35 mM SEZ (solution Z2) followed by a continuous interaction step in a spiral tube. The continuous interaction step provided complete cell lysis and denaturation of genomic DNA and proteins. Prior to collection in a cooled stirred vessel, a solution of lysed cells was mixed in a stream with a solution of 3 (33) chilled 3 M potassium acetate 2n. acetic acid. The addition of solution Z3 led to the precipitation of genomic DNA, RNA, proteins, and KOZ.

After that, lysate filtration was performed. Then the neutralized lysate was incubated at 5 ± 3 ° C for 2 to 24 hours without stirring and to remove the mass of the precipitated substance

- 34 011554 (turbid phase) was filtered through a 3.5 mm strainer, followed by deep filtration to improve the filtration stage. This resulted in a purified lysate with a supercoiled plasmid concentration of more than 90%.

Then anion exchange chromatography was performed. The solution of purified lysate was dissolved in purified water to a target conductivity of 50 mS / cm, filtered through a two-layer filter (3-0.8 μm) and applied to an anion-exchange chromatographic column. A 300 mm column was used, filled with 11.0 L of Pgac1ode1® TMAE H1Car (M) resin (Megsk; # 1.10316.5000). The purified lysate was applied to the column and elution was performed using a step gradient No. C1. The mass of contaminants bound to the column was eluted with a ΝηΟΊ solution at approximately 61 mS / cm, and plasmid DNA was eluted with a ΝηΟΊ solution at approximately 12 mS / cm. This resulted in an eluate of ion exchange chromatography with a high concentration of plasmid DNA.

After this, three-helix affinity chromatography was performed. The eluate from the anion exchange chromatographic column was dissolved in approximately 0.5 volumes of a 4.8 Μ ΝηΟΊ solution of 500 mM sodium acetate (pH 4.2) and pumped through a three-helix affinity chromatographic column balanced with 50 mM sodium acetate (pH 4.5) containing 2 M No. C1. The diameter of the column was 300 mm and it contained 10.0 L of TNAS 8eryasgu1 ™ 8-1000 gel (Ategkyat Vykshepsek; Rksaatau, N1). The column was washed with a solution containing 1 Μ ΝηΟΊ 50 mM sodium acetate (pH 4.5) and ΝΥ1ΡΟΡ was eluted with 100 mM Tgk (pH 9.0) containing 0.5 mM EDTA. This resulted in a three-helix affinity chromatography eluate with high plasmid concentration.

Then followed the hydrophobic interaction chromatography step. The affinity chromatographic column eluate was diluted in 3.6 volumes of a solution of 3.8 M ammonium sulfate in ThC (pH 8.0). After filtration through a 0.45 μm filter, the filtrate was applied at 60 cm / h to a hydrophobic interaction column (300 mm diameter) filled with 9.0 L of Touoreag1® Vi1u1-6508 resin (TokoN co., Sgoue Syu, OH). The column was washed with a solution of ammonium sulfate at approximately 240 mS / cm and eluted with Νν1ΡΟΡ ammonium sulfate at 220 mS / cm. This resulted in a relaxed free H1C eluate.

In a preferred embodiment, an additional diafiltration step has been performed. Conventional, commercially available diafiltration agents suitable for use in this method by standard techniques are known in the art. A preferred diafiltration method is diafiltration using an ultrafiltration membrane with a molecular weight exclusion limit in the range of 30,000 to 50,000 depending on the size of the plasmid. This diafiltration step involves changing the buffer and then determining the concentration. The eluate of stage 12 was 3-4 times concentrated by tangential flow filtration (membrane exclusion limit of 30 kDa) to a target concentration of approximately 2.5 to 3.0 mg / ml and the concentrate was a buffer replaced by diafiltration with a constant volume of 10 volumes of physiological saline and selected for the target plasmid concentration with saline. The concentration Νν1ΡΟΡ was calculated from the absorbance at 260 nm of samples of the concentrate. The Νν1ΡΟΡ solution was filtered through a 0.2 μm membrane filter and stored in containers in a cold room at 2-8 ° C until further processing. As a result, a purified plasmid DNA concentrate was obtained, the concentration of supercoiled plasmid being approximately 70, 75, 80, 85, 90, 95, and preferably 99%. The marginal yield of the plasmid according to this method is at least 35, 40, 45, 50, 55, 60, 65, 70 and 80% with an average yield of 60%.

Example 15

The method according to the above example, which includes the stage of ion exchange chromatography (AEC), the stage of three-helix affinity chromatography (TNAC) and the stage of hydrophobic chromatography (H1C), leads to a more purified preparation of plasmid DNA compared to previously known methods. This new method was compared with previously known methods and received in the preparations of plasmid DNA lower amounts of genomic DNA, RNA, protein and endotoxin. This is shown in FIG. 3. These experiments showed that AEC, TNAS and H1C provide a surprisingly higher cleaning yield compared to some of the 2-stage combinations for the effective removal of all contaminants. The combination of these stages provides a clear synergy in the efficiency of separation of plasmid DNA from other biological substances and contaminants, such as protein and endotoxin, RNA and genomic DNA, as well as open circular plasmids. In addition, a combination of synergistic stages, i.e. AEC / TNAC / H1C, according to the present invention, allows not only to obtain highly purified plasmid DNA of the pharmaceutical category, but also compositions of high purity and fully supercoiled, more than 80, 85, 90, 95 and more than 99% plasmid DNA.

Example 16

The method for producing a highly purified plasmid DNA preparation according to the above example, including an ion exchange chromatography step, a three-helix affinity chromatography step, and a hydrophobic chromatography step, was compared with previously known methods. What is it in

- 35 011554 is shown in FIG. 4, the method of the present invention unexpectedly produced pDNA preparations with much smaller amounts of genomic DNA, RNA, protein, and endotoxin in the range of less than ppm. Furthermore, as shown in FIG. 4, the method of the present invention shows the resulting amount of the product up to 10 g.

Example 17

The diafiltration step was carried out as described in Example 14, in accordance with the following conditions:

the buffer for stage (a) and for stage (b) was used to determine the best conditions for:

ίίί) the first diafiltration (stage (a)) versus 12.5–13.0 volumes of 50 mM TPZ-HC1, 150 mM No. C1, pH 7.4 (labeled buffer I), and ίν) the second diafiltration of the concentrate from the above stage (a) (stage (b)) versus from 3.0 to 3.5 volumes of salt carrier (150 mM Νπ ('Ί).

This alternative diafiltration step of the present invention efficiently and thoroughly removes ammonium sulfate and thoroughly EDTA. In addition, after these diafiltration steps, an appropriate target NaCl concentration of approximately 150 mM and a final Tg18 concentration between 400 μM and 1 mM are obtained. Examples of preparations of plasmid DNA compositions are presented in table. 6.

Table 6

Samples Final concentration 1st diafiltration 2nd diafiltration Active pharmaceutical ingredient Ammonium sulfate 10 μM <1 μM <1 μM EDTA 4 μm <1 μM <1 μM

TG15

IAs1

Example 18

The L806 labeled industrial batch of XU1PCP AP1 plasmid DNA (active pharmaceutical ingredients) was produced according to Examples 13 and 17 with the diafiltration step described in Example 17. First, the eluate was first diafiltered at approximately 2 mg AP1 / ml against approximately 13 volumes of buffer I and the resulting retentrate diafiltered against approximately 3 volumes of salt filler. Then the final retentrate was filtered through a 0.2 μm filter and adjusted to 1 mg / ml. The final AP1 (pH 7.24) was stored in a glass vial at 5 ° C until ΌΡ was obtained.

The stability study was carried out on samples of L806 stored in Oigap glass bottles (AP1), as well as in 8-ml bottles used in the manufacture of a pharmaceutical product. After 90 days at 5 ° C, the degree of depurinization and the content of open ring forms for all samples was a barely detectable amount (<0.3%). After 90 days at 25 ° C, the depurinization ratio and the content of open ring forms of L806 samples were also quite low. The ratios of depurinization and open ring forms calculated from this study were <1% per month (Fig. 5). This study demonstrated that in the preparation of the present invention, where the pH values were maintained from about 7.0 to 7.5, the stability profile of plasmid DNA IU1ECE is very stable. This clearly demonstrates that over a long period of time at 25 ° C, plasmid DNA remains stable in a non-degraded form with low ratios of depurinization and plasmid breaks.

Example 19

Labeled b804, b804, b806, b807 and bt05 lots of plasmid DNA HU1PCP AP1 (active pharmaceutical ingredients) were produced according to example 13 with the stage of the diafiltration process described in example 17. First, the eluate was subjected to the first diafiltration at approximately 2 mg AP1 / ml against approximately 13 volumes buffer I and the resulting retentrate were diafiltered against approximately 3 volumes of salt filler. Then the final retentrate was filtered through a 0.2 μm filter and adjusted to 1 mg / ml for storage in 8 ml vials. Received plasmid DNA with a concentration of NaCl approximately 150 mm and a final concentration of TP between 1 and 2 mm. The stability study was carried out on all of these samples stored in 8 ml vials used in the manufacture of a pharmaceutical product.

The stability study was carried out for 150 days at 25 ° C, the pH of the plasmid DNA compositions did not noticeably change, as shown in FIG. 6A. After 203 days, the pH of L804 was significantly reduced to 6.54 (-0.27 units).

For all batches, with the exception of L804, the ratios of depurinization and single-strand breaks at 25 ° C were approximately 1.0% per month and were linearly time-dependent after 140 days. The depurinization ratio L804 was significantly higher (2.7% per month) as a result of the significantly lower pH of this batch of AP1 (> 0.4 units at T ₀ ). Single strand break ratio

- 36 011554

L804 was significantly lower than its depurinization ratio (2.4% per month).

At 5 ° C, the pH of all solutions remained stable throughout the entire time and the degree of depurinization and single-strand breaks was very low (less than 0.5% after 200 days; Fig. 6B).

This study demonstrated that in the preparations of the present invention, the stability profile of plasmid DNA LU1ECE is very stable at all times at 5 and 25 ° C with very low ratios of depurinization and single-strand breaks.

The description should be understood in the light of the ideas of the references cited in the description. The embodiments in the description provide an illustration of embodiments of the invention and should not be construed as limiting the scope of the invention. A qualified person will immediately confirm that the invention includes many other embodiments. All publications and patents cited in this specification in full are hereby incorporated by reference. In cases where the material incorporated by reference is contrary to or not in accordance with this description, the description replaces any such material. The citation of any references herein does not acknowledge that such references are prior to the present invention.

Unless otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, etc., used in the description, including the claims, should be understood as changed in all cases by the term "approximately". Accordingly, unless otherwise indicated otherwise, the numerical parameters are approximate and may vary depending on the desired properties in the pursuit of the obtained parameters of the present invention. At least each numerical parameter should be considered in the light of a number of significant figures and the usual principles of rounding, but not as an attempt to limit the scope of the invention to the equivalent of the scope of the claims.

Unless otherwise indicated, the term “at least” precedes a series of elements, which should be understood with reference to each element in the series. A skilled person will find or be able to determine, using no more than a routine experiment, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the following claims.

A person skilled in the art can trust the contents of any of the links or documents referred to herein, and each link or document incorporated herein by reference in its entirety. However, none of the referenced herein or the document should change the meaning of any term or concept specifically indicated in this document. References and documents, as well as available to any specialist in this field of knowledge will allow you to make changes and options in the specific embodiments described here. The examples and specific embodiments described herein are not to be understood as limiting the scope or scope of the invention.

Claims (88)

1. A liquid composition that maintains stable plasmid DNA, containing plasmid DNA and a buffer solution, where the buffer is present in a concentration of less than 2 mM to maintain the pH of the composition between 6 and 9, and the composition contains a pre-purified supercoiled form of plasmid DNA. 2. The composition according to claim 1, where the plasmid DNA is stable at temperatures from about 4 to 25 ° C. 3. The composition according to any one of the preceding paragraphs, where the plasmid DNA is stable for several months, 1 year, 2, 3, 4, 5 years and up to 10 years. 4. The composition according to any one of the preceding paragraphs, where the plasmid DNA is stable at approximately 4 ° C for several months, 1 year, 2, 3, 4, 5, 10, 15 years and up to 20 years. 5. The composition according to any one of the preceding paragraphs, containing at least 80% supercoiled or closed circular form of plasmid DNA. 6. The composition according to any one of the preceding paragraphs, containing approximately 80%, approximately 85%, approximately 90%, approximately 95% and approximately or more than 99% of the supercoiled or closed circular form of plasmid DNA. 7. The composition according to any one of the preceding paragraphs, where the relationship of depurinization and single-strand breaks is less than 5% per month. 8. The composition according to any one of the preceding paragraphs, where the buffer is presented in a concentration of up to 2 mm. 9. The composition according to any one of the preceding paragraphs, where the buffer is presented in a concentration between 1 and 2 mm. 10. The composition of claim 8, where the buffer is presented in a concentration of less than 1 mm. 11. The composition of claim 10, where the buffer is presented at a concentration of between 250 μm and 1 mm. 12. The composition according to claim 11, where the buffer is presented at a concentration of approximately 400 μm. 13. The composition according to any one of the preceding paragraphs, where the buffer solution is present in such - 37 011554 concentration, to maintain the pH of the specified drug or composition between 6.2 and 8.5, or approximately ± 0.3 from one or both of these values. 14. The composition according to any one of the preceding paragraphs, where the buffer solution is present in such a concentration as to maintain the pH of the specified drug or composition between 6.2 and 8.5, or approximately ± 0.3 from one or both of these values, and the plasmid DNA has the ratio of depurinization and single-strand breaks 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 paragraphs, where the buffer solution is present in such a concentration as to maintain the pH of the specified drug or composition between 6.7 and 8.0, or approximately ± 0.3 from one or both of these values. 16. The composition according to any one of the preceding paragraphs, where the buffer solution is present in such a concentration as to maintain the pH of the specified drug or composition between 6.7 and 8.0, or approximately ± 0.3 from one or both of these values, and the plasmid DNA has the ratio of depurinization and single-strand breaks of less than 2% per year when stored at approximately 4 ° C and less than 2% per month when stored at approximately 25 ° C. 17. The composition according to any one of the preceding paragraphs, where the buffer solution is present in such a concentration as to maintain the pH of the specified drug or composition between 7.0 and 7.5, or approximately ± 0.3 from one or both of these values. 18. The composition according to any one of the preceding paragraphs, where the buffer solution is present in such a concentration as to maintain the pH of the specified drug or composition between 7.0 and 7.5, or approximately ± 0.3 from one or both of these values, and the plasmid DNA has the ratio of depurinization and single-strand breaks of less than 1% per year when stored at approximately 4 ° C and less than 1% per month when stored at approximately 25 ° C. 19. The composition according to any one of the preceding paragraphs, where the buffer solution contains: (a) TPC or lysine and an acid selected from a strong acid or weak acid; (b) Nerek and strong base; or (c) phosphate buffer. 20. The composition according to any one of the preceding paragraphs, where the buffer solution contains Tpc / HCl, lysine / HCl, Tpc / maleic acid, Tpc / malic acid, Tpc / acetic acid or Nerek / sodium hydroxide. 21. The composition according to any one of the preceding paragraphs, where the buffer solution is a TPC. 22. The composition according to any one of the preceding paragraphs, in addition, containing a salt excipient. 23. The composition of claim 22, wherein the salt excipient is NaCl. 24. The composition of claim 23, wherein the NaCl is present at a concentration of between 100 and 200 mM, and preferably about 150 mM. 25. The composition according to any one of the preceding paragraphs, where the plasmid DNA is highly purified or is a plasmid DNA of pharmaceutical quality. 26. A stable plasmid DNA composition comprising plasmid DNA and a buffer solution, where the buffer solution is present in a concentration sufficient to maintain plasmid DNA in a stable form at temperatures from about 4 to 25 ° C. 27. A stable plasmid DNA composition comprising plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration sufficient to maintain the plasmid DNA with at least 80% supercoiled plasmid DNA at a temperature of about 4 ° C for at least about 4 years. 28. A stable plasmid DNA composition comprising plasmid DNA and a buffer solution, where the buffer solution is present in a concentration sufficient to preserve plasmid DNA with ratios of depurinization and single strand breaks of less than 5% per year to 5% per month when stored at about 4 ° C. up to approximately 25 ° C. 29. A stable plasmid DNA salt composition containing plasmid DNA and a buffer solution, where the buffer solution is present in a concentration sufficient to maintain plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA from 4 to 25 ° C for a long period time. 30. A stable plasmid DNA composition comprising plasmid DNA and a buffer solution, where the buffer solution is present in a concentration sufficient to keep the plasmid DNA in a stable form with at least 80% supercoiled plasmid DNA at 4 to 25 ° C for up to 20 months. 31. The composition according to any one of paragraphs.26-30, further containing a salt excipient. 32. The composition of claim 31, wherein the salt excipient is NaCl present in a concentration of between 100 and 200 mM and preferably about 150 mM. 33. The composition according to any one of p-32, where the plasmid DNA is highly purified or is a plasmid DNA of pharmaceutical quality. - 38 011554 34. A method of storing plasmid DNA in a stable form in a composition, comprising: obtaining a purified sample of plasmid DNA; mixing the specified purified sample of plasmid DNA and a buffer solution at a concentration of up to 2 mm, sufficient to maintain the pH of the resulting composition between 6 and 9; and plasmid DNA storage. 35. The method according to clause 34, where the plasmid DNA contains at least 80% supercoiled plasmid DNA. 36. The method according to claims 34, 35, wherein the buffer solution is present in such a concentration as to maintain the pH of said composition between 6.2 and 8.5, or approximately ± 0.3 from one or both of these values. 37. The method according to clause 36, where the plasmid DNA is stored at temperatures from about 4 to 25 ° C with ratios of depurinization and single-strand breaks from less than 5% per month to less than 5% per year. 38. The method according to any of paragraphs 34-37, where the buffer solution is present in such a concentration as to maintain the pH of the specified composition between 6.7 and 8.0, or approximately ± 0.3 from one or both of these values. 39. The method according to § 38, where the plasmid DNA is stored at temperatures from about 4 to 25 ° C with ratios of depurinization and single-strand breaks from less than 2% per month to less than 2% per year. 40. The method according to any of paragraphs 34-39, where the buffer solution is present in such a concentration as to maintain the pH of the specified composition between 7.0 and 7.5, or approximately ± 0.3. 41. The method according to clause 40, where the plasmid DNA is stored at temperatures from about 4 to 25 ° C with ratios of depurinization and single-strand breaks from less than 1% per month to less than 1% per year. 42. The method according to any one of claims 34-41, wherein the buffer is added at a concentration of up to 2 mM. 43. The method according to § 42, where the buffer is added at a concentration between 1 and 2 mm. 44. The method according to § 42, where the buffer is added at a concentration of less than 1 mm. 45. The method according to item 44, where the buffer is added at a concentration of between 250 μm and 1 mm. 46. The method according to item 45, where the buffer is added at a concentration of approximately 400 μm. 47. The method according to claims 34-46, wherein a salt excipient is additionally added to the plasmid DNA and the buffer solution. 48. The method according to clause 47, where the salt excipient is a No. C1. 49. The method according to p, where Νηί ', Ί is added in a concentration between 100 and 200 mm and preferably approximately 150 mm. 50. The method according to any one of claims 34-49, wherein the highly purified plasmid DNA or pharmaceutical grade plasmid DNA is mixed with a buffer solution. 51. A method of storing 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, which includes obtaining a purified sample of plasmid DNA; mixing a purified sample of plasmid DNA and a buffer solution, where the buffer is present in a concentration of less than 2 mm; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 52. A method of storing 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, which comprises obtaining a purified plasmid DNA sample; mixing a purified sample of plasmid DNA and a buffer solution, where the buffer is present at a concentration between 1 and 2 mm; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 53. A method for storing 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, which comprises obtaining a purified plasmid DNA sample; mixing a purified sample of plasmid DNA and a buffer solution, where the buffer is present in 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 storing 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, which includes obtaining a purified plasmid DNA sample; mixing a purified sample of plasmid DNA and a buffer solution, where the buffer is present at a concentration between approximately 250 μM and 1 mm; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. - 39 011554 55. The method according to any of paragraphs 51-54, where the plasmid DNA is stored at temperatures from about 4 to 25 ° C with ratios of depurinization and single-strand breaks from less than 5% per month to less than 5% per year. 56. The method according to any one of claims 51 to 55, wherein a salt excipient is additionally added to the plasmid DNA composition. 57. The method according to p, where the salt excipient is ΝαΟΊ. 58. The method according to § 57, where No.-1C1 is present in a concentration between 100 and 200 mm and preferably approximately 150 mm. 59. A method of obtaining a stable plasmid DNA composition at a temperature of up to about 25 ° C, which includes a cell lysis step consisting of passing cells through (a) a turbulent flow device for quickly mixing a cell suspension with a cell lysing solution and (b) a device for laminar flow to ensure incubation of the formed mixture (a) without significant activation, where the formed mixture in (a) without significant mixing passes from the device for turbulent flow into the device for the lamin a stream, and in addition, optionally includes (c) a device for adding a neutralizing lysing solution of a second solution, the incubated mixture in (b) flows from a laminar flow device to a device for adding a second solution, thereby releasing plasmid DNA from cells ; one chromatography step for purification of the plasmid DNA thus isolated; mixing said purified plasmid DNA and buffer solution at a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6 and 9; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 60. A method for preparing a stable plasmid DNA composition at a temperature of up to about 25 ° C., comprising a cell lysis step consisting of passing cells through (a) a turbulent flow device for rapidly mixing a cell suspension with a cell lysing solution and (b) a device for laminar flow to ensure incubation of the formed mixture (a) without significant activation, where the formed mixture in (a) without significant mixing, where formed in (a) passes from the device for turbulent flow into a device for laminar flow, and, in addition, optionally includes (c) a device for adding a neutralizing lysing solution of a second solution, the incubated mixture in (b) flows from a device for laminar flow into a device for adding a second solution, thereby releasing plasmid DNA from cells; one chromatography step for purification of the plasmid DNA thus isolated; mixing said purified plasmid DNA and buffer solution at a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6.2 and 8.5, or approximately ± 0.3 from one or both of these values; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 61. A method of obtaining a stable plasmid DNA composition at a temperature of up to about 25 ° C, which includes a cell lysis step consisting of passing cells through (a) a turbulent flow device for quickly mixing a cell suspension with a cell lysing solution and (b) a device for laminar flow to ensure incubation of the formed mixture (a) without significant activation, where the formed mixture in (a) without significant mixing passes from the device for turbulent flow into the device for the lamin a stream, and in addition, optionally includes (c) a device for adding a neutralizing lysing solution of a second solution, the incubated mixture in (b) flows from a laminar flow device to a device for adding a second solution, thereby releasing plasmid DNA from cells ; one chromatography step for purification of the plasmid DNA thus isolated; mixing said purified plasmid DNA and buffer solution at a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 6.7 and 8.0, or approximately ± 0.3 from one or both of these values; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 62. A method of obtaining a stable plasmid DNA composition at a temperature of up to about 25 ° C, which includes a cell lysis step consisting of passing cells through (a) a turbulent flow device for quickly mixing a cell suspension with a cell lysing solution and (b) a device for laminar flow to ensure incubation of the formed mixture (a) without significant activation, where the formed mixture in (a) without significant mixing passes from the device for turbulent flow into the device for the lamin Nogo stream, and further optionally comprises (c) means for adding a second solution neutralizes the lysing solution, incubated in (b) flowing from the means for laminar flow into the means for adding a second races - 40 011554 creature, thereby releasing plasmid DNA from cells; one chromatography step for purification of the plasmid DNA thus isolated; mixing said purified plasmid DNA and buffer solution at a concentration of up to 2 mM sufficient to maintain the pH of the resulting composition between 7.0 and 7.5, or approximately ± 0.3 from one or both of these values; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 63. The method according to any one of paragraphs 59-62, where the buffer solution is present in a concentration of less than 2 mm. 64. The method according to any of paragraphs 59-63, where the buffer solution is present in a concentration of from about 1 to 2 mm. 65. The method according to any one of claims 59-64, wherein the buffer solution is added until a concentration of about 250 μM to less than 1 mM is reached in the plasmid DNA composition. 66. A method of obtaining a stable plasmid DNA composition at a temperature of up to about 25 ° C, which includes a cell lysis step consisting of passing cells through (a) a turbulent flow device for rapidly mixing a cell suspension with a cell lysing solution and (b) a device for laminar flow to ensure incubation of the formed mixture (a) without significant activation, where the formed mixture in (a) without significant mixing passes from the device for turbulent flow into the device for the lamin a stream, and in addition, optionally includes (c) a device for adding a neutralizing lysing solution of a second solution, the incubated mixture in (b) flows from a laminar flow device to a device for adding a second solution, thereby releasing plasmid DNA from cells ; one chromatography step for purification of the plasmid DNA thus isolated; mixing said purified plasmid DNA and a buffer solution, wherein the buffer solution is present in a concentration of less than 2 mM or less than 1 mM or between 250 μM and 1 mm and preferably 400 μM; and storing the plasmid DNA composition at a temperature of up to about 25 ° C. 67. The method according to any one of claims 59-66, wherein a salt excipient is additionally added to the plasmid DNA composition. 68. The method according to p, where the salt excipient is a No. C1. 69. The method of claim 68, wherein Ν; · ιί.Ί is added at a concentration between 100 and 200 mM, and preferably about 150 mM. 70. The method according to any one of claims 59-69, wherein the lysing solution is a solution containing a lysing agent selected from the group consisting of alkali, detergent, organic solvent and enzyme, or a mixture thereof. 71. The method according to any one of claims 59-70, wherein the plasmid DNA is purified by at least one chromatographic step including an anion exchange chromatography step, three-helix affinity chromatography or hydrophobic interaction chromatography. 72. The method according to p, where for the purification of plasmid DNA stage of anion exchange chromatography is combined with the stage of three-helix chromatography. 73. The method of claim 72, further comprising the step of hydrophobic interaction chromatography. 74. The method according to any one of claims 59-73, wherein the plasmid DNA is purified by a 3-step chromatography method including anion exchange chromatography, three-helix affinity chromatography and hydrophobic interaction chromatography carried out in this order. 75. The method according to any one of claims 59-74, wherein the first chromatography is performed prior to filtration of the lysate. 76. The method according to any one of claims 59-75, wherein the first chromatography is performed prior to the removal of the precipitate. 77. The method according to any one of claims 59-76, wherein the last chromatography steps follow the step of diafiltration and / or buffer replacement. 78. The method according to any one of paragraphs 59-77, where the removal of sediment is preceded by the passage of the solution through a strainer and through deep filtration. 79. The method according to any one of paragraphs 59-78, comprising the step of diafiltration to achieve the appropriate salt, buffer and pH target values. 80. The method according to any one of paragraphs 59-79, comprising the following stages: collecting the solution after the last stage of chromatography; conducting the first stage of diafiltration against Tp8 / No. 1C1 buffer; conducting the second stage of diafiltration against physiological saline under conditions acceptable for controlling the final buffer concentration and for stabilizing the pH of the final plasmid DNA preparation. - 41 011554 81. The method according to any one of claims 59-80, further comprising the step of sterilizing by filtration, preparing the composition and filling the vials of purified plasmid DNA. 82. The method according to any one of claims 59-81, wherein the chromatography steps remove impurities such as proteins, denatured genomic DNA, RNA, proteins, oligoribonucleotides, oligodeoxyribonucleotides, denatured plasmid DNA and lipopolysaccharides. 83. The method according to any one of paragraphs 59-81, wherein the chromatography steps are carried out on a solid support, which is any organic, inorganic or combined substance, porous, non-porous, super-porous or non-porous, suitable for chromatographic isolation, which is a derivative of polyalkylene glycols, alkanes, alkenes , alkynes, arenes, or other hydrophobic molecules imparting to the carrier. 84. The composition according to any one of claims 1 to 33, where the plasmid DNA contains a coding therapeutic and / or immunogenic sequence. 85. The composition of claim 84, wherein the therapeutic gene is a mammalian gene. 86. The use of the composition of claim 84 as a DNA vaccine. 87. The use of the composition of claim 84 or 85 as an agent for plasmid-based therapy, such as gene therapy. 88. The use of a composition according to any one of claims 1 to 33 in a method of treatment by treating a human or animal organism.