WO2002064752A1 - Method for the production of nucleic acids - Google Patents

Abstract

The invention relates to a method for the production of nucleic acids, in particular superspiral nucleic acids. The cultivation of the transformed bacterial cells to give a high cell density is preferably carried out in a batch process, in a given, synthetic aqueous medium, containing no complex components or components obtained from animals. The nucleic acids isolated from the bacterial cells and purified are suitable for use in non-viral gene therapy, cell therapy or genetic inoculation.

Description

 Process for the production of nucleic acids

The present invention relates to a method for producing bacterial biomass which contains supercoiled nucleic acids. The cultivation of the transformed bacterial cells to high cell densities is carried out in the batch process in a defined, synthetic aqueous medium which does not contain any complex components and / or components which are obtained from animals. The nucleic acids isolated and purified from the bacterial cells are suitable for use in non-viral gene therapy, cell therapy or genetic vaccination.

Background of the Invention The production of nucleic acids has gained great importance through the use of these biomolecules as transport systems (vectors) for therapeutic genes in modern forms of therapy such as cell therapy, gene therapy or genetic vaccination (DNA vaccination). Numerous nucleic acid vaccines and gene therapeutics are currently being tested in clinical studies. In these forms of therapy, nucleic acids are used as non-viral vectors. The gene transfer can take place by direct injection of the nucleic acids into tissue or cells or with the help of a gene gun. The DNA can be present as a "naked" nucleic acid or in formulation with other substances (e.g. liposomes) that increase gene transfer or efficiency.

Microorganisms have the property of being able to replicate extra-chromosomal, circular nucleic acids, such as plasmids or cosmids. This property can be used in large quantities to produce the nucleic acids.

Plasmids are extrachromosomal, mostly circular, double-stranded DNA molecules. They have a size between approx. 200 and 100,000 base pairs (bp), exist in the cells next to the bacterial chromosome and are replicated autonomously by this. Plasmids are often in multiple copies per cell. Their number varies between 2 to 20 copies per cell (low-copy plasmids) or several hundred copies per cell (high-copy plasmids). The additional genetic information provided by the plasmids gives the host organism a selection advantage over plasmid-free cells, such as resistance to an antibiotic. In biotechnology, plasmids have numerous applications as cloning and expression vectors. They have a replication system, one or more selection marker genes and a cloning section for accepting foreign gene sections.

The production of large quantities of these nucleic acids in high quality is of particular interest due to the increasing use of the nucleic acids in preclinical, clinical or veterinary applications in cell and gene therapy or DNA vaccination.

For the production of bacterial biomass for the production of nucleic acids of pharmaceutical quality, the bacterial cells are grown on nutrient media to high cell densities with a simultaneous high product concentration in the cells and high quality of the product. The media used here consist of carbon and nitrogen sources, as well as a mineral salt mixture. In the prior art methods, nutrient media are used whose carbon and / or nitrogen sources are one or more undefined complex components, such as extracts or hydrolysates of natural products (see, for example, Wan et al, US005487986 (1996) or Rainikainen et al, Biotechnol. Bioeng. 33 (1989), 386-393, O'Kennedy et al, J. Biotechnol. 76 (2000), 175-183). The prior art also uses nutrient media which contain components from animal sources. Other possible toxic substances can also get into the nutrient medium and thus come into contact with the nucleic acid product through the undefined, complex components. In addition, the formation of biomass and product varies greatly with the composition and quality of the complex components used, which can vary from batch to batch.

During cultivation, the metabolism of the media components generates biomass, the product as part of the cell mass and additional metabolic energy. In processes from the prior art, high substrate concentrations are also introduced at the beginning of the cultivation in order to generate high biomass and product concentrations. Instead, growth and product formation-inhibiting metabolic components such as acetate are formed (see Kleman et al, Appl. Environ. Microbiol. 60 (1994), 3952-3958). The formation of these undesirable metabolic by-products is at bypassing technology by using so-called inflow cultivation methods. The substrates are presented in lower concentrations at the beginning of the cultivation. After these substrates have been consumed, fresh, concentrated nutrient medium is supplied, which can be carried out continuously or in a controlled manner (see, for example, Chen et al, J. Ind. Microbiol. Biotechnol. 18 (1997), 43-48; Lahijani et al, Hu. Gen. Ther. 7 (1996), 1971-1980; Schmidt et al WO 99/61633 (1999)). The decisive disadvantage of these feed procedures is the complex, technical implementation of the control and regulation procedures for the feed of medium. In addition, the change between excess substrate and lack of substrate places a physiological stress on the bacterial cells, which can have a negative effect on the product formation process.

It was therefore an object of the present invention to provide a method for the production of nucleic acids, in particular superspiralized nucleic acids, of pharmaceutical quality.

Summary of the invention

The object is achieved by the method according to the invention, which completely dispenses with complex nutrient media components in the production of nucleic acids. The method according to the invention for the production of nucleic acids comprises the cultivation of bacterial cells containing the nucleic acid in a medium without complex components and the isolation of the nucleic acids. In a preferred embodiment, the medium without complex components comprises the following synthetic components:

(aa) organic carbon source,

(ab) inorganic nitrogen source

(ac) mineral salt mixture and

(ad) an additional, defined nitrogen-containing component that increases the product metabolism of the bacterial cells. The process according to the invention can be carried out particularly advantageously in the batch process and leads to large product quantities at high nutrient concentrations.

The method according to the invention enables the production of nucleic acids, especially supercoiled nucleic acids in large quantities and ensures high quality nucleic acids that can be used in pre-clinical, clinical or veterinary medical applications of cell and gene therapy or DNA vaccination.

At least 70-90%, in particular more than 90%, of superspiralized nucleic acids are preferably in the superspiralized ccc form. The ccc form (covalently closed circular) is a compact, covalently closed circular nucleic acid structure that is generated in microorganisms by topoisomerases.

In particular, contamination by undesirable substances that are difficult to detect and thus get into the nutrient medium is avoided by the method according to the invention, which e.g. is of fundamental importance in the course of the BSE problem.

The method according to the invention completely dispenses with the use of complex components.

The concentrations of the constituents in the medium are 1 to 500 g / L for the organic carbon source, preferably 10 to 300 g / L and in particular 50 to 100 g / L, 10 to 200 mM, preferably 20 to 80 mM and in particular for the inorganic nitrogen source 40 mM and for the additional, defined nitrogen-containing component 25 to 1000 mM, preferably 50 to 250 mM.

The additional, defined nitrogen-containing component that enhances the product metabolism is a preliminary step in the biosynthesis of the nucleotide bases of the nucleic acids.

The additional nitrogen-containing component is also a non-complex component.

The present invention therefore describes a method for producing nucleic acids. The cultivation of the transformed bacterial cells in one Medium, preferably carried out in the sentence process (= sentence cultivation process), which contains no complex components.

A batch cultivation method is a discontinuous cultivation method in which all components of the nutrient medium are present in the culture vessel before the microorganisms are added.

Complex components of nutrient media are undefined natural products or extracts obtained from them for the cultivation of microorganisms, such as yeast autolysate, yeast extract, peptones, malt or meat extracts.

The transformed bacterial cells are cultivated in a medium which contains the defined, synthetic constituents, organic carbon source, inorganic nitrogen source, mineral salt mixture and an additional, defined nitrogen-containing component which enhances the product metabolism. During cultivation, the transformed bacterial cells produce nucleic acids.

The special composition of the fully synthetic medium enables cultivation in the typesetting process to high cell densities or biomass concentrations (optical density (600 nm) ≥ 75) in conjunction with high product yields (plasmid concentration ≥ 65 mg / L), which were previously only possible within the scope of technology complex feed processes in mostly complex media were achieved. By using purely synthetic components and dispensing with complex and animal components, the medium is ideally suited for the production of nucleic acids in pharmaceutical quality for, for example, cell and gene therapy or genetic vaccination.

Detailed description of the invention

The present invention describes a method for producing nucleic acids, in particular superspiralized nucleic acids, the bacterial cells being cultivated preferably using the batch method. In the batch process, all nutrient sources are made available to the bacterial cells in the medium from the start of cultivation. However, the use of high substrate concentrations in processes which correspond to the prior art does not simultaneously lead to high cell densities and product quantities, in contrast to the present invention, because metabolic substances, such as acetate, are formed which inhibit further cell growth (see, for example, Kleman et al, Appl. Environ. Microbiol. 60 (1994), 3952-3958). Therefore, to avoid Soil inhibition is used in cultivation to high cell densities, so-called feed processes, in which only small amounts of nutrients are available to the bacterial cells at the beginning of the cultivation and when these nutrients are used fresh medium flows in. These feed processes are known and correspond to the prior art (see, for example, Chen et al, J. Ind. Microbiol. Biotechnol. 18 (1997), 43-48, Lahijani et al, Hum. Gen. Ther. 7 (1996), 1971-1980, Schmidt et al WO 99/61633 (1999)). Disadvantages of these methods are the complex, technical implementation of the control or regulation of the inflow of fresh medium, the physiological stress to which the bacterial cells are exposed due to alternating substrate excess and substrate deficiency conditions, the use of complex, undefined medium components or the small amounts of plasmid within the bacterial cells ( Plasmidmassenanteil). The balanced composition of the nutrient medium of the present invention enables cultivation in the batch process to high cell densities with a simultaneously high product concentration or high plasmid mass fraction without the growth-inhibiting by-product by-products being formed.

A particular advantage of the invention is that the medium for cultivating the bacterial cells does not contain any complex, undefined components, such as yeast extracts, peptones or animal hydrolyzates or other components that are obtained from animals that are used in methods according to the prior art ( see, for example, Wan et al, US005487986 (1996), O Kennedy et al, J. Biotechnol. 76 (2000), 175-183). Complex media components can be contaminated with undesirable substances that are difficult to detect and thus get into the nutrient medium. In the course of the BSE problem, the complete elimination of animal components in the production of pharmaceuticals is very advantageous.

Another disadvantage when using complex media components concerns the different quality of the starting material. Large differences in the composition can occur from batch to batch, so that the cultivation cannot be carried out reproducibly. The method according to the invention for producing plasmid-containing biomass in a synthetic medium is therefore superior to the prior art. The defined components, which are produced synthetically, can be used in the highest purity without contamination with undesirable substances and also do not vary in their composition. The balanced composition of the defined, synthetic medium from organic carbon source, inorganic nitrogen source, mineral salt mixture and an additional, defined nitrogen-containing component, which enhances the product metabolism, is a decisive advantage of the method according to the invention and allows the transformed bacterial cells to metabolize the substrates and Produce nucleic acids in large quantities without the growth-inhibiting by-products are formed.

In a preferred embodiment of the present invention, the bacterial cells are isolated from the culture medium. This can be done by filtration, centrifugation or other methods that correspond to the state of the art. After isolation, the bacterial cells can additionally be washed with a liquid which does not change the cells in order to remove residues of the culture medium.

The nucleic acids are produced within the bacterial cells. In a further preferred embodiment of the invention, the nucleic acids are therefore isolated from the bacterial cells and purified from the cells and other cell constituents. Cell lysis and purification can be carried out by methods described in the prior art, such as alkaline lysis followed by a chromatography step (see, for example, Marquet et al, Biopharm 8 (1995), 26-37) or Colpan et al, US 5990301, 1999 or Ferreira et al, J. Mol. Recogn. 11 (1998), 250-251 or Green et al, Biopharm 10 (1997), 52-62)). With the help of these methods, the nucleic acids can be isolated in pharmaceutical quality, so that they can be used as an active ingredient in cell or gene therapy and genetic vaccination.

In another preferred embodiment of the method of the invention, the cells are frozen or freeze-dried after cultivation. This is very useful if immediate isolation and purification of the nucleic acids is not desired and the bacterial cells are to be stored for a long time.

The bacterial cells can be gram-positive or gram-negative. In a preferred embodiment of the present invention, the transformed bacterial cells are Escherichia co // cells. In particular, an Exoli K12 strain with genotype recA ^' or recAl is used. These bacterial strains produce very homogeneous nucleic acids. It is therefore a great advantage of the method according to the invention that the nucleic acids in the cells are supercoiled, in particular more than 90% are in the ccc form. Another decisive advantage of the invention is that the purified nucleic acids can be used in pharmaceutical quality as an active ingredient in cell or gene therapy and genetic vaccination. No undesirable or toxic substances that exclude the use as a pharmaceutical in the clinic are used in the production.

A decisive advantage of the method of the invention relates to the fact that a large number of circular nucleic acids, such as plasmids, cosmids or even larger nucleic acid molecules, can be produced therewith. The size of the molecules has no influence on the yield or quality of the products.

The defined organic carbon source is preferably from the group of the monosaccharides, disaccharides, polysaccharides or poryols, in particular CHucose, maltose, raffinose, galactose, trehalose, sucrose, fructose, mannose, sorbose, fucose, rhamnose, arabinose, xylose, ribose, manitol , Sorbitol or glycerin used as an organic carbon source. In a preferred form of the process according to the invention, the carbon source in the medium is used in a concentration of from 1 to 500 g / L, preferably 10 to 300 g / L and in particular 50 to 100 g / L. The carbon sources can be chemically synthesized in the highest purity levels and are therefore not contaminated with undesirable substances or animal components.

In the process according to the invention, ammonium salts, such as, for example, ammonium chloride, ammonium phosphates, ammonium sulfate or ammonium nitrate or ammonia itself, are preferably used as the inorganic nitrogen source. In higher concentrations, ammonium ions together with phosphate and magnesium ions, which can also be contained in a defined, synthetic medium, form sparingly soluble magnesium ammonium phosphate. In order to avoid the formation of this salt precipitation during the preparation of the medium or during the cultivation, the ammonium salts are preferably used in a concentration of 10 to 200 mM, preferably 20 to 80 mM and in particular 40 mM.

Mineral salts are an important component of bacterial nutrient media. In the process according to the invention, at least water-soluble inorganic phosphates, chlorides, sodium salts,

Potassium salts and magnesium salts, preferably potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride and magnesium sulfate are used as the mineral salt mixture. In particular, the salts in the concentrations of potassium dihydrogen phosphate 1 to 50 g L, preferably 1.5 to 10 g L, dipotassium hydrogen phosphate 1 to 75 g / L, preferably 2.3 to 15 g / L, sodium chloride 1 to 20 g / L, preferably 2.5 to 10 g / L and magnesium sulfate 0.1 to 20 g / L, preferably 0.3 to 10 g / L.

In a special embodiment of the invention, the mineral salt mixture is additionally a trace element solution consisting of iron (ITI) chloride, zinc sulfate, manganese sulfate, cobalt sulfate, copper chloride, boric acid, sodium molybdate and citric acid, in particular iron (m) chloride in a concentration of 20 mM , Zinc sulfate in a concentration of 5 mM, manganese sulfate in a concentration of 11 mM, cobalt sulfate in a concentration of 2 mM, copper chloride in a concentration of 1 mM, boric acid in a concentration of 16 mM sodium molybdate in a concentration of 11 mM and citric acid in a concentration of 24 mM.

In a further embodiment of the invention, host strain-specific supplements, e.g. Vitamins or other components that the host strain used cannot synthesize itself are added. Numerous bacterial strains have, for example, thiamine auxotrophy, such as E. coli DH1 or E. coli DH5α, which is why the vitamin is added to the medium when these host strains are used.

With the method of the present invention, large amounts of biomass and nucleic acid product can be obtained in batch operation in a defined synthetic medium. With sentence cultivation methods from the prior art (see, for example, Wan et al, US005487986 (1996) or Rainikainen et al, Biotechnol. Bioeng. 33 (19989), 386-393, O Kennedy et al, J. Biotechnol. 76 (2000), 175-183) with complex nutrient media, significantly lower amounts of biomass and nucleic acid products are obtained because the nutrient sources used are also converted into metabolic products that can inhibit bacterial growth and product formation. Therefore, a decisive advantage of the method according to the invention is the use of an additional, defined nitrogen-containing component which increases the product metabolism.

The additional, defined nitrogen-containing component is ideally a precursor in the biosynthesis of the nucleotide bases of the nucleic acids and thus helps to build up the purine and

Pyrimidine bases in the metabolism of bacterial cells. In a preferred embodiment of the invention, the additional, defined nitrogen-containing component is selected from the Group consisting of amino acids, Pepnde, carbonic acid amides, and nucleotides or the phosphates of these compounds. In particular, the additional defined component is selected from the group consisting of: aspartate, glutamate, GHutamine, GHycin, adenosine, cytosine, guanine, thymine, or urea or the phosphates of these compounds. The use of these compounds is advantageous because they can be synthesized chemically. The concentration of the additional defined nitrogen-containing component in the medium is 25 to 1000 mM, preferably 50 to 250 mM

A particular advantage of the present invention is the fact that no antibiotics have to be added to the medium to increase the plasmid stability in order to cultivate the transformed bacterial cells. This is particularly advantageous for use of the purified nucleic acids as an active ingredient in cell and gene therapy or genetic vaccination.

In a preferred embodiment of the method according to the invention, the carbon and / or the nitrogen source can be completely metabolized. The bacterial cells can also be separated from the medium beforehand, but a separation after complete metabolism of the nutrient sources is more advantageous, because in this case the biomass and product concentration are highest.

A particular embodiment of the method of the invention is the cultivation in a temperature-controlled bioreactor. The cultivation is carried out in a temperature range of 25-42 ° C, preferably in the range of 36-38 ° C. In a special embodiment, the bacterial cells are supplied with oxygen during the cultivation by blowing air into the medium.

In the process according to the invention, the cultivation takes place in a pH range of 6.0-8.0, preferably in the range of 6.5 to 7.5. In particular, the pH is regulated during the cultivation by adding an acid and / or base. In addition, anti-foaming agents can optionally be added during the cultivation in order to avoid over-foaming.

Another decisive advantage of the method described relates to the fact that pre-cultivation can take place in a defined, synthetic medium. This completely prevents the bacterial cells from coming into contact with complex medium components. The invention is further illustrated by the following figures:

pictures

The pictures show

Figure 1 Dissolved oxygen concentration (pO ₂ ), stirrer frequency and

Carbon dioxide content of the reactor exhaust air in the cultivation of pUK21CMVß in E. coli DH5α in a defined, synthetic medium with 2 g L ^{* 1} ammonium chloride in a 7 L bioreactor.

Figure 2 Plasmid mass fraction, dry biomass and plasmid concentration during the cultivation of pUK21CMVß in E. coli DH5α in the synthetic medium in the 7 L bioreactor.

Figure 3 Course of the glutamate, glycerin, ammonium and acetate concentration of the cultivation of pUK21CMVß in E. coli DH5α in the synthetic medium in the 7 L bioreactor.

Figure 4 0.8% agarose gel of the pUK21CMVß samples from the cultivation in the synthetic medium in the 7 L bioreactor (M: λ-DNA, BStE π cut, R: pUK21CMVß reference).

Figure 5 Important operational variables in the cultivation of pUT649 in E. coli

DH5α in the synthetic medium with 2 g L ^'1 ammonium chloride in the 30 L bioreactor.

Figure 6 0.8% agarose gel of the pUT649 samples from the cultivation in the defined synthetic medium with 2 g L ^"1 ammonium chloride in a 30 L bioreactor after 19 and 22 h (M: λ-DNA, BStE II cut).

Figure 7 Quality analysis of plasmid pUT649 from cultivation in a 30 L bioreactor using capillary gel electrophoresis. Figure 8 plasmid concentration, plasmid mass fraction and dry biomass concentration in the cultivation of pUK21CMVß in E. coli DH5α in the synthetic medium in the 7 L bioreactor.

Figure 9 Glutamate, glycerin, ammonium ion and acetate concentration during the cultivation of pUK21CMVß in E. coli DH5α in the synthetic glycerin medium in the 7 L bioreactor.

Figure 10 plasmid mass fraction, dry biomass and plasmid concentration in the cultivation of E. coli DH5α with the plasmid pUK21CMVß in synthetic medium with 4 g L ^"1 ammonium chloride in a 7 L bioreactor.

Figure 11 Set cultivation of E. coli DH5α pUK21CMVß in M9 minimal medium in a 7 L bioreactor.

Figure 12 Plasmid, dry biomass concentration and plasmid mass fraction during the cultivation of pUK21CMVß in E. coli DH5α in semi-synthetic full medium with glutamate in the 7 L bioreactor.

The following examples serve to illustrate the invention and are not to be interpreted in a restrictive manner.

Examples

Example 1: Production of the plasmid pUK21CMVß by bacterial cells in a defined synthetic medium using the batch method in a 7 L bioreactor

The E. coΛ ^' strain DH5α (Clontech, Heidelberg, Germany, Cat. No. C2007-1) was used to produce the 7.6 kbp plasmid pUK21CMVß. The plasmid pUK21CMVß is a pUC21 derivative with kanamycin resistance (see Viera et al, Gene 100 (1991), 189-195) and contains the β-galactosidase from E. coli under the CMV (cytomegalus) promoter. The cultivation was carried out in a 7 L bioeactor (MBR, Wetzikon, Switzerland) in an aqueous, antibiotic-free, defined synthetic medium from 60 g L ^"1 GHycerin (87%), 2 g L ^{" 1} ammonium chloride, 30 g L ^"1 sodium L-GHutamate, 1.5 g L ¹ KH ₂ P04, 2.3 g L ^{" 1} K ₂ HPO ₄ , 2.5 g L ¹ NaCl, 5 g L ¹ citric acid x H ₂ 0, 1.0 g L ¹ MgSO ₄ x 7 H ₂ O, 10 mg L ^"1 thiamine hydrochloride and 2 mL L ^'1 trace element concentrate Trace element concentrate consisted of 5.4 g L ^"1 FeCl ₃ x 6 H ₂ O, 1.38 g L ^{" 1} ZnSO ₄ x 7 H ₂ O, 1.85 g L ^"1 MnSO ₄ x H ₂ O, 0.56 g L ¹ CoSO ₄ x 7 H ₂ O, 0.17 g L ^"1 CuCl ₂ , 1.0 g L ^{" 1} H3BO3, 2.5 g L ^'1 NaMoO ₄ x 2 H ₂ O and 5.0 g L ^"1 citric acid x H ₂ O. All media components with the exception of magnesium sulfate, thiamine hydrochloride and the trace element solution were heat sterilized in the bioreactor. The thiamine hydrochloride was taken up in water, sterile filtered and added to the sterile trace element solution. The magnesium sulfate was dissolved in water, separately heat sterilized and then likewise added to the sterile trace element solution Concentrate, magnesium sulfate and thiamine hydrochloride were added to the sterile medium in the bioreactor using a seeding flask. To prepare the inoculation culture, 200 ml of a sterile medium were inoculated with a glycerol culture of pUK21CMVß in DH5α. After 8 hours of incubation on a table shaker at n = 150 min ^"1 and a temperature of T = 37 ° C, 5 L of the sterile, synthetic medium in the 7 L bioreactor was inoculated with 50 mL of the inoculation culture. Cultivation in the bioreactor was carried out at 37 ° C and 0.2 bar internal reactor pressure. The air was supplied at 5 L min ^"1 and the pH was regulated to 7.0 by automatic addition of orthophosphoric acid (10%) and / or ammonia solution (25%). The speed of the stirrer was regulated from a minimum frequency of 150 min ^"1 by the dissolved oxygen concentration (pO ₂ ). The sampling for process control was carried out at intervals of 2 hours. The dry biomass concentration and the plasmid concentration were determined by capillary electrophoresis (see Schmidt et al, J Biotechnol 49 (1996), 219-229) after isolation with a commercially available kit according to the manufacturer's instructions, and the concentration of medium constituents was monitored, and the typical course of the most important cultivation parameters, stirrer frequency, dissolved oxygen concentration (pO ₂ ) and The course of the dry biomass, the plasmid concentration and the plasmid mass fraction during this cultivation is shown in Figure 2. The dry biomass concentration increased exponentially and reached a maximum value of X = 20 g L ^'1 concentration was P = 50 mg L ^'1 with a plasmid mass fraction of w _p = 3 mg g ^{' 1} . After 34 hours of cultivation, the substrates ammonium nitrogen, glutamate and glycerin were completely consumed (see Figure 3) and the bacterial cells reached the stationary phase, ie no further increase in biomass was obtained. The maximum product concentration of P = 50 mg L ^'1 was also reached at this time. The 0.8% agarose gel of the isolated plasmid DNA at different times of cultivation is shown in Figure 4. The plasmid DNA has a very high homogeneity and was almost exclusively in the ccc monomer form (approx. 94%) during the entire cultivation period. 250 mg of DNA could be isolated from 5 L of culture solution. This product yield is significantly higher than was previously obtained with synthetic media using the batch process (see Example 4).

Example 2: Preparation of the plasmid pUT649 by bacterial cells in a defined synthetic medium using the batch method in a 30 L bioreactor

The antibiotic-free, defined, synthetic medium described in Example 1 was used to produce the 4.6 kbp plasmid pUT649 (Eurogentec, Liege, Belgium) on a gram scale. As an inoculation culture, 200 ml of a sterile medium was inoculated with a glycerol culture of pUT649 in E. coli DH5α. After 8 hours of incubation on a table shaker at n = 150 min ^"1 and a temperature of T = 37 ° C, 20 L of the sterile medium, the composition of which is described in Example 1, were also in the 30 L bioreactor (MBR, Wetzikon, Switzerland) 200 ml of the inoculation culture were inoculated. The cultivation in the bioreactor was carried out at 37 ° C. and internal reactor pressure of 0.2 bar. The air was supplied at 30 L min ^"1 and the pH was brought to 7.0 by automatic addition of orthophosphoric acid and / or Regulated ammonia solution. At the beginning of the cultivation, the stirrer frequency was no = 150 min ¹ and was regulated to a target value of 40% using the dissolved oxygen concentration. Figure 5 shows the typical cultivation parameters of stirrer frequency, dissolved oxygen concentration and the carbon dioxide content of the reactor exhaust air. After reaching the stationary growth phase after 22 hours of cultivation, X = 21.9 g L ^"1 bio dry matter and a very high plasmid concentration of P = 68 mg L ^{" 1 were} reached. This corresponds to a plasmid mass fraction of w _p = 3.1 mg g ^'1 . The isolated DNA was very homogeneous, which is shown by the agarose gel in Figure 6. The

Quality analysis of the DNA using capillary gel electrophoresis shows that more than 97% of the DNA is in the ccc monomer form. This example shows that the invention

The method is applicable to different plasmids and also simply into a larger one Process scale can be transferred. With the aid of the invention, 1.3 g of very homogeneous plasmid DNA can be obtained on a cultivation scale of 20 L. The product concentration P = 68 mg L ^{"1 obtained} is the highest to date which has been obtained by batch cultivation processes on synthetic media. In the prior art, comparable high product yields are obtained only with the aid of feed cultivation processes.

Example 3: Influence of the ammonium concentration in the production of a plasmid by bacterial cells in a defined, synthetic medium in the batch process.

The following example illustrates the influence of the concentration of the inorganic nitrogen source in the batch cultivation of E. coli DH5α with the plasmid pUK21CMVß in the same synthetic medium from Examples 1 and 2 in the 7 L bioreactor. The cultivation was carried out under the same conditions as described in Example 1. Only the ammonium chloride concentration in the medium was changed before the cultivation. Figure 8 shows the course of the cultivation without the addition of ammonium chloride. Only by regulating the pH, small amounts of ammonia were added to the medium. The dry biomass concentration reached a maximum value of X = 17.5 g L ^"1 after 40 hours. The product concentration reached a maximum value of P = 20 mg L ^'1 after 30 hours, but fell to 15 mg L in the course of the following cultivation ^"1 from. The plasmid mass fraction did not remain constant, but initially rose to a maximum value of w _p = 2.7 mg g ^"1 and fell sharply after 22 h of cultivation time to values below w _p = 1 mg g ^{" 1} . Figure 9 shows the consumption of ammonium ions, glycerin and glutamate, as well as the formation of acetate. After 22 hours of cultivation, ammonium nitrate could no longer be detected in the medium. In parallel, the

Concentration of acetate from 0.3 g L ^" to 1 g L ^' . At this point, glutamate and glycerin were still sufficiently present so that ammonium was identified as the limiting substrate.

In a further cultivation, the amount of ammonium was therefore increased by using 4 g of L ^"

¹ ammonium chloride significantly increased. The dry biomass concentration after 34 h of cultivation was X = 26.6, g L ^"1 (see Figure 10). The product concentration rose in parallel with the dry biomass concentration and reached one after 30 hours maximum value of P = 41 mg L ^'1 , which corresponds to a plasmid mass fraction of w _p = 1.5 mg g ^"1. Compared with the cultivation with 2 g L ^{' 1} ammonium chloride (Examples 1 and 2), no further increase in the amount of product could be achieved so that there was no limiting influence.

Example 4: Preparation of the plasmid pUK21CMVß by bacterial cells in synthetic M9 minimal medium using the batch method in a 7 L bioreactor

This example shows set cultivation in a synthetic medium which corresponds to the prior art. To this was added as in Example 1 and 3, the plasmid pUK21CMVß in E. coli DH5a was obtained by culturing in the described in the literature, synthetic M9 minimal medium (Sambrook et al, Molecular Cloning, CSH Press, 2 ^nd edition, 1989, Cold Spring Harbor New York), consisting of 11 g L ^"1 glucose x H ₂ O, 1.0 g L ^{" 1} Na2HPO ₄ x 7 H ₂ O, 3.0 g L ^'1 KH ₂ PO ₄ , 0.5 g L ^"1 NaCl, 2 , 5 g L ^"1 (NH4) ₂ S04, 1.4 mg L ¹ ZnSO ₄ x 7 H ₂ O, 5.4 mg L ^'1 FeCl ₂ x 6 H ₂ O, 1.6 mg L ¹ MnSO ₄ , 0.167 mg L ^"1 CuCl ₂ , 0.56 g L ^{" 1} CoSO ₄ x 7 H ₂ O, 0.22 g L ^"1 MgCl ₂ x 7 H ₂ O and 14.7 mg L ^{" 1} CaCl ₂ in 7 L Bioreactor produces.

To prepare a preculture, 200 ml of sterile medium were inoculated with a glycerol culture and incubated at 37 ° C. for 8 hours with stirring on a table shaker at n = 150 min ^-1 . 5 L of sterile M9 medium were mixed with 50 ml of this preculture in a 7th L. Bioreactor inoculated. The cultivation in the bioreactor was carried out at 37 ° C. and 0.2 bar internal reactor pressure. The air was supplied at 5 L min ^"1 and the pH was regulated to 7.0 by automatic addition of orthophosphoric acid and / or ammonia solution ,

The course of the process parameters of dry biomass concentration, plasmid concentration and plasmid mass fraction over the entire cultivation period of 46 hours is shown in Figure 11. A biomass of X = 3.55 g L ^{'1 was} obtained during a cultivation time of 44 h. The product concentration to P = 4.65 mg L ^"1 after 44 hours when the stationary phase was reached. The plasmid mass fraction with w _p = 1.3 mg g ^{" 1} after 44 hours was significantly lower than with the method according to the invention in Examples 1 and 2. In these examples, a five times higher amount of biomass and even a ten times higher plasmid concentration was obtained. Example 5: Increasing the product yield

To illustrate the increase in the product yield by adding glutamate, the plasmid pUK21CMVß in E. coli DH5α was prepared in a 7 L bioreactor in a rich semi-synthetic medium containing glycerol, soybean peptone and yeast extract, with and without glutamate.

To prepare an inoculation culture, 200 ml of medium were inoculated with a glycerol culture and incubated on a table shaker at n = 150 min ^"1 and 37 ° C. for 10 hours. 5 L of the sterile medium were inoculated with 50 ml of the inoculation culture in the 7 L bioreactor Cultivation was carried out at 37 ° C. and 0.2 bar internal reactor pressure. The air was supplied at 5 L min ^"1 and the pH was adjusted to 7.0 by automatic addition of orthophosphoric acid and / or ammonia solution. Without the addition of glutamate, one became at the end of the cultivation after 20 hours

Bio-dry matter concentration of X = 7.6 g L ^'1 and a plasmid concentration of P = 26 mg L ^{' 1 were} obtained. By adding 30 g L ^"1 glutamate to the semisynthetic medium in another

Cultivation increased the product yield significantly (see Figure 12). To

The maximum dry matter concentration of X = 20 hours was cultivated

Received 23.9 g L ^"1 and the product concentration increased significantly to P = 45 mg L ^{" 1} .

This example shows that even with cultivation in semi-synthetic full media, the amount of product can be significantly increased by adding glutamate. Glutamate is an important media component in the production of nucleic acids and therefore an important one

Part of the process according to the invention.

Claims

Expectations 1. A method for producing nucleic acids comprising the following steps (a) culturing bacterial cells containing the nucleic acid in a medium without complex components and (b) Isolation of the nucleic acid. 2. The method of claim 1, wherein the medium comprises the following synthetic components: (aa) organic carbon source, (ab) inorganic nitrogen source (ac) mineral salt mixture and (ad) an additional, defined nitrogen-containing component that the Product metabolism increased. 3. The method according to claim 1 or 2, wherein no components are used for the preparation of the medium, which are obtained from animals. 4. The method according to any one of claims 1-3, wherein the nucleic acids are superspralized nucleic acids. 5. A method according to any one of claims 1-4, wherein isolating the nucleic acids, isolating the bacterial cells from the medium and optionally washing the bacterial cells, disrupting the bacterial cells and separating the nucleic acids from other cell contents. 6. A method according to claim 5, wherein the separation of the nucleic acids from other cell contents is carried out by chromatographic methods. 7. A method according to claim 5, wherein the bacterial cells are frozen or freeze-dried before or after the isolation of the bacterial cells from the medium. 8. A method according to any one of claims 1-7, wherein the transformed bacterial cells are Escherichia co // ^' ZeUen. 9. A method according to any one of claims 1-8, wherein an Ecoli K12 strain with genotype recA ^" or recAl is used. 10. A method according to any one of claims 1-9, wherein the nucleic acids are plasmids, cosmids or other larger circular nucleic acids. 11. A method according to any one of claims 1-10, wherein the purified nucleic acids can be used in pharmaceutical quality as an active ingredient in ZeU or gene therapy and genetic vaccination. 12. A method according to any one of claims 1-11, wherein the organic carbon source from the group of monosaccharides, disaccharides, polysaccharides or polyols. 13. A method according to any one of claims 1-12, wherein preferably glucose, maltose, raffinose, galactose, trehalose, sucrose, fructose, mannose, sorbose, fucose, rhamnose, arabinose, xylose, ribose, manitol, sorbitol or glycerol as an organic carbon source is used. 1 14. A method according to any one of claims 1-13, wherein the concentration of the carbon source in the medium is 1 to 500 g / L, preferably 10 to 300 g / L and in particular 50 to 100 g / L. 15. A method according to any one of claims 1-14, wherein ammonium salts such as ammonium chloride, ammonium phosphates, ammonium sulfate or ammonium nitrate or ammonia itself are used as an inorganic nitrogen source. 16. A method according to claims 1-15, wherein the concentration of the ammonium salt in the medium is 10 to 200 mM, preferably 20 to 80 mM and in particular 40 mM. 17. A method according to any one of claims 1-16, wherein the mineral salt mixture consists of at least water-soluble inorganic phosphates, chlorides, sodium salts, potassium salts and magnesium salts. 18. A method according to any one of claims 1-17, wherein the mineral salt mixture preferably at least potassium dihydrogen phosphate, dipotassium hydrogen phosphate, Contains sodium chloride and magnesium sulfate. 19. A method according to claims 1-18, wherein the mineral salts are used in the following concentrations: potassium dihydrogen phosphate 1 to 50 g / L, preferably 1.5 to 10 g / L, dipotassium hydrogen phosphate 1 to 75 g / L, preferably 2.3 to 15 g / L, sodium chloride 1 to 20 g / L, preferably 2.5 to 10 g / L and Magnesium sulfate 0.1 to 20 g / L, preferably 0.3 to 10 g / L. 20. A method according to any one of claims 1-19, wherein the mineral salt mixture additionally contains a trace element solution comprising iron (HT) chloride, zinc sulfate, manganese sulfate, cobalt sulfate, copper chloride, boric acid, sodium molybdate and citric acid. 21. A method according to claim 21, wherein the trace element solution iron (IQ) chloride in a concentration of 20 mM, zinc sulfate in a concentration of 5 mM, manganese sulfate in a concentration of 11 mM, cobalt sulfate in a concentration of 2 mM, copper chloride in a concentration of 1 mM, boric acid in a concentration of 16 mM, sodium molybdate in a concentration of 11 mM and citric acid in a concentration of 24 mM. 22. A method according to any one of claims 1-21, wherein the medium contains host-specific components, e.g. Vitamins or other components that the host strain used cannot synthesize itself can be added. 23. A method according to any one of claims 1-22, wherein the additional, defined nitrogen-containing component is a precursor in the biosynthesis of the nucleotide bases of the nucleic acids 24. A method according to any one of claims 1-23, wherein the additional, defined nitrogen-containing component is selected from the group consisting of Amino acids, peptides, carbonic acid amides, or nucleotides or the phosphates of these compounds. 25. A method according to any one of claims 1-24, wherein the additional, defined nitrogen-containing component is preferably selected from the group consisting of: aspartate, glutamate, glutamine, glycine, adenosine, cytosine, guanine, thymine, or Urea or the phosphates of these compounds. 26. A method according to any one of claims 1-25, wherein the concentration of the additional defined nitrogen-containing component in the medium is 25 to 1000 mM, preferably 50 to 250 mM. 27. A method according to any one of claims 1-26, wherein the medium is antibiotic-free. 28. A method according to any one of claims 1-27, wherein the carbon source and / or the nitrogen sources are completely metabolized. 29. A method according to any one of claims 1-28, wherein the cultivation is carried out in a temperature-controlled bioreactor. 30. A method according to any one of claims 1-29, wherein the cultivation is carried out in a temperature range of 25-42 ° C, preferably in the range of 36-38 ° C, in particular 37 ° C. 31. A method according to any one of claims 1-30, wherein the bacterial cells are supplied with oxygen during the cultivation by blowing air into the medium. 32. A method according to any one of claims 1-31, wherein the cultivation is carried out in a pH range of 6.0-8.0, preferably in the range of 6.5 to 7.5. 33. A method according to any one of claims 1-32, wherein the pH is regulated during the cultivation by adding an acid and or a base. 34. A method according to any one of claims 1-33, wherein anti-foaming agent is optionally added during the cultivation. 35. A method according to any one of claims 1-34, wherein the pre-cultivation takes place in a defined, synthetic medium. 36. The method according to any one of claims 1-35, wherein the cultivation takes place in the sentence method (sentence cultivation method).