MXPA99006453A - Method of purifying dna in a cross-flow centrifuge - Google Patents

Abstract

The invention concerns a method of purifying extrachromosomal DNA by passing an extrachromosomal DNA and fluid containing further cell components through a cross-flow centrifuge under given conditions, resulting in separation of the extrachromosomal DNA from the other cell components such that purified extrachromosomal DNA is obtained. The invention further concerns the use of the purified extrachromosomal DNA for cloning, transformation, transfection and microinjection into cells, for use in gene therapy processes, DNA vaccination and/or for polymerase chain reaction (PCR). The invention finally concerns the use of a cross-flow centrifuge for purifying extrachromosomal DNA.

Description

METHOD OF PURIFICATION OF DNA IN A CENTRIGUGA DE CRUZADO DE FLOW CRUZADO Description of the invention The present invention relates to a process for the purification of extrachromosomal DNA using a continuous flow centrifuge. The isolation of nucleic acids and in particular of plasmid DNA is of major importance in molecular biology and in modern medicine. Plasmid DNA refers to double extrachromosomal DNA molecules which usually have a size of 1 kb up to 200 kb ace, and are present in host cells in one to several hundred copies. Plasmid DNA is usually amplified in cells for example in gram-negative bacteria, in particular in E. coli. After which the cells are. lysed and the plasmid DNA is isolated from them. The isolated plasmid DNA can then be used for molecular biology applications or medical applications, for example to construct cloning vectors, to transform prokaryotic cells and to transfect eukaryotic cells. Various methods are known REF .: 30716 to lyse cells and to isolate plasmid DNA (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). In a process developed by Birnboim and Doly for the isolation of plasmid DNA from cells (Birnboim and Doly, Nucí Acid Res. 7 (1979) 1513-1523) the biomass is lysed with an NaOH / detergent solution and subsequently the pH value is adjusted to approximately 5.0 with potassium acetate. A precipitate forms in this process that contains mainly genomic DNA and fragments of the cell wall. In order to separate these impurities, the suspension is transferred to centrifuge buckets. The precipitate is then centrifuged with a cuvette centrifuge in order to obtain the supernatant containing the plasmid DNA. Centrifuges are also commonly used in fermentation processes in order to separate for example the supernatants of the fermenter from the cells and the cell fragments. Centrifuges with sieves and fixed wall centrifuges are usually used for this purpose (see Gerhartz W., Enzymes in industry: production and applications, 1990, VHC, Weinheim, Germany, chapter 3.2.1). With common centrifuges for laboratory the volume that can be processed is limited by the volume of the rotor and the buckets or tubes of the rotor, to less than ten liters (for example the Sorvall centrifuge, GSA rotor, 6 x 250 ml). Hence, this process can only be used to isolate the plasmid DNA in small quantities. An application of the method to large-scale processes is very problematic, due to the limited volume of the centrifuge. Large volumes can be processed by means of continuous flow centrifuges. International Patent WO92 / 12780 describes a technical design of a continuous flow centrifuge and its use for the separation of mixtures of macromolecules. In this process, for example, four standard proteins are prepared in a two-phase aqueous system at a maximum of 1,000 rpm, depending on the respective distribution coefficients of the proteins. The components of the mixture are obtained and separated from one another as a result of differences in elution times.

However, the demand for purified plasmid DNA for analytical and therapeutic applications in research and medicine is increasing due to the widespread use of molecular biology methods. Therefore, an object of the present invention was to provide a process that enables efficient and rapid purification of plasmid DNA in large quantities. A first aspect of the present invention relates to a process for the purification of extrachromosomal DNA, which is characterized in that an extrachromosomal DNA containing liquid and other cellular components, is passed through a continuous flow centrifuge under conditions that lead to a separation of the extrachromosomal DNA, from the insoluble cellular components and the purified extrachromosomal DNA, is isolated. In the prior art, continuous flow centrifuges have been used only for the separation of cells. It was now surprisingly found that continuous flow centrifuges can also be used to purify extrachromosomal DNA in large quantities, without damage to extrachromosomal DNA by the resulting shear forces. It was also surprising that the chromosomal DNA present in the suspension of the lysed cells is not fragmented during the continuous flow centrifugation, and can thus be quantitatively separated from the extrachromosomal DNA. The extrachromosomal DNA which is purified by the method according to the invention can be linear or circular, single stranded or double stranded. The DNA is preferably a circular and double-stranded plasmid DNA. The cell that contains extrachromosomal DNA can be a prokaryotic or eukaryotic cell; it is preferably a bacterial cell and in particular a gram-negative cell such as an E. coli cell. Optionally, cells containing the so-called artificial chromosomes can be used as extrachromosomal DNA. Artificial chromosomes are linear double-stranded DNA molecules, which are generally called YAC (yeast artificial chromosome) and are amplified in yeast cells. The liquid containing the extrachromosomal DNA that is used in the process according to the invention is preferably a cell lysate. The cell lysate is particularly and preferably prepared by alkaline lysis of the cells containing the extrachromosomal DNA and the subsequent acidification. However, it is also possible to use other common methods of cell lysis such as a combination of enzyme (lysozyme) and heat treatment. Any desired amount of cellular biomass can be used as an initial material for the process according to the invention. A biomass of 100 g to 50 kg is preferably lysed per batch. - The liquid containing extrachromosomal DNA is usually passed to a continuous flow centrifuge through a gradient and / or pumps. In the process according to the invention, a continuous flow centrifuge with a volume adapted to the lysis preparation is used. A volume of at least 0.1 to 50 liters is preferably used and a volume of 0.2 to 4 liters is particularly preferred. The centrifuge container is preferably cylindrical. The continuous flow centrifuge is operated at a suitable g number, preferably at 10,000 to 40,000 x g. Examples of commercially available continuous flow centrifuges are the CEPA high-speed centrifuges or the high-performance centrifuges of Carr Company (United States), which to date have a capacity of up to 9,000 liters / hour. The process according to the invention is generally carried out continuously. The suspension of the lysed biomass is passed to the continuous flow centrifuge from below. As a result of the rotation of the centrifuge container (10,000-40,0000 x g) solid components such as cell wall components and genomic DNA coupled to it are deposited on the wall of the centrifuge container. The solution containing the purified extrachromosomal DNA usually passes "out of the top of the continuous flow centrifuge, although it is also conceivable that the solution containing the extrachromosomal DNA will flow out of the sides, the bottom or other positions. continuous flow can be operated at different temperatures, the process is preferably carried out at 4 ° C to room temperature In the present process it is possible to purify the extrachromosomal DNA of different sizes, preferably extrachromosomal DNA is purified with a size of 1 kbp up to 200 kbp The extrachromosomal DNA is preferably linear, circular or supercoiled plasmid DNA After leaving the centrifuge the extrachromosomal DNA can be further purified, therefore, a RNase treatment is optionally carried out in order to eliminate the solution RNA.In addition, it is also possible to carry out the steps of p chromatographic urification such as anion exchange chromatography, affinity chromatography or hydroxyapatite chromatography. Examples of suitable materials for anion exchange chromatography are organic or inorganic polymers and copolymers such as polymeth acrylate (Macroprep-Biorad, Germany), polystyrene-divinylbenzene (Poros-Perseptive, HyperD-Biosepra, Source Pharmacia) or gel silica on the surface of which positively charged groups such as diethylaminoethyl (DEAE) or dimethylaminoethyl (DMAE) are linked. A particularly preferred material for anion exchange chromatography is Q-Sepharose. A particularly preferred material for affinity chromatography is hydroxyapatite. further, the DNA solution that is obtained may be subject to cross-flow filtration for further purification, concentration and / or re-dampening. In this cross-flow filtration it is also possible to achieve a substantial elimination of endotoxins from the DNA preparation. For this the DNA solution is guided tangentially beyond one or more semipermeable membranes whose exclusion size is chosen such that the DNA molecules are retained by the membranes and the substances with a lower molecular weight can pass through the membranes to obtain an endotoxin-free DNA solution. The extrachromosomal DNA obtained by the process according to the invention remains essentially undamaged and has essentially no single-strand or double-strand break. In particular, a plasmid DNA purified according to the invention shows only one dominant band after separation by gel electrophoresis, which corresponds to the conformation of the 'covalently closed circle. "Furthermore, there are no other bands besides the corresponding bands to the open circle and the conformations of the linearized circle.

The DNA obtained by the process according to the invention can be used directly for standard applications of molecular biology and medical applications, such as for cloning, for transformation, for transfection, for microinjection of cells, for use in methods of gene therapy, for vaccination with DNA and / or for the polymerase chain reaction (PCR). A further aspect of the present invention relates to the use of a continuous flow centrifuge for purifying extrachromosomal DNA.

Example In the experiment, a laboratory centrifuge CEPA, LE (open design) with a clarification cylinder made of stainless steel (1.4571, V4A) is used. Approximately 2000 g of biomass are lysed by the alkaline lysis method (modified method according to Birnboim and Doly, Birnboim and Doly, Nucí. "Acid Res. 7 (1979) 1513-1523). 1. Lysis of biomass E. coli 2000 g of wet E. coli biomass from the fermenter is filled into the depyrogenized vessels. Add 22.5 liters of resuspension buffer (50 mmol / 1 Tris-HCl, 10 mmol / l EDTA-Na2, pH 8 + 0.2) and stir slowly (approximately 35 rpm) for at least 24 hours 5 ± 4 ° C until the biomass is completely suspended. Then the temperature of the suspension increases slowly to 25 ° C. 22.5 liters 0.2 mol / l NaOH, 1% SDS are added to the suspension, while stirring at about 80 rpm, and incubated for 5 minutes at 25 ° C. 22.5 liters of potassium acetate buffer (3 mol / 1 of potassium acetate buffer pH 5.5) are added while stirring is maintained, and the temperature of the biomass is reduced as rapidly as possible to 4 ° C. The resulting lysate is clarified by filtration with the aid of a continuous flow centrifuge in continuous flow mode from side to side. 2. Centrifuging to continuous flow The viscous suspension is pumped into the continuous flow centrifuge through the inlet opening. During this period the centrifuge is operated at a g number of 10,000 - 18,000 x g. As soon as the liquid that flows outwards becomes turbid, the precipitate must be removed from the cylinder and the centrifugation is continued after the insertion of the clean cylinder. The clear solution of plasmid DNA that has been released from cellular impurities emerges from the top of the continuous flow centrifuge and is collected in a container. 3. Additional purification steps: Chromatography in Q-sepharose, hydroxyapatite chromatography and cross-flow filtration In a subsequent step, chromatography on Q-sepharose and hydroxyapatite is carried out. The decanted supernatant of the centrifuge is adjusted to a conductivity of 49 - 50 mS / cm by the addition of TE buffer (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA pH 8.5 ± 0.2) and cooled to 5 ± 4 ° C. The complete chromatography is carried out at this temperature. The supernatant from the centrifugation is absorbed onto the balanced column. Subsequently, the column is washed with approximately 8 CV 10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.65 mol / 1 NaCl pH 8.5 ± 0.2. A gradient is applied to the elution (5 CV buffer (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.65 mmol / 1 NaCl, pH 8.0 ± 2), 5 CV of buffer B (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.85 mol / 1 NaCl pH 8.0 ± 0.2)) to the column and the eluate is fractionated, detection is carried out at 254 nm. The prep (impurities) is separated from the main peak (plasmid DNA) by collecting the main peak in a separate container, starting from the rising flank. Subsequently, a chromatography on hydroxyapatite (HA ceramic) is carried out at 5 ± 4 ° C. Balance absorber: 0.1 mol / l of potassium phosphate, 6 mol / l of urea pH 7.0 ± 0.2. Shock absorber 1: 0.15 mol / 1 potassium phosphate, 6 mol / 1 urea pH 7.0 ± 0.2.

Washing buffer 2: 0.02 mol / 1 of potassium phosphate buffer pH 7.0 ± 0.2. Elution buffer: 0.5 mol / l of potassium phosphate pH 7.0 ± 0.2. The detection is carried out at 254 nm using a UV detector / recorder unit. A solution of 1% product (plasmid DNA) is used as a calibration solution that was measured with a calibrated photometer. The combined Q-sepharose is adjusted to a final concentration of 1.1 mmol / 1 of calcium chloride and absorbed onto the equilibrated column. Then the column is successively washed with: 1. 0.1 mol / l of potassium phosphate, 6 mol / l of urea pH 7.0 ± 0.2 until the absorbance is no longer detectable in the detector. 2-4 HP, 0.15 mol / l of potassium phosphate, 6 mol / l of urea pH 7.0 ± 0.2 5 CV, 0.02 mol / l of potassium phosphate pH 7.0 ± 0. 2.

This is eluted with 0.5 mol / l of potassium phosphate buffer pH 7.0 ± 0.1 after the washing steps at a flow rate of 5-6 HP / hour. The peak is combined and concentrated to approximately 50 ml with a cross-flow filtration. The CFF is carried out at a retentate flow rate of 100-200 l / h »m2, a transmembrane pressure of approximately 0.8 bar and a cross-flow pressure of approximately 1.2 bar. The retentate is subsequently diafiltered by flow against the TE buffer (10 mmol / l of Tris-HCl, 1 mmol / 1 of EDTA, pH 8.0 until the values for the pH and the conductivity of the retentate and of the TE buffer are in accordance After completion of the diafiltration process, the retentate is adjusted to a plasmid DNA concentration of 1 mg / ml by dilution with diafiltration buffer. 4. Gel electrophoresis The intact quality of the plasmid DNA that was obtained is verified by means of agarose gel electrophoresis. For this, an aliquot of the plasmid DNA at various concentrations is applied to an agarose gel. The agarose gel illustrated in Figure 1 shows the length of the standard DNA No. II (fragment sizes: 125, 564, 2027, 2322, 4361, 6557, 9416, 23130 bp) in bands 1 and 10 and the standard No III of DNA length (fragment sizes: 125, 564, 831, 947, 1375, 1584, 1904, 2027, 3530, 4268, 4973, 5148, 21226 bp) in bands 2 and 9. pBR322 (4162 bp) it is applied as a reference plasmid in band 3, which was purified by a conventional gradient method in cesium chloride. It is known that the plasmid DNA purified by this method contains essentially plasmid DNA corresponding to the covalently closed circle conformation (dominant supercoiled band). The plasmid DNA (pCMV-CAT) purified by the method according to the invention is applied to different amounts in the bands 4, 5 and 6. This plasmid DNA was subsequently purified after the process according to the invention, by means of chromatography in Q-sepharose and hydroxyapatite, and by cross-flow filtration.

Caption: 1% agarose gel Lane 1: DNA II Length Standard (Boehringer Mannheim GmbH, Cat. No. 236250) Band 2: DNA III Length Standard (Boehringer Mannheim GmbH, Cat. No. 528552) Band 3: pBR322 (Boehringer Mannheim GmbH, Cat. No. 481238) (0.4 μg) Band 4: pCMV-CAT after CFF, 0.19 μg (solution of active substance in bulk) Band 5 : pCMV-CAT after CFF, 0.45 μg (solution of active substance in bulk) Band 6: pCMV-CAT after CFF, 0.71 μg (solution of active substance in bulk) Band 7: buffer TE Band 8: pBR322 (Boehringer Mannheim GmbH, Cat. No. 481238) (0.4 μg) Band 9: DNA III standard length (Boehringer Mannheim GmbH, Cat. No. 528552) Band 10: DNA length standard II (Boehringer Mannheim GmbH, Cat. No. 236250).

The purified plasmid DNA according to the invention, such as the reference plasmid DNA (lane 3), essentially shows a dominant band. This shows that the plasmid DNA, isolated according to the invention, is not damaged and retains its original conformation. In addition to the absence of additional bands on the agarose gel, it shows that the chromosomal DNA contained in the suspension of cells used is not fragmented during continuous flow centrifugation, but can be completely separated as a precipitated macromolecule from the plasmid DNA.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (12)

1. RE I INDI CACI ONES Having described the invention as above, the content of the following claims is claimed as property: .- Process for the purification of extrachromosomal DNA, characterized in that a liquid that is a cellular lysate, that contains AD? Extrachromosomal and other cellular components, is passed through a continuous flow centrifuge operated at an acceleration of 10,000 to 40,000 x g without previous centrifugation steps in a continuous process and the conditions that tend to the separation of AD? extrachromosomal of insoluble cellular components and the AD? purified extrachromosomal is isolated.
2. 2. Process according to claim 1, characterized in that the lysis is an alkaline lysis.
3. 3. - Process according to one of the preceding claims, characterized in that the cell containing AD? Extrachromosomal is a bacterial cell, preferably an E.Coli cell.
4. 4. Process according to one of the preceding claims, characterized in that the liquid phase in the centrifuge is obtained by lysing from 100 to 50 kg of biomass.
5. 5. Process according to one of the preceding claims, characterized in that the liquid containing AD? Extrachromosomal is passed in the centrifugal continuous flow by means of a gradient and / or pumps. H.
6. H . - Process according to one of the preceding claims, characterized in that a continuous flow centrifuge is used whose centrifugal container has a volume of at least 0.1-50 1.
7. 7. - Process according to one of the preceding claims, characterized in that use a centrifugal continuous flow centrifugal container has a volume of at least 0.2-4 1.
8. 8. Process according to one of the preceding claims, characterized in that the size of the extrachromosomal DNA is 1 pba 200 bp.
9. 9. Process according to one of the preceding claims, characterized in that the extrachromosomal DNA is linear, circular or superspiral plasmid DNA.
10. 10. Process according to one of the preceding claims, characterized in that the solution containing purified extrachromosomal DNA can be further purified.
11. 11. Process according to claim 10, characterized in that a further purification step consists of an anion exchange chromatrography, an affinity chromatography, a hydroxyapatite chromatography, a RNase treatment and / or a cross flow filtration.
12. 12. Process according to one of the preceding claims, characterized in that an extrachromosomal DNA is isolated that has no breaks in the essential pounds. Use of an extrachromosomal DNA according to one of the previous claims for cloning, transforming, transfecting, microinjection in cells, for use in gene therapy methods and / or for the reaction in DNA polymerase (PCR). Use of a continuous flow centrifuge to purify extrachromosomal DNA.