EP1713910A1 - Procede de separation chromatographique d'un melange d'acides nucleiques - Google Patents

Procede de separation chromatographique d'un melange d'acides nucleiques

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Publication number
EP1713910A1
EP1713910A1 EP05706998A EP05706998A EP1713910A1 EP 1713910 A1 EP1713910 A1 EP 1713910A1 EP 05706998 A EP05706998 A EP 05706998A EP 05706998 A EP05706998 A EP 05706998A EP 1713910 A1 EP1713910 A1 EP 1713910A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
conductivity
acid mixture
plasmid dna
kci
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05706998A
Other languages
German (de)
English (en)
Inventor
Markus Müller
Jörg HUCKLENBROICH
Lothar Breitkopf
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Priority to EP05706998A priority Critical patent/EP1713910A1/fr
Publication of EP1713910A1 publication Critical patent/EP1713910A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a method for the chromatographic separation of a nucleic acid mixture, in particular for the separation and purification of plasmid DNA from other constituents of the nucleic acid mixture, in particular other nucleic acids.
  • the method according to the invention is characterized in particular by the fact that plasmid DNA can be separated from contaminating RNA without the addition of ribonucleases and by the use of inexpensive and environmentally friendly components. These parameters make it possible to use this method for the production of plasmid DNA on a large scale.
  • the present invention comprises the use of the plasmid DNA obtained by means of the method according to the invention for the production of a plasmid DNA-containing agent for use in gene therapy and genetic vaccination.
  • a fundamental problem with the purification of plasmids is the removal of other nucleic acid species from the product. This problem arises above all in areas in which a particularly pure plasmid DNA preparation is required, for example when the plasmid DNA is used in gene therapy.
  • the other nucleic acid species mentioned are, above all, the various RNAs, but also genomic DNA and ssDNA (single stranded), etc.
  • a particular difficulty is the removal of the RNA.
  • the removal of the RNA using ribonucleases is known from the prior art. The RNA is degraded to ribonucleotides by means of the ribonucleases, which can be separated from the plasmid DNA much more easily in a subsequent chromatographic separation process.
  • the massive disadvantage of this method is the use of an RNase, which is usually a foreign protein.
  • the RNase is obtained from animal material, usually from cattle.
  • the addition of animal proteins in production processes must be ruled out due to possible contamination of the product with bacterial, viral or proteinogenic pathogens. This is particularly true for bovine proteins due to the BSE problem.
  • the use of RNase and also alcohol-containing buffers is a major cost factor. This is a cost factor that should not be underestimated, particularly in the production of plasmid DNA on a large scale, that is to say in ranges of approximately> 2 g.
  • alcohol there is an additional burden on the employees involved and the environment as a significant factor.
  • the alkaline lysis described in principle by Bimborn and Dohly (Nucl. Acids Res. 7, pp. 1513-1522; 1979) is preferred, but not limited thereto.
  • Other options are lysis by heat or lysis in the presence of detergents. Lysis by high pressure (French press) has proven to be unsuitable because the high shear forces that arise result in very small fragments of genomic DNA, which are practically no longer separable from the plasmid DNA.
  • Chromatographic methods are known from the prior art for purifying the nucleic acids from such a lysate. A distinction is generally made between two methods. First, the method according to Gillespie and Vogelstein (Proc. Natl. Acad. Sei., USA, 76 pp. 615 - 619; 1979) is known from the prior art. In this method, the nucleic acid is purified by binding to silica gel or diatomaceous earth in the presence of chaotropic salts, e.g. GuHCI, NaI, etc. In contrast to the anion exchanger, the
  • Proteins are not suitable for use in gene therapy.
  • the technical problem on which the method according to the invention is based is the purification of plasmid DNA from a nucleic acid mixture and the improvement in the separation of contaminants such as RNA, ssDNA and genomic DNA without using an RNase.
  • Another object of the invention is to provide a method which allows plasmids to be purified inexpensively and in an environmentally friendly manner even on a large scale.
  • the technical problem on which the invention is based is solved by a method according to the claims.
  • a) the nucleic acid mixture with one or more alkali metal salts and / or alkaline earth metal salts in an aqueous solution is adjusted to a conductivity which has a conductivity of Corresponds to 70 mS to 95 mS at a pH of 4.8 to 5.4 and at a temperature of 20 ° C, and b) the nucleic acid mixture is brought into contact with a chromatographic support material, and a) the support material is then contacted at least once with a Solution containing an alkali salt in a concentration range of 900 mM to 1800 mM based on a pH of 7 to 7.4 and / or an alkaline earth metal salt in a concentration range of 100 mM to 240 mM
  • the cells which may be prokaryotic or eukaryotic cells, must first be lysed. This can be done in the manner described above.
  • the alkaline lysis described in principle by Birnborn and Dohly (Nucl. Acids Res. 7, pp. 1513-1522; 1979) is preferred, but not limited thereto.
  • Other options are lysis by heat or lysis in the presence of detergents. Lysis by high pressure (French press) has proven to be unsuitable, since the high shear forces created result in very small fragments of genomic DNA which are practically no longer separable from the plasmid DNA.
  • a nucleic acid mixture in the sense of the invention can be a cell lysate as well as a pre-cleaned or clarified lysate, but can also be an artificial mixture in which plasmid DNA is contaminated with at least one further nucleic acid species and possibly other contaminants.
  • the nucleic acid mixture is a prokaryotic clarified lysate.
  • the method according to the invention ensures the chromatographic separation of the contaminants mentioned and provides a plasmid DNA which meets the purity requirements for use in gene therapy or genetic vaccination.
  • the person skilled in the art understands chromatography as a collective term for the physical-chemical separation of substance mixtures due to their different distribution between a stationary phase and a mobile phase.
  • an anion exchange material is used to separate the plasmid DNA from the contaminants.
  • the commercially available material QIAGEN ® QIAGEN GmbH, Hilden, Germany
  • This material enables a very efficient separation of the RNA by means of the method according to the invention, but also from eg ssDNA, from plasmid DNA.
  • RNA and ssDNA elute in a distinct peak which, in the method according to the invention, is very far from the likewise very distinct peak of the plasmid DNA.
  • the risk of co-elution of plasmid DNA and RNA or ssDNA is thus significantly reduced compared to the methods known from the prior art.
  • the chromatographic support material available under the name QIAGEN ® (QIAGEN GmbH, Hilden, Germany) is a modified porous inorganic material.
  • QIAGEN ® QIAGEN GmbH, Hilden, Germany
  • silica gel, diatomaceous earth, glass, aluminum oxides, titanium oxides, zirconium oxides, hydroxylapatite and organic support materials such as dextran, agarose, acrylamide, polystyrene resins or copolymers of the monomeric constituents of the materials mentioned are suitable as inorganic support materials.
  • the anion exchanger which is preferably used in the process according to the invention is, for example, by reacting one of the abovementioned support materials in a first step with a silanizing reagent of the general formula I.
  • R 1 is an alkoxy radical with 1 to 10 C atoms, in particular -OCH 3 , -OC 2 H 5 or - OC 3 H, or a halogen atom, in particular -Cl, or a dialkylamino group with identical or different alkyl radicals with 1 to 6 C- atoms;
  • R 2 and R 3 independently of one another are a hydrocarbon radical with 1 to 10 C atoms, in particular -CH 3) -C 2 H 5 or -C 3 H 7 , or an alkoxy radical with 1 to 10 C atoms, in particular -OCH 3 , -OC 2 H 5 or -OC 3 H 7 , or a halogen atom or an alkyl radical with 4 to 20 C atoms interrupted by at least one oxygen atom or an amino group, this radical also being a mono- or can be substituted several times by halogen, cyano, nitro, amino, monoalkylamino, dialkylamino, hydroxy or aryl
  • X is an amino, hydroxyl, epoxy group or a halogen atom
  • R is a hydrocarbon chain with 2 to 20 C atoms or an alkyl radical interrupted by at least one oxygen atom or an amino group, this radical also one or more times with halogen, cyano, Nitro, amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl and / or epoxy may be substituted
  • Y is a hydrocarbon radical with functional groups forming anion exchangers with 1 to 10 carbon atoms, one or more times with amino, monoalkylamino, dialkylamino -, Trialkylammonium can be, is, as also in EP 0 743 949, pages 4 to 5, to which reference is made here.
  • the nucleic acid mixture according to the optional step a) of the above-described method according to the invention one or more alkali salts and / or alkaline earth salts in an aqueous solution to a conductivity that corresponds to a conductivity of 70 mS to 95 mS at a pH of 4.8 to 5.4 and at a temperature of 20 ° C.
  • the salts preferably used in the process according to the invention are alkali metal salts, i.e. salts in which the cationic component or part of the cationic component originates from an element of the first main group of the Periodic Table of the Elements, and / or are alkaline earth metal salts, i.e. salts in which the cationic component or part of the cationic component comes from an element of the second main group of the periodic table of the elements.
  • the alkali salts are particularly preferably alkali halides and the alkaline earth salts are alkaline earth halides.
  • alkali halides KCl, NaCl, CsCl and / or LiCl and the alkaline earth halide CaCl 2 is particularly preferred.
  • an ammonium salt pseudoalkali metal salt
  • an ammonium salt of a carboxylic acid particularly preferably ammonium acetate
  • the salts used are very particularly preferably KCl and / or NaCl.
  • mixtures of various alkali salts and / or alkaline earth metal salts can also be used in the process according to the invention.
  • alkali salts in a concentration range from 900 mM to 1800 mM based on a pH from 7 to 7.4 and / or alkaline earth metal salts in a concentration range from 100 mM to 240 mM based on a pH of 7 to 7.4 used.
  • all aqueous solutions that appear sensible to a person skilled in the art can be used for the washing step, for example buffered systems such as, for example, but not limited to, tris, potassium acetate, borate or MOPS buffered systems, or alternatively unbuffered systems, ie the salts are only dissolved in water. Different pH values can potentially result from different buffer systems or these can be set.
  • the concentration ranges selected here relate to a pH value of 7 to 7.4, but in principle the pH value of the washing solution can be varied in the above-mentioned pH range. It is known to the person skilled in the art that when the pH of such a washing solution is changed, the concentration of the salts contained therein must also be changed in order to achieve the same effect, in this case, therefore, the elution of the contaminants, that is to say when the method is carried out to a shift in the elution points of contaminants (eg RNA) and plasmid DNA, with the same pH value of the washing and elution solution but not to a shift in the ratio of the elution points, which means that the distance between the elution peaks of the different nucleic acid species is advantageously always the same remains.
  • contaminants eg RNA
  • the person skilled in the art can carry out the parameters required for this on the basis of his specialist knowledge without inventive step.
  • the method according to the invention comprises at least one washing step, but it is also possible to carry out a number of washing steps, which seem sensible in number to the person skilled in the art, also with washing buffers according to the invention which differ from one another.
  • At least one washing step is carried out with a solution containing KCI in a concentration range from 1100 mM to 1800 mM based on a pH of 7 to 7.4, particularly preferably at least one washing step is carried out with a solution containing KCI in a concentration range from 1300 mM to 1700 mM based on a pH of 7 to 7.4.
  • At least one washing step is carried out with a solution containing NaCl in a concentration range from 950 mM to 1200 mM based on a pH of 7 to 7.4, particularly preferably at least one washing step is carried out with a solution containing NaCl in a concentration range from 1100 mM to 1150 mM based on a pH of 7 to 7.4.
  • aqueous solutions which appear sensible to the person skilled in the art can be used, for example buffered systems such as, but not limited to, tris, potassium acetate, borate or MOPS-buffered systems, or alternatively unbuffered systems, ie the salts are only dissolved in water.
  • buffered systems such as, but not limited to, tris, potassium acetate, borate or MOPS-buffered systems, or alternatively unbuffered systems, ie the salts are only dissolved in water.
  • buffered systems such as, but not limited to, tris, potassium acetate, borate or MOPS-buffered systems, or alternatively unbuffered systems, ie the salts are only dissolved in water.
  • buffered systems such as, but not limited to, tris, potassium acetate, borate or MOPS-buffered systems, or alternatively unbuffered systems, ie the salts are only dissolved in water.
  • Different pH values can potentially result from different buffer systems or these can
  • the elution step is carried out with a solution containing KCI in a concentration of 1900 mM or higher, based on a pH of 7 to 7.4.
  • the upper limit of KCI is only limited by its solubility in the solution used.
  • the elution step is carried out with a solution containing NaCl in a concentration of 1300 mM or higher, based on a pH of 7 to 7.4.
  • the NaCI concentration is only limited by its solubility in the solution used.
  • the setting of the conductivity of the nucleic acid mixture before bringing the nucleic acid mixture into contact with the chromatographic support material is also carried out, as already mentioned above, with alkali metal salts and / or alkaline earth metal salts.
  • the nucleic acid mixture is adjusted with KCI to a conductivity which corresponds to a conductivity of 70 mS to 85 mS at a pH of 4.8 to 5.4 and at a temperature of 20 ° C., very particularly preferably one Conductivity that corresponds to a conductivity of 70 mS to 80 mS at a pH of 4.8 to 5.4 and at a temperature of 20 ° C.
  • the nucleic acid mixture is adjusted with NaCl to a conductivity which corresponds to a conductivity of 70 mS to 95 mS at a pH of 4.8 to 5.4 and at a temperature of 20 ° C., very particularly preferably a conductivity that corresponds to a conductivity of 85 mS to 95 mS at a pH of 4.8 to 5.4 and at a temperature of 20 ° C.
  • the alkali halide KCI is used in the process according to the invention at least in the washing step of the chromatographic support material identified above with c).
  • room temperature means that the process is carried out under normal process conditions, corresponding approximately to a range from 18 ° C to 25 ° C.
  • the method can be carried out at all temperatures which appear sensible to the person skilled in the art.
  • the method according to the invention is preferably suitable for the purification of plasmid DNA.
  • plasmids of different sizes do not show any significant differences in the elution points, ie in the salt concentrations at which the plasmid DNA is eluted from the chromatographic support material. It is therefore not necessary to adapt the parameters of the method, such as salt concentrations or pH values, to different plasmid sizes. Since a method is available with the subject matter of the invention in which large scale plasmid can also be obtained for the production of a plasmid DNA-containing agent for use in gene therapy or genetic vaccination, endotoxin removal can advantageously be incorporated into the method without any problems become.
  • the clarified lysate can be mixed with an endotoxin removal buffer known from the prior art (for example containing Triton X 100, Triton X 114, polymyxin, etc.) and used without change in the method according to the invention.
  • an endotoxin removal buffer known from the prior art (for example containing Triton X 100, Triton X 114, polymyxin, etc.) and used without change in the method according to the invention.
  • Illustration 1
  • the absorbance is plotted against the KCI concentration at 254 nm of the flow through an HPLC column filled with QIAGEN ® chromatography material. The elution of different nucleic acid species with increasing KCI concentration is shown. The test conditions are explained in more detail in Example 2.
  • 1 kg of biomass was obtained from 30 L of an overnight fermentation culture of E. coli DH5 ⁇ , containing pCMVß plasmid, by centrifugation.
  • the biomass was resuspended in 15 L of a resuspension buffer (10 mM EDTA; 50 mM Tris / HCl pH 8) and then incubated with 15 L of a lysis buffer (200 mM NaOH; 1% (w / v) SDS) for 10 minutes at room temperature.
  • 15 L of a neutralization buffer (3 M potassium acetate, pH 5.5) were then added.
  • the precipitate formed in this step (proteins, membrane components, genomic DNA, etc.) was then removed.
  • the lysate thus pre-clarified was then filtered, whereby a clarified lysate was produced.
  • the clarified lysate indicated consequently a pH of 5.2 and was adjusted at a temperature of 20 ° C with 3 M KCI to a conductivity of 80 mS.
  • a chromatography column was equilibrated with QIAGEN ® -Chromatographiematerial filled (column volume ca. 7 L) and washed with 10 column volumes of a Aquilibr michspuffers (20 mM potassium acetate) at a flow rate of 3.3 cm / min.
  • the clarified lysate was loaded onto the column after equilibration of the chromatography material, and the run was carried out at a flow rate of 1.1 cm / min. 5 column volumes of equilibration buffer (20 mM potassium acetate) were then again passed over the column at a flow rate of 3.3 cm / min.
  • the column was then washed directly with 10 column volumes of a KCI solution (1350 mM KCI; 50 mM Tris / HCl, pH 7.2) at a flow rate of 3.3 cm / min.
  • the plasmids were then eluted with a column volume of an elution buffer (1600 mM NaCl; 50 mM Tris / HCl, pH 7.2). After subsequent ultrafiltration / diafiltration and final sterile filtration, a yield of approximately 400 mg pCMVß resulted.
  • a Tris buffer was passed over the column in a continuous gradient (50 mM Tris / HCl; pH 7.2; gradient 0 to 3 M KCI) and the elution of DNA and RNA was determined using a photometer (absorbance measurement at 254 nm). It could be shown that partially degraded and short-chain RNA is eluted in a distinct peak (maximum at 780 mM KCI), followed by an only slightly diffuse peak of longer-chain RNA (maximum at 1120 mM KCI, end of elution at 1310 mM KCI) , The elution of the plasmid DNA reaches a maximum at 1900 mM KCI and ends at 2150 mM KCI. The results are shown as superimposed tracks in Figure 1. It is clear that the plasmids advantageously elute regardless of their size.

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Abstract

L'invention concerne un procédé de séparation chromatographique d'un mélange d'acides nucléiques, notamment pour séparer et épurer un ADN plasmidique des autres constituants d'un mélange d'acides nucléiques, en particulier des autres acides nucléiques. L'invention est caractérisée en ce que l'ADN plasmidique est séparé d'ARN contaminants sans apport de ribonucléases, en utilisant des éléments peu coûteux et respectueux de l'environnement. Ces paramètres permettent d'appliquer ce procédé pour produire des ADN plasmidiques à grande échelle ( production de série'). La présente invention porte également sur l'utilisation de l'ADN plasmidique obtenu selon ce procédé pour réaliser un agent contenant un ADN plasmidique pour la thérapie génique et la vaccination génétique.
EP05706998A 2004-01-29 2005-01-25 Procede de separation chromatographique d'un melange d'acides nucleiques Withdrawn EP1713910A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP05706998A EP1713910A1 (fr) 2004-01-29 2005-01-25 Procede de separation chromatographique d'un melange d'acides nucleiques

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP04001864A EP1559783A1 (fr) 2004-01-29 2004-01-29 Procédé pour la séparation chromatographique d'un mélange d'acides nucléiques
PCT/EP2005/000693 WO2005073376A1 (fr) 2004-01-29 2005-01-25 Procede de separation chromatographique d'un melange d'acides nucleiques
EP05706998A EP1713910A1 (fr) 2004-01-29 2005-01-25 Procede de separation chromatographique d'un melange d'acides nucleiques

Publications (1)

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EP1713910A1 true EP1713910A1 (fr) 2006-10-25

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EP04001864A Withdrawn EP1559783A1 (fr) 2004-01-29 2004-01-29 Procédé pour la séparation chromatographique d'un mélange d'acides nucléiques
EP05706998A Withdrawn EP1713910A1 (fr) 2004-01-29 2005-01-25 Procede de separation chromatographique d'un melange d'acides nucleiques

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US (1) US20070275920A1 (fr)
EP (2) EP1559783A1 (fr)
JP (1) JP2007519407A (fr)
CN (1) CN1914319B (fr)
AU (1) AU2005209389A1 (fr)
WO (1) WO2005073376A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP2638156B1 (fr) * 2010-11-09 2016-01-27 Qiagen GmbH Procédé et dispositif pour l'isolement et la purification d'acides nucléiques double brin
WO2014047141A1 (fr) * 2012-09-19 2014-03-27 Beckman Coulter, Inc. Utilisation d'ions divalents, de protéases et de détergents sous un faible ph pour l'extraction d'acides nucléiques
CN104232687A (zh) * 2014-09-17 2014-12-24 许瑞安 一种重组腺相关病毒rAAV载体的分离纯化方法
GB201709531D0 (en) * 2017-06-15 2017-08-02 Ge Healthcare Bio Sciences Ab Method and apparatus for determining one or more buffer composition recipes
US11198879B2 (en) * 2018-10-25 2021-12-14 Viet Nam National University Ho Chi Minh City Mixture of cell extract and method for site-directed cloning

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DE3639949A1 (de) * 1986-11-22 1988-06-09 Diagen Inst Molekularbio Verfahren zur trennung von langkettigen nukleinsaeuren
DE4432654C2 (de) * 1994-09-14 1998-03-26 Qiagen Gmbh Verfahren zur Isolierung von Nukleinsäuren aus natürlichen Quellen
US5990301A (en) * 1994-02-07 1999-11-23 Qiagen Gmbh Process for the separation and purification of nucleic acids from biological sources
WO1995021179A1 (fr) * 1994-02-07 1995-08-10 Qiagen Gmbh Procede de reduction ou d'elimination d'endotoxines
US5981735A (en) * 1996-02-12 1999-11-09 Cobra Therapeutics Limited Method of plasmid DNA production and purification
EP1168918A4 (fr) * 1999-03-03 2002-08-21 Univ Pennsylvania Compositions vaccinales et de therapie genique, methodes de production et d'utilisations de celles-ci
GB9927904D0 (en) * 1999-11-25 2000-01-26 Amersham Pharm Biotech Ab A method fro obtaining a nucleic acid variant
DE19962577A1 (de) * 1999-12-23 2001-07-12 Tittgen Biotechnologie Dr Chromatographiematerial und Verfahren unter Verwendung desselben
US6406892B1 (en) * 2001-05-23 2002-06-18 Bio-Rad Laboratories, Inc. Acetate-free purification of plasmid DNA on hydroxyapatite

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JP2007519407A (ja) 2007-07-19
EP1559783A1 (fr) 2005-08-03
US20070275920A1 (en) 2007-11-29
AU2005209389A1 (en) 2005-08-11
CN1914319A (zh) 2007-02-14
WO2005073376A1 (fr) 2005-08-11
CN1914319B (zh) 2010-06-09

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