EP0961826A2 - Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique - Google Patents

Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique

Info

Publication number
EP0961826A2
EP0961826A2 EP98904051A EP98904051A EP0961826A2 EP 0961826 A2 EP0961826 A2 EP 0961826A2 EP 98904051 A EP98904051 A EP 98904051A EP 98904051 A EP98904051 A EP 98904051A EP 0961826 A2 EP0961826 A2 EP 0961826A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
solution
membranes
cross
flow filtration
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Withdrawn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
EP98904051A
Other languages
German (de)
English (en)
Inventor
Wolfgang Kuhne
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.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics 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 Roche Diagnostics GmbH filed Critical Roche Diagnostics GmbH
Priority to EP98904051A priority Critical patent/EP0961826A2/fr
Publication of EP0961826A2 publication Critical patent/EP0961826A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

  • the present invention relates to a process for the purification and / or for the concentration of nucleic acids in a solution, a process for the separation of endotoxins from a nucleic acid preparation and the use of a cross-flow filtration system for the purification or / and for the concentration of nucleic acids in a solution and for the separation of endotoxins from a nucleic acid preparation.
  • nucleic acid purification processes are 5 common methods.
  • the obtained biological material for example E.coli bacterial cells
  • the disruption usually lysis with lysozyme or ultrasound
  • the supernatant is shaken out with phenol o.
  • Ultracentrifugation is then carried out on a cesium chloride gradient (Birnboim & Doly, Nucl. Acid Res. 7 (1979) 1513-1523, Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842).
  • cesium chloride gradient Barnboim & Doly, Nucl. Acid Res. 7 (1979) 1513-1523, Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842.
  • such preparations generally contain bacterial endotoxins, phenol, cesium chloride and / or ethidium bromide as impurities.
  • Endotoxins are bacterial toxins that are found in all Enterobacteriaceae, eg. B. Salmonella, Shigella and E.coli can be found. Endotoxins act as pyrogens in 0 mammals and cause numerous other pathological effects in addition to fever. Lipid A is particularly responsible for the toxic effects of endotoxins.
  • nucleic acid purification process after alkaline lysis of the biological material, for example E. coli cells, according to Birnboim & Doly, chromatography of the centrifugation supernatant is carried out under high salt conditions using anion exchange columns (e.g. Mono-Q, Source-Q from Pharmacia , Macroprep-Q by Biorad, Poros-Q by Perspective Biosystems or HyperD-Q by Biosepra, see Chandra et al., Analyt. Bio-chem. 203 (1992) 169-172; Dion et al., J. Chrom 535 (1990) 127-147). Even after this cleaning step, the plasmid preparation still contains impurities such as proteins and, in particular, considerable amounts of endotoxins. O
  • the cleaning methods mentioned have a final desalination and concentration step in common.
  • Isopropanol / ethanol precipitation of the nucleic acid with subsequent centrifugation and resuspension of the nucleic acid pellet in buffer is usually used (cf. e.g. Sambrook J. et al. (1989), Molecular Cloning; A Laboratory Manual, 2nd 5 Ed., Cold Spring Harbor Laboratory Press).
  • the precipitate of the nucleic acid which forms is then centrifuged off and the supernatant is discarded.
  • the pellet which contains the nucleic acid, is taken up in 70% ethanol in a subsequent step, centrifuged again, the supernatant is discarded and, after the pellet has dried, it is resuspended in a desired buffer.
  • the isopropanol / ethanol precipitation method is only practical for applications in the laboratory where relatively small volumes are used.
  • the isopropanol / ethanol precipitation method described has further significant disadvantages. For reasons of operational safety and environmental protection, it is very unfavorable to use the isopropanol / ethanol volumes required on an industrial process scale. In addition, the isopropanol / ethanol precipitation method does not meet the technical requirements for providing therapeutically usable nucleic acids, since endotoxins contained in the nucleic acid solution cannot be completely removed with this method.
  • WO 95/21177 describes a method for isolating and purifying nucleic acids for use in gene therapy, the purification essentially by centrifuging, filtering, affinity chromatography or chromatography on an inorganic chromatography material and chromatography on an ion exchanger he follows .
  • the nucleic acid is treated with an "endotoxin removal buffer" which contains 10% Triton ® X 100 and 40 mmol / 1 MOPS buffer (3-morpholino-1-propanesulfonate).
  • endotoxin removal buffer which contains 10% Triton ® X 100 and 40 mmol / 1 MOPS buffer (3-morpholino-1-propanesulfonate).
  • GW Rembhotkar and GS Khatri describe the purification of ⁇ -phage lysate using "tangential flow filtration".
  • a subsequent ⁇ phage DNA preparation is carried out using customary methods using chlorophore, phenol-chlorophore treatment and ethanol precipitation.
  • WO96 / 36706 and 96/02658 AI describes a process for the isolation and purification of plasmid DNA from microorganisms which have been produced on an industrial scale. The cells are disrupted by adding a lysis solution and heating to 70 ° C. to 100 ° C.
  • a clear cell supernatant is obtained by batch centrifugation and diafiltration.
  • the diafiltration is carried out in the "dead-end mode", but not by tangential overflowing of the membrane in accordance with a cross-flow fraction process.
  • This is followed by a further purification by means of anion exchange chromatography and reversed phase HPLC. In this process, only the cell disruption is carried out in a continuous process step, the further purification of the plasmid DNA is carried out in batches in conventional centrifuges and diafiltration devices.
  • EP-A 96 101 628.4 it is proposed to purify nucleic acid preparations via anion exchangers by means of a high salt gradient in order to obtain nucleic acid solutions which have a protein content of less than 0.1%, free of impurities such as Ethidium bromide, phenol, cesium chloride and detergents are and are largely free of endotoxins.
  • the present invention relates to a method for purifying and / or concentrating nucleic acids in a solution, which is characterized in that the solution containing nucleic acid is passed tangentially past one or more semipermeable membranes so that the nucleic acid molecules of are retained in the membranes and substances with a lower molecular weight can pass through the membranes, a purified or / and concentrated nucleic acid solution being obtained.
  • the present invention relates to a method for separating endotoxins from a nucleic acid preparation, which is characterized in that the preparation containing nucleic acid is guided tangentially past one or more semipermeable membranes, so that the nucleic acid molecules are retained by the membranes and substances with a lower molecular weight can pass through the membranes and / or be adsorbed by the membrane, an essentially endotoxin-free nucleic acid solution being obtained.
  • the method of the present invention can be used to clean and concentrate nucleic acid solutions using a cross-flow filtration system.
  • the nucleic acids are not damaged by cross-flow filtration (CFF). So far it has always been assumed that the shear forces occurring at the CFF would lead to damage to nucleic acids, in particular to strand breaks. For this reason, the CFF has so far only been used for the concentration and diafiltration of proteins.
  • the method of the invention can be used not only to isolate nucleic acids in large quantities Gen and desired purity are obtained, but the method of the present invention also avoids the use of organic solvents, which is of great advantage from a toxicological point of view as well as from a safety and environmental point of view.
  • the method of the present invention can also be used to obtain nucleic acid preparations which are largely freed of impurities, in particular endotoxins, so that they can be used in therapeutic processes or applications.
  • the present method thus solves a problem which has arisen as a result of the rapidly increasing amount of nucleic acids which can be used therapeutically, in particular DNA which can be used therapeutically, and which is expected to increase further in the future.
  • the retentate flow rate (RF) can be varied over a wide range, preferably an RF of 100 l / h * m 2 to 4,000 l / h «m 2 is used.
  • the process can also be carried out at varying temperatures, preferably in a temperature range from 4 ° C to 25 ° C.
  • membranes such as membranes made of modified polyether sulfone (PES), polyvinylidene difluoride, are used to separate contaminants and in particular from endotoxins
  • a plasmid DNA purified according to the invention shows only a dominant band after electrophoretic separation, which band corresponds to the "covalently closed circle” conformation. Furthermore, in addition to small proportions of the "Open Circle” and “Linearized Circle” conformations, there are no other bands.
  • the present invention relates to d ng the use of a cross-flow filtration system for the separation of endotoxins from a nucleic acid preparation.
  • the preceding invention relates to the use of the nucleic acids purified or / and concentrated by cross-flow filtration for cloning, for transformation, for transfection, for microinjection into cells, for use in methods of gene therapy, DNA - vaccination and / or for polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • 2,000 g of moist E.coli biomass is filled from the fermenter into depyrogenated centrifuge beakers.
  • 22.5 1 resuspension buffer (50 mmol / 1 Tris-HCl, 10 mmol / 1 EDTA-Na 2 , pH 8 ⁇ 0.2) are added and slowly (approx. 35 RPM) at 5 ⁇ 4 ° C for at least 24 hours. stirred until the biomass is completely suspended. The temperature of the suspension is slowly increased to 25 ° C.
  • 22.5 1 0.2 mol / 1 NaOH, 1% SDS are added to the suspension with stirring at approx. 80 RPM and incubated at 25 ° C. for 5 minutes.
  • 22.5 l of potassium acetate buffer (3 mol / 1 potassium acetate buffer pH 5.5) are added with stirring and the temperature of the biomass is reduced to 4 ° C. as quickly as possible.
  • the biomass is centrifuged for 30 minutes at 26,000 x g and 4 ° C.
  • the supernatant, which contains the plasmid DNA is isolated and clear filtered through a 5 ⁇ m filter candle.
  • the next step is a chromatography on Q-Sepharose.
  • the decanted centrifuge supernatant is adjusted to 49-50 mS / cm conductivity by adding TE buffer (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA pH 8.5 ⁇ 0.2) and cooled to 5 ⁇ 4 ° C . All chromatography is carried out at this temperature.
  • the centrifugation supernatant is drawn onto the equilibrated column. The column is then washed with about 8 SV 10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.65 mol / 1 NaCl pH 8.5 + 0.2.
  • Equilibration buffer 0.1 mol / 1 potassium phosphate, 6 mol / 1 urine substance pH 7.0, 0 ⁇ 0.2.
  • Wash buffer 2 0.02 mol / 1 potassium phosphate buffer pH 7.0 ⁇ 0.2.
  • the plasmid DNA starting solution has a plasmid DNA concentration of approx. 200 ⁇ g / ml and a volume of approx. 3750 ml.
  • the CFF is at a retentate flow rate of 100-200 l / h »m 2 , a transmembrane pressure of approx. 0.8 bar and an overflow pressure of approx. 1.2 bar.
  • the volume is concentrated to approx. 50 ml and the retentate is then fluently diafiltered against TE buffer (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, pH 8.0) until the values for pH and Conductivity of retentate and TE buffer match.
  • the retentate is adjusted to a plasmid DNA concentration of 1 mg / ml by dilution with diafiltration buffer.
  • a sample of the plasmid DNA solution prepared is taken and analyzed as described under point 3.1.
  • a plasmid DNA solution with a volume of 100 ml is increased with 1000 EU of the E-Toxate ® endotoxin standard solution to 10 EU / ml and then used as the starting solution in the experiment.
  • the solution is diluted to 1000 ml with TE buffer and then concentrated again to the starting volume of 100 ml using the CFF (concentrate 1).
  • the dilution and concentration step is repeated four times in a row. After each concentration step, a sample is taken from the respective concentrate (samples: concentrate 2, 3, 4, 5) and the endotoxin concentration of the sample is analyzed using the Limulus amebocyte lysate method.
  • the plasmid DNA solution can be easily concentrated and diafiltered using the CFF.
  • the results of the investigation are summarized in the table below.
  • agarose gel shows lanes 1 and 10 of DNA length standard No. II (fragment sizes: 125, 564, 2027, 2322, 4361, 6557, 9416, 23130 bp) and lanes 2 and 9 of DNA length standard No. III (fragment sizes : 125, 564, 831, 647, 1375, 1584, 1904, 2027, 3530, 4268, 4973, 5148, 21226 bp).
  • Lane 3 shows pBR322 (4162bp) as a comparative plasmid, which was purified by conventional cesium chloride gradient method.
  • Lane 1 DNA length standard II (Boehringer Mannheim GmbH,
  • Lane 3 pBR322 (Boehringer Mannheim GmbH, Cat.No. 481 238) (0.4 / vg)
  • Lane 4 pCMV-CAT according to CFF, 0, 1 9 yg (bulk drug solution)
  • Lane 6 pCMV-CAT according to CFF, 0.71 ⁇ g (bulk drug solution)
  • the following table shows that the endotoxins are largely purified after the first concentration step.
  • the further CFF reduces the endotoxin concentration up to the detection limit of the test method.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé de purification et/ou de concentration d'acides nucléiques dans une solution, selon lequel la solution contenant l'acide nucléique est guidée de manière tangentielle pour passer sur une ou plusieurs membranes semi-perméables, de manière que les molécules d'acide nucléique soient retenues par les membranes et que des substances présentant un poids moléculaire moins important puissent passer à travers les membranes et/ou soient adsorbées au niveau de la membrane. On obtient ainsi une solution d'acide nucléique purifiée et/ou concentrée. On procède de la même manière pour séparer des endotoxines contenues dans une préparation d'acide nucléique. L'invention concerne en outre l'utilisation d'un système de filtration tangentielle pour purifier et/ou concentrer des acides nucléiques dans une solution, ainsi que pour séparer des endotoxines contenues dans une préparation d'acide nucléique. L'invention concerne par ailleurs l'utilisation des acides nucléiques purifiés ou concentrés par filtration tangentielle, à des fins de clonage, de transformation, de transfection, de micro-injection dans des cellules, d'application dans des procédés de thérapie génique, de vaccination par ADN et/ou de réaction en chaîne de la polymérase (PCR).
EP98904051A 1997-01-10 1998-01-09 Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique Withdrawn EP0961826A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP98904051A EP0961826A2 (fr) 1997-01-10 1998-01-09 Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP97100331A EP0853123A1 (fr) 1997-01-10 1997-01-10 Purification d'ADN à l'aide de 'cross-flow-filtration'
EP97100331 1997-01-10
EP98904051A EP0961826A2 (fr) 1997-01-10 1998-01-09 Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique
PCT/EP1998/000103 WO1998030685A2 (fr) 1997-01-10 1998-01-09 Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique

Publications (1)

Publication Number Publication Date
EP0961826A2 true EP0961826A2 (fr) 1999-12-08

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EP97100331A Withdrawn EP0853123A1 (fr) 1997-01-10 1997-01-10 Purification d'ADN à l'aide de 'cross-flow-filtration'
EP98904051A Withdrawn EP0961826A2 (fr) 1997-01-10 1998-01-09 Purification et/ou concentration d'adn par filtration tangentielle, separation d'endotoxines contenues dans une preparation d'acide nucleique

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EP97100331A Withdrawn EP0853123A1 (fr) 1997-01-10 1997-01-10 Purification d'ADN à l'aide de 'cross-flow-filtration'

Country Status (8)

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EP (2) EP0853123A1 (fr)
JP (1) JP2000513580A (fr)
KR (1) KR100322398B1 (fr)
CN (1) CN1243543A (fr)
BR (1) BR9807062A (fr)
CA (1) CA2277165C (fr)
TR (1) TR199901840T2 (fr)
WO (1) WO1998030685A2 (fr)

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JP2003505077A (ja) 1999-07-23 2003-02-12 ジェネンテック・インコーポレーテッド 平行流ろ過法を用いるｒｎａｓｅ−及び有機溶媒−非含有プラスミドｄｎａの精製方法
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Also Published As

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KR20000069943A (ko) 2000-11-25
AU723817B2 (en) 2000-09-07
CA2277165A1 (fr) 1998-07-16
KR100322398B1 (ko) 2002-03-18
EP0853123A1 (fr) 1998-07-15
WO1998030685A3 (fr) 1998-10-22
JP2000513580A (ja) 2000-10-17
TR199901840T2 (xx) 2000-02-21
BR9807062A (pt) 2000-05-02
AU6208498A (en) 1998-08-03
CN1243543A (zh) 2000-02-02
CA2277165C (fr) 2004-03-30
WO1998030685A2 (fr) 1998-07-16

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