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WO2007115046A1 - Préparation d'acide nucléique à faible teneur d'endotoxine - Google Patents

Préparation d'acide nucléique à faible teneur d'endotoxine Download PDF

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Publication number
WO2007115046A1
WO2007115046A1 PCT/US2007/065379 US2007065379W WO2007115046A1 WO 2007115046 A1 WO2007115046 A1 WO 2007115046A1 US 2007065379 W US2007065379 W US 2007065379W WO 2007115046 A1 WO2007115046 A1 WO 2007115046A1
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Prior art keywords
nucleic acid
endotoxin
binding agent
degrading enzyme
bead
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PCT/US2007/065379
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English (en)
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John Nicholas Price
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Invitrogen Corporation
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Publication of WO2007115046A1 publication Critical patent/WO2007115046A1/fr

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    • 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
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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

Definitions

  • inventions related to the preparation of biological materials.
  • the disclosed inventions relate specifically to methods and materials for reducing endotoxin contamination or removing endotoxins from nucleic acid preparations.
  • Endotoxins are lipopoly-saccharides (LPSs) present in the outer membrane of Gram-negative host cells ⁇ e.g., Escherichia coli), and can co-purify with and contaminate nucleic acids isolated from such cells. This can be because endotoxins can form micelle type structures similar in density, size and charge distribution to nucleic acids.
  • LPSs lipopoly-saccharides
  • Endotoxin contamination of plasmid DNA preparations can reduce transfection efficiency (Weber et al, (1995) BioTechniques 19(6):930-939), and can negatively affect non-viral therapeutic gene transfer and nucleic acid vaccination (Morrison and Ryan, (1997) Ann. Rev. Med. 38:417-32; Boyle et al, (1998) DNA Cell Biol. 17:343-48).
  • Minimizing endotoxin contamination of plasmid DNA is very important for transfection purposes, particularly if the aim is therapeutic, for example in gene therapy, where it is very important that inflammatory or necrotic side reactions due to impurities such as endotoxins do not occur.
  • Known methods for preparing plasmid DNA can involve the use of, e.g., silica, glass powder, hydroxyl-apatite, pH-switchable moieties, and/or reverse-phase, affinity, size-exclusion or anion-exchange chromatography.
  • Some popular methods for reducing endotoxin levels in plasmid preparations involve the use of surfactants (e.g., as described in WO 95/21177, WO 95/21179).
  • surfactants e.g., as described in WO 95/21177, WO 95/21179.
  • Such methods generally require labor-intensive phase separation steps that can compromise yield, and can be complex, time-consuming, and of limited suitability to purification of large-scale preparations of DNA.
  • such methods have proven ineffective for some researchers (Montbriand et al, (1996) J. Biotechnol. 44:43-46; Manthorpe et al, (1993) Hum. Gene Ther. 4: 419-
  • inventive methods and materials for reducing endotoxin contamination of nucleic acid preparations involve the use of an enzymatic endotoxin degrading agent to rapidly and efficiently prepare nucleic acids suitable for a variety of applications, including transfection of cultured cells.
  • the invention relates to methods for isolating nucleic acid from a biological source, the methods involving: contacting a medium comprising the nucleic acid to be isolated with an endotoxin degrading enzyme and a nucleic acid binding agent, such that the nucleic acid binds to the binding agent; separating the nucleic acid-bound binding agent from the medium; and separating the nucleic acid from the binding agent.
  • the medium can be contacted with the endotoxin degrading agent before contacting the medium with a nucleic acid binding agent.
  • the medium also can be contacted with the endotoxin degrading agent after contacting the medium with a nucleic acid binding agent.
  • the invention also relates to methods for isolating nucleic acid from a biological source, the methods involving: contacting a medium comprising the nucleic acid to be isolated with a nucleic acid binding agent, such that the nucleic acid binds to the binding agent; separating the nucleic acid-bound binding agent from the medium; separating the nucleic acid from the binding agent; and contacting the separated nucleic acid with an endotoxin degrading enzyme.
  • the invention also relates to methods for isolating nucleic acid from a biological source, the methods involving: contacting a medium comprising the nucleic acid to be isolated with a nucleic acid binding agent, such that the nucleic acid binds to the binding agent; separating the nucleic acid-bound binding agent from the medium; contacting the separated nucleic acid with an endotoxin degrading enzyme; and separating the nucleic acid from the binding agent.
  • the invention also relates to a method for reducing endotoxin contamination, the method involving contacting plasmid DNA with an endotoxin degrading enzyme outside of cells.
  • the invention also relates to a method for reducing endotoxin contamination, the method involving contacting a lysate of gram-negative bacterial cells with an endotoxin degrading enzyme.
  • the medium comprising the nucleic acid to be isolated includes Gram-negative bacteria or lysed Gram-negative bacteria.
  • the biological source is a genetically engineered or biotechnological source, hi some embodiments of the above- described methods the nucleic acid is plasmid DNA.
  • the nucleic acid binding agent is silica, diatomaceous earth, hydroxyapatite, cellulose (e.g., DEAE cellulose, nitrocellulose, cellulose acetate, diazocellulose), an anion exchange material, a charge switch material, or a nucleic acid capture probe.
  • the nucleic acid binding agent includes, incorporates or is provided in association with polyester, polyolefin, scintered polyethylene, glass, controlled pore glass, polysaccharide, agar, agarose, dextran, Sepharose, polystyrene, polyvinylchloride, polypropylene, polyethylene, nylon, ceramic, porous ceramic, plastic, porous plastic, aluminum oxide, titanium oxide, zirconium oxide, acrylic amide or any combination of any of the aforementioned.
  • the nucleic acid binding agent is provided in a flat, wafer, cylindrical, porous, non-porous, matrix, bead (e.g., magnetic bead, latex bead, paramagnetic bead, superparamagnetic bead, polystyrene bead), gel, slurry, resin, polymer, cartridge, plug, membrane, filter, strand, or woven fiber form.
  • the endotoxin degrading enzyme is capable of cleaving phosphate groups of Lipid A.
  • the endotoxin degrading enzyme is capable of cleaving acyl chains of Lipid A.
  • the endotoxin degrading enzyme is alkaline phosphatase, lipase, or a combination thereof, hi some embodiments of the above-described methods the isolated nucleic acid is substantially free of other cellular biomolecules. In some embodiments of the above-described methods the isolated nucleic acid is substantially free of endotoxin contamination.
  • the invention also relates to compositions that include an endotoxin degrading enzyme, and one or more of the following: (a) cell lysate of Gram-negative bacteria; (b) plasmid DNA, or (c) nucleic acid binding agent, hi some embodiments, the endotoxin degrading enzyme is capable of cleaving phosphate groups of Lipid A. In some embodiments, the endotoxin degrading enzyme is capable of cleaving acyl chains of Lipid A. In some embodiments, the endotoxin degrading enzyme is alkaline phosphatase, lipase, or a combination thereof. In some embodiments the composition includes (a) and (b).
  • the composition includes (a) and (c). In some embodiments the composition includes (b) and (c). In some embodiments the composition includes (a), (b) and (c).
  • the nucleic acid binding agent is silica, diatomaceous earth, hydroxyapatite, cellulose (e.g., DEAE cellulose, nitrocellulose, cellulose acetate, diazocellulose), an anion exchange material, a charge switch material, or a nucleic acid capture probe.
  • the nucleic acid binding agent includes, incorporates or is provided in association with polyester, polyolefin, scintered polyethylene, glass, controlled pore glass, polysaccharide, agar, agarose, dextran, Sepharose, polystyrene, polyvinylchloride, polypropylene, polyethylene, nylon, ceramic, porous ceramic, plastic, porous plastic, aluminum oxide, titanium oxide, zirconium oxide, acrylic amide or any combination of any of the aforementioned.
  • the nucleic acid binding agent is provided in a flat, wafer, cylindrical, porous, non-porous, matrix, bead (e.g., magnetic bead, latex bead, paramagnetic bead, superparamagnetic bead, polystyrene bead), gel, slurry, resin, polymer, cartridge, plug, membrane, filter, strand, or woven fiber form.
  • bead e.g., magnetic bead, latex bead, paramagnetic bead, superparamagnetic bead, polystyrene bead
  • gel e.g., slurry, resin, polymer, cartridge, plug, membrane, filter, strand, or woven fiber form.
  • Nucleic acids purified using the inventive methods and materials disclosed herein are sufficiently free of endotoxins and can be used for additional processing, analysis, or therapeutic administration.
  • inventive methods and materials disclosed herein are sufficiently free of endotoxins and can be used for additional processing, analysis, or therapeutic administration.
  • the present disclosure describes methods and materials for reducing endotoxin contamination of biological materials, such as nucleic acid preparations.
  • the reduction of endotoxin contamination is achieved by contacting the biological material with an endotoxin degrading enzyme.
  • the methods described herein for reducing endotoxin contamination i.e., reducing endotoxin levels or removing endotoxins
  • biological materials are compatible with, and can be integrated into, any plasmid purification technique, including large-scale preparation of nucleic acids.
  • endotoxin degrading enzymes can be used at any step in a nucleic acid preparation method from addition to cells to a final "polishing" step, or anywhere in between.
  • the methods disclosed herein are particularly well suited for preparing nucleic acid for use in techniques that require low levels of endotoxin contamination, for example transfection of cells, non-viral therapeutic gene transfer, gene therapy, gene silencing, microinjection, and nucleic acid vaccination.
  • Endotoxins are associated with the outer membrane of pathogenic and nonpathogenic Gram-negative bacteria.
  • endotoxin is occasionally used to refer to any cell-associated bacterial toxin, it properly refers to the lipopolysaccharide (LPS) complex associated with the outer membrane of Gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, and Haemophilus.
  • LPSs are amphiphilic molecules that have a molecular weight of about 1OkDa. LPSs have three components or regions: Lipid A, an R polysaccharide, and an O polysaccharide.
  • Lipid A is the lipid component of LPS. It contains the hydrophobic, membrane-anchoring region of LPS, and consists of a phosphorylated N- acetylglucosamine (NAG) dimer and associated fatty acids. All fatty acids in Lipid A are saturated. Some fatty acids are attached directly to the NAG dimmer, while others are esterified to the 3 -hydroxy fatty acids that are characteristically present.
  • the structure of Lipid A is highly conserved among Gram-negative bacteria, and is virtually constant among Enter obacteriaceae.
  • R antigen or R polysaccharide is attached to the 6 position of an NAG.
  • the R polysaccharide consists of a short chain of sugars. Two unusual sugars are usually present in the core polysaccharide, heptose and 2-keto-3-deoxyoctonoic acid (KDO). KDO has been used as an indicator in assays for LPS (endotoxin).
  • the R polysaccharide is common to all members of a bacterial genus ⁇ e.g., Salmonella), but also is structurally distinct in other genera of Gram-negative bacteria. For example, Salmonella, Shigella and Escherichia have similar, but not identical, R polysaccharides.
  • Somatic (O) antigen or O polysaccharide is attached to the R polysaccharide. It consists of repeating oligosaccharide subunits made up of about 3-5 different sugars, with individual chains varying in length, ranging up to about 40 repeat units.
  • the O polysaccharide is much longer than the R polysaccharide, and it maintains the hydrophilic domain of the LPS molecule.
  • a major antigenic determinant of the Gram- negative bacterial cell wall resides in the O polysaccharide. There is great variation in the composition of the sugars in the O side chain between species and even strains of Gram-negative bacteria.
  • sugars are known to occur and many of these sugars are characteristically unique dideoxyhexoses, which occur in nature only in Gram-negative cell walls.
  • Variations in sugar content of the O polysaccharide contribute to the wide variety of antigenic types of Salmonella and E. coli, and presumably other strains of Gram-negative species.
  • Particular sugars in the structure, especially the terminal ones confer immunological specificity of the O antigen, in addition to the "smoothness" (colony morphology) of the strain. Loss of the O specific region by mutation results in the strain becoming a "rough"(colony morphology) or R strain.
  • Toxicity is associated with the lipid component (Lipid A) of LPS, while immunogenicity is associated with the polysaccharide components. Both Lipid A and the polysaccharide side chains act as determinants of virulence in Gram-negative bacteria. LPS also elicits a variety of inflammatory responses in an animal. Mild hydrolysis of LPS yields Lipid A plus polysaccharide. Endotoxins are toxic to most mammals, and regardless of the bacterial source, all endotoxins produce the same range of biological effects in the animal host. For example, endotoxins contribute to the typical phenomena which accompany a bacterial intoxication, such as inflammatory reactions and fever, as well as endotoxic shock.
  • Lipid A Since Lipid A is embedded in the outer membrane of bacterial cells, it probably only exerts its toxic effects when released from multiplying cells in a soluble form, or when bacteria are lysed e.g., as a result of autolysis, complement and the membrane attack complex (MAC), ingestion and killing by phagocytes, or killing with certain types of antibiotics. It has been reported that the enzymatic cleavage of the phosphate groups of Lipid A neutralizes toxicity (Ulrich and Myers, (1995) Pharm. Biotechnol. 6:495-524; Gustafson and Rhodes, (1992) Biochem Biophys Res Commun. 182:269-75).
  • the present disclosure relates to methods and materials for reducing endotoxin levels or removing endotoxins in biological materials, e.g., materials comprising nucleic acids to be isolated.
  • biological materials comprising nucleic acids can be or be derived from natural, genetically engineered, or biotechnological sources, such as prokaryotic cell cultures.
  • Nucleic acids to be isolated can be, e.g., single-stranded or double-stranded RNA or DNA, RNA/DNA hybrids, DNA fragments, oligonucleotides, amplified DNA or RNA, BACs, or plasmid DNA.
  • Extrachromosomal nucleic acid molecules can be isolated from cellular sources. Such extrachromosomal nucleic acids include plasmids, vectors, phagemids, cosmids, cDNA molecules, and RNA (e.g., mRNA or rRNA) molecules. Extrachromosomal nucleic acids can be single-stranded or double stranded, linear or circular, supercoiled, and which can be DNA or RNA molecules. Nucleic acids to be isolated can be, e.g., between 6 base pairs and 1000 kilobase pairs in length.
  • a nucleic acid source can be a prokaryotic cell, such as members of the genera Escherichia (particularly E. coli), Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia, Bordetella, Legionella, Pseudomonas, Mycobacterium, Helibacter, Agrobacterium, Collectorichum, Rhizobium, and Streptomyces.
  • an endotoxin degrading enzyme is a peptide that is capable of degrading endotoxins, preferably in a catalytic manner. Some endotoxin degrading enzymes cleave the phosphate groups of Lipid A ⁇ e.g., alkaline phosphatase). Other endotoxin degrading enzymes cleave the acyl chains of Lipid A ⁇ e.g., lipase).
  • Alkaline phosphatases, lipases and similar endotoxin degrading enzymes suitable for use in the disclosed methods can be easily identified by those skilled in the art using well-known and freely accessible protein homology databases such as PFAM (http://www.sanger.ac.uk/Software/Pfam/), PALI (http://pauling.mbu.iisc.ernet.in/ ⁇ pali/), and SCOP (http://scop.mrc-lmb.cam.ac.uk/scop/index.html).
  • An endotoxin degrading enzyme can be attached to an immobilized matrix and exposed to a medium that contains the nucleic acid to be isolated.
  • Methods for isolating nucleic acids from cells are well known to those of skill in the art, and usually involve three basic steps: (1) preparing a medium that contains the nucleic acid to be isolated; (2) binding the nucleic acid to a nucleic acid binding agent; and (3) separating the bound nucleic acid from the nucleic acid binding agent.
  • nucleic acids can be contaminated by endotoxins from the host cell, hi some cell types, the number of endotoxins present in the cells is approximately 3.5x10 6 copies per cell (Escherichia CoIi and Salmonella Typhimurium Cell, and MoI. Biology, J. L. Ingraham et al. Eds., 1987, ASM), in some cases exceeding the number of plasmid DNA molecules in the cells by a factor of more than 10 4 .
  • plasmid DNA obtained from Gram- negative cells can be contaminated with large amounts of endotoxins.
  • Endotoxin degrading enzymes can be used in a number of ways in connection with known methods of nucleic acid isolation to reduce the amount of endotoxin in isolated nucleic acids.
  • an endotoxin degrading enzyme can be used before, during, or after preparing a medium that contains the nucleic acid to be isolated; before, during or after binding the nucleic acid to a nucleic acid binding agent; and/or before, during or after nucleic acid isolation.
  • an endotoxin degrading enzyme treatment can be used as a "front-end step” by adding endotoxin degrading enzyme to a medium that contains the nucleic acid to be isolated, before any nucleic acid binding or isolation steps.
  • An endotoxin degrading enzyme treatment also can be used as a "polishing step” after nucleic acid isolation. Endotoxin degrading enzyme treatments also can be used at any point in between such "front-end” and “polishing" treatments.
  • Media that contain a nucleic acid to be isolated can be prepared by those of skill in the art using well known, and routine methods. Such media can be obtained, for example, from natural, genetically engineered, or biotechnological sources by any means, including cell lysis, homogenization, centrifugation, filtration, and/or precipitation.
  • Whole cells and cell lysates can serve as a media for use in the disclosed nucleic acid isolation methods, as can crude or semi-purified samples of nucleic acid.
  • Methods for cell lysis are well known to those of skill in the art. Such methods can involve alkaline lysis (Birnborn and Dohly, (1979) Nucl. Acids Res. 7:1513-22), high pressure boiling lysis, mechanical lysis, French Press, lithium chloride treatment, the use of anionic, cationic, zwitterionic, polymeric and/or non-polymeric detergents, and/or digestion by enzymes such as lysozyme and proteinase K.
  • a cell lysis agent as used herein refers to a composition or a component of a composition that affects lysis, rupture, or poration of cells used as a nucleic acid source, such that the nucleic acid molecules that are contained in the cells are released from the cells. Any known component and/or method can be used to lyse cells suitable for use in the present disclosure. Medium containing the nucleic acid that is obtained by cell lysis is referred to as a "cell lysate.”
  • a "cleared lysate” is a cell lysate that has been processed (e.g., by precipitation, filtration, and/or centrifugation) to substantially or entirely remove insoluble materials such as proteins and genomic DNA.
  • a cleared lysate can be contaminated by relatively small cell constituents, chemicals from the preceding treatment steps (if any), RNA, proteins, and endotoxins. The removal of these impurities can require a plurality of subsequent purification steps.
  • Media that has been prepared or processed in some way can serve as a media for use in the disclosed nucleic acid isolation methods.
  • Such media include cleared lysates (described above), and crude or semi-purified samples of nucleic acid.
  • a suitable medium can be obtained from biological material from cleared lysates from which cellular RNA and/or soluble proteins have been removed; from pre- purified nucleic acids present in a buffer and/or bound to a nucleic acid binding agent; and from solutions formed after nucleic acid amplification that contain endotoxin impurities.
  • Cell lysates can be centrifuged or filtered to remove cellular debris.
  • Another conventional technique for removing cellular debris is "salting-out," in which proteins and other contaminants are precipitated from the medium using high concentrations of salt such as potassium acetate or ammonium acetate (see, e.g., Miller et ai, (1988) Nucl. Acids Res. 16:1215).
  • the precipitates can be removed by centrifugation or filtration, and the nucleic acids can be recovered by alcohol precipitation.
  • the resultant cleared lysates can be concentrated or pre-purified by known methods, such as dialysis, filtration and precipitation.
  • Preparation of nucleic acids from cellular sources also can involve the use of an extraction reagent containing organic solvent, such as phenol, chloroform or isoamyl alcohol to separate the nucleic acids from cellular debris (see, e.g., Murmur, (1961) J. MoI. Biol. 3:208-28; Gross-Bellard et al, (1973) Eur. J. Biochem. 36:32-38; Blin et al, (1976) Nucl. Acids Res. 3:2303-08; U.S. Patent No. 4,623,627; Sambrook and Russell (2001) Molecular Cloning — A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, New York).
  • organic solvent such as phenol, chloroform or isoamyl alcohol
  • E. coli that contain a plasmid DNA can be lysed with alkaline detergent solution.
  • an acidic potassium-containing neutralization buffer can be used to form a precipitate of SDS, proteins, membranes, and chromosomal DNA that can be removed by centrifugation or filtration.
  • This cleared lysate can be subject to treatment with endotoxin degrading enzyme.
  • An endotoxin degrading enzyme treatment can also be used at any point prior to preparing a cleared lysate (e.g., by addition to whole cells), or at any time thereafter in the preparation of nucleic acid from the cleared lysate.
  • Methods of the invention involve contacting a medium that contains the nucleic acid to be isolated with a nucleic acid binding agent (i.e., a chemical or material that binds or traps nucleic acid molecules).
  • Nucleic acid binding agents include silica, diatomaceous earth, hydroxyapatite, cellulose (e.g., DEAE cellulose, nitrocellulose, cellulose acetate, diazocellulose), anion exchange materials, charge switch materials (U.S. Pat. 6,914,137 and PCT/GB98/03602, each incorporated herein by reference), and nucleic acid capture probes.
  • a nucleic acid binding agent can include, incorporate or be provided in association with polyester, polyolefin, scintered polyethylene, glass, controlled pore glass, polysaccharide, agar, agarose, dextran, Sepharose, polystyrene, polyvinylchloride, polypropylene, polyethylene, nylon, ceramic, porous ceramic, plastic, porous plastic, aluminum oxide, titanium oxide, zirconium oxide, and acrylic amide.
  • a nucleic acid binding agent can be provided in any form including flat, wafer, cylindrical, porous, non-porous, matrix, bead (e.g., magnetic bead, latex bead, paramagnetic bead, superparamagnetic bead, polystyrene bead), gel, slurry, resin, polymer, cartridge, plug, membrane, filter, strand, and woven fiber.
  • Nucleic acid binding agents can be used in chromatographic methods. For example, a nucleic acid to be isolated can be applied to a column containing a substrate that comprises a nucleic acid binding agent ⁇ e.g., hydroxyapatite, anion exchange material, charge switch material, silica or diatomaceous earth).
  • a washing step to remove impurities can follow application of the nucleic acid.
  • nucleic acid bound to the binding agent is eluted from the column using a buffer that causes the nucleic acid to be separated from the nucleic acid binding agent.
  • An anionic exchange chromatography substrate permits interaction between the negatively charged phosphates of the nucleic acid and positively charged nucleic acid binding agents of the substrate.
  • the nucleic acid to be isolated is applied to the substrate under low-salt conditions that permit binding of the nucleic acid to the substrate.
  • Impurities such as cellular proteins, and metabolites can be washed away using a wash buffer.
  • High-salt buffer conditions are used to elute the nucleic acid from the substrate.
  • 4,997,932 and 6,297,371) can involve the use of a substrate comprising diethylamino ethyl (DEAE) sepharose, DEAE sephadex, DEAE spherodex, DEAE spherosil, diethylaminopropyl groups, dimethylaminoethyl (DMAE), dimethylaminopropyl groups, or polyetyrene/DVB.
  • DEAE diethylamino ethyl
  • DEAE sephadex DEAE sephadex
  • DEAE spherodex DEAE spherosil
  • diethylaminopropyl groups diethylaminopropyl groups
  • DMAE dimethylaminoethyl
  • DMAE dimethylaminopropyl groups
  • polyetyrene/DVB polyetyrene/DVB
  • Silica-based nucleic acid preparation involves the selective adsorption of nucleic acids to a silica material (e.g., kieselbig, siloid, diatomaceous earth, glass, silica gel, alumina, titania, hydroxyapatite), typically in the presence of high concentrations of chaotropic salts.
  • a silica material e.g., kieselbow, siloid, diatomaceous earth, glass, silica gel, alumina, titania, hydroxyapatite
  • nucleic acids typically are applied to silica materials under high-salt conditions, and elution is performed under low-salt conditions.
  • Some exemplary plasmid purification kits that are available commercially include PureYieldTM Plasmid Midiprep System, Wizard® Plus SV Minipreps DNA Purification System, Wizard® SV 96 and SV 9600 Plasmid DNA Purification System, Wizard® MagneSilTM Plasmid Purification System, Wizard® MagneSilTM TfxTM DNA Purification System, Wizard® Purefection Plasmid DNA Purification System, Wizard® Plus DNA Purification System (from Promega Corp.); PurelinkTM HiPure Plasmid Kits, PurelinkTM Quick Plasmid Kits, PurelinkTM HQ Plasmid Kits, PurelinkTM Plasmid Purification Systems, ChargeSwitch® NoSpin Plasmid Kits, S.N.A.P.
  • MiniPrep and MidiPrep Kits from Invitrogen Corp.
  • An endotoxin degrading enzyme can be used to reduce or eliminate endotoxin contamination in any plasmid purification method or kit (including those described herein), at any step of the purification.
  • Nucleic acid can be substantially separated and isolated from a nucleic acid binding agent by any means known in the art. Separation and isolation of nucleic acids from a nucleic acid binding agent can involve changing the chemical and/or physical conditions of the environment of the nucleic acid bound nucleic acid binding agent; applying magnetic and/or physical force; and/or using centrifugation, vacuum, suction, pressure, filtration, precipitation, and/or dialysis.
  • Separated and isolated nucleic acids can be purified further by any known purification method and/or can be manipulated using any known molecular biology techniques known in the art, such as precipitation and resuspension, sequencing, amplification, endonuclease digestion, nucleic acid synthesis, transformation, transfection, and the like. Such further treatment and manipulation will depend on the intended use of the nucleic acid. For some applications, high concentrations of salts can be removed from separated and isolated nucleic acids, e.g. by adsorbing the nucleic acid to a mineral material and subsequently desorbing the nucleic acids with solutions of low ionic strength or demineralized, endotoxin-free water.
  • Some methods for further reducing endotoxins from nucleic acids involve chromatographic steps (e.g., metal-chelating chromatographic methods), gel filtration (e.g., with ultrafiltration membranes or a microporous anion exchange membrane), or precipitation with isopropanol, ammonium acetate, or polyethylene glycol.
  • chromatographic steps e.g., metal-chelating chromatographic methods
  • gel filtration e.g., with ultrafiltration membranes or a microporous anion exchange membrane
  • Nucleic acid treated with an endotoxin degrading enzyme can be further treated to remove degraded endotoxin.
  • nucleic acid treated with an endotoxin degrading enzyme can be subjected to chromatography (e.g., gel chromatography, affinity chromatography, inorganic chromatography, High Performance Liquid Chromatography (HPLC), molecular sieve chromatography, or affinity chromatography), filtration, precipitation, polyacrylamide gel electrophoresis, and treatment with non-ionic detergents.
  • chromatography e.g., gel chromatography, affinity chromatography, inorganic chromatography, High Performance Liquid Chromatography (HPLC), molecular sieve chromatography, or affinity chromatography
  • Nucleic acids purified using the methods disclosed herein can be substantially free of endotoxin contamination.
  • substantially free of endotoxin contamination is an endotoxin content of less than about 5 EU (endotoxin units) per ⁇ g plasmid DNA, e.g. to between 5 and 4, 3, 2, 1, 0.5, 0.1, 0.05 or 0.01 EU/ ⁇ g plasmid DNA, or to between 1 and 0.5, 0.1, 0.05 or 0.01 EU/ ⁇ g plasmid DNA, or to between 0.1 and 0.05 or 0.01 EU/ ⁇ g plasmid DNA.
  • Assaying endotoxin levels can be performed using the standard assay described in Levin, J.& Bang, F.B.
  • Plasmid DNA was obtained using the anion exchange based PureLink HiPure kit (Cat. No. K2100-05, Invitrogen Corp.). The manufacturer's protocol was followed, except that the salt concentration of the plasmid-containing eluate was adjusted to 1 M. 4 U of ApexTM Heat Labile Alkaline Phosphatase (AP49050; Epicentre Biotechnologies) and 1/1 Oth volume of 1OX alkaline phosphatase reaction buffer were added to the salt- adjusted eluate. The mixture was incubated at 37 0 C for 30 min to degrade endotoxin, then at 70 0 C for 5 min to inactivate the alkaline phosphatase. Isopropanol (0.7 vol.) was added to precipitate the plasmid DNA, and the rest of the protocol followed per manufacturer's instructions.
  • Endotoxin levels were determined using the assay described in Levin, J.& Bang, F. B. (1968) Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin, Thromb. Diath. Haemorrh. 19:186-97.
  • Treatment with alkaline phosphatase reduced endotoxin levels to 0.02 EU/ ⁇ g from 0.18 EU/ ⁇ g (untreated sample).
  • Plasmid DNA obtained using the silica based PureLink HQ Mini-prep kit (Cat. No. K2100-01, Invitrogen Corp.). The manufacturer's protocol was followed, except that 4U of ApexTM Heat Labile Alkaline Phosphatase (AP49050; Epicentre Biotechnologies) and 1/10 th volume 1OX Alkaline Phosphatase reaction buffer was added to the plasmid- containing eluate. The mixture was incubated at 37 0 C 30 min, then at 70 0 C for 5 min to inactivate the alkaline phosphatase. Plasmid DNA was precipitated with 1/10* volume NaOAc and 2.5 volumes ethanol. Precipitated DNA was washed with 70% ethanol, dried, and resuspended in the same volume pyrogen- free water.
  • AP49050 ApexTM Heat Labile Alkaline Phosphatase
  • 1OX Alkaline Phosphatase reaction buffer was added to the plasmi
  • Endotoxin levels were determined using the assay described in Levin, J.& Bang, F. B. (1968) Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin, Thromb. Diath. Haemorrh. 19:186-97.
  • Treatment with alkaline phosphatase reduced the endotoxin levels of the isolated plasmid DNA approximately 17 fold, to 3 EU/ ⁇ g DNA.
  • Treatment with water, heat-inactivated alkaline phosphatase or BSA instead of alkaline phospatase did not substantially reduce the Endotoxin level of isolated plasmid DNA.
  • Varying the amount of alkaline phosphatase from 1 to 4 U, the alkaline phosphatase incubation temperature from 37 to 50 ° C, or the alkaline phosphatase incubation time from 30 min to 10 min did not significantly affect the results. Omission of alcohol precipitation following heat inactivation of alkaline phosphatase also did not significantly affect the results.
  • Plasmid DNA was obtained using the ChargeSwitch ® NoSpin Plasmid Kit (Cat. No. CS 10201 , Invitrogen Corp.). Alkaline phosphatase was tested for its efficiency in removing endotoxin at several steps in the procedure. The manufacturer's protocol was followed, except that 1 U of ApexTM Heat Labile Alkaline Phosphatase (AP49050; Epicentre Biotechnologies) in 1/10* volume alkaline phosphatase reaction buffer were added to (i) clarified lysate, (ii) plasmid-bound ChargeSwitch ® magnetic beads, or (iii) plasmid containing eluate.
  • AP49050 ApexTM Heat Labile Alkaline Phosphatase

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Abstract

La présente invention concerne des procédés et des matières permettant de réduire la contamination par des endotoxines de matières biologiques, comme par exemple des préparations d'acides nucléiques. Les procédés impliquent l'utilisation d'agent de dégradation d'une endotoxine enzymatique pour préparer rapidement et efficacement des acides nucléiques appropriés pour une variété d'applications, incluant la transfection de cellules cultivées et des applications thérapeutiques. L'utilisation d'un agent de dégradation des endotoxines est aisément intégrée à de nombreux protocoles et procédés existants de purification d'ADN de plasmides.
PCT/US2007/065379 2006-03-31 2007-03-28 Préparation d'acide nucléique à faible teneur d'endotoxine WO2007115046A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3228710A1 (fr) 2016-04-06 2017-10-11 AXAGARIUS GmbH & Co. KG Purification d'acides nucléiques d'un échantillon comprenant des acides nucléiques et de l'endotoxin
US9988338B2 (en) 2014-09-24 2018-06-05 Basf Se Method for producing diesters of terephthalic acid
US10207978B2 (en) 2014-09-24 2019-02-19 Basf Se Method for producing diesters of terephthalic acid with a dehydration of recirculated alcohol
US10239818B2 (en) 2014-09-24 2019-03-26 Basf Se Method for producing diesters of terephthalic acid with circulation of the reaction mixture
US10266477B2 (en) 2014-09-24 2019-04-23 Basf Se Method for producing diesters of terephthalic acid with enrichment of recirculated alcohol
US11274292B2 (en) 2011-03-29 2022-03-15 Phynexus, Inc. Devices and methods for plasmid purification

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020032324A1 (en) * 1994-02-07 2002-03-14 Qiagen Gmbh Process for the preparation of endotoxin-free or endotoxin-depleted nucleic acids and/or oligonucleotides for gene therapy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020032324A1 (en) * 1994-02-07 2002-03-14 Qiagen Gmbh Process for the preparation of endotoxin-free or endotoxin-depleted nucleic acids and/or oligonucleotides for gene therapy

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BENTALA H. ET AL.: "Removal of phosphate from lipid A as a strategy to detoxify lipopolysaccharide", SHOCK, vol. 18, no. 6, December 2002 (2002-12-01), pages 561 - 566 *
POELSTRA K. ET AL.: "Dephosphorylation of endotoxin by alkaline phosphatase in vivo", AM. J. PATHOL., vol. 151, no. 4, October 1997 (1997-10-01), pages 1163 - 1169 *
TRENT M.S. ET AL.: "A PhoP7PhoQ-induced Lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium", J. BIOL. CHEM., vol. 276, no. 12, 23 March 2001 (2001-03-23), pages 9083 - 9092, XP002319844 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11274292B2 (en) 2011-03-29 2022-03-15 Phynexus, Inc. Devices and methods for plasmid purification
US9988338B2 (en) 2014-09-24 2018-06-05 Basf Se Method for producing diesters of terephthalic acid
US10207978B2 (en) 2014-09-24 2019-02-19 Basf Se Method for producing diesters of terephthalic acid with a dehydration of recirculated alcohol
US10239818B2 (en) 2014-09-24 2019-03-26 Basf Se Method for producing diesters of terephthalic acid with circulation of the reaction mixture
US10266477B2 (en) 2014-09-24 2019-04-23 Basf Se Method for producing diesters of terephthalic acid with enrichment of recirculated alcohol
EP3228710A1 (fr) 2016-04-06 2017-10-11 AXAGARIUS GmbH & Co. KG Purification d'acides nucléiques d'un échantillon comprenant des acides nucléiques et de l'endotoxin
US10457931B2 (en) 2016-04-06 2019-10-29 Axagarius Gmbh & Co. Kg Purification of nucleic acid from a sample containing nucleic acid and endotoxin

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