WO2003080834A2 - Procedes de purification pour des oligonucleotides et leurs analogues - Google Patents

Procedes de purification pour des oligonucleotides et leurs analogues Download PDF

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Publication number
WO2003080834A2
WO2003080834A2 PCT/GB2003/001161 GB0301161W WO03080834A2 WO 2003080834 A2 WO2003080834 A2 WO 2003080834A2 GB 0301161 W GB0301161 W GB 0301161W WO 03080834 A2 WO03080834 A2 WO 03080834A2
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WIPO (PCT)
Prior art keywords
target oligonucleotide
anion exchange
oligonucleotide
solution
exchange composition
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PCT/GB2003/001161
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English (en)
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WO2003080834A3 (fr
Inventor
Jack T. Johansen
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Avecia Biotechnology Inc.
Avecia Limited
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Application filed by Avecia Biotechnology Inc., Avecia Limited filed Critical Avecia Biotechnology Inc.
Priority to US10/508,799 priority Critical patent/US20060035224A1/en
Priority to CA002479901A priority patent/CA2479901A1/fr
Priority to AU2003216830A priority patent/AU2003216830A1/en
Priority to KR10-2004-7014747A priority patent/KR20040108672A/ko
Priority to EP03712366A priority patent/EP1490488A2/fr
Priority to JP2003578560A priority patent/JP2005520547A/ja
Publication of WO2003080834A2 publication Critical patent/WO2003080834A2/fr
Publication of WO2003080834A3 publication Critical patent/WO2003080834A3/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
    • 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
    • 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

  • Synthetic oligonucleotides have emerged as important biomolecules for a wide variety of applications. Such applications include the use of synthetic oligonucleotides as hybridization probes, linkers, primers for DNA sequencing, amplification reactions (e.g., polymerase chain reactions, reverse transcriptase reactions), potential therapeutics in antisense and related technology investigations and diagnostic tools for the detection of genetic and viral diseases.
  • synthetic oligonucleotide analogs have received approval of the FDA for the treatment of CMV and at present, several oligonucleotide analogs are undergoing clinical trials.
  • TLC thin layer chromatography
  • PAGE polyacrylamide gel electrophoresis
  • HPLC high-performance liquid chromatography
  • a hydrophobic 5' protecting group e.g., a 5'-O-trityl group
  • the hydrophobic 5'-protecting group can then be used to separate the full-length "trityl on" target oligonucleotide from shorter failure sequences which do not possess the hydrophobic 5'-protecting group (i.e., "trityl off' sequences).
  • the trityl group can be cleaved from the target oligonucleotide with acid. While this is an effective approach, it is time consuming and laborious and requires subsequent hydrolysis and extraction steps to isolate the purified product.
  • an anion exchange composition containing fixed positive charges is used to bind the target oligonucleotide and failure sequences (both of which are polyanions).
  • the strength of the binding of the target oligonucleotide and failure sequences to the anion exchange composition is directly proportional to their length.
  • a salt gradient is typically used to weaken the interaction of the oligonucleotides with the anion exchange composition.
  • the shortest oligonucleotides elute first, while the longer oligonucleotides elute at higher salt concentrations.
  • the use of a salt gradient in anion exchange separation of oligonucleotides is disadvantageous for a number of reasons.
  • the desired eluted oligonucleotide typically has to be desalted prior to use in downstream applications. Consequently, this necessitates subsequent desalting steps which increase the time, labour and expense required for separation.
  • the use of high concentrations of salt in eluting solutions can also lead to increased corrosion of stainless steel parts (e.g., HPLC pumps, fittings, valves, columns, tubing) in the machinery used for anion exchange separations.
  • the present invention is drawn to methods of separating oligonucleotides from impurities.
  • a target oligonucleotide in a mixture comprising the target oligonucleotide and an impurity, is separated from the impurity using a titratable anion exchange composition.
  • the target oligonucleotide is bound to the titratable anion exchange composition and an eluting solution which increases in pH over time is passed through the titratable anion exchange composition with the target oligonucleotide bound thereon.
  • the eluting solution does not substantially increase in salt concentration.
  • the target oligonucleotide is then eluted, thereby separating it from the impurity which elutes at a different pH than the target oligonucleotide.
  • one or more washing steps can be performed prior to eluting the target oligonucleotide.
  • the methods of the invention can be used with a variety of titratable anion exchange compositions, including titratable anion exchange compositions which comprise a primary amine, a secondary amine or a tertiary amine.
  • Suitable titratable anion exchange compositions include anion exchange compositions comprising polyimizadole, polyhistidine, polylysine or polyethyleneimine.
  • the titratable anion exchange composition is conjugated to a support, for example, a synthetic polymer support, such as silica gel, a polysaccharide, a polystyrene, especially a styrene- divinylbenzene copolymer, a polyethylene, a polypropylene, a polyacrylate, or an agarose, for example those agaroses available under the trade name Sepharose.
  • the titratable anion exchange composition is covalently bonded to a support, optionally via a linker group.
  • the support may be a functionalised support. Examples of such functionalised supports are well known in the art, and include for example, the hydroxy or amino-functionalised supports, especially hydroxy or amino-functionalised polystyrene.
  • the methods of the invention can be used to separate a variety of oligonucleotides, including, for example, single-stranded and/or double-stranded nucleic acids.
  • oligonucleotides include naturally-occurring oligonucleotides, such as deoxyribonucleic acids (DNA) and ribonucleic acids (RNA).
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • the methods of the invention can be used to separate synthetic oligonucleotides, for example, phosphates, phosphorothioates, phosphorodithioates, methyl phosphonates, phosphoramidates and chimeras.
  • the target oligonucleotide to be separated is 5'-O-protected (for example, with a 5'-O-trityl, such as a 5'-O-dimethoxytrityl, protecting group).
  • a 5'-O-trityl such as a 5'-O-dimethoxytrityl, protecting group.
  • the 5'-O-protecting group is cleaved from the target oligonucleotide prior to elution, while the target oligonucleotide is bound to the titratable anion exchange composition.
  • Cleavage of the 5'-O-trityl protecting group is achieved by passing through the titratable anion exchange composition with target oligonucleotide bound thereon, a sufficient amount of an acidic solution (e.g., a solution comprising aqueous acetic acid (e.g., 20%-80% v/v acetic acid)), prior to elution of the target oligonucleotide.
  • an acidic solution e.g., a solution comprising aqueous acetic acid (e.g., 20%-80% v/v acetic acid)
  • eluting solutions can be used to elute the target oligonucleotide provided that they increase pH. Separation of a target oligonucleotide from an impurity can be achieved without substantially increasing the salt concentration over time. Suitable eluting solutions are those that begin at a pH suitable for binding of the target oligonucleotide to the titratable anion exchange composition, and over time, increase in pH so that the titratable anion exchange composition cannot bind the target oligonucleotide. In one embodiment, the eluting solution is substantially free of metal salts.
  • the methods of the invention can be used to separate the target oligonucleotide from a variety of impurities.
  • impurities include any composition which possesses a different molecular structure than the target oligonucleotide.
  • the impurity to be separated from the target oligonucleotide is one or more oligonucleotides having a shorter length than the target oligonucleotide (e.g., one or more failure sequences).
  • the impurity i.e., one or more oligonucleotides having a shorter length than the target oligonucleotide
  • the impurity to be separated from the target oligonucleotide comprises one or more salts, especially metal salts.
  • the methods of the invention can also be used to increase the concentration of a target oligonucleotide.
  • the target oligonucleotide is concentrated by eluting the target oligonucleotide with a volume of solution which is less than the volume that the target oligonucleotide was originally contained within.
  • the methods of the present invention are advantageous in that they avoid the more laborious and time-consuming conventional separation steps which are typically required to separate or purify oligonucleotides.
  • the present invention provides a rapid, simplified, more efficient and less expensive method for large-scale purification of oligonucleotides.
  • the present invention is drawn to methods of separating or purifying an oligonucleotide from an impurity.
  • separating and purifying are used interchangeably and refer to a process by which an oligonucleotide having a particular molecular structure is physically segregated from an impurity having a different molecular structure. Such segregation can be partial or complete.
  • oligonucleotide comprises an oligomer or polymer of nucleotides which are covalently linked by optionally modified phosphodiester bonds. Nucleotides have a common structure comprising an optionally modified phosphate group which is linked to a pentose which in turn is linked to an organic base. If the pentose is ribose, the nucleic acid is RNA and the nucleotides are ribonucleotides. If the pentose is 2'-deoxyribose, the nucleic acid is DNA and the nucleotides are deoxyribonucleotides.
  • oligonucleotides are polyanions which possess a net negative charge which is approximately proportional to their length.
  • bases may be attached to the pentose, but the five that predominate in naturally-occurring DNA and RNA are adenine ("A"), thymine ("T”, primarily in DNA), uracil ("U”, primarily in RNA), guanine ("G”), and cytosine ("C”).
  • oligonucleotide and “polynucleotide” are interchangeable and refer to a nucleotide multimer or oligomer having from a few, e.g., 2- 20, to many, e.g., 20 to several hundred or more, for example up to 250, nucleotides.
  • Oligonucleotides include double-stranded and single-stranded nucleic acids, e.g., single- stranded or double-stranded DNA, RNA or DNA-RNA hybrids. Oligonucleotides further include both naturally-occurring oligonucleotides and synthetic oligonucleotides.
  • Naturally-occurring oligonucleotides are nucleic acids that are found in an organism, for example, nucleic acids including but not limited to, genomic DNA, complimentary DNA (cDNA), chromosomal DNA, plasmid DNA, mRNA, tRNA and rRNA. Such naturally-occurring nucleic acids can also include altered nucleic acids, for example, naturally-occurring nucleic acids which contain additions, deletions or modifications of one or more nucleotides (e.g., polymorphic or allelic variants). Naturally- occurring oligonucleotides include nucleic acids which are isolated from an organism, for example, using methods described herein and/or other known methods.
  • Synthetic oligonucleotides are oligonucleotides which are prepared by artificial means, rather than isolated from an organism.
  • synthetic oligonucleotides include but are not limited to, oligonucleotides which are prepared on solid phases using well-known and/or commercially-available procedures (e.g., using an automated nucleic acid synthesizer or other chemical synthesis method).
  • Synthetic oligonucleotides further include oligonucleotides which comprise one or more modified nucleotides.
  • a modified nucleotide is a nucleotide that has been structurally altered so that it differs from a naturally-occurring nucleotide.
  • modified nucleotides include nucleotides which contains a modified sugar moiety, a modified phosphate moiety and/or a modified nucleobase.
  • Modification of the sugar moiety includes, but is not limited to, replacement of the ribose ring with a hexose, cyclopentyl or cyclohexyl ring.
  • the D-ribose ring of a naturally-occurring nucleic acid can be replaced with an L-ribose ring or the ⁇ -anomer of a naturally-occurring nucleic acid can be replaced with the ⁇ -anomer.
  • Modified phosphate moieties include phosphorothioates, phosphorodithioates, methyl phosphonates, alkylphosphonates, alkylphosphonothioates, methyl phosphates, phosphoramidates, and the like, or combinations thereof.
  • Oligonucleotides which comprise such modified phosphate linkages can have improved properties when compared to corresponding oligonucleotides comprising only phosphate diester linkages. For example, oligonucleotides comprising modified linkages can have increased resistance to degradation by nucleases which may be present in an organism (e.g., when used in antisense applications).
  • Modified nucleobases include 7-deazaguanine, 7-deaza-8-azaguanine, 5- propynylcytosine, 5-propynyluricil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-6- oxopurine, 6-oxopurine, 3-deazaadenosine, 2-oxo-5-methylpyrimidine, 2-oxo-4- methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5- methylpyrimidine, 2-amino-purine, 5-fluorouricil, 2,6-diaminopurine, 8-aminopurine, 4- triazolo-5-methylthymine, and 4-triazolo-5-methyluricil. Modified nucleobases can also include abasic moieties.
  • oligonucleotide analogs which comprise one or more modified sugar moieties, phosphate moieties and/or nucleobases are well known to those of skill in the art.
  • Chimeric oligonucleotides for example, an oligonucleotide that contains both phosphodiester and phosphorothioate linkages are also encompassed by the present invention.
  • a modified nucleotide can be produced by a chemical modification either prior to, during, or subsequent to incorporation into an oligonucleotide, for example, using methods that are well known in the art.
  • a modified nucleotide can be produced by incorporating a modified nucleoside triphosphate into a nucleic acid polymer chain during an amplification reaction (e.g., using a polymerase chain reaction (PCR)).
  • PCR polymerase chain reaction
  • modified nucleotides include but are not limited to, dideoxynucleotides, biotinylated nucleotides, amine-modified nucleotides, alkylated nucleotides, fluorophore-labeled nucleotides, radiolabeled nucleotides, phosphorothioates, phosphoramidites, phosphites, ring atom-modified derivatives and the like. Oligonucleotides containing multiple modified nucleotides and/or any combination of modified nucleotides are also encompassed by the invention.
  • Oligonucleotides further encompass oligonucleotide polymers which possess a modified backbone, for example, protein-nucleic acids (PNAs) or PNA hybrids. Methods for producing such modified oligonucleotides or oligonucleotide polymers are well known to those of skill in the art.
  • PNAs protein-nucleic acids
  • the methods of the invention are used to separate a synthetic oligonucleotide from an impurity.
  • the synthetic oligonucleotide to be separated has a preferred length, for example, from about 2 to about 100 nucleotides, from about 2 to about 75 nucleotides, or from about 4 to about 50 nucleotides.
  • Many synthetic oligonucleotides of current therapeutic interest comprise from 8 to about 40 nucleotides.
  • the oligonucleotide to be separated has a length of from about 8 to about 40 nucleotides.
  • Naturally-occurring oligonucleotides can be obtained from various biological materials including but not limited to, organisms, tissues and/or cells from veterinary or human clinical test samples (e.g., test samples collected for diagnostic and/or prognostic purposes). Methods for obtaining such naturally occurring oligonucleotides are well known in the art.
  • cells can be lysed and the resulting lysate can be processed using techniques familiar to one of skill in the art to obtain an aqueous solution of nucleic acid (e.g., DNA and/or RNA) (see, for example, Ausebel, F., et al., Current Protocols in Molecular Biology, Wiley, New York (1988); Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)).
  • nucleic acid e.g., DNA and/or RNA
  • oligonucleotides either naturally-occurring or synthetic, can be produced using biological methods, chemical methods or a combination of biological and chemical methods.
  • oligonucleotides can be generated biologically using recombinant nucleic acid methodology, artificial recombination (e.g., the polymerase chain reaction (PCR)) or various cloning strategies (e.g., those that employ vectors and/or restriction enzymes). Oligonucleotides can also be generated chemically, for example, using an automated nucleic acid synthesizer or other known chemical synthesis method. Today, the vast majority of oligonucleotides are produced using an automated synthesizer.
  • PCR polymerase chain reaction
  • an oligonucleotide is synthesized 3' to 5', by reacting, step-wise and in a predetermined order, 5'-protected nucleotides (activated at their respective phosphate group) with the deprotected 5'-position in a terminal nucleotide residue of a growing oligonucleotide chain, which is itself attached to a solid support.
  • the most popular protecting groups for the 5'-position have been strongly hydrophobic, for example, 5'-O-trityl protecting groups (e.g., 5'-O-dimethoxytrityl).
  • Oligonucleotides can also be subjected to various molecular biological and/or separation techniques, prior to and/or subsequent to, being utilized in the methods of the invention.
  • separation techniques include, but are not limited to, affinity separation (e.g., nucleic acid hybridization), electrophoretic separation (e.g., using size- fractionation agarose or polyacrylamide gels) and/or chromatographic separation (e.g., HPLC, ion exchange chromatography, reverse-phase chromatography) (see, for example, Ausebel, F., et al., Current Protocols in Molecular Biology, Wiley, New York (1988); Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).
  • the methods of the present invention can also be utilized in combination with other separation processes (for example, anion exchange chromatography, reverse-phase chromatography).
  • the oligonucleotide is present in a mixture.
  • Mixtures comprising oligonucleotides include solutions which comprise a target oligonucleotide.
  • Such mixtures include samples which contain naturally-occurring oligonucleotides (e.g., samples containing nucleic acids found in a living or dead naturally- occurring or artificially-grown unicellular or multicellular organism).
  • the mixtures can include samples which contain synthetic oligonucleotides.
  • the mixture comprising the target oligonucleotide further comprises one or more oligonucleotides having a shorter length than the target oligonucleotide.
  • the mixture is a synthetic mixture of oligonucleotides, for example, a synthetic mixture obtained using an automated synthesizer.
  • the mixture in addition to comprising the target oligonucleotide, further comprises truncated failure sequences.
  • Other mixtures which can contain oligonucleotides include but are not limited to, buffered solutions, e.g., Tris-based solutions, MOPS-based solutions, HEPES-based solutions, acetate-based solutions and phosphate-based solutions.
  • Mixtures which comprise solutions typically used in chemical or biological applications are also encompassed. Such mixtures include samples obtained in synthesizing a target oligonucleotide (e.g., samples comprising a PCR product, samples obtained from an automated synthesizer), samples obtained in sequencing a target oligonucleotide or samples obtained in separating an oligonucleotide (e.g., samples obtained from a chromatographic separation). Mixtures which contain a combination of more than one type of oligonucleotide (e.g., a mixture comprising both naturally-occurring and synthetic oligonucleotides) are also encompassed.
  • a target oligonucleotide e.g., samples comprising a PCR product, samples obtained from an automated synthesizer
  • samples obtained in sequencing a target oligonucleotide or samples obtained in separating an oligonucleotide e.g., samples obtained from a chromatographic separation.
  • the methods of the invention separate a target oligonucleotide from an impurity.
  • An impurity as defined herein, is a composition which possesses a different molecular structure than the target oligonucleotide. Separation of the impurity from the target oligonucleotide can be partial or substantially complete.
  • the impurity to be separated from the target oligonucleotide comprises one or more oligonucleotides having a shorter length than the target oligonucleotide.
  • the impurity to be separated from the target oligonucleotide comprises one or more failure sequences generated during the synthesis of the target oligonucleotide.
  • failure sequences are generated because in the production of synthetic oligonucleotides, during each stepwise addition of a monomer to the nascent oligonucleotide chain, approximately 1-2% of the coupling reactions may fail (i.e., no monomer addition occurs). Consequently, the resulting products are generally a heterogeneous mixture of oligonucleotides of varying length where the amount of undesired impurities or contaminants (e.g., failure sequences) is proportional to the length of the desired product and overall yield of the synthesis. For most downstream applications, it is often necessary to separate the target oligonucleotide from such failure sequences.
  • the impurity to be separated from the target oligonucleotide comprises a salt (i.e. an ionic salt), particularly a metal salt.
  • Salts are common impurities which are often found in samples containing a target oligonucleotide, for example, in samples obtained using anion exchange chromatography.
  • the presence of salts in a sample containing a target oligonucleotide typically necessitates one or more subsequent separation or desalting steps in order to remove the salts prior to utilizing the target oligonucleotide.
  • the methods of the invention are advantageous in that they do not require a salt gradient to elute the oligonucleotide and therefore may not require subsequent desalting steps.
  • Salts that can be separated from an oligonucleotide using the methods of the invention include, but are not limited to, NaCI, KCI, MgCI 2 , CaCI 2 , NaHCO 3 , Na 2 CO 3 , NaOH, NH 4 CI, NaOC(O)CH 3 and NaCIO 4 .
  • Other salts which can be separated using the methods of the invention are known to those of skill in the art.
  • the salt can be separated from the target oligonucleotide by two mechanisms.
  • the salt does not bind to the target oligonucleotide or the anion exchange composition, it will simply pass through the titratable anion exchange composition and therefore will be separated from the target oligonucleotide which binds and is subsequently eluted at a higher pH.
  • the salt binds to the target oligonucleotide or the anion exchange composition, it can be removed by washing the anion exchange composition with target oligonucleotide bound thereon, with a suitable washing solution prior to eluting the target oligonucleotide.
  • the anion exchange composition with target oligonucleotide bound thereon is exposed to one or more washing steps prior to elution.
  • Washing solutions that are suitable for the methods of the invention are those that have a suitable pH and can remove impurities without causing the target oligonucleotide to release from the titratable anion exchange composition.
  • Such washing solutions include but are not limited to, water and buffered solutions (e.g., Tris- based solutions, MOPS-based solutions, HEPES-based solutions, acetate-based solutions, phosphate-based solutions).
  • Other suitable solutions which can be used to wash the anion exchange composition with oligonucleotide bound thereon are well known in the art.
  • an anion exchange composition is a composition which contains positive charge and displaceable ions (counterions) of negative charge.
  • an anion exchange composition is capable of binding molecules of negative charge (for example, oligonucleotides) and thereby displacing the negatively-charged counterions.
  • the anion exchanger compositions of the present invention are preferably solid compositions.
  • the anion exchange compositions used in the present invention are titratable.
  • a "titratable anion exchange composition” is an anion exchange composition which is capable of losing positive charge as pH increases.
  • the titratable anion exchange compositions used in the present invention lose positive charge as the pH increases from about 7.0 to about 12.0.
  • Titratable anion exchange compositions include compositions comprising a primary amine, a secondary amine or a tertiary amine.
  • Suitable titratable anion exchange compositions include anion exchange compositions comprising polyimizadole, polyhistidine, polylysine, polyethyleneimine, polypropyleneimine, modified polyethyleneimine and poly(ethylene-imine/oxyethylene).
  • the titratable anion exchange composition is polyethyleneimine, which loses positive charge as the pH increases from about 8 to about 11.
  • the titratable anion exchange compositions used in the invention possess atoms (e.g., nitrogen atoms) which are protonated and therefore positively charged at neutral and low pH.
  • atoms e.g., nitrogen atoms
  • polyethyleneimine possesses nitrogen atoms which are positively charged at a pH of about 5-8 (or lower pH).
  • the nitrogen atoms of polyethyleneimine begin to become deprotonated.
  • the use of polyethyleneimine allows the failure sequences, which are shorter (and therefore have less negative charge) than the target oligonucleotide, to release from the polyethyleneimine at a lower pH than the longer, more negatively-charged target oligonucleotide.
  • the pH increases, more and more of the nitrogen atoms of polyethyleneimine become deprotonated which allows the longer failure sequences and eventually the target oligonucleotide to release.
  • all of the nitrogen atoms of polyethyleneimine become deprotonated and therefore cannot retain negatively- charged oligonucleotides.
  • a titratable anion exchange composition e.g., polyethyleneimine
  • a salt gradient to elute bound oligonucleotides and provides a more rapid, efficient and economical method of oligonucleotide separation.
  • the titratable anion exchange composition is conjugated to a support.
  • Conjugation of a titratable anion exchange composition to a support can be by methods that are known in the art (e.g. covalently bonding, optionally via a linker group, such as a divinyl sulphone linker or an epichlorohydrin linker).
  • conjugation is performed such that the fixed positive charges and displaceable ions of negative charge (counterions) of the titratable anion exchange composition are accessible to the oligonucleotide (i.e., are surface exposed).
  • Suitable supports include solid or semi-solid supports, for example, synthetic polymer supports, such as silica (e.g., silica gel), polysaccharides, synthetic polyolefins including polystyrenes (e.g., styrene-divinylbenzene copolymer), polyethylenes, polypropylenes, polyacrylics (e.g., polyacrylamides, polyacrylates) and agaroses, for example the agaroses commercially available under the trade name Sepharose.
  • synthetic polymer supports such as silica (e.g., silica gel), polysaccharides, synthetic polyolefins including polystyrenes (e.g., styrene-divinylbenzene copolymer), polyethylenes, polypropylenes, polyacrylics (e.g., polyacrylamides, polyacrylates) and agaroses, for example the agaroses commercially available under the trade name Sepharose.
  • the titratable anion exchange composition is polyethyleneimine-derivatized silica gel or polyethyleneimine-derivatized styrene-divinyl benzene copolymer.
  • the titratable anion exchange composition is not conjugated to a support. Rather, the solid titratable anion exchange composition itself acts as a support to which the oligonucleotide can be bound and released.
  • concentration and amount of titratable anion exchange composition used in the methods of the invention depend on a number of factors, e.g., the quantity and nature of the mixture comprising the oligonucleotide, the concentration and nature of the oligonucleotide in the mixture and the concentration and nature of the impurities in the mixture.
  • concentration and nature of the mixture comprising the oligonucleotide depend on a number of factors, e.g., the quantity and nature of the mixture comprising the oligonucleotide, the concentration and nature of the oligonucleotide in the mixture and the concentration and nature of the impurities in the mixture.
  • concentration and nature of the oligonucleotide in the mixture depend on a number of factors, e.g., the quantity and nature of the mixture comprising the oligonucleotide, the concentration and nature of the oligonucleotide in the mixture and the concentration and nature of the impurities in the mixture.
  • the separation can proceed relatively quickly, e.g., using an increased flow rate.
  • Another important variable which can be altered to achieve suitable separation is the rate of change in pH of the eluting solution.
  • a suitable pH elution profile e.g., continuous pH gradient, step-wise pH gradient, rapid rate of change in pH, slow rate of change in pH.
  • eluting solutions which increase in pH can be used in the methods of the invention including solutions which are suitably buffered to achieve the desired pH, e.g., tris-based solutions, MOPS-based solutions, HEPES-based solutions, acetate-based solutions, phosphate-based solutions.
  • Other solutions which are typically used in anion exchange separations and are known in the art are also encompassed.
  • the eluting solution can also contain one or more ionic salts, e.g., NaCI, KCI, MgCI 2 , CaCI 2 , NaHCO 3 , Na 2 CO 3 , NaOH, NH 4 CI, NaOH and the like, however, as described herein, the eluting solutions do not utilize a salt gradient.
  • the volume of the eluting solution used in the methods of the present should be sufficient to saturate the titratable anion exchange composition.
  • the eluting solution which is passed through the titratable anion exchange composition with the target oligonucleotide bound thereon increases in pH over time but does not substantially increase its salt concentration over the same period of time.
  • the lack of a "substantial increase" in salt concentration means that any increase in salt concentration which occurs does not significantly affect the elution of the target oligonucleotide from the titratable anion exchange composition.
  • the eluting solutions used in the present invention have a substantially constant salt concentration.
  • a substantially constant salt concentration means that any variations in salt concentration (increase or decrease) are small enough that they do not significantly affect elution of the target oligonucleotide from the titratable anion exchange composition.
  • the salt concentration of the eluting solution can decrease over time.
  • the eluting solution is substantially free of metal salts.
  • substantially free of metal salts refers to the fact that the solution contains an insubstantial amount of metal salts (i.e., no metal salts or so little metal salts as to not affect downstream applications which utilize the oligonucleotide).
  • the methods of the present invention utilize a pH gradient and not a salt gradient for elution.
  • the eluting solutions used in the present invention include solutions that increase in pH in a step-wise manner as well as solutions that increase in pH in a continuous manner.
  • the solution used to elute the target oligonucleotide increases in pH in a linear manner over time.
  • the solution used to elute the target oligonucleotide increases from a pH of about 8 to a pH of about 1 1 .
  • the titratable anion exchange composition which is utilized comprises polyethyleneimine.
  • the eluting solutions can also comprise one or more buffering agents for suitably altering the pH.
  • the eluting solution comprises one or more of NaHCO 3 , Na 2 CO 3 , NaOH, NH 4 OH and/or NH 4 HCO 3 .
  • the eluting solution is free from metal salts, and advantageously comprises salts which are volatile on drying, such as NH 4 HCO 3 and especially NH 4 OH.
  • the elution rate is generally not critical, the solution used to elute the target oligonucleotide is generally administered at a flow rate of about 350 to 550 cm/hr and more commonly at a rate of between about 420 to 450 cm/hr.
  • the eluting solutions which comprise salts have a salt concentration substantially lower than the salt concentrations employed in salt-gradient elution.
  • concentration of the salts in a pH gradient eluting solution is no more than 0.1 M, such as from about 0.01 to about 0.07M, for example about 0.05M.
  • the overall increase in pH with the pH gradient does not increase the salt concentration in the eluting solution by more than about 100%.
  • the salt concentration may remain substantially constant or even decrease.
  • the salt concentration of the eluting solution commonly does not exceed 0.2M, and preferably is no more than 0.1 M.
  • the salt concentrations are typically at least about 0.5M, commonly increasing to 2M or even higher, such as 3 to 4M.
  • the methods of the invention can be used for the separation of 5'-O-protected oligonucleotides.
  • the 5'-O-protecting group can be cleaved from the target oligonucleotide while it is still bound to the titratable anion exchange composition.
  • Suitable 5'-O-protecting groups are known in the art and include, for example, substituted or unsubstituted trityl groups (e.g., 4,4'-dimethoxytrityl (DMT)).
  • the methods of the invention are used for the separation of a 5'-O- trityl protected oligonucleotide (e.g., a 5'-O-dimethoxytrityl protected oligonucleotide).
  • Cleavage of the protecting group from the bound oligonucleotide i.e., prior to eluting off of the titratable anion exchange composition
  • a suitable reagent is a sufficient amount of an acidic solution.
  • a trityl protecting group (e.g., 5'-O-DMT) can be cleaved from the target oligonucleotide using aqueous acetic acid, for example 20%-80% v/v aqueous acetic acid.
  • aqueous acetic acid for example 20%-80% v/v aqueous acetic acid.
  • the methods of the present invention are advantageous in that they avoid the more laborious and time-consuming conventional separation steps which are typically required to separate or purify oligonucleotides. In eliminating the necessity of utilizing a salt gradient in anion exchange separations, the methods of the invention reduce the number of steps which is typically required to separate oligonucleotides. Thus, the present invention provides a rapid, simplified, more efficient and less expensive method for large-scale purification of oligonucleotides.
  • the methods of the invention decrease damage to stainless steel and/or moving parts of chromatographs and other anion exchange separation machinery (e.g., HPLC pumps, fittings, valves, columns, tubing).
  • anion exchange separation machinery e.g., HPLC pumps, fittings, valves, columns, tubing.
  • Polyethyleneimine-derivatized silica gel (Matrex Ion Exchange Silica PEI-300-15; product number S674 (Millipore, Bedford, MA); Buffer A; 50 mM NaHCO 3 solution; pH 8.2;
  • Buffer B 50 mM NaHCOa Na 2 CO 3 solution; pH adjusted to 1 1.1 with 0.1 M NaOH;
  • the oligonucleotide TCG-TCG-TGT-TTT-CTA-TTT-TCG-UTT (SEQ ID NO. 1 ) was synthesized using solid support and phosphoramidite chemistry. The 5'-O-trityl protecting group was removed at the end of oligonucleotide synthesis using 3% dichloroacetic acid in methylene chloride. The oligonucleotide was then released from the support and protecting groups were removed with concentrated ammonium hydroxide solution. The solution containing the target oligonucleotide was then adjusted to pH 7.5 by adding 1.0 M phosphoric acid solution (total oligonucleotide was 121 ,950 OD).
  • the target oligonucleotide was separated using a pH gradient (pH 8.2 to 11.1 ) which was obtained by using Buffer A and Buffer B (0 to 80 % Buffer B in 20 column volumes at a flow rate of 435 cm/hr). The fractions were collected and assayed for the desired oligonucleotide using analytical anion exchange HPLC. Fractions with purity greater than 90% were pooled. Concentration of the oligonucleotide product and desalting were either carried out using UF membrane filtration or by binding to PEI-derivatized silica gel. The oligonucleotide product was then lyophilized.
  • the column was regenerated by washing it with Buffer C (1 column volume), Buffer D (2 column volumes) and finally with Buffer A (until washes attained pH less than 8.5). The column is then suitable for additional separations.
  • Reagents Polyethyleneimine-derivatized silica gel (Matrex Ion Exchange Silica PEI-300-15; product number S674 (Millipore, Bedford, MA); Buffer A; 0.25 M NH 4 HHCO 3 solution; pH 7.5; Buffer B; 0.1 M NH 4 OH; Buffer C; MILLI-Q Water Buffer D; 0.1 M NH 4 OH, 2 M NaCI solution; pH 7.5
  • a slurry of PEI-derivatized silica gel (100 mL) was made in Buffer D and was packed in a glass column at a flow rate of 8 mL/minute. The column was subsequently washed with two column volumes of Buffer D. The column was then re-equilibrated with Buffer A until the pH was less than 8.
  • the oligonucleotide TCG-TCG-TGT-TTT-CTA-TTT-TCG-UTT (SEQ ID NO. 1 ) was synthesized as described in Example 1. After synthesis, the target oligonucleotide was separated from the by-products by ion exchange chromatography using standard means, resulting in solution with a pH 12 that contained the purified oligonucleotide and about 1.5- 2.0 M sodium chloride. The pH of this solution was then adjusted to 7.5 with 1.0 M acetic acid. The sample was then loaded onto the column at a flow rate of 8 mUminute (1000 OD/mL resin).
  • the loaded column was first washed with five column volumes of Buffer A, followed by washing with Buffer C until the conductivity was less than 0.01 millisiemons/cm.
  • the oligonucleotide was then eluted from the column with Buffer B. After elution, the column was regenerated by washing with two column volumes of Buffer A.
  • Example 3 An 18-mer fully phosphorothioated deoxyribonucleotide containing 66% full length product (FLP) was purified 5'-dimethoxytrityl on using the method of Example 1 , except that Buffer A had a pH of 6.0. Analysis of the purified, eluted nucleotide showed a product purity of >94% FLP. Comparable results were also achieved when a PEI-derivatised polystyrene bead was employed as the titratable anion-exchange support.
  • FLP full length product

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Abstract

L'invention concerne des procédés de séparation d'oligonucléotides et d'impuretés. Selon les procédés de l'invention, un oligonucléotide cible, contenu dans un mélange comprenant ledit oligonucléotide cible et une impureté, est séparé de l'impureté, au moyen d'une composition à échange d'anions, pouvant être titrée. Ledit oligonucléotide cible est lié à la composition à échange d'anions, pouvant être titrée et une solution d'élution, dont le pH augmente dans le temps, s'écoule à travers la composition à échange d'anions liée avec l'oligonucléotide. De préférence, la concentration en sel de la solution d'élution n'augmente pas sensiblement. Ledit oligonucléotide cible est élué puis séparé de l'impureté qui est éluée soit à un pH plus faible soit à un pH plus élevé que ledit oligonucléotide cible.
PCT/GB2003/001161 2002-03-21 2003-03-19 Procedes de purification pour des oligonucleotides et leurs analogues WO2003080834A2 (fr)

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CA002479901A CA2479901A1 (fr) 2002-03-21 2003-03-19 Procedes de purification pour des oligonucleotides et leurs analogues
AU2003216830A AU2003216830A1 (en) 2002-03-21 2003-03-19 Purification methods for oligonucleotides and their analogs
KR10-2004-7014747A KR20040108672A (ko) 2002-03-21 2003-03-19 올리고뉴클레오티드 및 그 유사체의 정제방법
EP03712366A EP1490488A2 (fr) 2002-03-21 2003-03-19 Procedes de purification pour des oligonucleotides et leurs analogues
JP2003578560A JP2005520547A (ja) 2002-03-21 2003-03-19 オリゴヌクレオチド及びその類似体のための精製方法

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WO2006132588A1 (fr) * 2005-06-10 2006-12-14 Quiatech Ab Methode pour purifier des oligonucleotides synthetiques contenant au moins une etiquette
WO2007065950A1 (fr) * 2005-12-09 2007-06-14 Qiagen Gmbh Procede pour realiser un enrichissement en acides nucleiques a chaine courte
GB2445442A (en) * 2006-09-26 2008-07-09 Ge Healthcare Bio Sciences Nucleic acid purification using anion exchange
US7655793B2 (en) 2006-09-26 2010-02-02 Ge Healthcare Bio-Sciences Corp. Nucleic acid purification method
US7655794B2 (en) 2006-09-26 2010-02-02 Ge Healthcare Bio-Sciences Corp. Nucleic acid purification method
EP2513335A2 (fr) * 2009-12-14 2012-10-24 Betty Wu Procédé et matériaux pour séparer des matériaux d'acide nucléique
WO2013045434A1 (fr) * 2011-09-26 2013-04-04 Qiagen Gmbh Procédés de séparation d'acides nucléiques par tailles
WO2014122288A1 (fr) 2013-02-08 2014-08-14 Qiagen Gmbh Procédé de séparation de l'adn suivant la taille
AU2012205204B2 (en) * 2005-12-09 2015-03-12 Qiagen Gmbh Method for enriching short-chain nucleic acids
US9663779B2 (en) 2008-12-23 2017-05-30 Qiagen Gmbh Nucleic acid purification method
WO2020055922A1 (fr) * 2018-09-11 2020-03-19 Amgen Inc. Procédés de purification d'oligonucléotides riches en guanine
WO2022015801A1 (fr) * 2020-07-14 2022-01-20 Waters Technologies Corporation Kits et procédés utiles pour séparer des oligonucléotides de composants matriciels
US11912983B2 (en) 2019-12-05 2024-02-27 Waters Technologies Corporation Polyanionic acids to improve recovery and minimize system loss

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JP5914338B2 (ja) * 2009-09-24 2016-05-11 キアジェン ゲイサーズバーグ インコーポレイテッド 陰イオン交換材料を使用した核酸の単離および分析のための組成物、方法およびキット
CN103833797A (zh) * 2012-11-26 2014-06-04 福建金山生物制药股份有限公司 一种纯化硫代寡核苷酸的工业化方法
CN106632558B (zh) * 2016-08-25 2019-09-13 淮阴师范学院 一种寡聚核苷酸的纯化方法及应用
CN114051499A (zh) * 2019-07-09 2022-02-15 豪夫迈·罗氏有限公司 用于寡核苷酸的脱保护的方法
CN111394427B (zh) * 2020-04-22 2023-04-11 宁波市博坤生物科技有限公司 一种含泥土检材中微量dna的提取方法
WO2024089953A1 (fr) * 2022-10-27 2024-05-02 住友化学株式会社 Procédé de production d'oligonucléotide

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WO2006132588A1 (fr) * 2005-06-10 2006-12-14 Quiatech Ab Methode pour purifier des oligonucleotides synthetiques contenant au moins une etiquette
AU2006323954B2 (en) * 2005-12-09 2012-05-10 Qiagen Gmbh Method for enriching short-chain nucleic acids
WO2007065950A1 (fr) * 2005-12-09 2007-06-14 Qiagen Gmbh Procede pour realiser un enrichissement en acides nucleiques a chaine courte
EP3524678A1 (fr) * 2005-12-09 2019-08-14 QIAGEN GmbH Procede pour realiser un enrichissement en acides nucleiques a chaine courte
EP3064583A1 (fr) * 2005-12-09 2016-09-07 Qiagen GmbH Procédé pour réaliser un enrichissement en acides nucléiques à chaine courte
AU2012205204B2 (en) * 2005-12-09 2015-03-12 Qiagen Gmbh Method for enriching short-chain nucleic acids
US7977109B2 (en) 2005-12-09 2011-07-12 Qiagen Gmbh Method for enriching short-chain nucleic acids
US7655794B2 (en) 2006-09-26 2010-02-02 Ge Healthcare Bio-Sciences Corp. Nucleic acid purification method
GB2445442A (en) * 2006-09-26 2008-07-09 Ge Healthcare Bio Sciences Nucleic acid purification using anion exchange
US7655792B2 (en) 2006-09-26 2010-02-02 Ge Healthcare Bio-Sciences Corp. Nucleic acid purification method
US7655793B2 (en) 2006-09-26 2010-02-02 Ge Healthcare Bio-Sciences Corp. Nucleic acid purification method
US9663779B2 (en) 2008-12-23 2017-05-30 Qiagen Gmbh Nucleic acid purification method
EP2513335A4 (fr) * 2009-12-14 2013-09-11 Betty Wu Procédé et matériaux pour séparer des matériaux d'acide nucléique
EP2513335A2 (fr) * 2009-12-14 2012-10-24 Betty Wu Procédé et matériaux pour séparer des matériaux d'acide nucléique
WO2013045434A1 (fr) * 2011-09-26 2013-04-04 Qiagen Gmbh Procédés de séparation d'acides nucléiques par tailles
WO2014122288A1 (fr) 2013-02-08 2014-08-14 Qiagen Gmbh Procédé de séparation de l'adn suivant la taille
US10745686B2 (en) 2013-02-08 2020-08-18 Qiagen Gmbh Method for separating DNA by size
WO2020055922A1 (fr) * 2018-09-11 2020-03-19 Amgen Inc. Procédés de purification d'oligonucléotides riches en guanine
US11912983B2 (en) 2019-12-05 2024-02-27 Waters Technologies Corporation Polyanionic acids to improve recovery and minimize system loss
WO2022015801A1 (fr) * 2020-07-14 2022-01-20 Waters Technologies Corporation Kits et procédés utiles pour séparer des oligonucléotides de composants matriciels
US20220017887A1 (en) * 2020-07-14 2022-01-20 Waters Technologies Corporation Compositions, kits and methods useful for separating oligonucleotides from matrix components

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