CA2449552A1 - Processes of purifying oligonucleotides - Google Patents

Processes of purifying oligonucleotides Download PDF

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Publication number
CA2449552A1
CA2449552A1 CA002449552A CA2449552A CA2449552A1 CA 2449552 A1 CA2449552 A1 CA 2449552A1 CA 002449552 A CA002449552 A CA 002449552A CA 2449552 A CA2449552 A CA 2449552A CA 2449552 A1 CA2449552 A1 CA 2449552A1
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oligonucleotide
solution
aggregating agent
aggregate
treated
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CA002449552A
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CA2449552C (en
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Max N. Moore
John Charles Arthur
Kent Vansooy
Anthony N. Scozzari
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Ionis Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Methods for preparing purified oligonucleotides by treating a solution comprising an oligonucleotide with an aggregating agent and a precipitation enhancer under conditions sufficient to form an oligonucleotide aggregate and isolating the oligonucleotide to produce a purified oligonucleotide.

Claims (61)

  1. What is Claimed is:

    A method for preparing a purified oligonucleotide comprising the steps of a) providing a solution comprising an oligonucleotide;

    b) treating said solution with an aggregating agent and a precipitation enhancer under conditions sufficient to form an oligonucleotide aggregate; and c) isolating said oligonucleotide aggregate to form said purified oligonucleotide.
  2. The method of claim 1 wherein said solution comprises a deprotected oligonucleotide.
  3. 3. The method of claim 2 wherein said solution is acidic.
  4. 4. The method of claim 3 wherein said solution is prepared by treatment of a 5'-protected oligonucleotide in a solvent with a deprotecting agent effective to remove the 5'-protecting group.
  5. 5. The method of claim 4 wherein said 5'-protecting group is selected from the group consisting of trimethoxytrityl, dimethoxytrityl, monomethoxytrityl, 9-phenylxanthen-9-yl, and 9-(p-methoxyphenyl)xanthen-9-yl.
  6. 6. The method of claim 5 wherein said protecting group is dimethoxytrityl.
  7. 7. The method of claim 2 wherein the concentration of said deprotected oligonucleotide in said solution is at least about 2250 OD/mL.
  8. 8. The method of claim 7 wherein the concentration of said deprotected oligonucleotide in said solution is from about 2500 OD/mL to about 7500 OD/mL.
  9. 9. The method of claim 8 wherein the concentration of said deprotected oligonucleotide in said solution is from about 4500 OD/mL to about 6500 OD/mL.
  10. 10. The method of claim 1 wherein said solution is prepared by reconstituting an isolated, deprotected oligonucleotide in water.
  11. 11. The method of claim 1 wherein said aggregating agent comprises an alcohol.
  12. 12. The method of claim 11 wherein said alcohol is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol and denatured ethanol.
  13. 13. The method of claim 1 wherein said precipitation enhancer comprises a salt.
  14. 14. The method of claim 13 wherein said salt is selected from the group consisting of sodium salts, lithium salts, ammonium salts, potassium salts, magnesium salts, cesium salts and zinc salts.
  15. 15. The method of claim 14 wherein said salt is sodium acetate.
  16. 16. The method of claim 14 wherein said salt is sodium hydroxide.
  17. 17. The method of claim 1 wherein said oligonucleotide is treated with said aggregating agent at a temperature of between about 15°C and about 25°C.
  18. 18. The method of claim 1 wherein said oligonucleotide is treated with said aggregating agent at a temperature from about 18°C to about 20°C.
  19. 19. The method of claim 1 wherein said oligonucleotide is treated with said precipitation enhancer prior to treating said oligonucleotide with said aggregating agent.
  20. 20. The method of claim 1 wherein said oligonucleotide is treated with said aggregating agent prior to treating said oligonucleotide with said precipitation enhancer.
  21. 21. The method of claim 1 wherein said oligonucleotide is treated with a mixture of said precipitation enhancer and said aggregating agent.
  22. 22. The method of claim 1 wherein said solution is treated with said aggregating agent in a ratio of about 1 part solution to at least about 1.5 parts aggregating agent by volume.
  23. 23. The method of claim 22 wherein said solution is treated with said aggregating agent in a ratio of about 1 part solution to between about 2 parts and about 4 parts aggregating agent by volume.
  24. 24. The method of claim 22 wherein said solution is treated with said aggregating agent in a ratio of about 1 part solution to between about 2.5 parts and about 4.5 parts aggregating agent by volume.
  25. 25. The method of claim 1 wherein said oligonucleotide aggregate is isolated by centrifugation.
  26. 26. The method of claim 25 wherein said centrifugation is conducted at a speed of less than about 3000 rotations per minute.
  27. 27. The method of claim 26 wherein said centrifugation is conducted at a speed of less than about 2500 rotations per minute.
  28. 28. The method of claim 25 wherein said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to about 5 parts aggregating agent by volume.
  29. 29. The method of claim 28 wherein said aggregating agent is ethanol and said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to between about 2 and about 4 parts aggregating agent by volume.
  30. 30. The method of claim 29 wherein said aggregating agent is selected from the group consisting of 1-propanol, isopropyl alcohol and denatured ethanol and said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to about 3 parts aggregating agent by volume.
  31. 31. The method of claim 1 wherein said oligonucleotide is isolated by gravitational settling.
  32. 32. The method of claim 31 wherein said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to 5 parts aggregating agent by volume.
  33. 33. The method of claim 32 wherein said aggregating agent is ethanol and said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to between about 2 and about 4.5 parts aggregating agent by volume.
  34. 34. The method of claim 27 wherein said aggregating agent is ethanol and said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to between about 2 and about 3.5 parts aggregating agent by volume.
  35. 35. The method of claim 34 wherein said aggregating agent is selected from the group consisting of 1-propanol, isopropyl alcohol and denatured ethanol and said oligonucleotide is treated with said aggregating agent in a ratio of 1 part oligonucleotide to between about 3 parts aggregating agent by volume.
  36. 36. The method of claim 1 wherein said oligonucleotide is isolated by filtration.
  37. 37. The method of claim 36 wherein an amount of said oligonucleotide remaining in said solution after said oligonucleotide is isolated from said solution is not more than about 3.5%.
  38. 38. The method of claim 37 wherein an amount of said oligonucleotide remaining in said solution after said oligonucleotide is. isolated from said solution is not more than about 1.5%.
  39. 39. The method of claim 38 wherein an amount of said oligonucleotide remaining in said solution after said oligonucleotide is isolated from said solution is not more than about 1 %.
  40. 40. A method for preparing a purified oligonucleotide comprising the steps of a) providing a solution comprising an oligonucleotide;

    b) treating said solution with ethanol, wherein said ethanol is at a temperature of between about 15°C and about 25°C and a sodium salt under conditions sufficient to form an oligonucleotide aggregate; and c) isolating said oligonucleotide aggregate to form said purified oligonucleotide.
  41. 41. The method of claim 40 wherein said sodium salt is sodium acetate or sodium hydroxide.
  42. 42. A method for preparing a purified oligonucleotide comprising the steps of a) providing a solution comprising an oligonucleotide;
    b) treating said solution with a precipitation enhancer with subsequent treatment with an aggregating agent under conditions sufficient to form an oligonucleotide aggregate; and c) isolating said oligonucleotide aggregate to form said purified oligonucleotide.
  43. 43. The method of claim 42 wherein said precipitation enhancer is sodium acetate.
  44. 44. The method of claim 42 wherein said aggregating agent is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol and denatured ethanol.
  45. 45. A method for preparing a purified oligonucleotide comprising 'the steps of treating a first solution comprising a 5'-protected oligonucleotide with an aggregating agent under conditions sufficient to form a first oligonucleotide aggregate;

    isolating said first oligonucleotide aggregate;

    dissolving the isolated first oligonucleotide aggregate in a solvent thereby forming a second solution;

    treating said second solution with a deprotecting reagent effective to remove said 5'-protecting groups;

    treating said second solution with an aggregating agent and a precipitation enhancer under conditions sufficient to form a second oligonucleotide aggregate;

    isolating said second oligonucleotide aggregate;

    dissolving said second oligonucleotide aggregate in a solvent to give a third solution; and treating said third solution with an aggregating agent and a precipitation enhancer under conditions sufficient to form a third oligonucleotide aggregate; and isolating said third oligonucleotide aggregate to give said purified oligonucleotide.
  46. 46. The method of claim 45 wherein said first solution is effluent obtained from chromatography of crude oligonucleotide.
  47. 47. The method of claim 46 wherein said chromatography is high pressure liquid chromatography.
  48. 48. The method of claim 47 wherein said high pressure liquid chromatography is performed using a column loaded with reverse phase media or strong anion exchange resin.
  49. 49. The method of claim 45 wherein said isolating of said first oligonucleotide aggregate or said second oligonucleotide aggregate is performed by gravitational settling or centrifugation.
  50. 50. The method of claim 45 wherein said isolating of said third oligonucleotide aggregate is performed by filtration.
  51. 51. The method of claim 45 wherein said purified oligonucleotide is at least about 90% pure.
  52. 52. The method of claim 51 wherein said purified oligonucleotide is at least about 98% pure.
  53. 53. A method for preparing a purified oligonucleotide comprising the steps of treating a first solution comprising an oligonucleotide with an aggregating agent and a precipitation enhancer under conditions sufficient to form a first oligonucleotide aggregate;

    isolating said first oligonucleotide aggregate;
    dissolving the isolated first oligonucleotide aggregate in a solvent thereby forming a second solution;

    treating said second solution with an aggregating agent and a precipitation enhancer under conditions sufficient to form a second oligonucleotide aggregate; and isolating said second oligonucleotide aggregate to give said purified oligonucleotide.
  54. 54. The method of claim 53 wherein said purified oligonucleotide is at least about 90% pure.
  55. 55. The method of claim 54 wherein said purified oligonucleotide at least about 98% pure.
  56. 56. The method of claim 53 wherein said oligonucleotide of said first solution is a 5'-deprotected oligonucleotide.
  57. 57. The method of claim 56 wherein said first solution is prepared by acidification of HPLC effluent containing a 5'-protected oligonucleotide.
  58. 58. The method of claim 57 wherein said HPLC effluent results from HPLC
    purification of a cleaved and base deblocked 5'-protected oligonucleotide.
  59. 59. The method of claim 53 wherein said isolating of said first oligonucleotide aggregate is performed by gravitational settling or centrifugation.
  60. 60 The method of claim 53 wherein said isolating of said second oligonucleotide aggregate is performed by filtration.
  61. 61. The method of claim 49 wherein said solvent is water.
CA2449552A 2001-06-07 2002-06-05 Processes of purifying oligonucleotides Expired - Fee Related CA2449552C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/876,242 US6632938B2 (en) 2001-06-07 2001-06-07 Processes of purifying oligonucleotides
US09/876,242 2001-06-07
PCT/US2002/017915 WO2002100873A1 (en) 2001-06-07 2002-06-05 Processes of purifying oligonucleotides

Publications (2)

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CA2449552A1 true CA2449552A1 (en) 2002-12-19
CA2449552C CA2449552C (en) 2011-03-29

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CA2449552A Expired - Fee Related CA2449552C (en) 2001-06-07 2002-06-05 Processes of purifying oligonucleotides

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US (1) US6632938B2 (en)
EP (1) EP1399457B1 (en)
KR (1) KR20040065994A (en)
CN (1) CN1514842A (en)
CA (1) CA2449552C (en)
WO (1) WO2002100873A1 (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7125945B2 (en) * 2003-09-19 2006-10-24 Varian, Inc. Functionalized polymer for oligonucleotide purification
BRPI0607080A2 (en) * 2005-01-28 2009-12-01 Girindus Ag method for the purification of a protected oligonucleotide
US9045522B2 (en) 2006-07-31 2015-06-02 Wanli Bi Nucleic acid amplification using a reversibly modified oligonucleotide
US8334099B2 (en) 2006-07-31 2012-12-18 Wanli Bi Nucleic acid amplification using a reversibly modified oligonucleotide
CN103833797A (en) * 2012-11-26 2014-06-04 福建金山生物制药股份有限公司 Industrial method for purifying phosphorothioate oligodeoxynucleotide
CN110066789B (en) * 2019-05-17 2021-04-20 通用生物系统（安徽）有限公司 Improved HPLC purification method of long-chain DNA primer
CN114805459A (en) * 2022-05-20 2022-07-29 江苏集萃工业生物技术研究所有限公司 Separation and extraction method of UMP sodium salt
CN115710165B (en) * 2022-11-17 2023-05-26 北京擎科生物科技有限公司 Method for preparing 4,4' -dimethoxy trityl chloride by utilizing oligonucleotide synthesis waste liquid

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US4948882A (en) * 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
GB8920097D0 (en) * 1989-09-06 1989-10-18 Ici Plc Amplification processes
US5419966A (en) * 1991-06-10 1995-05-30 Microprobe Corporation Solid support for synthesis of 3'-tailed oligonucleotides
US5304603A (en) * 1991-06-18 1994-04-19 The Population Council Leydig cell stimulator
US5210264A (en) 1992-01-10 1993-05-11 Isis Pharmaceuticals, Inc. S-(2,4-dichlorobenzyl)-β-cyanoethyl phosphorothioate diester
US5656741A (en) * 1992-03-30 1997-08-12 Barrskogen, Inc. Process and reagents for processing synthetic oligonucleotides
US5925517A (en) * 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
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US6238905B1 (en) * 1997-09-12 2001-05-29 University Technology Corporation Thermophilic polymerase III holoenzyme
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AU2001257604A1 (en) * 2000-04-11 2001-10-23 Micrologix Biotech Inc. HPV-specific short-mers

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EP1399457A1 (en) 2004-03-24
CN1514842A (en) 2004-07-21
US6632938B2 (en) 2003-10-14
EP1399457A4 (en) 2006-08-23
CA2449552C (en) 2011-03-29
US20030055241A1 (en) 2003-03-20
WO2002100873A1 (en) 2002-12-19
KR20040065994A (en) 2004-07-23
EP1399457B1 (en) 2014-04-23

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