IP Library › Granted Patent US 12,275,983
Granted Patent B2
US 12,275,983 · App. 16/833,724 · Granted Apr 15, 2025

Processes for the production of oligonucleotides

Inventors: Andreas Crameri (Stevenage, GB); Malcolm Leithhead Hill (Stevenage, GB); David Graham Tew (Stevenage, GB)
Assignee: GlaxoSmithKline Intellectual Property Development Limited
C12Q1/6811C07H1/00C07H21/00C12N9/93C12Q1/6855C12Q1/686C12Y605/01001C07K2319/20C12Q2600/106C12Q2600/156G01N2030/027
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,275,983
App. No.
16/833,724
Granted
Apr 15, 2025
Kind
B2
Abstract

Disclosed herein are novel processes for the production of oligonucleotides that are suitable for use in the production of chemically modified oligonucleotides, such as those for use in therapy.

Claims (28)

1. A continuous or semi-continuous flow process for purifying a single-stranded oligonucleotide product from impurity oligonucleotide strands, wherein the single-stranded oligonucleotide product has at least one modified nucleotide residue, wherein the modification is chosen from: modification at the 2′ position of the sugar moiety of the nucleotide residue, modification of the nucleobase of the nucleotide residue, and modification of the backbone of the nucleotide residue, the process comprising:

a) providing a template oligonucleotide (I) complementary to the sequence of the single-stranded oligonucleotide product, said template having properties that allow it to be separated from the single-stranded oligonucleotide product;

b) providing a pool of oligonucleotides (II) comprising the single-stranded oligonucleotide product and impurity oligonucleotide strands;

c) contacting the template oligonucleotide (I) and the pool of oligonucleotides (II) in conditions to allow annealing, thereby producing (i) annealed template and impurity oligonucleotide strands and (ii) annealed template and single-stranded oligonucleotide product;

d) changing the conditions to separate any impurity oligonucleotide strands, comprising denaturing the annealed template and impurity oligonucleotide strands;

e) separating the impurity oligonucleotide strands from the annealed template and single-stranded oligonucleotide product;

f) changing the conditions to separate the single-stranded oligonucleotide product, comprising denaturing the annealed template and single-stranded oligonucleotide product;

g) separating the single-stranded oligonucleotide product from the template; and

h) recycling the template.

2. The process according to claim 1 , whereby the denaturing results from a temperature increase, changing pH, or changing a salt concentration in a buffering solution.

3. The process according to claim 1 , including two steps of increasing temperature: i) to denature any annealed impurities and ii) to denature annealed product.

4. The process according to claim 1 , wherein the single-stranded oligonucleotide product is 10 to 200 nucleotides long.

5. The process according to claim 1 , wherein a property that allows the template to be separated from the single-stranded oligonucleotide product is that the template is attached to a support material.

6. The process according to claim 5 , wherein the support material is a soluble support material.

7. The process according to claim 6 , wherein the soluble support material is selected from the group consisting of polyethylene glycol, a soluble organic polymer, DNA, a protein, a dendrimer, a polysaccharide, an oligosaccharide, and a carbohydrate.

8. The process according to claim 5 , wherein the support material is an insoluble support material.

9. The process according to claim 8 , wherein the support material is selected from the group consisting of: a glass bead, a polymeric bead, a fibrous support, a membrane, a streptavidin coated bead, cellulose, and a reaction vessel.

10. The process according to claim 5 , wherein multiple, repeated copies of the template are attached in a continuous manner via a single attachment point to the support material.

11. The process according to claim 1 , wherein a property that allows the template to be separated from the product is the molecular weight of the template.

12. The process according to claim 1 , wherein the modification is selected from the group consisting of 2′-Fluoro (2′-F), 2′-O-methyl (2′-OMe), 2-O-methoxyethyl (2′-MOE), and 2′-amino, or wherein the oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a bridged nucleic acid (BNA).

13. The process according to claim 1 , wherein the modification is selected from the group consisting of a 5-methyl pyrimidine, a 7-deazaguanosine and an abasic nucleotide.

14. The process according to claim 1 , wherein the modification is selected from the group consisting of phosphorothioate, phosphoramidate and phosphorodiamidate.

15. The process according to claim 1 , wherein the single-stranded oligonucleotide product is at least 90% pure.

16. The process according to claim 1 , wherein the process is for purifying a therapeutic oligonucleotide.

17. The process according to claim 1 , wherein the product is purified at gram or kilogram scale and/or the process is carried out in a 1 L or larger reactor.

18. The process according to claim 1 , wherein the single-stranded oligonucleotide product is 20 to 30 nucleotides long.

19. The process according to claim 1 , wherein the single-stranded oligonucleotide product is at least 95% pure.

20. The process according to claim 1 , wherein the single-stranded oligonucleotide product is at least 98% pure.

Assignments (2)
CHANGE OF ADDRESS Recorded Oct 8, 2025
From: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
To: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
Reel/Frame 073032/0390 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 30, 2020
From: CRAMERI, ANDREAS; HILL, MALCOLM LEITHHEAD; LOVELOCK, SARAH LOUISE; SCHOBER, MARKUS; TEW, DAVID GRAHAM; THOMAS, PAMELA JOAN
To: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
Reel/Frame 052257/0730 →
Priority Claims (1)
GB 1612011 · Jul 11, 2016 · national
Continuity (2)
Continuation 15643535 · Jul 7, 2017
Related Publication 20200277659A1 · Sep 3, 2020
References Cited (68)
US 5516664A · Hyman · 1996 [cited by applicant]
US 5547843A · Studier et al. · 1996 [cited by applicant]
US 5602000A · Hyman · 1997 [cited by applicant]
US 5998175A · Akhavan-Tafti · 1999 [cited by applicant]
US 6004826A · Segev · 1999 [cited by applicant]
US 6110668A · Strizhov et al. · 2000 [cited by applicant]
US 6660229B2 · Cantor · 2003 [cited by applicant]
US 6867294B1 · Sanghvi et al. · 2005 [cited by applicant]
US 7033753B1 · Kool · 2006 [cited by applicant]
US 7695906B2 · Schatz · 2010 [cited by applicant]
US 8148065B1 · Wallace · 2012 [cited by applicant]
US 10640812B2 · Crameri et al. · 2020 [cited by applicant]
US 20030008306A1 · Turnbull et al. · 2003 [cited by applicant]
US 20030073081A1 · Mukai et al. · 2003 [cited by applicant]
US 20030120035A1 · Gao et al. · 2003 [cited by applicant]
US 20030228602A1 · Parker et al. · 2003 [cited by applicant]
US 20040248104A1 · Yakhini et al. · 2004 [cited by applicant]
US 20160108382A1 · Efcavitch et al. · 2016 [cited by applicant]
CN 1350581A · 2002 [cited by applicant]
CN 101889096A · 2010 [cited by applicant]
CN 105506125A · 2016 [cited by applicant]
EP 1130113A1 · 2001 [cited by applicant]
JP 2019525917A · 2019 [cited by applicant]
WO 1989012696A1 · 1989 [cited by applicant]
WO 2000005412A1 · 2000 [cited by applicant]
WO WO0050870A1 · 2000 [cited by applicant]
WO 0127326A2 · 2001 [cited by applicant]
WO 0129211A2 · 2001 [cited by applicant]
WO 0164864A2 · 2001 [cited by applicant]
WO WO0171037A1 · 2001 [cited by applicant]
WO 2003106679A1 · 2003 [cited by applicant]
WO 2004029223A2 · 2004 [cited by applicant]
WO 2006071568A2 · 2006 [cited by applicant]
WO 2009043112A1 · 2009 [cited by applicant]
WO WO2009097673A1 · 2009 [cited by applicant]
WO 2012010711A1 · 2012 [cited by applicant]
WO 2014041337A1 · 2014 [cited by applicant]
WO 2018011067A2 · 2018 [cited by applicant]
International Preliminary Report on Patentability dated Nov. 26, 2018 for PCT/EP2017/067049. [cited by applicant]
International Patent Application No. PCT/EP2017/067049 Written Response to the EPO dated Aug. 13, 2018. [cited by applicant]
Karkare et al. “Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino” 71 Applied Microbiology and Biotechnology 575-586 (2006). [cited by applicant]
Frank et al. “A new generation for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports” 11(13) Nucleic Acids Research 4365-4377 (1983). [cited by applicant]
Toy et al. “Soluble Polymer-Supported Organic Synthesis” 33(8) Accounts of Chemical Research 546-554 (2000). [cited by applicant]
El-Sagheer, et al., “Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in [cited by applicant]
Qiu, et al., “Solid phase click ligation for the synthesis of very long oligonucleotides”, Chemical Communications; Issue 62; 49; 2013; pp. 6959-6961. [cited by applicant]
Hili et al., “DNA ligase-mediated translation of DNA into densely functionalized nucleic acid polymers.”, Journal of the American Chemical Society, American Chemical Society, United States, United States, (Dec. 20, 2012… [cited by applicant]
Lei et al., “A High-Fidelity Codon Set for the T4 DNA Ligase-Catalyzed Polymerization of Modified Oligonucleotides.”, ACS Combinatorial Science Dec. 14, 2015, (Dec. 14, 2015), vol. 17, No. 12, doi:10.1021/acscombsci.5b0… [cited by applicant]
Lundin et al., “Oligonucleotide Therapies: The Past and the Present”, Human Gene Therapy, Mary Ann Liebert, Inc. Publishers, GB, GB , (Aug. 1, 2015), vol. 26, No. 8, doi:10.1089/hum.2015.070, ISSN 1043-034, pp. 475-485,… [cited by applicant]
Goodchild, “Conjugates of Oligonucleotides and Modified Oligonucleotides: A Review of their Synthesis and Properties,” Bioconjugate Chemistry, American Chemical Society, US, US , (May 1, 1990), vol. 01., No. 03., doi:10… [cited by applicant]
Verma et al., “Modified oligonucleotides: synthesis and strategy for users.”, Annual Review of Biochemistry, Palto Alto, CA, US, US, (Jan. 1, 1998), vol. 67, doi:10.1146/annurev.biochem.67.1.99, ISSN 0066-4154, pp. 99-1… [cited by applicant]
Zhao et al., “Effects of 2′-O-methyl nucleotide on ligation capability of T4 DNA ligase”, Acta Biochimica Biophysica Sinica, Blackwell Publishing, Inc., Malden, MA, US, US , (Sep. 1, 2014), vol. 46, No. 9, doi:10.1093/a… [cited by applicant]
Suzuki et al., “Simple and Rapid Enzymatic Method for the Synthesis of Single-Strand Oligonucleotides Containing Trifluorothymidine”, Nucleosides, Nucleotides & Nucleic Acids, Taylor & Francis, US, US , (Nov. 30, 2010),… [cited by applicant]
Pengpumkiat et al., “Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library”, PLoS One, (Mar. 1, 2016), vol. 11, No. 3, doi:10.1371/journal.… [cited by applicant]
Chen et al., “Template-directed chemical ligation to obtain 3′-3′ and 5′-5′ phosphodiester DNA linkages”, Scientific reports, Nature Publishing Group, England, England , doi:10.1038/srep04595, (Apr. 4, 2014), p. 4595, S… [cited by applicant]
Stark et al., “An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs”, RNA, Cambridge University Press on behalf of the RNA Society, (Jan. 1, 2006), vol. 12, No. 11, doi:10.1261/rna.93506, IS… [cited by applicant]
Vanmeert et al., “Rational design of an XNA ligase through docking of unbound nucleic acids to toroidal proteins”, Nucleic Acids Research, Oxford University Press, GB, GB , (Jul. 26, 2019), vol. 47, No. 13, doi:10.1093/… [cited by applicant]
Kershaw et al., “Splint Ligation of RNA with T4 DNA Ligase”, Christopher J. Kershaw, Raymond T. O'keefe, Boegel, Sebastian [HerausgeberIn], Bioinformatics for Cancer Immunotherapy : Methods and Protocols, New York, NY, … [cited by applicant]
Noll et al., “Purification of Small Interfering RNA Using Nondenaturing Anion-Exchange Chromatography”, Nucleic Acid Therapeutics, Mary Ann Liebert, Inc. Publishers, US, US , (Dec. 1, 2011), vol. 21, No. 6, doi:10.1089/… [cited by applicant]
ATDBIO: “Gene Synthesis,” Jan. 5, 2016, [Retrieved on Feb. 8, 2019] Retrieved from URL: https://www.aidbio.com/content/63/Gene-synthesis. [cited by applicant]
Barany F., “The Ligase Chain Reaction in a PCR World,” PCR Methods Applications, Cold Spring Harbor Laboratory Press, US, Aug. 1, 1991, vol. 1, No. 1, pp. 5-16, ISSN: 1054-9803. [cited by applicant]
Brill W K D., “Facile Methods to Recycle Nucleosides During Solid Phase Synthesis of Oligonucleotides”, Tetrahedron Letters, 1994, vol. 35, No. 19, pp. 3041-3044. [cited by applicant]
Knunyants I.L., Chemical Encyclopedia, v. 2, Soviet Encyclopedia Publishing House, Moscow, V.Z, 1990, pp. 664-665. [cited by applicant]
Kramer M., et al., “Enzyme-Free Ligation of 5'-Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure,” Chemistry Biodiversity, Aug. 11, 2017, vol. 14, No. 9, p. e1700315, ISSN: 1612-1872. [cited by applicant]
Kukwikila M., et al., “Assembly of a Biocompatible Triazole-linked Gene by One-pot Click-DNA Ligation,” Nature Chemistry, Sep. 11, 2017, vol. 9, No. 11, pp. 1089-1098, ISSN: 1755-4330. [cited by applicant]
Ling M.M., et al., “Approaches to DNA Mutagenesis An Overview,” Analytical Biochemistry, 1997, vol. 254, No. 2, pp. 157-178. [cited by applicant]
Shivalingam A., et al., “Synthesis of Chemically Modified DNA,” Biochemical Society Transactions, Jun. 15, 2016, vol. 44, No. 3, pp. 709-715, ISSN: 0300-5127. [cited by applicant]
Tarantul, Explanatory Dictionary of Molecular and Cellular Biotechnology, Moscow, 2015, vol. 1, p. 411. [retrieved online https://www.ncbi.nlm.nih.gov/nlmcatalog/101685659] English Title only. [cited by applicant]
Taylor J.W., et al., 1985, vol. 13, N24, pp. 8749-8764. [cited by applicant]