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US-12616921-B2 - Process for the deprotection of oligonucleotides

US12616921B2US 12616921 B2US12616921 B2US 12616921B2US-12616921-B2

Abstract

The invention relates to a new process for the purification of oligonucleotides which comprises the removal of an acid labile 5′hydroxy protecting group at the 5′-O-oligonucleotide terminus of the oligonucleotide by means of an on-column de-protection with an acid.

Inventors

  • Yong Rag CHOI
  • Younggoo KANG
  • Sung Won KIM
  • Kyeong Eun JUNG
  • Pascal SCHMIDT

Assignees

  • HOFFMANN-LA ROCHE INC.

Dates

Publication Date
20260505
Application Date
20220106
Priority Date
20190709

Claims (14)

  1. 1 . A method for purifying oligonucleotides comprising removing an acid labile 5′hydroxy protecting group at the 5′-O-oligonucleotide terminus of the oligonucleotide by way of an on-column de-protection with acetic acid, comprising the steps: a. passing a buffer solution comprising a phosphate salt and a polar aprotic solvent to an anion-exchange column as a first equilibration step; b. charging a diluted aqueous ammonia solution of a crude oligonucleotide onto the column; c. passing a buffer solution comprising a phosphate salt and a polar aprotic solvent to the column as a second equilibration step; d. washing the column with a buffer solution comprising a phosphate salt, a polar aprotic solvent and an alkali halide; e. passing a buffer solution comprising a phosphate salt and a polar aprotic solvent to the column as a third equilibration step; f. passing an acid solution to the column to remove an acid labile 5′hydroxy protecting group at the 5′-O-oligonucleotide terminus of the oligonucleotide; g. passing a buffer solution comprising a phosphate salt and a polar aprotic solvent to the column as a fourth equilibration step; h. eluting the de-protected oligonucleotide with a buffer solution comprising a phosphate salt, a polar aprotic solvent and an alkali halide.
  2. 2 . The method of claim 1 , wherein the acid labile 5′hydroxy protecting group is 4,4′-dimethoxytrityl, 4-methoxytrityl, trityl, 9-phenyl-xanthen-9-, 9-(p-tolyl)-xanthen-9-yl or tert-butyldimethylsilyl.
  3. 3 . The method of claim 1 , wherein the phosphate salt in the at least one buffer solution is an alkali phosphate or mixtures thereof, mono sodium phosphate or di sodium phosphate or mixtures thereof.
  4. 4 . The method of claim 1 , wherein the phosphate salt content in at least one buffer solution is selected between 10 mM and 40 mM, or between 20 mM and 30 mM.
  5. 5 . The method of claim 1 , wherein the aqueous ammonia solution, which is charged on the column has a total oligonucleotide content of 8 to 20 g per L column volume, or 10 to 15 g per L column volume.
  6. 6 . The method of claim 1 , wherein the polar aprotic solvent in at least one buffer solution is acetonitrile.
  7. 7 . The method of claim 1 , wherein the alkali halide is sodium chloride.
  8. 8 . The method of claim 1 , wherein the buffer solution in washing step d) comprises 0.2 M to 1.0 M, or 0.4 M to 0.7 M sodium chloride.
  9. 9 . The method of claim 1 , wherein the buffer solution in elution step h) comprises 1.5.M to 3.0 M, or 1.8 M to 2.5 M sodium chloride.
  10. 10 . The method of claim 1 , wherein the flow rate of the acetic acid in step f) is between 1.5 L/min and 2.5 L/min.
  11. 11 . The method of claim 1 , wherein the flow rate of the buffer solution in steps a) to e) and g) to h) is between 2.0 L/min and 3.0 L/min.
  12. 12 . The method of claim 1 , wherein the method further comprising i. washing the filtrate from step h) with purified water via tangential flow filtration as a desalting and concentration step; and j. lyophilizing the filtrate obtained from the desalting and concentrating step i).
  13. 13 . The method of claim 1 , wherein the oligonucleotide consists of modified DNA, RNA or LNA nucleoside monomers or combinations thereof and is 10 to 40, or 10 to 25 nucleotides in length.
  14. 14 . The method of claim 1 , wherein the acetic acid is an aqueous acetic acid with a concentration of acetic acid in water of 50 to 95% by weight, 70 to 90% by weight, or 75 to 85% by weight.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Application No. PCT/EP2020/068922, filed Jul. 6, 2020, which claims priority to European Patent Application No. 19185225.0, filed Jul. 9, 2019, which are incorporated herein by reference in its entirety. SEQUENCE LISTING This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 21, 2022, is named P35629-US-SL.txt and is 655 bytes in size. The invention relates to a new process for the purification of oligonucleotides which comprises the removal of an acid labile 5′hydroxy protecting group at the 5′-O -oligonucleotide terminus of the oligonucleotide by way of an on-column de-protection with an acid. The oligonucleotide which is typically prepared via solid phase synthesis, after its cleavage from the solid support, still contains a significant amount of impurities. For standard monomers of a 15- to 20-mer length the API purity is at best in the range of 70 to 80%. For chemically modified monomers or for longer sequences the API content is typically even lower. Selective separation methods have been developed to prepare high purity oligonucleotides which satisfy the specifications for a therapeutic application. In one method the oligonucleotide, after its cleavage from the solid support, is left with the acid labile 5′hydroxy protecting group at the 5′-O-oligonucleotide terminus. The hydrophobicity of this group allows the application of effective chromatography techniques for purification. It is common strategy that the crude oligonucleotide is passing the following steps (for instance Krotz et al, Organic Process Research & Development 2003, 7, 47-52): a) reversed phase chromatography b) concentration and desalting c) removal of the acid labile 5′hydroxy protecting group in solution and d) further concentration and desalting It was found that this known processes require substantial operation time due to the number of single operation steps a) to d). Object of the invention was to reduce the number of purification steps and with that reduce the operation time and also attempt to reach a higher overall yield. It was found that the object of the invention could be reached with the novel process for the purification of oligonucleotides as outlined above. The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein. The term acid labile 5′hydroxy protecting group is defined as a protecting group which is cleavable with the help of a suitable acid and which has a hydrophobic character. Typical acid labile 5′hydroxy protecting groups are selected from 4,4′-dimethoxytrityl, 4-methoxytrityl, trityl, 9-phenyl-xanthen-9-, 9-(p-tolyl)-xanthen-9-yl or from tert-butyldimethylsilyl, preferably from 4,4′-dimethoxytrityl, 4-methoxytrityl or trityl or even more preferably from 4,4′-dimethoxytrityl. The term oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleotides. For use as a therapeutically valuable oligonucleotide, oligonucleotides are typically synthesized containing 10 to 40 nucleotides, preferably 10 to 25 nucleotides in length. The oligonucleotides may consist of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof. The LNA nucleoside monomers are modified nucleosides which comprise a linker group or a bridge between C2′ and C4′ of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. Optionally modified as used herein refers to nucleosides modified as compared to the equivalent DNA, RNA or LNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase moiety. In a preferred embodiment the modified nucleoside comprises a modified sugar moiety, and may for example comprise one or more 2′ substituted nucleosides and/or one or more LNA nucleosides. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”. The DNA, RNA or LNA nucleosides are as a rule linked by a phosphodiester (P═O) and/or a phosphorothioate (P═S) internucleoside linkage which covalently couples two nucleosides together. Accordingly, in some oligonucleotides all internucleoside linkages may consist of a phosphodiester (P═O), in other oligonucleotides all internucleoside linkages may consist of a phosphorothioate (P═S) or in still other oligonucleotides the sequence of internucleoside linkages vary and comprise both phosphodiester (P═O) and phosphorothioate (P═S) internucleoside linkages. The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, w