PROCESS FOR THE DEPROTECTION OF OLIGONUCLEOTIDES

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.

Claims

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 an acid.

2. The method of claim 1, wherein the column is an ion— exchange chromatography column.

3. 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.

4. The method of purifying oligonucleotides comprising: a. passing a first buffer solution to an anion-exchange column as a first equilibration step, wherein the first buffer solution includes a phosphate salt and an organic solvent; b. charging a diluted aqueous ammonia solution of a crude oligonucleotide onto the column; c. passing the first buffer solution to the column as a second equilibration step; d. washing the column with a second buffer solution, wherein the second buffer includes a phosphate salt, an organic solvent and an alkali halide; e. passing the first buffer solution 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 the first buffer solution to the column as a fourth equilibration step; eluting the de-protected oligonucleotide with the second buffer solution.

5. The method of claim 4, wherein the phosphate salt in the first or second buffer solution is an alkali phosphate or mixtures thereof, mono sodium phosphate or di sodium phosphate or mixtures thereof.

6. The method of claim 4, wherein the phosphate salt content in the first or second buffer solution is selected between 10 mM and 40 mM, or preferably between 20 mM and 30 mM.

7. The method of claim 4, 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.

8. The method of claim 4, wherein the organic solvent in the first or second buffer solution is selected from a polar protic or a polar aprotic solvent.

9. The method of claim 4, wherein the alkali halide is sodium chloride.

10. The method of claim 4, wherein the second buffer solution in the washing step d) comprises 0.2 M to 1.0 M, preferably or 0.4 M to 0.7 M sodium chloride.

11. The method of claim 4, wherein the second buffer solution in the elution step h) comprises 1.5.M to 3.0 M, or 1.8 M to 2.5 M sodium chloride.

12. The method of claim 1 or 4, wherein the acid is a protic acid.

13. The method of claim 1 or 4, wherein the acid is acetic acid.

14. The method of claim 4, wherein the flow rate of the acid solution in step f) is selected between 1.5 L/min and 2.5 L/min.

15. The method of claim 4, 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.

16. The method of claim 4, 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).

17. The method of claim 1 or 4, 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.

18. The method of claim 2, wherein the ion—exchange chromatography column is an anion—exchange chromatography column.

19. The method of claim 13, wherein the 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.

20. The method of claim 8 wherein the organic solvent is acetonitrile.

Description

EXAMPLES

Abbreviations:

[0083] Ac.sub.2O=acetic acid anhydride [0084] (d)A=(deoxy) adenosine [0085] (d)C=(deoxy) cytidine [0086] (d)G=(deoxy) guanosine [0087] DCA=dichloroacetic acid [0088] DCI=4,5-dicyanoimidazole [0089] DMT=4,4′-dimethoxytrityl [0090] CV=column volume [0091] Et.sub.3N=triethylamine [0092] EtOH=ethanol [0093] MeCN=acetonitrile [0094] MOE=2-methoxyethyl [0095] NA=not applicable [0096] NaOAc=sodium acetate [0097] NMI=N-methyl imidazole [0098] PADS=phenylacetyldisulfide [0099] PhMe=Toluene [0100] T=thymidine [0101] U=uridine [0102] underlined nucleosides are 2′-MOE nucleosides

Example 1

[0103]

TABLE-US-00002 a) Synthesis of (SEQ ID NO: 1) 5′-DMT-.sup.MeC.sub.S.sup.MeU.sub.O.sup.MeC.sub.OA.sub.OG.sub.ST.sub.SA.sub.SA.sub.S.sup.MeC.sub.SA.sub.ST.sub.ST.sub.SG.sub.SA.sub.S.sup.MeC.sub.SA.sub.O.sup.MeC.sub.O .sup.MeC.sub.OA.sub.S.sup.MeC-3′ 19 NH.sub.3

[0104] The title compound was synthesized as “DMT-on” using an ÄKTA oligopilot-100 on a 72 mmol scale.

[0105] The following phosphoramidites have been used in each cycle:

TABLE-US-00003 Cycle P-amidite 1 MOE.sup.MeC .sup.(Bz) 2 MOEA .sup.(Bz) 3 MOE.sup.MeC .sup.(Bz) 4 MOE.sup.MeC .sup.(Bz) 5 MOEA .sup.(Bz) 6 .sup.MedC .sup.(Bz) 7 dA .sup.(Bz) 8 dG (tBu) 9 T 10 T 11 dA .sup.(Bz) 12 .sup.MedC .sup.(Bz) 13 dA .sup.(Bz) 14 dA .sup.(Bz) 15 T 16 MOE-G .sup.(iBu) 17 MOEA .sup.(Bz) 18 MOE.sup.MeC .sup.(Bz) 19 MOE.sup.MeU 20 MOE.sup.MeC .sup.(Bz)

[0106] Synthesis parameters Cycles 3-5, 17-19

TABLE-US-00004 Detritylation DCA in PhMe, then MeCN Coupling Phosphoramidite (in MeCN), DCI and NMI (in MeCN), recycling loop, then MeCN Oxidation I.sub.2 (in pyridine/H.sub.2O), then MeCN Capping Ac.sub.2O (in MeCN), NMI (in pyridine), then PhMe

[0107] Synthesis parameters Cycles 1-2, 6-16:

TABLE-US-00005 Detritylation DCA in PhMe, then MeCN Coupling Phosphoramidite (in MeCN), DCI and NMI (in MeCN), recycling loop, then MeCN Sulfurization PADS (in 3-picoline/MeCN), then MeCN Capping. Ac.sub.2O (in MeCN), NMI (in pyridine), recycling loop (only cycle 1), then PhMe

[0108] Synthesis parameters Cycle 20:

TABLE-US-00006 Detritylation. DCA in PhMe, then MeCN Coupling Phosphoramidite (in MeCN), DCI and NMI (in MeCN), recycling loop, then MeCN Sulfurization PADS (in 3-picoline/MeCN), then MeCN

TABLE-US-00007 Backbone a) Et.sub.3N (in MeCN), recycling loop, deprotection and then PhMe cleavage/ b) aq. NH.sub.4OH, 55° C.; filtration; deprotection: H.sub.2O wash

[0109] b) On-column detritylation and formation of

TABLE-US-00008 (SEQ ID NO: 1) 5′-.sup.MeC.sub.S.sup.MeU.sub.O.sup.MeC.sub.OA.sub.OG.sub.ST.sub.SA.sub.SA.sub.S.sup.MeC.sub.SA.sub.STs.sub.ST.sub.SG.sub.SA.sub.S.sup.MeC.sub.SA.sub.O.sup.MeC.sub.O.sup.MeC.sub.OA.sub.S.sup.MeC- 3′ 19 Na

[0110] A solution of the crude material (338.87 g) obtained from example 1a) was subjected to the purification step which consists of several equilibration steps, a column wash step, an on-column detritylation step and a final product elution step.

[0111] First, the crude product from solid phase organic synthesis was loaded onto the pre-equilibrated AEX column, followed by a re-equilibration. DMT-on shortmers, capped failure sequences and other small molecules were removed by washing the column with a buffer containing a low concentration of sodium chloride. After column equilibration, the DMT group of the bound oligonucleotide was removed by aqueous acetic acid (referred to as on-column detritylation) followed by another column equilibration to remove residual acid and to establish the starting conditions of the elution step. In the last step, the product was eluted applying a high concentration sodium chloride gradient. The elution profile was monitored by ultraviolet (UV) absorption spectroscopy. The full-length DMT-off product peak was collected in several fractions. The fractions were tested for purity, organic impurities and pooled accordingly. The purification parameters are outlined in the table below:

TABLE-US-00009 Parameter Conditions Stationary Phase Monosized, rigid polystyrene/divinyl benzene polymer, 30 μm, Source 30Q, Cytiva Column Volume (CV) 30.0 L Eluent A 25 mM sodium phosphate.sup.1, 10% MeCN Eluent B 25 mM sodium phosphate.sup.1, 10% MeCN, 2M NaCl Eluent C 80% acetic acid .sub.(aq) Eluent D 25 mM sodium phosphate.sup.1, 10% MeCN, 0.5M NaCl Flow Rate (detritylation) 2.0 L/min Flow Rate (other steps) 2.5 L/min Temperature (gradient elution) 37° C. UV Detection Wavelength 260 nm Loading Solution from 10 L (total oligonucleotide content 32 mg/g solution) oligonucleotide synthesis Gradient Step % A % B % C % D CV Equilibration 1.sup.2 100 0 0 0 2 Equilibration 2 100 0 0 0 2 Wash 100 0 0 0 3 0 0 0 100 0 0 0 100 5 Equilibration 3 100 0 0 0 2 Detritylation 0 0 100 0 20 Equilibration 4 100 0 0 0 5 Gradient Elution 100 0 0 0 20 0 100 0 0 Isocratic Wash 0 100 0 0 2 .sup.1mixture of sodium mono- and di-phosphate) .sup.2Prior to loading the crude oligonucleotide solution.

[0112] c) Tangential flow Filtration/Lyophylization

[0113] Two purification batches (example 1b) were combined for the tangential flow filtration/lyophilization step.

[0114] The combined solutions (541.76 g, purity 89.2%) were concentrated and afterwards desalted by tangential flow filtration with purified water. The end of desalting was detected by conductivity of the removed permeate. The desalted solution was concentrated again and the concentration of the oligonucleotide in solution was adjusted by addition of purified water to 60-100 mg/mL. The solution was filtered through a 0.2 μm filter (Sartopore-2, Sartorius) and then lyophilized. The conditions of the performed lyophilization cycle is depicted in the table below:

TABLE-US-00010 Primary Secondary Step Freezing Drying Sequence Drying Sequence Temp (° C.) −45 −20 −20 0 20 25 Vacuum (bar) NA 0.27 0.01

[0115] The solution obtained after rinsing was lyophilized to obtain 508.26 g of the title product purity 90.9%).

Example 2 Comparison Example

[0116]

TABLE-US-00011 a) Synthesis of (SEQ ID NO: 1) 5′-DMT-.sup.MeC.sub.S.sup.MeU.sub.O.sup.MeC.sub.OA.sub.OG.sub.ST.sub.SA.sub.SA.sub.S.sup.MeC.sub.SA.sub.ST.sub.ST.sub.SG.sub.SA.sub.S.sup.MeC.sub.SA.sub.O.sup.MeC.sub.O .sup.MeC.sub.OA.sub.S.sup.MeC-3′ 19 NH.sub.3

[0117] The title compound has been prepared in accordance to example 1a.

[0118] b) HPLC

[0119] A portion of crude material was purified by HPLC (column with Amberchrom XT20 resin) according to the parameters in the Table below:

TABLE-US-00012 Step Mobile Phase A.sup.1 Mobile Phase B.sup.2 Column Volume (%) (%) (CV) Gradient 90 10 2 60 40 20 10 90 2 .sup.150 mM triethylammonium acetate (TEAA) pH 6.5-7.5 .sup.2Acetonitrile

[0120] The purified solution showed 97.2% purity and 90.8% yield.

[0121] c) Detritylation in solution

[0122] The pH of the combined fractions from example 2b was adjusted to pH 3 using glacial acetic acid as the detritylation agent followed by re-adjusting the pH to 5 with 10 M NaOH. The title compound was precipitated by adding the pH adjusted solution to ethanol. The yield obtained was 66.0%.