WO2023067038A1

WO2023067038A1 - Process for oligonucleotide purification

Info

Publication number: WO2023067038A1
Application number: PCT/EP2022/079156
Authority: WO
Inventors: Dennis Jul HANSEN; Inna LARSEN APPELDORFF
Original assignee: Roche Innovation Center Copenhagen A/S
Priority date: 2021-10-22
Filing date: 2022-10-20
Publication date: 2023-04-27

Abstract

The present invention relates to a process for purifying an oligonucleotide from a mixture comprising said oligonucleotide and a solid support, the process comprising desalting the mixture by size exclusion chromatography in which simultaneous with desalting, at least a portion of the solid support is removed by filtration.

Description

PROCESS FOR OLIGONUCLEOTIDE PURIFICATION

TECHNICAL FIELD

The present invention relates to processes for preparing and purifying oligonucleotides. The process of the invention is ideally suited for small-scale and intermediate-scale production and is significantly more cost and/or time effective than methods used in the art to date.

BACKGROUND

Recent developments in DNA/RNA technology, and in particular, antisense therapeutics, have meant that the production/purification of synthetic oligonucleotides has become of increasing importance. The purification challenges are significant and wide-ranging; given that large numbers of oligonucleotides must be purified in smaller quantities for instance for high throughput screening.

The principles of oligonucleotide synthesis are well known in the art (see e.g., Oligonucleotide synthesis; Wikipedia; https://en.wikipedia.org/wiki/Oligonucleotide synthesis, of March 15, 2016). Larger scale oligonucleotide synthesis nowadays is carried automatically using computer-controlled synthesizers. Oligonucleotides which are typically prepared via solid phase synthesis, after cleavage from the resin, still contain 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.

Processes for purification of oligonucleotides are reported in inter alia W02020083898, W02012010711 and WO2020173845.

Processes which require filtration and evaporation steps, e.g. of concentrated ammonia, may require substantial both operation and waiting time. The process of the present invention is a more efficient process compared to other methods used to date.

SUMMARY

It has been found by the present inventors that improved efficiency in oligonucleotide synthesis and purification can be achieved. This invention relates to a process for purification oligonucleotides from a crude aqueous mixture comprising said oligonucleotide and a solid support, said process comprising the step of: a. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide.

A process is also described for providing a purified oligonucleotide, said process comprising the steps of: a. synthesising said oligonucleotide on a solid support; b. cleaving the oligonucleotide from said solid support to provide a first solution comprising said oligonucleotide and said solid support; c. deprotecting said oligonucleotide while in said first solution, to provide a second solution comprising deprotected oligonucleotide and solid support; d. diluting the second solution comprising said deprotected oligonucleotide and said solid support with a solvent to provide a crude aqueous mixture comprising said deprotected oligonucleotide and said solid support, e. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide, and f. evaporating solvent from said desalted solution so as to provide said purified oligonucleotide.

Further details of the invention are provided in the following description and dependent claims.

FIGURES

Figure 1 illustrates a conventional process for oligonucleotide purification (open arrows to the right), and the process according to the invention (shaded arrows to the left). DETAILED DISCLOSURE

Scale of oligonucleotide synthesis: When referring to the scale of oligonucleotide synthesis we refer to the molar amount of oligonucleotide product present in the crude mixture. The process according to the present invention is applicable across a broad scale range of oligonucleotide amount. In some embodiments, the scale is greater than 1 pmol, such as greater than 5 pmol, such as greater than 10 pmol, such as greater than 100 pmol, such as greater than 200 pmol, such as greater than 500 pmol, such as greater than 1000 pmol (1 mmol), such as greater than 2 mmol, such as greater than 5 mmol, such as greater than 10 mmol, such as greater than 50 mmol or greater than 100 mmol or greater than 200 mmol. A preferred scale is between 10 pmol and 100 pmol. Particularly preferred is a scale of around 20 pmol.

Product purity: The purified oligonucleotide product obtained from the method of the invention may, in some embodiments, be at least about 75% pure, such as at least about 80% pure, such as at least about 85% pure, such as at least about 90% pure, such as at least about 95% pure. Purity of the oligonucleotide may be determined using standard assays known in the art, such as HPLC, LC-MS, or UPLC.

As noted above, a process is described for providing a purified oligonucleotide, said process comprising the steps of: a. synthesising said oligonucleotide on a solid support; b. cleaving the oligonucleotide from said solid support to provide a first solution comprising said oligonucleotide and said solid support; c. deprotecting said oligonucleotide while in said first solution, to provide a second solution comprising deprotected oligonucleotide and solid support; d. diluting the second solution comprising said deprotected oligonucleotide and said solid support with a solvent, preferable an aqueous solution, to provide a crude aqueous mixture comprising said deprotected oligonucleotide and said solid support, e. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide, and f. evaporating solvent from said desalted solution so as to provide said purified oligonucleotide.

Synthesis

The oligonucleotides described herein may be prepared using standard solid phase oligonucleotide synthesis. Suitable methodology will be familiar to the skilled artisan (see, for example, Sanghvi et al, 1999, Chemical synthesis and purification of phosphorothioate antisense oligonucleotides, in "Manual of Antisense Methodology" (G. Hartman and S Endres, eds), p2-23, Kluwer Academic Publishers, NY; Deshmukj; Large Scale Chromatographic Purification of Oligonucleotides; Handbook of Bioseparations; 2000; Vol 2, p51 1-534; Capaldi, D.C., Scozzari, A.N., Manufacturing and Analytical Processes for 2'-O-(2- Methoxyethyl)-Modified Oligonucleotides, in Antisense Drug Technology, 2.ed., Crooke, ST., ed, CRC Press, 2008, Chapter 14, p401-434). By way of example, the oligonucleotide may be prepared using a solid phase synthesizer such as, for example, MerMade 12 or 192 synthesiser from Biosearch, an ABI-type bench synthesizer, a Millipore 8800 DNA synthesizer or a Cytiva Oligopilot or OligoProcess synthesizer. The scale of oligonucleotide synthesis may be varied by selection of the appropriate oligonucleotide synthesizer, for example for lOOmmol scale synthesis an Oligo Process (Cytiva, formerly GE Healthcare) may be used.

The oligonucleotide synthesis in principle is a stepwise addition of nucleotide residues to the 5 '-terminus of the growing chain until the desired sequence is assembled. As a rule, each addition is referred to as a synthetic cycle and in principle consists of the chemical reactions al) de -blocking the protected hydroxyl group on the solid support, a2 ) coupling the first nucleoside as activated phosphoramidite with the free hydroxyl group on the solid support, a3 ) oxidizing or sulfurizing the respective P-linked nucleoside to form the respective phosphodiester (P=O) or the respective phosphorothioate (P=S); a4 ) optionally, capping any unreacted hydroxyl groups on the solid support; a5) de-blocking the 5' hydroxyl group of the first nucleoside attached to the solid support; a6 ) coupling the second nucleoside as activated phosphoramidite to form the respective P-linked dimer; a7 ) oxidizing or sulfurizing the respective P-linked dinucleoside to form the respective phosphodiester (P=O) or the respective phosphorothioate (P=S); a8) optionally, capping any unreacted 5' hydroxyl groups; a9 ) repeating the previous steps until the desired sequence is assembled.

Preferably, the oligonucleotides are prepared using phosphoramidite coupling chemistry of 5’- protected nucleotides. More preferably, the 5'-protecting group is a 4,4'-dimethoxytrityl (DMT) protecting group. Other protecting groups useful in oligonucleotide synthesis are also suitable and will be familiar to the skilled person. Oligonucleotides

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. The oligonucleotides may consist of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof.

In particular, the oligonucleotide may comprise at least one modified nucleotide, such as at least one Locked Nucleic Acid (LNA). "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".

LNA nucleoside monomers are modified nucleosides which comprise a linker group (referred to as a biradical 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.

Further examples of modified nucleoside units are 2'-O-alkyl-RNA unit, 2'-OMe-RNA unit, 2'- amino-DNA unit, 2'-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2'MOE RNA unit. The term 2'fluoro-DNA refers to a DNA analogue with a substitution to fluorine at the 2' position (2'F). 2'fluoro-DNA is a preferred form of 2'fluoro-nucleotide. 2'-deoxy-2'- fluoro-arabinonucleic acid (FANA) is another example.

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.

The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are described with capital letters A, T, G and Me C (5-methyl cytosine) for LNA nucleoside and with small letters a, t, g, c and Me c for DNA nucleosides. Modified nucleobases include but are not limited to nucleobases carrying protecting groups such as tert-butylphenoxyacetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl or dimethylformamidino (see Wikipedia, https://en.wikipedia.org/wiki/Nucleoside_phosphoramidite).

The oligonucleotides prepared by the process of the invention are single stranded oligonucleotides which optionally comprise one or more nucleotide analogues, such as LNA, which form part of, or the entire contiguous nucleotide sequence of the oligonucleotide.

In some embodiments, the oligonucleotide prepared by the process of the invention is 100% complementary to a miRNA sequence, such as a human microRNA sequence, or one of the microRNA sequences referred to in WO 2009043353.

In the context of the present invention the oligonucleotide is single stranded, this refers to the situation where the oligonucleotide is in the absence of a complementary oligonucleotide, i.e., it is not a double stranded oligonucleotide complex, such as an siRNA. It will be recognised that once purified according to the present invention, the oligonucleotide may be hybridised with other oligonucleotides which may be complementary to part of or all of the oligonucleotide prepared according to the present invention, to form, for example, a siRNA.

High affinity nucleotide analogues are nucleotide analogues which result in oligonucleotides having a higher thermal duplex stability with a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide. This may be determined by measuring the melting temperature of the duplex (Tm).

Oligonucleotide Design

In certain embodiments, the oligonucleotide of the invention is a gapmer. A gapmer oligonucleotide is an oligonucleotide which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region B (B), wherein region B is flanked both 5' and 3' by regions of affinity enhancing nucleotide analogues, such as from 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions A (A) and C (C) respectively. Suitably, the oligonucleotide is a LNA gapmer oligonucleotide (i.e. having a gapmer structure in which at least one nucleotide is LNA). In one embodiment, all of the nucleotide units in the oligonucleotide are Locked Nucleic Acid (LNA).

Preferably the gapmer comprises a (poly)nucleotide sequence of formula (5' to 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (A) (5' region) consists of or comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region B (B) consists of or comprises at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region C (C) (3'region) consists of or comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region D (D), when present consists of or comprises 1, 2 or 3 nucleotide units, such as DNA nucleotides.

In some embodiments, region A consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units. In some embodiments B consists of or comprises 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleotides which are capable of recruiting RNAse, or from 6-10, or from 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNAse. In some embodiments, region B consists of or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably from 4- 12 DNA units, more preferably from 6-10 DNA units, such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.

In some embodiments, region A consist of 3 or 4 nucleotide analogues, such as LNA, region B consists of 7, 8, 9 or 10 DNA units, and region C consists of 3 or 4 nucleotide analogues, such as LNA. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8- 3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 nucleotide units, such as DNA units.

In other embodiments, the oligonucleotide of the invention is a "mixmer" A mixmer is an oligonucleotide comprising both DNA and LNA. Chemical modifications in a mixmer may be mixed in almost random order. Typically, a mixmer consists of alternating short stretches of LNA and DNA. A mixmer according to the invention may comprise several other nucleotide analogues at the same time e.g. DNA-inverted, 2'OMe, 2'F, DNA, in a single oligonucleotide sequence.

In other embodiments, the oligonucleotide of the invention consists of a single type of sugar moiety e.g. 100% DNA sequence or 100% 2'MOE RNA.

Length

When referring to the length of a nucleotide molecule as referred to herein, the length corresponds to the number of monomer units, i.e. nucleotides, irrespective as to whether those monomer units are nucleotides or nucleotide analogues. With respect to nucleotides, the terms monomer and unit are used interchangeably herein.

The process of the present invention is suitable for purifying oligonucleotides which are for example, 40 nucleotide units in length or less, such as 30 nucleotide units in length or less, more preferably 6 to 25 nucleoside units in length. In some embodiments, the oligonucleotide has a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

For use as a therapeutically valuable oligonucleotide, oligonucleotides are typically synthesized as 5 to 40 nucleotides, preferably 10 to 25 nucleotides in length.

In one embodiment, the present invention is particularly suitable for the purification of oligonucleotides, for example, consisting of 14 to 25 nucleotide units. The process of the present invention is particularly suitable for purifying oligonucleotides, in which at least 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or such as 100% of the nucleotide units of the oligonucleotide are DNA nucleotide units.

In some embodiments, the oligonucleotide is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 75 %, preferably at least 80 %, preferably at least 85%, preferably at least 90%, preferably at least 95%, or 100% of the nucleotides are Locked Nucleic Acid (LNA) nucleotide units.

In some embodiments, the contiguous nucleotide sequence comprises or consists of 7, 8, 9 or 10, preferably contiguous, LNA nucleotide units. In some embodiments, at least 80% (for example, such as 85%, 90%, 95%, or 100%) of the internucleotide bonds in the oligonucleotide of the invention are phosphorothioate bonds.

In other embodiments, at least 80% (for example, such as 85%, 90%, 95%, or 100%) of the internucleotide bonds in the oligonucleotide of the invention are phosphodiester bonds.

Cleavage and deprotection

The oligonucleotide is cleaved from the solid phase and the protecting groups are removed, e.g. using standard techniques which are well known in the art. Suitably, steps of cleaving and deprotection are carried out essentially simultaneously, to provide a solution comprising deprotected oligonucleotide and solid support.

The steps of cleaving and deprotection may for example be carried out via treatment (of the solid-supported oligonucleotide) with a basic aqueous solution, preferably an aqueous ammonia solution.

In one preferred aspect, the steps of cleaving and deprotection are carried out via treatment with an aqueous ammonia solution, and wherein said process does not comprise a step of ammonia evaporation between prior to a desalting step. In conventional purification processes, ammonia evaporation is one of the most time-consuming steps (e.g. overnight). The realisation by the present inventors that ammonia evaporation can be avoided runs counter to prevailing understanding, and allows the process to proceed directly to subsequent steps of dilution and desalting, while maintaining good levels of purity in the oligonucleotide product.

Dilution

After cleavage and deprotection, the second solution comprising said deprotected oligonucleotide and said solid support is diluted with a solvent, preferable an aqueous solution, to provide a crude aqueous mixture comprising said deprotected oligonucleotide and said solid support.

In some embodiments, the crude oligonucleotide is dissolved in a metal salt solution, such as saline (NaCI solution). Hence, the crude aqueous mixture may comprise at least one metal salt. Preferably, the metal salt solution is a buffer which is suitable for HPLC. Optionally, the pH of the crude aqueous oligonucleotide solution is adjusted with a base, for example, aqueous sodium hydroxide solution.

When adjusted, the pH is preferably adjusted to about 7 to about 8. Preferably, the pH is adjusted by the addition of an aqueous solution of sodium hydroxide (for example, using a 10 mM NaOH solution).

Purification of Oligonucleotides

As noted, a process is provided for purifying an oligonucleotide from a crude aqueous mixture comprising said oligonucleotide and a solid support, said process comprising the step of: desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide.

The crude aqueous mixture obtained e.g. from the synthesis, cleavage, deprotection and dilution steps, is typically an unpurified product from oligonucleotide synthesis which typically comprises the oligonucleotide product as well as truncated versions of the oligonucleotide, deletion fragments as well as cleaved protection groups.

As used herein, the term "desalting" refers to a process by which impurities, such as inorganic salts, are removed from a mixture. When using a dried (lyophilized) crude oligonucleotide, as part of an initial step of the desalting process, the oligonucleotide is either dissolved in an electrolyte solution, or is dissolved in a suitable solvent, and an electrolyte is subsequently added to the oligonucleotide solution. Water is typically used as the solvent. The electrolyte may, for example, be a metal salt, such as a sodium or potassium salt, such as a sodium or potassium halogen salt, such as KCI, NaCI, or NaBr. The metal salt acts as a counter ion for the oligonucleotide anion. In some embodiments, the pH of the oligonucleotide solution may be adjusted to a pH of 7 or above, such as a pH of between about 7 to about pH 8. The suitable pH of the oligonucleotide solution may be achieved by using a basic solvent to dissolve the oligonucleotide, or by adjusting the pH of the oligonucleotide solution to a pH of 7 or higher.

The step of desalting suitably removes at least ammonium, sodium and chloride ions from said crude aqueous mixture.

The desalting step takes place on a size exclusion chromatography column. Suitable desalting columns are e.g. those sold under the "Cytiva HiPrep" brand. One example of a suitable size exclusion chromatography column comprises optionally-functionalised cross-linked dextran, known as Sephadex.

In a preferred embodiment, the desalting step takes place on a single size exclusion chromatography column. This is enabled by appropriately adjusting the liquid volumes used in the process of the present invention. For instance, the volume of the aqueous ammonia solution and I or the volume and I or concentration of the aqueous sodium chloride solution can be adjusted, so that the final volume of the solution does not exceed the capacity of one column. This lower volume can thus be purified using a single column, instead of 2 or more columns in series, which saves time and material. Experiments have shown that by reducing the number of columns from two to one (i.e. going from method 2a to 2b, below) the purification time was also reduced by approx. 50%.

Suitably, the desalting step takes place using a size exclusion chromatography column equipped with an inlet filter. The inlet filters can be end-of-line filters for tubing for single use, HPLC solvent filters or exchangeable suction frits made of porous PTFE, for example BOLA Suction Filters.

Further steps

After the desalting step, the next step in the process is evaporating solvent from said desalted solution so as to provide said purified oligonucleotide.

In the present context, the term "evaporating solvent" means that at least a portion of a solvent is removed. "Lyophilisation" requires evaporation of solvent to dryness, typically producing a powder or crystalline solid.

The purification process is typically followed by quality control, QC.

The procedure shown in Figure 1, right-hand process (open arrows), is applied for direct desalting procedure:

1. Synthesis

2. Cleavage and deprotection

3. Filtration 4. Evaporation or upconcentration

5. Dissolution

6. Purification by desalting

7. Evaporation or lyophilization

The filtration of oligonucleotides from the insoluble resin and evaporation of concentrated aqueous ammonia to yield crude oligonucleotide are both time-demanding steps that can take up to 24 hours depending on the instrument used. When the oligonucleotide is concentrated or evaporated to be tightly packed material, it can be very difficult to dissolve it again in order to continue with further purification. The dissolution step can take several hours and require assistive device such as heating baths in order to provide complete solution.

By introducing a filtration during the application of the sample to the instrument for purification by desalting, it is possible to remove the time demanding evaporation/ lyophilization and dissolution steps and the associated difficulties. It was found that the oligonucleotides in concentrated aqueous ammonia can be simultaneously filtered from the insoluble resin and loaded into the LC instrument for further purification by using a filter at the end of the inlet tubing (cf. Figure 1, left hand process, indicted by solid arrows).

By doing simultaneously filtration and loading into the LC instrument, the new optimized procedure is now more time and/or cost effective.

New optimized procedure according to the present invention as shown in Figure 1, left-hand process (shaded arrows) :

1. Synthesis

2. Cleavage and deprotection

3. Filtration and purification by desalting

4. Evaporation or lyophilization

Again, the process is typically followed by quality control, QC. Example:

Three different oligonucleotides were selected and synthesized as DMT-off using MerMade 12 synthesizer on 20 pmol scale.

Each compound A, B, C was synthesized in three batches (1, 2, 3) in order to take each sequence through three different purification process to compare the yield.

After the synthesis, each compound was treated with 20 ml of concentrated aqueous ammonia and 60 C for at least 5 to 24 hours for cleavage and deprotection. After this point, each sequence was taken through different purification methods:

Method 1 (comparative) :

Separation of oligonucleotide from the resin was done using sample filtration columns e.g. Kinesis TELOS. The filter was also washed using approx. 10 ml 50 % ethanol in water.

The filtrate containing oligonucleotide, ammonia and ethanol was evaporated using SpeedVac Vacuum Concentrator to give a crude oligonucleotide sample.

Crude oligonucleotide was dissolved in approx. 20 ml solution with 10 mM NaOH + 2 M NaCI.

The solution was loaded into LC system with two columns in series, for instance using two HiPrep 26/10 Desalting columns.

Method 2a :

The non-uniform suspension with oligonucleotide and the insoluble resin suspended in concentrated aqueous ammonia was used as is for the following steps.

Approx. 10 ml solution with 10 mM NaOH + 2 M NaCI was added directly to the suspension. The solution was loaded into LC system with two columns in a series, for instance using two HiPrep 26/10 Desalting columns, using an inlet line tubing equipped with a wet inlet filter for solvents.

Method 2b:

The same as in method 2a, but only one HiPrep 26/10 Desalting column was used.

Overview over all samples synthesized and the method used for purification:

The specification for the desalting program on LC system:

Instrument: Akta Pure 25

Solvent: water

Flow: 15 ml/min

Temperature: ambient

Column: Cytiva HiPrep 26/10 Desalting columns (size exclusion chromatography/gel filtration) MW cut-off 5000 Da.

The solutions obtained after desalting were lyophilized using GeneVac Centrifugal Evaporator. The yield of each compound after lyophilization:

To compare the quality of the products from the three different purification flows, all samples were sent to the elemental analysis:

Water content was determined by Thermogravimetric analysis (TGA).

CHNSO was determined by combustion analysis.

Sodium was determined by atomic absorption spectroscopy (AAS).

All the results are corrected for water content using the results from TGA analysis. Oxygen results from elemental analysis are not included. Results are shown as a percentage of found vs theoretical results.

The elemental analyses show no significant difference between the different purification methods.

Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

1. A process for purifying an oligonucleotide from a crude aqueous mixture comprising said oligonucleotide and a solid support, said process comprising the step of: a. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide.

2. The process according to claim 1, wherein the step of desalting removes at least ammonium, sodium and chloride ions from said crude aqueous mixture.

3. The process according to any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide, such as at least one Locked Nucleic Acid (LNA).

4. The process according to any one of the preceding claims, wherein the oligonucleotide is a LNA gapmer oligonucleotide.

5. The process according to any one of claims 1-2, wherein all of the nucleotide units in the oligonucleotide are DNA nucleotide units.

6. The process according to any one of the preceding claims, wherein the nucleotide units are linked by phosphodiester linkages or phosphorothioate linkages, or a mixture thereof.

7. The process according to any one of the preceding claims, wherein said crude aqueous mixture comprises at least one metal salt.

8. The process according to any one of the preceding claims, wherein the pH of the crude aqueous solution is adjusted with a base to about 7 to about 8.

9. The process according to any one of the preceding claims, wherein said oligonucleotide consists of 5 to 40 contiguous nucleotide units, preferably 10 to 25, such as 14 to 25 contiguous nucleotide units.

10. The process according to any one of the preceding claims, wherein said oligonucleotide is 5'-deprotected, prior to the desalting step.

11. The process according to any one of the preceding claims, wherein said desalting step takes place on a single size exclusion chromatography column.

12. A process for providing a purified oligonucleotide, said process comprising the steps of: a. synthesising said oligonucleotide on a solid support; b. cleaving the oligonucleotide from said solid support to provide a first solution comprising said oligonucleotide and said solid support; c. deprotecting said oligonucleotide while in said first solution, to provide a second solution comprising deprotected oligonucleotide and solid support; d. diluting the second solution comprising said deprotected oligonucleotide and said solid support with a solvent to provide a crude aqueous mixture comprising said deprotected oligonucleotide and said solid support, e. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, in a process according to any one of the preceding claims, to provide a desalted solution comprising said oligonucleotide, and f. evaporating solvent from said desalted solution so as to provide said purified oligonucleotide.

13. The process according to claim 12, wherein steps (b) and (c) are carried out essentially simultaneously, to provide a second solution comprising deprotected oligonucleotide and solid support.

14. The process according to any one of claims 12-13, wherein steps (b) and (c) are carried out via treatment with a basic aqueous solution, preferably an aqueous ammonia solution.

15. The process according to any one of claims 12-14, wherein steps (b) and (c) are carried out via treatment with an aqueous ammonia solution, and wherein said process does not comprise a step of ammonia evaporation between prior to step (e). 19

16. The process according to any one of claims 12-15, wherein steps (a)-(e), and preferably steps (a)-(f), of said process are carried out sequentially, without any intervening steps.

17. The process according to any one of claims 12-16, wherein said desalting step takes place on a single size exclusion chromatography column.

18. The process according to any one of the preceding claims, wherein said desalting step takes place using a size exclusion chromatography column equipped with an inlet filter.

19. The process according to any one of the preceding claims, wherein the oligonucleotide consists of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof and is 5 to 40, preferably 10 to 25 nucleotides in length.