阅读说明：本技术 用于纯化寡核苷酸的方法 (Method for purifying oligonucleotides ) 是由 J·利尔 F·斯拉多耶维奇 于 2019-10-22 设计创作，主要内容包括：本发明涉及一种用于纯化寡核苷酸的新方法,所述方法包括通过用酸性缓冲溶液进行切向流过滤,除去所述寡核苷酸的5’-O-寡核苷酸末端的酸不稳定的5’羟基保护基。所述方法需要的步骤较少且使自动化程度更高。(The present invention relates to a novel method for purifying oligonucleotides comprising removing acid-labile 5 'hydroxyl protecting groups at the 5' -O-oligonucleotide end of said oligonucleotides by tangential flow filtration with an acidic buffer solution. The method requires fewer steps and allows a higher degree of automation.)

1. A method for purifying an oligonucleotide comprising removing an acid-labile 5 'hydroxyl protecting group at a 5' -O-oligonucleotide terminus of the oligonucleotide by tangential flow filtration with an acidic buffer solution.

2. The process of claim 1 wherein the acid-labile 5' hydroxy protecting group is 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.

3. The method according to any one of claims 1 to 3, wherein the pH value of the acidic buffer solution is in the range of 2 to 6, preferably in the range of 2.5 to 4.0.

4. The method of any one of claims 1 to 4, wherein the acidic buffer is an aqueous solution of a weak acid and its conjugate base, which is acidified with a protic acid to bring the pH within a desired range.

5. The method of claim 4, wherein the acidic buffer is an acetate buffer and the protic acid is hydrochloric acid.

6. The process according to any one of claims 1 to 5, wherein the acidic buffer solution additionally comprises a polar protic or polar aprotic organic solvent.

7. The method of any one of claims 1 to 6, wherein the tangential flow filtration is performed at a transmembrane pressure of 0.5 to 10.0 bar.

8. The method according to any one of claims 1 to 7, wherein the tangential flow filtration is between 5 and 50l/h m²At a rate in between.

9. The process of any one of claims 1 to 8, wherein the tangential flow filtration is performed with a Molecular Weight Cut Off (MWCO). ltoreq.5 kDA membrane.

10. The method according to any one of claims 1 to 9, wherein the oligonucleotide content in the acidic buffer solution is selected between 1.0mg/l and 100.0 mg/l.

11. The method of any one of claims 1 to 10, wherein the method further comprises the step of, after removal of the acid-labile 5 'hydroxyl protecting group at the 5' -O-oligonucleotide terminus of the oligonucleotide:

a. a neutralization step comprising neutralizing the resulting filtrate with a base by tangential flow filtration; and

b. a desalting step comprising washing the filtrate resulting from said neutralization by tangential flow filtration with water or a mixture of water and a polar protic or polar aprotic organic solvent; and

c. optionally freeze-drying the filtrate obtained from the desalting step.

12. The method of any one of claims 1 to 11, wherein the method further comprises the step of prior to removing the acid labile 5 'hydroxyl protecting group at the 5' -O-oligonucleotide terminus of the oligonucleotide:

a. the crude oligonucleotide obtained after cleavage from the resin and deprotection of the phosphate is subjected to reverse phase high performance liquid chromatography or anion exchange chromatography.

13. The method according to any one of claims 1 to 12, wherein the oligonucleotide consists of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof and is 10 to 40, preferably 10 to 25 nucleotides in length.

Technical Field

The present invention relates to a novel method for purifying oligonucleotides comprising removing acid-labile 5 'hydroxyl protecting groups from the 5' -O-oligonucleotide ends of oligonucleotides by tangential flow filtration with acidic buffer solutions.

Background

Oligonucleotides, which are usually prepared by solid phase synthesis, still contain a large amount of impurities after cleavage from the resin. For standard monomers of 15 to 20 monomer lengths, the API purity is at most in the range of 70% to 80%. For chemically modified monomers or for longer sequences, the API content is usually even lower.

Disclosure of Invention

Selective separation methods have been developed to produce high purity oligonucleotides that meet specifications for therapeutic applications.

In one method, the oligonucleotide leaves an acid-labile 5 'hydroxyl protecting group at the end of the 5' -O-oligonucleotide after cleavage from the resin. The hydrophobicity of this group allows for purification using efficient chromatographic techniques.

A common strategy is to subject the crude oligonucleotide to the following steps (e.g., Krotz et al, Organic Process Research & Development 2003,7,47-52)

a) Reversed phase chromatography

b) Concentration and desalination

c) Removing the acid-labile 5' hydroxy protecting group from solution, and

d) further concentration and desalination

It has been found that this known method requires a large amount of operating time due to the number of individual operating steps a) to d).

The aim of the invention is to reduce the number of steps, to achieve a higher degree of automation and thus to reduce the overall operating time.

It has been found that the objects of the present invention can be achieved with the novel process for the purification of oligonucleotides as outlined above.

Detailed Description

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 aid of a suitable acid and has hydrophobic properties.

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 a molecule comprising two or more covalently linked nucleotides as is commonly understood by the skilled person. For use as therapeutically valuable oligonucleotides, oligonucleotides are generally synthesized in lengths of 10 to 40 nucleotides, preferably 10 to 25 nucleotides.

The oligonucleotide may be composed of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof.

LNA nucleoside monomers are modified nucleosides that contain a linker group (called a diradical or bridge) between C2 'and C4' of the ribose sugar ring of the nucleotide. These nucleosides are also referred to in the literature as bridged nucleic acids or Bicyclic Nucleic Acids (BNA).

As used herein, optionally modified refers to a nucleoside that is modified compared to an equivalent DNA, RNA or LNA nucleoside by the introduction of one or more modifications of a sugar moiety or a 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 interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer".

Typically, DNA, RNA or LNA nucleosides are linked by phosphodiester (P ═ O) and/or phosphorothioate (P ═ S) internucleoside linkages, which covalently couple the two nucleosides together.

Thus, in some oligonucleotides, all internucleoside linkages may consist of phosphodiester (P ═ O), in other oligonucleotides all internucleoside linkages may consist of phosphorothioate (P ═ S), or in other oligonucleotides, the sequence of internucleoside linkages is different and comprises both phosphodiester (P ═ O) and phosphorothioate (P ═ S) nucleosides.

The nucleobase portion may be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C or U, wherein each letter may beTo optionally include modified nucleobases with equivalent functions. For example, in the exemplary oligonucleotides, for LNA nucleosides, the capital letters A, T, G and^Mec (5-methylcytosine) describes the nucleobase moiety and, for DNA nucleosides, the lower case letters a, t, g, C and^Mec describes the nucleobase moiety. Modified nucleobases include, but are not limited to, nucleobases with protecting groups such as tert-butylphenoxyacetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl or dimethylcarboxamido (see Wikipedia, Phosphoramidi t-Synthesis, https:// de. Wikipedia. org/wiki/Phosphoramid-Synthesis, 24/3/2016).

Preferably, the oligonucleotide consists of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof and is 10 to 40, preferably 10 to 25 nucleotides in length.

The principles of Oligonucleotide synthesis are well known in the art (see, e.g., Oligonucleotide synthesis; Wikipedia, the free encyclopedia; https:// en. Wikipedia. org/wiki/Olig Oligonucleotide synthesis, 2016, 3, 15).

Today, larger scale oligonucleotide synthesis can be automated using computer controlled synthesizers.

Typically, oligonucleotide synthesis is solid phase synthesis, wherein the assembled oligonucleotide is covalently bound to a solid support material through its 3' -terminal hydroxyl group and remains attached thereto throughout the chain assembly. Suitable supports are commercially available macroporous polystyrene supports, for example, from GE Healthcare's Primer support 5G or KinovateAnd (3) a carrier.

Oligonucleotide synthesis is principally the stepwise addition of nucleotide residues to the 5' -end of a growing strand until the desired sequence is assembled.

In general, each addition is called a synthesis cycle, which in principle consists of the following chemical reactions

a₁₎Deblocking the protected hydroxyl groups on the solid support,

a₂) Coupling a first nucleoside as an activated phosphoramidite to a free hydroxyl group on a solid support,

a₃) Oxidizing or sulfurizing the corresponding P-linked nucleoside to form the corresponding phosphodiester (P ═ O) or the corresponding phosphorothioate (P ═ S);

a₄) Optionally, capping any unreacted hydroxyl groups on the solid support;

a₅) Deblocking the 5' hydroxyl group of the first nucleoside attached to the solid support;

a₆) Coupling a second nucleoside as an activated phosphoramidite to form respective P-linked dimers;

a₇) Oxidizing or sulfurizing the corresponding P-linked dinucleoside to form the corresponding phosphodiester (P ═ O) or the corresponding phosphorothioate (P ═ S);

a₈) Optionally, capping any unreacted 5' hydroxyl groups;

a₉) Repeating the previous step a₅To a₈Until the desired sequence is assembled.

Subsequently, the resin can be cleaved from the resin with concentrated ammonia. During this cleavage, the protecting groups on the phosphate and nucleotide bases are also removed.

The cleaved crude oligonucleotide leaves an acid labile 5 'hydroxyl protecting group at the end of the 5' -O-oligonucleotide.

The method is characterized in that the acid-labile 5 'hydroxyl protecting group at the 5' -O-oligonucleotide end of the oligonucleotide is removed by tangential flow filtration with an acidic buffer solution.

The term acid-labile 5' hydroxy protecting group is defined above as a protecting group which is cleavable with the aid of a suitable acid and has hydrophobic properties.

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 (DMT).

Acidic buffers are aqueous solutions of weak acids and their conjugate bases that are acidified with protic acids to bring the pH within the desired range.

Typical acidic buffers are acetate or citrate buffers, which consist of acetic acid or citric acid as weak acid and its sodium salt as conjugate base.

The protic acid used for the acidification of the acidic buffer may be selected from aqueous mineral acids, such as from hydrochloric acid, phosphoric acid, sulfuric acid or nitric acid, but is typically selected from aqueous hydrochloric acid.

The desired pH range is desirably between 2 and 6, preferably between 2.5 and 4.0, even more preferably between 2.8 and 3.5.

The acidic buffer solution may also contain polar protic or polar aprotic organic solvents in an amount of 5% (V) to 50% (V), preferably in an amount of 20% (V) to 40% (V).

Suitable polar protic solvents are primary aliphatic alcohols, such as methanol, ethanol or isopropanol, preferably ethanol.

Suitable polar aprotic solvents are acetonitrile, dimethyl sulfoxide or N-methyl-2-pyrrolidone, but preferably acetonitrile.

In a preferred embodiment, the acid buffer is an acetate buffer, which is acidified with aqueous hydrochloric acid to a pH in the range of 2.8 to 3.5, and then diluted with ethanol to form a buffer solution containing 35% (V) to 45% (V) ethanol.

The concentration of the acetate salt can be chosen in the range from 10mmol/l to 1mol/l, preferably in the range from 50mmol/l to 250 mmol/l.

Tangential flow filtration is characterized by the feed passing through the filtration membrane at a positive pressure (tangentially) relative to the permeate side. A portion of the material smaller than the membrane pore size passes through the membrane as permeate or filtrate; all other material is retained on the feed side of the membrane as retentate.

Suitable membranes are commercially available, for example under the trade name Pellicon from Merck Millipore^TMOr from Sartorius under the trade name Hydrosart^TMThe film of (1).

The process of the invention advantageously functions with a membrane having a molecular weight cut-off (MWCO) of less than or equal to 5kDA, preferably less than or equal to 2.5kDA, more preferably from 0.5 to 2.5kDA, even more preferably from 1.8 to 2.2 kDA.

The tangential flow filtration is performed at a transmembrane pressure of 0.5 to 10.0 bar, more preferably 1.0 to 4 bar, even more preferably 1.5 to 2.0 bar.

The flux, which represents the flow per unit area, is generally between 5 and 50l/h m²In the range of 9 to 15l/h m, preferably in the range of²Is selected within the range of (1).

The content of oligonucleotide in the acidic buffer solution is chosen between 1.0mg/l and 100.0mg/l, preferably between 5.0mg/l and 50.0 mg/l.

In another embodiment of the invention, the method further comprises the step of performing after removing the acid labile 5 'hydroxyl protecting group at the 5' -O-oligonucleotide terminus of the oligonucleotide:

a neutralization step comprising neutralizing the resulting retentate with a base by tangential flow filtration; and

a desalting step comprising washing the retentate obtained from the neutralization step by tangential flow filtration with water or a mixture of water and a polar protic or polar aprotic organic solvent; and

optionally freeze-drying the retentate obtained from the desalting step.

Suitable bases for neutralising the retentate are aqueous inorganic bases, such as aqueous alkali metal hydroxide solutions, such as aqueous sodium hydroxide solution or alkaline buffers.

The subsequent washing of the filtrate can be carried out with water or a mixture of water and a polar protic organic solvent selected from primary aliphatic alcohols such as methanol, ethanol or isopropanol, preferably ethanol.

Typically, the desalting step is carried out in a gradient, starting with a mixture of water and a polar protic organic solvent and ending with water.

The volume ratio of water to polar protic organic solvent in the mixture is generally between 1:1 and 6:1, preferably between 4:1 and 3: 2.

The parameters used for tangential flow filtration during neutralization, as well as the parameters in the desalting step, i.e. transmembrane pressure, permeate flow rate and membrane type, generally correspond to those described for the previous step, including removal of the acid-labile 5' hydroxyl protecting group.

After the desalting step, the retentate obtained may be subjected to a lyophilization step or further purification, such as to remove residual solvent from the oligonucleotides.

In the most preferred embodiment, the method is characterized in that

Removal of acid-labile 5 'hydroxy protecting groups at the ends of oligonucleotide 5' -O-oligonucleotides by tangential flow filtration with acidic buffer solution

A neutralization step comprising neutralizing the resulting retentate with a base by tangential flow filtration; and

a desalting step comprising washing the retentate obtained from the neutralization step by tangential flow filtration with water or a mixture of water and a polar protic or polar aprotic organic solvent; and

optionally freeze-drying the retentate obtained from the desalting step.

The deprotection, neutralization and desalting steps are sequential tangential flow filtration steps and can be automated and performed by the same software controlled apparatus without any manual intervention.

As described above, after the oligonucleotide is formed on the resin, it is usually cleaved from the resin with concentrated ammonia. In addition to the acid labile 5 'hydroxyl protecting group at the 5' -O-oligonucleotide end of the oligonucleotide, phosphate and protecting groups on the nucleotide base are also removed during this cleavage.

In a preferred embodiment, the method further comprises a step prior to removing the acid labile 5 'hydroxyl protecting group at the 5' -O-oligonucleotide terminus of the oligonucleotide. This prior step is intended to remove shorter length (non-5' -protected) truncates of the desired oligonucleotide.

Reversed-phase high performance liquid chromatography or anion exchange chromatography of the crude oligonucleotide obtained after cleavage from the resin and deprotection of the phosphate.

Anion exchange chromatography is based on the competitive interaction of the charged ions of the sample solution with the buffer medium used. This can be done using conventional commercially available anion exchange resins, preferably those with trimethylammonium functionalization. These phase materials can be obtained, for example, from GE Healthcare, Tosoh Bioscience, Bio-Rad or Merck. Particularly good results have been achieved with the anion exchange resin TSKgel Super Q-5PW (QAE) available from Tosoh Bioscience.

Reverse phase chromatography can be performed using conventional, commercially available phase materials, typically C8 or C18 phase materials, such as a modified silica gel adsorbent as the stationary phase and a suitable organic solvent (e.g., acetonitrile) and buffer (if applicable). Suitable modified silica gel type phase materials may be selected from Kromasil^TMC18、Kromasil^TMC8, YMC Triart C18, and YMC Triart C8.

Illustratively, the oligonucleotide may be selected from the group consisting of:

5'-(^MeC*)T*T*(^MeC*)t*t*c*t*a*t*c*t*a*(^MeC*)g*c*A*T*-3'

wherein represents a phosphorothioate bridge; A. g, T and^Mec (5-methylcytosine) is an LNA nucleoside monomer, and a, t, C, g are DNA nucleoside monomers.

The compounds disclosed herein have the following nucleobase sequences

SEQ ID No.1：cttctctatctacgcat'

Examples of the invention

Abbreviations:

ACN ═ acetonitrile

Ac₂Acetic anhydride (O ═ acetic anhydride)

CV is the column volume

DAC ═ dichloroacetic acid

DCM ═ dichloromethane

DMT-4, 4' -dimethoxytrityl

EtOH ═ ethanol

NaOAc ═ sodium acetate

NMI ═ N-methylimidazole?

Example 1

a. Synthesis of 5' - ()^MeC*)T*T*(^MeC*)t*t*c*t*a*t*c*t*a*(^MeC*)g*c*A*T*-3'

Use ofThe title compound was synthesized as "DMT-on" in a ratio of 1.9mmol to oligopilot-100. The synthesis parameters are given in table 1.

Table 1: forSynthesis parameters of OP-100.

b. Cleavage from solid support and RP-HPLC purification

The crude material was cleaved from the resin and deprotected by dissolving the dried resin in 30% aqueous ammonia (190mL) and stirring at 65 ℃ for 5 h. The solid support was filtered off and the aqueous solution was concentrated to about 60mL by rotary evaporation. The solution was further treated with H₂O was diluted to a final volume of 100mL and solid Na was added₂CO₃To obtain 50mM Na₂CO₃Solution (crude oligomer content of 124 mg/mL). A portion of the crude material (80mL) was purified by RP-HPLC according to the parameters in Table 2.

Table 2: RP-HPLC purification parameters.

c. DMT deprotection, desalination and concentration by tangential flow filtration

Fractions from RP purification were collected to yield 2.5L (2mg/mL of oligomer) and washed with H₂O diluted to a final volume of 4.2L. In a Hydrosart Sartocon cassette (0.1 m) equipped with two 2kD cut-offs²) Is/are as followsThe material was processed on a cross-flow machine to trigger DMT deprotection, concentration and tangential flow filtration. The program parameters are detailed in table 3.

Table 3:detailed parameters of the cross-flow procedure.

The solution obtained after washing was lyophilized to obtain 4.00g of the title product (42% overall yield based on synthesis scale, 88% UV purity).