WO2017115652A1 - Separation and analysis method - Google Patents

Abstract

In order to use synthetic oligonucleotides in pharmaceutical product applications, a method is provided for advanced separation and analysis of impurities such as oligonucleotides of incomplete length, said impurities being included in oligonucleotide reaction mixtures. The method separates and analyses 31-mer or longer synthetic oligonucleotides, by reversed-phase chromatography using an ion-pair reagent. The ion-pair reagent includes a dialkyl ammonium salt.

Description

Separation analysis method

The present invention relates to a method for advanced separation analysis of synthetic oligonucleotides.
This application claims priority based on Japanese Patent Application No. 2015-256976 filed in Japan on December 28, 2015, the contents of which are incorporated herein by reference.

Synthetic oligonucleotides (synthetic oligonucleic acids) are used in various molecular biological experiments such as polymerase chain reaction primers, diagnostic probes, single nucleotide polymorphism (SNP) detection, and DNA sequencing. Synthetic oligonucleotides are synthesized primarily by a continuous reaction using phosphoramidite nucleic acid monomers on a solid support.

In recent years, there has been an increasing interest in the application of synthetic oligonucleotides to the medical field. Examples include ribozymes, aptamers, antisense, and RNA interference (RNAi), which are called nucleic acid pharmaceuticals.

Various factors such as binding strength, target specificity, serum stability, nuclease resistance, and cell permeability need to be considered for stable transport of synthetic oligonucleotides in the body. Examples of techniques for improving these include skeletal modifications such as modification of phosphorothioate and methylphosphonate, use of locked nucleic acid (LNA), and the like.
While these skeletal modifications impart nuclease resistance, they can exhibit non-sequence-specific effects, reduced permeation performance, and even cytotoxicity.

Oligonucleotide synthesis methods are optimized to the level that does not require purification when used for PCR primers, but more sophisticated purification and analysis are required for application to pharmaceuticals. In particular, long-chain DNA and modified / backbone variants are prone to unreacted coupling and side reactions during synthesis. Thus, a synthetic oligonucleotide reaction mixture contains mainly incomplete oligonucleotides or incomplete oligonucleotides. For example, a fragment in which a nucleic acid is deleted from a target oligonucleotide, often called an nx fragment (n is the number of target nucleotides (nucleic acids), x is a number deleted, n, x are positive integers, and n> x, For example, n-1, n-2, etc.) and n + 1 fragments incorporated in duplicate are included as impurities. Furthermore, incomplete or inaccurately deprotected fragments and isomers having partial chirality are also included (Patent Document 1).

Generally, gel electrophoresis, gel filtration chromatography, reverse phase chromatography, and ion exchange chromatography are selected as methods for separating highly synthetic oligonucleotides. Chromatography is excellent in operability, and in particular, reverse phase chromatography that does not require desalting is widely used.
In reverse-phase chromatography of synthetic oligonucleotides, an ion-pairing reagent having a positive dissociation group and a hydrophobic functional group in the molecule is generally used (Patent Document 1). The ion-pairing reagent forms a neutral ion pair with the negatively dissociated oligonucleotide, increasing the retention in the reverse-phase filler, and the difference in hydrophobicity between long-chain oligonucleotides and modified / backbone variants In many cases, the separation analysis of oligonucleotides and impurities of interest is small.

For example, Non-Patent Document 1 and Non-Patent Document 2 describe a method by reverse phase chromatography using Hydroshare C18 manufactured by YMC. Hydrosphere C18 is a particulate filler in which octadecyl groups are bonded to a silica substrate having a diameter of 12 nm and a diameter of 3 μm. By adding an ion pair reagent such as triethylamine or dibutylamine to an acetic acid buffer as an eluent, it is possible to highly separate dimer to 30mer synthetic oligonucleotides.

Also, Non-Patent Document 3 describes a method by reverse phase chromatography using XBridge OST C18 manufactured by WATERS. XBridge OST C18 is a particle type filler in which octadecyl groups are bonded to an ethylene-crosslinked hybrid silica base material having a pore diameter of 130 mm and a diameter of 2.5 μm. By adding triethylamine as an ion-pairing reagent to acetic acid or HFIP as an eluent, it is possible to highly separate 10 to 60-mer synthetic oligonucleotides.

International Publication No. 2006/107775

YMC DATA SHEET F21018A YMC APPLICATION DATA COLLECTIONS (5) (P.39) WATERS XBRIDGE OST C18 METHOD GUIDELINES

An object of the present invention is to provide a technique for highly separating and analyzing impurities such as incomplete length oligonucleotides contained in an oligonucleotide reaction mixture in order to apply synthetic oligonucleotides to pharmaceuticals.

As a result of intensive studies on the precise separation of the oligonucleotide reaction mixture, the present inventors have highly separated a 31-mer or more synthetic oligonucleotide by reverse phase chromatography using an ion-pairing reagent containing a dibutylammonium salt. The inventors have found that analysis is possible and have arrived at the present invention. The present invention is as follows.

[1] A method for separating and analyzing 31-mer or more synthetic oligonucleotides by reverse phase chromatography using an ion-pairing reagent, wherein the ion-pairing reagent contains a dialkylammonium salt.
[2] The method for separation analysis according to [1], wherein the dialkylammonium salt is a dibutylammonium salt.
[3] The reverse phase chromatography is column chromatography, and the separation and analysis method is performed by equilibrating with a first eluent containing the ion pair reagent and water or the ion pair reagent, water and a water-soluble organic solvent in the column. A step of dissolving the synthetic oligonucleotide in the first eluent, preparing a sample for analysis, and injecting the analysis sample into the column equilibrated with the sample in the equilibration step, And a step of selectively eluting the synthetic oligonucleotide using a second eluent having a higher content of the water-soluble organic solvent than the first eluent. The separation analysis method according to [1] or [2], which is characterized.
[4] The separation analysis method according to any one of [1] to [3], wherein the column contains a non-porous filler.

According to the present invention, there is provided a method for separating and analyzing synthetic oligonucleotides, which can be separated and analyzed to a high degree by reverse phase chromatography even for long-chain synthetic oligonucleotides of 31-mer or more.

2 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Example 1. FIG. 2 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Example 2. FIG. 2 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Comparative Example 1. 6 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Comparative Example 2. 6 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Comparative Example 3.

Hereinafter, the best mode for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

The method for separating and analyzing a synthetic oligonucleotide according to the present invention is a method for highly separating and analyzing impurities contained in a synthetic process of a 31-mer or more synthetic oligonucleotide by reverse phase chromatography using an ion-pairing reagent containing a dibutylammonium salt. It is a technique to do.

The method for separating and analyzing a synthetic oligonucleotide according to one embodiment of the present invention is preferably reverse phase column chromatography. In that case, the separation analysis method of one embodiment of the present invention includes a step of equilibrating a column with a first eluent containing the ion-pairing reagent, water, and a water-soluble organic solvent; and the synthetic oligonucleotide in the first eluent And preparing a sample for analysis; injecting the analytical sample into the column equilibrated in the equilibration step and fixing the sample to the column; and at least from the first eluent And a step of selectively eluting the synthetic oligonucleotide using a second eluent containing a large amount of the water-soluble organic solvent.

The “synthetic oligonucleotide” is generally synthesized by continuously reacting a nucleic acid monomer on a solid phase carrier, and a nucleic acid oligomer synthesized by a phosphoramidite method is preferable. The target chain length of the synthetic oligonucleotide is 10 to 110 mer, preferably 10 to 70 mer, more preferably 31 to 62 mer.

“Reverse Phase Chromatography” uses the difference in hydrophobicity of the target object to determine the movement speed due to the difference in partitioning between the hydrophobic stationary phase (filler) and the non-hydrophobic mobile phase (eluent). Reverse phase partition chromatography based on the difference. Depending on the form of the stationary phase (packing agent) and the mobile phase (eluent), for example, there is column reverse phase chromatography.

The “ion pair reagent” is a reagent having a hydrophobic functional group that forms an ion pair with an ionic target object, and increases the retention of the formed target object in the hydrophobic column and improves the separation.
An ion-pairing agent having a positive dissociation group and a hydrophobic functional group can be used for nucleotide (nucleic acid) separation. Typical ion pair reagents include trialkylammonium salts of organic or inorganic acids, for example, triethylammonium acetate described in Non-Patent Document 2 above.

“Impurity contained in the synthesis process” means an impurity having a molecular weight different from that of the target synthetic oligonucleotide, for example, an incomplete length oligonucleotide (oligonucleic acid) often referred to as an nx fragment (n is the target nucleotide (nucleic acid ) Number, x is a missing number, n, x is a positive integer, n> x, for example n-1, n-2, etc.), n + 1 fragment incorporated in duplicate, acetyl group, isobutyryl group, cyanoethyl Insufficient backbone modifications such as oligonucleotides with insufficient deprotection of functional groups that protect nucleic acid monomers such as groups and methyl groups, incomplete oligonucleotides such as depurines and depyrimidines, and phosphodiester impurities in phosphorothioates These include oligonucleotides and isomers having partial chirality.

The method for separating and analyzing a synthetic oligonucleotide according to one embodiment of the present invention is as follows. That is, in column reverse phase chromatography, an eluent containing an ion-pairing reagent containing dibutylammonium salt is passed in advance through a column containing a packing with a hydrophobic functional group immobilized thereon, and equilibrated. The solution in which the synthetic oligonucleotide is dissolved is passed through to bind the synthetic oligonucleotide into the column. Subsequently, the impurities contained in the synthetic oligonucleotide synthesis process and the target synthetic oligonucleotide are separated and eluted by a gradient that gradually increases the concentration of the organic solvent in the eluent to be passed, that is, a so-called gradient. Pass the eluent that was passed again during equilibration to regenerate the column.

As the filler according to one embodiment of the present invention, a filler composed of silica or a polymer having a hydrophobic functional group fixed can be used. Silica is particularly preferable as the filler. The shape is preferably non-porous particles, and the particle size is preferably 3 μm or less, more preferably 2 μm or less. Furthermore, the hydrophobic functional group to be fixed is preferably a butyl group, a phenyl group, an octyl group or an octadecyl group, and particularly preferably an octadecyl group.

The eluent of one embodiment of the present invention is not particularly limited except that an ion-pairing reagent containing a dibutylammonium salt is used, and an aqueous eluent containing a salt of dibutylamine and an acid and a C1 to 3 alcohol or nitrile. It is preferable to combine with an organic solvent of the system. The acid is preferably carbonic acid, acetic acid, formic acid, trifluoroacetic acid or propionic acid. A combination of an aqueous eluent containing dibutylamine and acetic acid and acetonitrile is most preferred. The salt concentration of the aqueous eluent is not particularly limited, but is preferably 1 to 100 mM, more preferably 5 to 50 mM, and particularly preferably 10 to 20 mM. Further, the pH of the aqueous eluent is not limited as long as it can be bound to and eluted from the column, preferably 6 to 8, and more preferably 6.5 to 7.5.

As the ion pair reagent of one embodiment of the present invention, for example, an ion pair reagent containing a dibutylammonium salt and an alkylammonium salt other than the dibutylammonium salt, for example, a triethylammonium salt can be used.
In that case, the ratio of the dibutylammonium salt to the ion-pairing reagent is preferably 30% mol or more, more preferably 40%, and even more preferably 50% or more. Most preferably, the ion pair reagent is a dibutylammonium salt.

The column temperature during the separation analysis of one embodiment of the present invention is preferably 20 to 100 ° C, more preferably 30 to 80 ° C, and most preferably 40 to 70 ° C.

For the method for separating and analyzing a synthetic oligonucleotide according to one embodiment of the present invention, a commercially available reverse phase chromatography apparatus can be used, and examples include analytical apparatuses such as Shimadzu Corporation Prominence and Waters Acquity.

As the column used in the method for separating and analyzing a synthetic oligonucleotide according to one embodiment of the present invention, a commercially available analytical or separation reversed phase column can be used. For example, MICRA (registered trademark) NPS ODS-1 (EPROGEN) Examples include a column for analysis or fractionation such as a particle size of 1.5 μm, 33 × 4.6 mm ID) Waters X Bridge OST C18 (particle size 1.7 μm, pore size 135 mm, 50 × 4.6 mm ID).

Examples of the detection method used in the method for separating and analyzing a synthetic oligonucleotide according to one embodiment of the present invention include a detector using ultraviolet / visible absorption such as a synthetic oligonucleotide, a detector using a mass spectrum of a synthetic oligonucleotide, and the like. Can be mentioned. In the case of an ultraviolet / visible absorption detector, the detection wavelength is, for example, 254 nm.
By such a method, the synthetic oligonucleotide can be highly separated and analyzed.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
Example 1
Separation analysis of synthetic oligonucleotides using ion-pairing reagents containing dibutylammonium salts

(1) Equilibration step of column EPROGEN's MICRA (registered trademark) NPS ODS-1 (particle size: 1.5 μm, 33 × 4.6 mm ID) was used for the column. A system controller SCL-10AVP, liquid feeding unit LC10-AD, autosampler SIL-10ADVP, column oven CTO-AC10VP, detector SPD-M10A (manufactured by Shimadzu Corporation) are used as chromatographs, column temperature is 40 ° C., and acetonitrile is used as an eluent. A dibutylammonium acetate aqueous solution to which was added was used. Acetonitrile was added to 10 mM dibutylammonium acetate aqueous solution (pH 7.0), and 9.6 mM dibutylammonium acetate aqueous solution containing acetonitrile having a final concentration of 4.5 v / v% was prepared as the first eluent. The column was equilibrated by passing 10 mL or more. The mobile phase flow rate was 1.0 mL / min. NPS ODS-1 is a column containing a non-porous silica particle filler to which octadecyl groups are bonded.

(2) Preparation step of sample for analysis Synthetic oligonucleotide 62-mer purchased from Eurofin Genomics (trade name: standard oligo 50 nmol scale product salt-free grade; SEQ ID NO: 1 (5′-CATGAGAAGTATGAACAACAGCCCCACACCGGCTGTTTGTCATACTTTCTCATGGGTTCTTCGGAA-3 ′) The sample for analysis was prepared such that the concentration of the synthetic oligonucleotide was 0.1 mg / mL by dissolving in 10 mM dibutylammonium acetate aqueous solution.

(3) Synthetic oligonucleotide binding step 10 uL of the prepared analytical sample was injected into the column equilibrated in (1), and the synthetic oligonucleotide was bound to the column using the first eluent. The mobile phase flow rate was 1 mL / min.

(4) Separation and Elution Step of Synthetic Oligonucleotide and Impurities The mobile phase is a first stage from the first eluent, and the linear gradient has a gradient that increases the final concentration of acetonitrile to 18 v / v% in the first 7.5 minutes. , Followed by a two-stage gradient including a second-stage gradient with a gradient that increased the final concentration of acetonitrile to 31.5 v / v% in 32.5 minutes. Synthetic oligonucleotides and impurities bound to the column were separated and eluted by a linear gradient gradient that gradually increased the acetonitrile concentration.

The detector is an ultraviolet / visible (uv / vis) detector, and the detection wavelength is 254 nm.

As a result, as shown in FIG. 1, a peak of the synthetic oligonucleotide was observed at a retention time of 29 minutes, and a peak of what was considered to be an impurity was observed before that.

(Example 2)
Separation and analysis of synthetic oligonucleotides using an ion-pairing reagent containing dibutylammonium salt (1) Column equilibration step The same procedure as in Example 1 was performed.

(2) Preparation step of sample for analysis Synthetic oligonucleotide 62-mer purchased from Eurofin Genomics (trade name: standard oligo 50 nmol scale product salt-free grade; SEQ ID NO: 1 (5′-CATGAGAAGTATGACAACAGCCCCACACCGGCTGTTTCATGTGTCTCTTCGAGA-3 ′) 40-mer (trade name: standard oligo 50 nmol scale product salt-free grade; SEQ ID NO: 2 (base sequence represented by 5′-CCACACCCGCGTTGTCATACTTCTCCATGGTTCTTCGGAA-3 ′)), 80-mer (trade name: Standard oligo 50 nmol scale salt-free grade; SEQ ID NO: 3 (5′-GTT CATGTTGTTGGGATTGAGTTTTGAACTCGGCAACAAGAAACTGCCTGAGTTACATCAGTCGGTTTTCGTCGAGGGC-3 ') nucleotide sequence represented by), 100-mer (trade name: Standard oligo 50nmol-scale product salt-free grade; SEQ ID NO: 4 (5'-GCTGATCTGTGGCTTAGGTAGTTTCATGTTGTTGGGATTGAGTTTTGAACTCGGCAACAAGAAACTGCCTGAGTTACATCAGTCGGTTTTCGTCGAGGGC-3') nucleotide sequence represented by), 110-mer (trade name: standard oligo 50 nmol scale product salt-free grade; SEQ ID NO: 5 (5′-TGCTCGCTCAGCCTGATCTGTGGC The nucleotide sequence) represented by TAGGTAGTTTCATGTTGTTGGGATTGAGTTTTGAACTCGGCAACAAGAAACTGCCTGAGTTACATCAGTCGGTTTTCGTCGAGGGC-3 ') was dissolved in 10mM dibutyl ammonium acetate aqueous solution, a final concentration of synthetic oligonucleotides each chain length were prepared samples for analysis to be 0.01 mg / mL.

(3) Synthetic oligonucleotide binding step The procedure was performed in the same manner as in Example 1 except that the injection amount of the prepared analytical sample into the column was 20 uL.

(4) Separation and elution step of synthetic oligonucleotide and impurities Example except that the final concentration of acetonitrile was increased to 42.3 v / v% in the second stage gradient of 58.5 minutes so as not to change the gradient. 1 was carried out.

The detector was implemented in the same manner as in Example 1.

As shown in FIG. 2, the peak of the synthetic oligonucleotide 40-mer at the retention time of 24 minutes, the peak of the synthetic oligonucleotide 62-mer at the retention time of 29 minutes, and the synthetic oligonucleotide 80-mer at the retention time of 32 minutes. A peak of the synthetic oligonucleotide 100-mer was observed at a retention time of 35 minutes, and a peak of the synthetic oligonucleotide 110-mer was observed at a retention time of 37 minutes.

(Comparative Example 1)
Separation and analysis of synthetic oligonucleotide using triethylammonium salt as ion-pairing reagent. Except that the eluent used in the column equilibration step (1) of Example 1 was 10 mM triethylammonium acetate (pH 7.0). Performed as in Example 1.
A synthetic oligonucleotide peak was observed at a retention time of 8.5 minutes. However, the retention on the column is weak, and the impurity peak detected before the main peak as seen in Example 1 cannot be separated, and the separation of the synthetic oligonucleotide is insufficient. It was impossible.
The results are shown in FIG.

(Comparative Example 2)
Separation and analysis of synthetic oligonucleotide using triethylammonium salt as ion-pairing reagent The eluent used in the column equilibration step (1) of Example 1 was 100 mM triethylammonium acetate (pH 7.0). The linear gradient of the binding step (3) was carried out in the same manner as in Example 1 except that the linear gradient was increased to 30% in 30 minutes to increase the final concentration of acetonitrile to 31.5 v / v%.
A synthetic oligonucleotide peak was observed at a retention time of 15 minutes. However, it could not be separated from the impurity peak detected before the main peak as seen in Example 1, the synthetic oligonucleotide was insufficiently separated, and high-level separation analysis was impossible.
The results are shown in FIG.

(Comparative Example 3)
Separation and analysis of synthetic oligonucleotide using triethylammonium salt as ion-pairing reagent. Except that the eluent used in the column equilibration step (1) of Example 2 was 10 mM triethylammonium acetate (pH 7.0). Performed as in Example 2.
A synthetic oligonucleotide peak was observed at a retention time of 8.5 minutes. However, the retention on the column was weak, and the five main peaks as seen in Example 2 could not be separated and eluted together. Therefore, the separation of the synthetic oligonucleotide was insufficient and advanced separation analysis was impossible.
The results are shown in FIG.

Claims (4)

A method for separating and analyzing 31-mer or more synthetic oligonucleotides by reverse-phase chromatography using an ion-pairing reagent,
A separation analysis method, wherein the ion pair reagent contains a dialkylammonium salt. The method for separation and analysis according to claim 1, wherein the dialkylammonium salt is a dibutylammonium salt. The reverse phase chromatography is column chromatography;
The separation analysis method includes:
Equilibrating the column with a first eluent containing the ion pair reagent and water or the ion pair reagent, water and a water-soluble organic solvent;
Dissolving the synthetic oligonucleotide in the first eluent to prepare a sample for analysis;
Injecting the analytical sample into the column equilibrated in the equilibration step and fixing the sample to the column;
The method further comprises the step of selectively eluting the synthetic oligonucleotide using a second eluent having a higher content of the water-soluble organic solvent than the first eluent. The separation analysis method according to 2. The separation analysis method according to any one of claims 1 to 3, wherein the column contains a non-porous packing material.

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