阅读说明：本技术 寡核苷酸的混合物的分离分析方法 (Method for separating and analyzing mixture of oligonucleotides ) 是由 贵家润治 青木裕史 小木户谦 于 2018-06-21 设计创作，主要内容包括：本发明涉及通过液相色谱对寡核苷酸的混合物进行分离分析的方法,上述液相色谱使用了填充了在由交联有机高分子形成的多孔质粒子的表面固定二醇而成的填充剂的柱。根据该方法,与以硅胶作为基材的柱相比,能够以高灵敏度进行分离分析,并且可以将柱进行利用碱性溶液的洗涤。(The present invention relates to a method for separating and analyzing a mixture of oligonucleotides by liquid chromatography using a column packed with a filler in which a diol is immobilized on the surface of porous particles made of a crosslinked organic polymer. According to this method, separation and analysis can be performed with higher sensitivity than a column using silica gel as a base material, and the column can be washed with an alkaline solution.)

1. One method is a method of separating and analyzing a mixture of oligonucleotides by liquid chromatography using a column packed with a packing material in which a diol is immobilized on the surface of a porous particle made of a crosslinked organic polymer.

2. The method of claim 1, wherein the diol has a structure comprising a structure of formula (I),

R-O-CH₂-CH(OH)-CH₂(OH) (I)

wherein R represents a structural portion of the porous particle.

3. The method according to claim 1 or 2, wherein the crosslinked organic polymer is crosslinked polyvinyl alcohol.

4. The method according to any one of claims 1 to 3, wherein the mobile phase of the liquid chromatography is a mixture of a volatile saline solution and a water-soluble organic solvent.

5. The method according to any one of claims 1 to 4, wherein the components separated and eluted by the column are detected by an ultraviolet-visible light detector and/or a mass spectrometer.

6. The method according to any one of claims 1 to 5, wherein the liquid chromatography is carried out by gradient elution with a mobile phase.

7. The method according to any one of claims 1 to 6, comprising a step of washing the column by introducing an alkaline solution having a pH of 10.0 to 13.0 into the column before or after the liquid chromatography is performed.

8. The method of any one of claims 1 to 7, wherein the oligonucleotide is a synthetic oligonucleotide.

Technical Field

The present invention relates to a method for separating and analyzing a mixture of oligonucleotides by liquid chromatography.

Background

In recent years, interest in the application of synthetic oligonucleotides to the medical field has increased. Examples thereof include ribozymes, aptamers, antisense, and RNA interference (RNAi), which are called nucleotide drugs.

Such oligonucleotides are generally synthesized by the phosphoramidite method. The product in this method contains oligonucleotides having various chain lengths and impurities resulting from side reactions, in addition to the target oligonucleotide. Therefore, various methods for separating and analyzing synthetic oligonucleotides have been studied.

Methods employing hydrophilic interaction chromatography (HILIC) can be carried out with eluents containing low concentrations of volatile salts. Therefore, a mass spectrometer can be used as a detector, and highly sensitive analysis can be performed. In addition, when separation is performed for the purpose of separating oligonucleotides, it is also excellent in that the step of desalting and the like in the separation of oligonucleotides from an eluate is easy.

Non-patent document 1 describes a method using an Ascentis Silica column manufactured by シグマアルドリッ チ. The column was packed with a porous unmodified silica base material (pore diameter 12nm) having a diameter of 3 μm. It has been reported that 2-to 10-mer synthetic oligonucleotides are highly separated by a gradient method of reducing the acetonitrile concentration using an ammonium formate buffer/acetonitrile mixed solution as an eluent.

Non-patent document 2 describes a method using an Amide-80 column available from imperial ソ ー. The column had a filler in which an amide group-containing structure was bonded to a porous silica substrate (pore diameter: 8nm) having a diameter of 3 μm. It has been reported that synthetic oligonucleotides of 15-to 20-mer, which are phosphorothioated, are highly separated by applying a gradient of decreasing acetonitrile concentration by mixing the eluent with an aqueous ammonium formate solution/acetonitrile.

Disclosure of Invention

Problems to be solved by the invention

In a conventional column filled with a filler composed of a silica base material, the silica base material is dissolved, and therefore, the alkali solution cannot be passed through the column. Therefore, washing with a strong base, particularly an aqueous sodium hydroxide solution, effective for performance recovery by removing proteins, oligonucleotides, side reactions, and the like when the separation performance of the column is lowered due to adsorption of these, reduction of the residue to the elution fraction when continuously used, sterilization of the column and the apparatus, and the like, cannot be performed.

In addition, if further utilization of oligonucleotides in the medical field or the like is considered, a more excellent method for separation and analysis of oligonucleotides is desired.

Accordingly, an object of the present invention is to provide a method which enables washing with an alkaline solution and separation and analysis of oligonucleotides with high sensitivity.

Means for solving the problems

The present invention provides the following separation analysis method.

[1] One method is a method of separating and analyzing a mixture of oligonucleotides by liquid chromatography using a column packed with a packing material in which a diol is immobilized on the surface of a porous particle made of a crosslinked organic polymer.

[2] The method according to 1, wherein the diol has a structure represented by formula (I).

R-O-CH₂-CH(OH)-CH₂(OH)(I)

(wherein R represents a structural moiety of the porous particle.)

[3] The method according to 1 or 2, wherein the crosslinked organic polymer is crosslinked polyvinyl alcohol.

[4] The method according to any one of the above 1 to 3, wherein the mobile phase (eluent) in the liquid chromatography is a mixture of a volatile saline solution and a water-soluble organic solvent.

[5] The method according to any one of the above 1 to 4, wherein the components separated and eluted by the column are detected by an ultraviolet-visible light detector and/or a mass spectrometer.

[6] The method according to any one of the above 1 to 5, wherein the liquid chromatography is carried out by gradient elution of a mobile phase.

[7] The method according to any one of 1 to 6, comprising a step of washing the column by passing an alkaline solution having a pH of 10.0 to 13.0 through the column before or after the liquid chromatography is performed.

[8] The method according to any one of the above 1 to 7, wherein the oligonucleotide is a synthetic oligonucleotide.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, separation and analysis of oligonucleotides can be performed with high sensitivity as compared with a method using a column using a silica-based filler.

In addition, since the column can be washed with an alkaline solution, the column can be easily washed and sterilized, and further, the separation of the oligonucleotide for pharmaceutical purposes becomes easier.

Drawings

FIG. 1 is a chromatogram of the ultraviolet detection method (wavelength 260nm) and a mass spectrometry mass spectrum obtained in example 1.

FIG. 2 is a chromatogram of the ultraviolet detection method (wavelength 260nm) and a mass spectrometry mass spectrum obtained in comparative example 1.

Detailed Description

In one embodiment of the present invention, a method for separating and analyzing a mixture of oligonucleotides is a method for separating and analyzing a mixture of oligonucleotides by liquid chromatography, and a packing material in which a diol structure is fixed to the surface of a porous particle made of a crosslinked organic polymer is used as a packing material for a column for liquid chromatography.

The mixture of oligonucleotides is typically a reaction mixture obtained by synthesis of oligonucleotides. Examples thereof include nucleotide oligomers synthesized by successively reacting nucleotide monomers on a solid support and synthesized by the phosphoramidite method. The target chain length of the synthetic oligonucleotide ranges from 10-110 mer. In the method of the present invention, it is preferable to isolate 10 to 70-mer, and more preferably 19 to 62-mer as a sample.

The crosslinked organic polymer used as a base material of the filler for the column is preferably a crosslinked (meth) acrylic resin, a crosslinked polyvinyl alcohol (PVA), or the like. In the case of using a crosslinked (meth) acrylic resin, the monomer unit needs to contain a monomer unit having a hydroxyl group or a functional group capable of being converted into a hydroxyl group. Crosslinked polyvinyl alcohol is more preferable in that it can be used even under strongly alkaline conditions.

Crosslinked polyvinyl alcohol is obtained by copolymerizing vinyl acetate with a crosslinkable monomer having a plurality of unsaturated double bonds such as triallyl isocyanurate, and then saponifying the resultant. The mass ratio of the monomer composition of the copolymer is not particularly limited, and in order to exhibit sufficient hydrophilicity by the subsequent reaction treatment for introducing a diol group, the ratio of the crosslinkable monomer to the total monomers is preferably 90 mass% or less, more preferably 80 mass% or less, and still more preferably 70 mass% or less. In order to ensure practical mechanical strength when the porous particles are used as a filler for liquid chromatography, the ratio of the crosslinkable monomer to the total monomers is preferably 10% by mass or more, more preferably 20% by mass or more, and still more preferably 30% by mass or more. If the mechanical strength is insufficient, the filler may be deformed and the column may be clogged by the pressure generated when the mobile phase is transported, thereby exceeding the pressure range in which the apparatus can be used.

The density of the hydroxyl group of the crosslinked polyvinyl alcohol obtained by saponification is preferably in the range of 1.2mmol/g to 10.5mmol/g, more preferably in the range of 1.6mmol/g to 9.3mmol/g, and still more preferably in the range of 2.0mmol/g to 8.0 mmol/g. When the amount is within this range, glycidol for introducing a glycol can be sufficiently added to the particle surface, and an appropriate hydrophilicity can be imparted to the filler.

The density of the hydroxyl groups can be determined by calculating the mass increase of the porous particles generated by the reaction of the reagent reacting with the hydroxyl groups. For example, the method shown in the examples and the like can be preferably used.

The following suspension polymerization method is generally used as a method for obtaining the crosslinkable organic polymer as porous particles: an oil phase obtained by mixing a monomer, a non-polymerizable organic solvent compatible with the monomer, and a polymerization initiator is suspended in an aqueous phase to form oil droplets having a desired size, and the oil droplets are heated and stirred to obtain particles. In addition, a method of forming particles of a desired size by dropping an oil phase through a porous membrane typified by an SPG (Shirasu porous glass) membrane or a microchannel formed on a quartz substrate into an aqueous phase is used. In all particle formation methods, pores are formed by the volume occupied by the non-polymerizable organic solvent mixed with the oil phase. The oil phase granulated by the above-mentioned operation is then heated and stirred in the water phase to cause a polymerization reaction to proceed, thereby imparting strength as a crosslinkable polymer. After the polymerization reaction, the non-polymerizable organic solvent and the like are removed by washing with an organic solvent, and the porous body is obtained.

The particles are preferably spherical.

The particle size of the particles is preferably 1 to 30 μm as a volume average particle size in order to obtain sufficient separation performance and high sensitivity. Considering that excessive pressure rise is unlikely to occur, the volume average particle size is more preferably 3 to 10 μm, and particularly preferably 3 to 5 μm.

The volume average particle diameter can be measured by a coulter counter or an image analysis type particle size distribution meter. In order to obtain a desired particle size, sieve classification using a sieve or particle size control using an air classifier may be performed.

The size of the pores to be formed into a porous structure is preferably 3 to 30nm as an average pore diameter for achieving both separation performance and mechanical strength, and more preferably 10 to 25nm in terms of obtaining sufficient separation performance. If the pore diameter is too small, the specific surface area becomes small, and the ability to exhibit hydrophilic interaction may not be sufficiently obtained. If the pore diameter is too large, the mechanical strength is not maintained, and the particles may be broken by the pressure generated in the column.

The average pore diameter can be measured using a gas adsorption type specific surface area measuring instrument.

The filler for a column in one embodiment of the present invention has a structure in which a diol is fixed to the surface of the porous particles made of the crosslinked organic polymer. The structure of a preferred diol is one in which 1 hydroxyl group is bonded to an adjacent carbon atom. More preferably, the particles have a structure represented by formula (I) on the surface thereof.

R-O-CH₂-CH(OH)-CH₂(OH)(I)

(wherein R represents a structural moiety of the porous particle.)

The amount of the diol structure immobilized on the surface of the particle is preferably 0.2 to 8mmol/g, more preferably 0.5 to 4mmol/g, based on the mass of the porous particle.

This structure can be produced by reacting a compound having a hydroxyl group and an epoxy group, such as glycidol, with porous particles formed of a crosslinked organic polymer having a hydroxyl group on the surface. The reaction of glycidol can be carried out both under basic conditions and under acidic conditions.

As a mobile phase (eluent) used for liquid chromatography, a mixed solution of a volatile saline solution and a water-soluble organic solvent is suitably used. Examples of the volatile salt include ammonium formate, ammonium acetate, and ammonium hydrogen carbonate. The water-soluble organic solvent is preferably an alcohol compound or nitrile compound having 1 to 3 carbon atoms. A preferred mixture is a combination of aqueous ammonium formate and acetonitrile.

The concentration of the volatile saline solution is not particularly limited, but is preferably 1 to 100mM, more preferably 5 to 70mM, and particularly preferably 20 to 50 mM. The pH of the volatile saline solution is not limited as long as the oligonucleotide is eluted, and is preferably 5.0 to 9.0, more preferably 6.5 to 7.5.

The mixing ratio of the volatile aqueous salt solution and the water-soluble organic solvent is not particularly limited. The mixing ratio is expressed using the volume before mixing. The ratio of the water-soluble organic solvent can be increased to such an extent that problems such as precipitation of salts and solubility of the oligonucleotide do not occur. In addition, the ratio of the water-soluble organic solvent can be reduced to such an extent that the hydrophilic interaction sufficiently functions. In general, the ratio of the water-soluble organic solvent is preferably 30% to 90%.

Typical separation analysis methods of the present invention are as follows.

The column packed with the packing material is set in a liquid chromatograph, and an appropriate eluent (mobile phase) is introduced in advance to equilibrate the column. Then, a solution obtained by dissolving the mixture of oligonucleotides as the sample in the eluent is injected and liquid chromatography is performed.

A so-called gradient elution method in which the concentration of the water-soluble organic solvent in the eluent to be passed through is gradually decreased may also be employed. By performing the gradient elution, impurities contained in the oligonucleotide synthesis process can be more easily separated from the target oligonucleotide. Then, the eluent which had passed through the column during the re-equilibration was passed through the column to equilibrate the column.

The column temperature is preferably 25 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 40 to 50 ℃ in the measurement.

As the apparatus for liquid chromatography, commercially available products can be used. Examples thereof include Nexera (registered trademark) manufactured by Shimadzu corporation, and Acquisty (registered trademark) manufactured by ウォーターズ corporation.

As the column, any commercially available column can be used as long as it is within the above-mentioned predetermined range. For example, Shodex (registered trademark) HILICPak (registered trademark) VN-502D (inner diameter 2.0 mm. times. length 150mm, particle diameter 5 μm) manufactured by Showa Denko K.K. can be used.

Examples of the detection by liquid chromatography include an ultraviolet/visible light detector (UV/Vis detector), a Mass Spectrometer (MS) using a mass spectrometer, and the like. In the case of an ultraviolet/visible light detector, the detection wavelength is, for example, 260 nm.

The uv/vis detectors can be used primarily for quantitative analysis, the mass spectrometers can be used primarily for qualitative analysis, or a combination of these can be used.

The filler used in one embodiment of the present invention has such a property as to be stable under alkaline conditions. Therefore, after performing separation analysis by liquid chromatography, the column can be washed by passing an alkaline solution therethrough. This allows proteins, oligonucleotides, side-products, and the like that have been mixed into the column and adsorbed to the packing material to be decomposed and removed, or allows the interior of the column to be sterilized, and is therefore suitable when liquid chromatography is performed for the purpose of separating oligonucleotides.

The introduction of the alkaline solution may be performed before or after the liquid chromatography, as required.

A filler using silica gel as a base material is generally not usable under a condition of pH 8 or more because silica gel dissolves. So that alkaline solutions cannot be used. Since the filler used in one embodiment of the present invention is formed of a crosslinked organic polymer, an alkaline solution having a pH corresponding to the properties of the crosslinked organic polymer to be used can be used. In particular, since a filler comprising crosslinked polyvinyl alcohol as a base material is not generally deteriorated by an alkaline solution used, the alkaline solution can be used for column washing or the like.

The alkaline solution used for washing is not particularly limited as long as it is a solution exhibiting alkalinity of pH10.0 to 13.0. In consideration of the washing effect and economy, an aqueous solution of 0.01 to 0.1M sodium hydroxide is suitable.

The reason why high separation can be obtained when a filler having a diol structure immobilized on the surface of porous particles formed of a crosslinked organic polymer is used is not clear as compared with a filler using silica gel, but in a column using silica gel, it is considered that a weak ionic interaction derived from silanol acts simultaneously, and the like, and this is considered to be a possibility of causing deterioration of separation in hydrophilic interaction chromatography using a difference in hydrophilicity.