Ion pair liquid chromatography analysis method for oligonucleotide diastereoisomer without DMT removal

Abstract

The invention provides an ion pair liquid chromatography analysis method for an oligonucleotide diastereoisomer without DMT removal, and relates to the technical field of instrument analysis. The analysis method comprises the following steps: detecting a sample solution containing the DMT-free oligonucleotide diastereoisomers through an ion pair reversed-phase ultra-high performance liquid chromatograph to obtain the number and separation condition of the DMT-free oligonucleotide diastereoisomers; wherein the mobile phase A for detection comprises dibutylammonium acetate, acetonitrile and water, and the mobile phase B comprises dibutylammonium acetate, acetonitrile and water. According to the method, the ion pair reversed-phase ultra-high performance liquid chromatography is adopted for analysis, the method has the advantages of being good in component peak separation effect, high in analysis precision, easy and convenient to operate and the like, and the purpose of effectively controlling the quality of the oligonucleotide DMT-on diastereoisomer is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of instrumental analysis, in particular to a method for ion-pair liquid chromatography analysis of non- enantiomeric oligonucleotide without removing DMT (dimethoxytrityl). BACKGROUND

[0002] Synthetic oligonucleotides are usually chemically modified to enhance their ability to resist endonuclease and exonuclease degradation in vivo. In addition, appropriate modifications can also improve their cellular uptake efficiency and target binding properties. One of the widely used modification methods is to modify the phosphate bond between nucleotides. For example, replacing one of the non-bridging oxygen atoms in the phosphate group with a sulfur atom forms a phosphorothioate bond (i.e. phosphorothioate structure). This modification introduces a chiral center in the phosphate group, resulting in the formation of 2z non- enantiomeric isomers (z is the number of phosphorothioate modification sites). Because these isomers are difficult to completely separate, phosphorothioate oligonucleotides usually exhibit broader chromatographic peaks than their natural phosphodiester counterparts in chromatographic analysis. The existing separation technology is not sufficient to effectively separate thousands of non- enantiomeric isomers. Therefore, there is an urgent need to develop a new method that can inhibit the separation of phosphorothioate oligonucleotide non- enantiomeric isomers while retaining the separation ability between full-length phosphorothioate oligonucleotides (n) and shorter synthetic impurities (such as n-1, n-2…n-x, where x represents the number of truncated nucleotides).

[0003] DMT (dimethoxytrityl) is a commonly used protecting group in DNA chemical synthesis, which effectively prevents unnecessary polymerization of nucleosides by covalently binding to the 5'-hydroxyl group of nucleotides. During the synthesis process, the DMT group can be removed by treating with trichloroacetic acid (TCA) in dichloromethane, thereby exposing the free 5'-hydroxyl group for subsequent condensation reactions.

[0004] In ion-pair reversed phase (IPRP) liquid chromatography, the retention mechanism is complex and can be described by electrostatic theory. The core lies in the interaction between the positively charged ion-pair reagent (such as alkylamine) and the negatively charged (phosphorothioate) phosphate group. The neutral complex formed by the two can further be adsorbed by the hydrophobic stationary phase, thereby producing a retention behavior similar to ion exchange. This also explains why the retention time of the target analyte usually extends with the increase of the hydrophobicity of the ion-pair reagent (such as alkylamine).

[0005] In view of the above, the present application is proposed. SUMMARY

[0006] The present application aims to provide a method for analyzing non-enantiomeric oligonucleotide ions without DMT by ion pair liquid chromatography. The analysis method of the present application uses ion pair reversed-phase ultra-high performance liquid chromatography (IPRP-UHPLC) method for analysis, which has the advantages of good component peak separation effect, high analysis precision, simple operation, etc., and achieves the purpose of effectively controlling the quality of oligonucleotides.

[0007] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted: In a first aspect, the present application provides a method for analyzing non-enantiomeric oligonucleotide ions without DMT by ion pair liquid chromatography, which comprises: The sample solution containing non-enantiomeric oligonucleotide ions without DMT is detected by an ion pair reversed-phase ultra-high performance liquid chromatograph to obtain the number and separation of non-enantiomeric oligonucleotide ions without DMT; Wherein, the mobile phase A for detection comprises dibutylammonium acetate, acetonitrile and water, and the mobile phase B comprises dibutylammonium acetate, acetonitrile and water.

[0008] Further, the oligonucleotide without DMT is an 18 nt oligonucleotide sample without 5'-DMT deprotection.

[0009] Further, the sample solution containing non-enantiomeric oligonucleotide ions without DMT is prepared by the following steps: The oligonucleotide without DMT is dissolved in water to obtain a sample solution; Further, the concentration of the sample solution is 0.5-1.5 mg / mL.

[0010] Further, in the mobile phase A, the concentration of dibutylammonium acetate is 4-6 mM, and the volume percentage content of acetonitrile is 10-20%.

[0011] Further, in the mobile phase B, the concentration of dibutylammonium acetate is 4-6 mM, and the volume percentage content of acetonitrile is 80-90%.

[0012] Further, the pH of the mobile phase A and the mobile phase B is independently 7.0-8.0.

[0013] Further, the gradient elution conditions of the detection are as follows: 0 min, mobile phase A: 60-80%, mobile phase B: 20-40%; 2 min, mobile phase A: 60-80%, mobile phase B: 20-40%; 47 min, mobile phase A: 30-50%, mobile phase B: 50-70%; 50 min, mobile phase A: 60~80%, mobile phase B: 20~40%; 55 min, mobile phase A: 60~80%, mobile phase B: 20~40%.

[0014] Further, the gradient elution condition of the detection is as follows: 0 min, mobile phase A: 70%, mobile phase B: 30%; 2 min, mobile phase A: 70%, mobile phase B: 30%; 47 min, mobile phase A: 40%, mobile phase B: 60%; 50 min, mobile phase A: 70%, mobile phase B: 30%; 55 min, mobile phase A: 70%, mobile phase B: 30%.

[0015] Further, the chromatographic column used in the detection is an OST BHE C18 chromatographic column.

[0016] Further, the specification parameters of the chromatographic column used in the detection include: the pore size inside the filler particles is 120~140 Å, the particle size is 1.5~2.0 µm, the inner diameter is 1.5~2.5 mm, and the column length is 100~150 mm.

[0017] Further, the chromatographic column used in the detection is a Waters ACQUITY Premier OST BHE C18 chromatographic column, with a specification of 130 Å, 1.7 µm, 2.1×100 mm.

[0018] Further, the wavelength used in the detection is 250~270 nm.

[0019] Further, the flow rate of the detection is 0.1~0.2 mL / min.

[0020] Further, the injection volume of the detection is 5~15 µL.

[0021] Further, the column temperature of the detection is 20~30℃.

[0022] Further, the temperature of the injection tray of the detection is 5~15℃.

[0023] Further, after the preparation of the mobile phase A and the mobile phase B, the following post-processing steps are required: The mobile phase A and the mobile phase B are respectively filtered through a microporous membrane and then degassed by ultrasonic.

[0024] Further, the pore size of the microporous membrane is 0.20~0.25 µm.

[0025] Further, the power of the ultrasonic degassing is 100-300 W, and the time of the ultrasonic degassing is 1-10 min.

[0026] Compared with the prior art, the present application has the following beneficial effects: The analysis method of the present application adopts ion pair reversed phase ultra-high performance liquid chromatography (IPRP-UHPLC) method to analyze, under specific mobile phase conditions, high-precision separation of DMT-on oligonucleotide diastereoisomers, achieving the purpose of effectively controlling the quality of oligonucleotides. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0028] Figure 1 The figure of the analysis sample detection results provided for Example 1.

[0029] Figure 2 The figure of the analysis sample detection results provided for Example 2.

[0030] Figure 3 The figure of the analysis sample detection results provided for Example 3.

[0031] Figure 4 The figure of the analysis sample detection results provided for Example 4.

[0032] Figure 5 The structure diagram of the sample to be tested provided for Examples 1-4. DETAILED DESCRIPTION

[0033] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Clear indications to the contrary are necessary to overcome the common meaning of the term when it appears alone or can be defined in accordance with its conventional usage from proper context. In this application, unless indicated otherwise, the use of "or" means "and / or". Furthermore, the use of the term "including" as well as other forms such as "include", "includes" for claiming consists of, is non-limiting.

[0034] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0035] The technical solutions of the present application will be described clearly and completely below in connection with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0036] In a first aspect, the present application provides a method for analyzing non- enantiomeric isomers of DMT-on oligonucleotide by ion-pair liquid chromatography, the method comprising: dissolving the DMT-on oligonucleotide in water to obtain a sample solution; detecting the sample solution by ion-pair reversed-phase ultra-high performance liquid chromatography to obtain the number and separation of non-enantiomeric isomers of DMT-on oligonucleotide; wherein the mobile phase A comprises dibutylammonium acetate, acetonitrile and water, and the mobile phase B comprises dibutylammonium acetate, acetonitrile and water.

[0037] It should be noted that the present application aims to systematically investigate the effects of different chromatographic parameters on the separation behavior of non- enantiomeric isomers of DMT-on oligonucleotide in ion-pair systems. The specific goals include: (i) screening ion-pair systems that can promote the separation of non- enantiomeric isomers to support the analysis needs of partially sulfurized DMT-on oligonucleotide isomers; (ii) studying the effects of flow rate on the separation of DMT-on oligonucleotide non-enantiomeric isomers in selected ion-pair systems; (iii) exploring the effects of column temperature on the separation of DMT-on oligonucleotide non-enantiomeric isomers in selected ion-pair systems.

[0038] Based on the fact that there are few methods for analyzing non-enantiomeric oligonucleotides without DMT in prior researches and it is difficult to effectively separate non-enantiomeric oligonucleotides without DMT, the present application provides a method for analyzing non-enantiomeric oligonucleotides without DMT by ion pair reversed phase; under specific mobile phase conditions, the method can separate DMT-on oligonucleotide non-enantiomers with high precision, has the advantages of good separation effect of component peaks, high analysis precision, simple operation, etc., and achieves the purpose of effectively controlling the quality of oligonucleotides.

[0039] As an optional embodiment, the oligonucleotide without DMT is an 18 nt oligonucleotide sample without 5'-DMT deprotection.

[0040] As an optional embodiment, the sample solution containing non-enantiomeric oligonucleotides without DMT is prepared by the following steps: The oligonucleotide without DMT is dissolved in water to obtain a sample solution.

[0041] As an optional embodiment, the concentration of the sample solution is 0.5-1.5 mg / mL, for example, it can be 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, etc.

[0042] As an optional embodiment, in the mobile phase A, the concentration of dibutylammonium acetate is 4-6 mM, for example, it can be 4 mM, 4.2 mM, 4.4 mM, 4.6 mM, 4.8 mM, 5 mM, 5.2 mM, 5.4 mM, 5.6 mM, 5.8 mM, 6 mM, etc., and the volume percentage of acetonitrile is 10-20%, for example, it can be 10%, 12%, 14%, 16%, 18%, 20%, etc.

[0043] As an optional embodiment, the pH of the mobile phase A is independently 7.0-8.0, for example, it can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, etc.

[0044] As an optional embodiment, in the mobile phase B, the concentration of dibutylammonium acetate is 4-6 mM, for example, it can be 4 mM, 4.2 mM, 4.4 mM, 4.6 mM, 4.8 mM, 5 mM, 5.2 mM, 5.4 mM, 5.6 mM, 5.8 mM, 6 mM, etc., and the volume percentage of acetonitrile is 80-90%, for example, it can be 80%, 82%, 84%, 86%, 88%, 90%, etc.

[0045] As an optional embodiment, the pH of the mobile phase B is each independently 7.0-8.0, for example, can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, etc.

[0046] As an optional embodiment, the gradient elution condition of the detection is as follows: 0 min, mobile phase A: 60-80% (for example, can be 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, etc.), mobile phase B: 20-40% (for example, can be 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, etc.); 2 min, mobile phase A: 60-80% (for example, can be 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, etc.), mobile phase B: 20-40% (for example, can be 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, etc.); 47 min, mobile phase A: 30-50% (for example, can be 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, etc.), mobile phase B: 50-70% (for example, can be 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, etc.); 50 min, mobile phase A: 60-80% (for example, can be 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, etc.), mobile phase B: 20-40% (for example, can be 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, etc.); 55 min, mobile phase A: 60-80% (for example, can be 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, etc.), mobile phase B: 20-40% (for example, can be 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, etc.).

[0047] As a preferred embodiment, the gradient elution condition of the detection is as follows: 0 min, mobile phase A: 70%, mobile phase B: 30%; 2 min, mobile phase A: 70%, mobile phase B: 30%; 47 min, mobile phase A: 40%, mobile phase B: 60%; 50 min, mobile phase A: 70%, mobile phase B: 30%; 55 min, mobile phase A: 70%, mobile phase B: 30%.

[0048] As an optional implementation, the wavelength used in the detection is 250-270 nm, for example, it can be 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 262 nm, 264 nm, 268 nm, 270 nm, etc.

[0049] As an optional implementation, the flow rate of the detection is 0.1-0.2 mL / min, for example, it can be 0.1 mL / min, 0.11 mL / min, 0.12 mL / min, 0.13 mL / min, 0.14 mL / min, 0.15 mL / min, 0.16 mL / min, 0.17 mL / min, 0.18 mL / min, 0.19 mL / min, 0.2 mL / min, etc.

[0050] As an optional implementation, the injection volume of the detection is 5-15 μL, for example, it can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, etc.

[0051] As an optional implementation, the column temperature of the detection is 20-30℃, for example, it can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, etc.

[0052] As an optional implementation, the temperature of the injection tray of the detection is 5-15℃, for example, it can be 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, etc.

[0053] As an optional implementation, the chromatographic column used in the detection is an OST BHE C18 chromatographic column.

[0054] As an optional implementation, the specification parameters of the chromatographic column used in the detection include: the pore size in the filler particle is 120-140 Å (for example, it can be 120 Å, 125 Å, 130 Å, 135 Å, 140 Å, etc.), the particle size is 1.5-2.0 μm (for example, it can be 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, etc.), the inner diameter is 1.5-2.5 mm (for example, it can be 1.5 mm, 1.6 mm, 1.8 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.5 mm, etc.), and the column length is 100-150 mm (for example, it can be 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, etc.).

[0055] As a preferred implementation, the chromatographic column used in the detection is a Waters ACQUITY Premier OSTBHE C18 chromatographic column, and the specification is 130 Å, 1.7 μm, 2.1 x 100 mm.

[0056] As an optional implementation, after the mobile phase A and the mobile phase B are prepared, the following post-processing steps are required; The mobile phase A and the mobile phase B are respectively filtered through a microporous membrane and then degassed by ultrasonic.

[0057] As an optional implementation, the pore size of the microporous membrane is 0.20-0.25 μm, for example, it can be 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, etc.

[0058] As an optional implementation, the power of the ultrasonic degassing is 100-300 W, for example, it can be 100 W, 150 W, 200 W, 250 W, 300 W, etc., and the time of the ultrasonic degassing is 1-10 min, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0059] As an optional implementation, the detection further includes the step of detecting the sample solution by using a high-performance liquid chromatograph.

[0060] The application will be further described by examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0061] Example 1 The embodiment provides an ion pair liquid chromatography analysis method of non-diastereoisomer of oligonucleotide without DMT removal, and the analysis method comprises the following steps: (1) accurately take 10 mg of sample and place it in a 10 mL volumetric flask, add a small amount of purified water to dissolve, and then dilute to volume, wherein the concentration of the sample solution is 0.5 mg / mL, and the oligonucleotide without DMT removal is an 18 nt oligonucleotide sample without 5'-DMT removal; (2) the prepared mobile phase A and mobile phase B are filtered through a microporous membrane (pore size is 0.22 μm), and are degassed by ultrasonic degassing at a power of 200 W for 5 min, and then are placed on a high performance liquid chromatograph for degassing; after the peak is generated, the area normalization method is used for diastereoisomer ratio calculation; Wherein, the detection uses a Waters premier ultra-high performance liquid chromatograph with a UV detector; Wherein, the detection uses a chromatographic column which is Waters ACQUITY Premier OST BHE C18, 130Å, 1.7µm, 2.1×100 mm; Wherein, the detection uses mobile phase A which comprises dibutylammonium acetate (5 mM), acetonitrile (15%) and ultrapure water, and the pH is 7.4; the detection uses mobile phase B which comprises dibutylammonium acetate (5 mM), acetonitrile (85%) and ultrapure water, and the pH is 7.4; The gradient elution conditions are shown in Table 1 as follows: Table 1

[0062] The detection conditions are set as follows: detection wavelength 260 nm; flow rate 0.15 mL / min; injection volume 10 μL; column temperature 25℃; injection tray temperature 10℃; diluent / blank: ultrapure water; (3) observe the number of diastereoisomers and the separation situation (such as Figure 1 ).

[0063] Embodiment 2 The embodiment provides an ion pair liquid chromatography analysis method of non-diastereoisomer of oligonucleotide without DMT removal, and the analysis method comprises the following steps: (1) accurately take 10 mg of sample and place it in a 10 mL volumetric flask, add a small amount of purified water to dissolve, and then dilute to volume, wherein the concentration of the sample solution is 0.5 mg / mL, and the oligonucleotide without DMT removal is an 18 nt oligonucleotide sample without 5'-DMT removal; (2) The prepared mobile phase A and mobile phase B were filtered through a microporous membrane (pore size 0.22 μm) and degassed at a power of 200 W for 5 min, and then placed on a high performance liquid chromatograph for degassing. After the peaks were out, the area normalization method was used to calculate the diastereoisomer ratio; The detection used a Waters premier ultra-high performance liquid chromatograph equipped with an ultraviolet detector. The detection used a Waters ACQUITY Premier OST BHE C18 column with a pore size of 130 Å, a particle size of 1.7 μm, and a length of 2.1 x 100 mm. The detection used mobile phase A including dibutylammonium acetate (5 mM), acetonitrile (15%), and ultrapure water, with a pH of 7.4; and mobile phase B including dibutylammonium acetate (5 mM), acetonitrile (85%), and ultrapure water, with a pH of 7.4. The gradient elution conditions are shown in Table 2 below: Table 2

[0064] The detection conditions were set as follows: detection wavelength 260 nm; flow rate 0.25 mL / min; injection volume 10 μL; column temperature 60℃; injection tray temperature 10℃; diluent / blank: ultrapure water. (3) The number of diastereoisomers and the separation were observed (such as Figure 2 ).

[0065] Example 3 The present embodiment provides a method for analyzing ion pair liquid chromatography of DMT-unremoved oligonucleotide diastereoisomers, which comprises the following steps: (1) 10 mg of sample was accurately weighed and placed in a 10 mL volumetric flask, and a small amount of purified water was added for dissolution and constant volume, wherein the concentration of the sample solution was 0.5 mg / mL, and the DMT-unremoved oligonucleotide was an 18 nt oligonucleotide sample without 5'-DMT deprotection; (2) The prepared mobile phase A and mobile phase B were filtered through a microporous membrane (pore size 0.22 μm) and degassed at a power of 200 W for 5 min, and then placed on a high performance liquid chromatograph for degassing. After the peaks were out, the area normalization method was used to calculate the diastereoisomer ratio; The detection used a Waters premier ultra-high performance liquid chromatograph equipped with an ultraviolet detector. The detection uses a Waters ACQUITY Premier OST BHE C18 column, 130 Å, 1.7 μm, 2.1*100 mm; The detection uses mobile phase A including dibutylammonium acetate (5 mM), acetonitrile (15%) and ultrapure water, and the pH is 7.4; and mobile phase B including dibutylammonium acetate (5 mM), acetonitrile (85%) and ultrapure water, and the pH is 7.4; The gradient elution conditions are shown in Table 3 below: Table 3

[0066] The detection conditions are set as follows: detection wavelength 260 nm; flow rate 0.25 mL / min; injection volume 10 μL; column temperature 25°C; injection tray temperature 10°C; diluent / blank: ultrapure water; (3) The number of diastereoisomers and the separation are observed (for example, as shown in FIG. 2). Figure 3

[0067] Example 4 The present example provides a method for ion pair liquid chromatography analysis of non-DMT-deprotected oligonucleotide diastereoisomers, which comprises the following steps: (1) 10 mg of sample is accurately weighed and placed in a 10 mL volumetric flask, and a small amount of purified water is added for dissolution, and then the volume is adjusted, wherein the concentration of the sample solution is 0.5 mg / mL, and the non-DMT-deprotected oligonucleotide is an 18 nt oligonucleotide sample without 5'-DMT deprotection; (2) The prepared mobile phase A and mobile phase B are respectively filtered through a microporous membrane (pore size 0.22 μm), and then degassed by ultrasonic degassing at a power of 200 W for 5 min, and then placed on a high performance liquid chromatograph for degassing. After the peak is out, the area normalization method is used to calculate the diastereoisomer ratio; The detection uses a Waters premier ultra-high performance liquid chromatograph equipped with a UV detector; The detection uses a Waters ACQUITY Premier OST BHE C18 column, 130 Å, 1.7 μm, 2.1*100 mm; The detection uses mobile phase A including dibutylammonium acetate (5 mM), acetonitrile (15%) and ultrapure water, and the pH is 7.4; and mobile phase B including dibutylammonium acetate (5 mM), acetonitrile (85%) and ultrapure water, and the pH is 7.4; The gradient elution conditions are shown in Table 4 below: Table 4 ​

[0068] The detection condition is set as follows: detection wavelength 260 nm; flow rate 0.15 mL / min; injection volume 10 μL; column temperature 25°C; injection disc temperature 10°C; diluent / blank: ultrapure water; (3) The number of diastereoisomers and the separation are observed (for example, as shown in Figure 4 ).

[0069] The isomer peaks 1-4 of each of the above examples and comparative examples are shown in Table 5 below: Table 5

[0070] Note: Figure 5 The structure diagram of the sample to be tested provided for Examples 1-4; peaks 1-4 represent four products of the same structure but different stereoisomers.

[0071] As shown in Table 5, under the optimal detection conditions of ion pair liquid chromatography of the DMT-on diastereoisomers of the oligonucleotide without DMT removal according to the present application, the liquid phase analysis results are stable and accurate, and the best separation effect of DMT-on diastereoisomers of various oligonucleotides is achieved.

[0072] As shown in Figure 1 , the detection condition separates the main peak into four peaks, and the first two peaks are basically baseline separated, and the third peak is separated into one peak, and from the separation degree of the peaks and the number of peaks, it can be seen that under the conditions of the column temperature, flow rate and gradient of Example 1, the separation effect of DMT-on diastereoisomers is better, and therefore, when separating the non-DMT-on enantiomers of oligonucleotides, lower column temperature, lower flow rate and longer gradient can be considered; the error is small, the liquid phase analysis results are stable and accurate, and the best separation effect of DMT-on diastereoisomers of various oligonucleotides is achieved.

[0073] As shown in Figure 2 , the detection condition of Example 2 can only separate the main peak into three peaks, of which two peaks are only split apart; as shown in Figure 3 , the detection condition of Example 3 can only separate the main peak into three peaks, of which the splitting degree of two peaks is increased, and the third peak has a tendency to separate; as shown in Figure 4 , the main peak is separated into three peaks, of which the splitting degree of two peaks is increased compared to the detection condition (b), and the third peak is separated into a shoulder peak.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An ion-pair liquid chromatography method for the analysis of diastereomers of oligonucleotides without DMT removal, characterized in that, The analytical method includes: The sample solution containing oligonucleotide diastereomers without DMT was detected by ion-pair reversed-phase ultra-high performance liquid chromatography to obtain the number of oligonucleotide diastereomers without DMT and the separation status. The mobile phase A for detection comprises dibutylammonium acetate, acetonitrile, and water, and the mobile phase B comprises dibutylammonium acetate, acetonitrile, and water.

2. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1, characterized in that, The oligonucleotides that have not been deprotected from DMT are 18 nt long oligonucleotide samples that have not been deprotected from 5'-DMT.

3. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1, characterized in that, The sample solution containing the oligonucleotide diastereomers that have not been de-DMT-removed was prepared by the following steps: The oligonucleotides that have not been de-DMT-removed were dissolved in water to obtain a sample solution; Preferably, the concentration of the sample solution is 0.5~1.5 mg / mL.

4. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1, characterized in that, In the mobile phase A, the concentration of dibutylammonium acetate is 4-6 mM, and the volume percentage of acetonitrile is 10-20%. Preferably, in the mobile phase B, the concentration of dibutylammonium acetate is 4-6 mM, and the volume percentage of acetonitrile is 80-90%. Preferably, the pH of mobile phase A and mobile phase B are each independently 7.0 to 8.

0.

5. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1, characterized in that, The gradient elution conditions for the detection are as follows: 0 min, mobile phase A: 60~80%, mobile phase B: 20~40%; 2 min, mobile phase A: 60~80%, mobile phase B: 20~40%; 47 min, mobile phase A: 30-50%, mobile phase B: 50-70%; 50 min, mobile phase A: 60-80%, mobile phase B: 20-40%; 55 min, mobile phase A: 60~80%, mobile phase B: 20~40%.

6. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 5, characterized in that, The gradient elution conditions for the detection are as follows: 0 min, mobile phase A: 70%, mobile phase B: 30%; 2 min, mobile phase A: 70%, mobile phase B: 30%; 47 min, mobile phase A: 40%, mobile phase B: 60%; 50 min, mobile phase A: 70%, mobile phase B: 30%; 55 min, mobile phase A: 70%, mobile phase B: 30%.

7. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1, characterized in that, The chromatographic column used for the detection was an OST BHE C18 column; Preferably, the specifications of the chromatographic column used for the detection include: the pore size of the packing particles is 120~140 Å, the particle size is 1.5~2.0 µm, the inner diameter is 1.5~2.5 mm, and the column length is 100~150 mm; Preferably, the chromatographic column used for the detection is a Waters ACQUITY Premier OST BHE C18 column with dimensions of 130 Å, 1.7 µm, and 2.1 × 100 mm.

8. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1, characterized in that, The detection uses a wavelength of 250~270 nm; Preferably, the detection flow rate is 0.1~0.2 mL / min; Preferably, the injection volume for detection is 5~15 μL; Preferably, the column temperature for detection is 20~30℃; Preferably, the temperature of the sample injection plate for detection is 5~15℃.

9. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 1 or 4, characterized in that, After preparing the mobile phase A and mobile phase B, the following post-processing steps are required; Mobile phase A and mobile phase B were filtered through a microporous membrane and then degassed by ultrasonication.

10. The ion-pair liquid chromatography method for analyzing diastereomers of oligonucleotides without DMT removal according to claim 9, characterized in that, The microporous membrane has a pore size of 0.20~0.25 μm; Preferably, the ultrasonic degassing power is 100~300 W, and the ultrasonic degassing time is 1~10 min.

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