Method for detecting purity of double-stranded oligonucleotide

Abstract

The invention discloses a double-stranded oligonucleotide purity detection method, and belongs to the technical field of oligonucleotide detection.The method comprises the following steps that S1, a double-stranded oligonucleotide sample to be detected is dissolved or diluted with water, and a test solution is prepared; s2, carrying out liquid chromatography detection on the test solution; a mobile phase for chromatographic detection is composed of a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution of 5-50 mM NaCl, 100 mM HFIP and 20 mM DIPEA; the mobile phase B is methanol; s3, performing quantitative analysis on the purity of the oligonucleotide compounds in the sample according to the liquid chromatography detection result. The method is suitable for detecting the purity of oligonucleotide double strands, and the DS purity can be accurately detected by adjusting the type of the mobile phase and avoiding DS melting.

Description

Technical Field

[0001] The present invention belongs to the technical field of oligonucleotide detection, and particularly relates to a method for detecting the purity of double-stranded oligonucleotides. Background Art

[0002] The non-denaturing purity of an oligonucleotide duplex refers to the purity of the target product in the oligonucleotide duplex under non-denaturing conditions. In biochemical and molecular biology experiments, denaturing conditions usually refer to the destruction of the secondary and tertiary structures of biological macromolecules (such as nucleic acids or proteins) by changing temperature, pH value, or adding denaturing agents (such as urea, formamide, etc.), so that they are in a relatively "loose" state. Non-denaturing conditions, on the other hand, maintain the native structure of biological macromolecules. For oligonucleotide duplexes, it means keeping their double-stranded structure under normal physiological conditions (such as temperature, pH value, etc. close to those in vivo). Generally, the temperature under non-denaturing conditions is in the range of normal temperature to moderate temperature (such as about 20 - 50 °C), and the pH value is neutral or close to neutral (about pH 7).

[0003] Under non-denaturing conditions, an oligonucleotide duplex can maintain its normal base pairing relationship. Bases are connected by hydrogen bonds. For example, two hydrogen bonds are formed between adenine (A) and thymine (T), and three hydrogen bonds are formed between guanine (G) and cytosine (C). This stable double-stranded structure is crucial for its biological functions (such as acting as primers, probes, etc.).

[0004] Non-denaturing ion pair reversed-phase high performance liquid chromatography (IP-RP-HPLC): is a technique for detecting the purity of oligonucleotides. Non-denaturing conditions mean that during the detection process, the double-stranded structure of the oligonucleotide will not be destroyed and can maintain its native secondary structure. This technique is mainly used to analyze the purity of oligonucleotides, especially to detect the relative contents of the target double-stranded oligonucleotide (DS) and other impurities (such as single-stranded sense strand SS and antisense strand AS). However, when using non-denaturing purity detection, the situation where AS, SS, and DS coexist is also observed. In this case, it is impossible to judge the true proportion of DS. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting the purity of double-stranded oligonucleotides to solve the problem of inaccurate detection of the purity of double-stranded oligonucleotides.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The present application proposes a method for detecting the purity of double-stranded oligonucleotides, including the following steps:

[0008] S1. Dissolve or dilute the double-stranded oligonucleotide sample to be tested with water to prepare a test solution.

[0009] S2. First, perform liquid chromatography detection on the test sample solution;

[0010] Composition of the mobile phase for chromatography detection: Mobile phase A is an aqueous solution of 5 - 50 mM NaCl + 100 mM HFIP + 20 mM DIPEA; Mobile phase B is methanol;

[0011] S3. According to the liquid chromatography detection results, perform quantitative analysis on the purity of the oligonucleotide compounds in the sample.

[0012] Furthermore, the conditions for chromatography detection include a column temperature of 20 - 40 °C; a flow rate of 0.1 - 0.3 mL / min; an injection temperature of 10 - 20 °C.

[0013] Furthermore, chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7μm, 2.1×150mm, P / N: 186009486 or an equivalent chromatographic column.

[0014] Furthermore, the gradient elution program for liquid chromatography detection is as follows:

[0015] At 0 min, mobile phase A is 70%, mobile phase B is 30%;

[0016] At 12 min, mobile phase A is 55%, mobile phase B is 45%;

[0017] At 14 min, mobile phase A is 55%, mobile phase B is 45%;

[0018] At 15 min, mobile phase A is 70%, mobile phase B is 30%;

[0019] At 20 min, mobile phase A is 70%, mobile phase B is 30%.

[0020] Furthermore, the injection sequence for liquid chromatography detection:

[0021] Number of injection needles for the blank solution ≥ 1 needle;

[0022] Number of injection needles for the AS positioning solution is 1 needle;

[0023] Number of injection needles for the SS positioning solution is 1 needle;

[0024] Number of injection needles for the test sample solution is 1 needle.

[0025] Furthermore, the resolution of liquid chromatography detection is greater than 1.5.

[0026] Furthermore, the concentration of the test sample solution is 0.5 - 1 mg / L.

[0027] Further, during the preparation of the mobile phase for liquid chromatography detection, it is filtered with a 0.22 μm filter membrane and degassed by ultrasonic treatment; during the preparation of mobile phase B, ultrasonic degassing is carried out.

[0028] Further, the concentration of the AS positioning solution is 0.5 - 1 mg / L; the concentration of the SS positioning solution is 0.5 - 1 mg / L; the blank solution is ultrapure water.

[0029] Further, the preparation steps of the AS positioning solution are as follows: dissolving and shaking the AS strand with a diluent;

[0030] The preparation steps of the SS positioning solution are as follows: dissolving the SS strand with a diluent;

[0031] The diluent is ultrapure water.

[0032] Advantages of the present invention:

[0033] The present invention provides a method for detecting the purity of double-stranded oligonucleotides, which is used for the non-denaturing purity detection of oligonucleotide double strands. When using non-denaturing purity detection, sometimes the DS is denatured, and the situation where AS (antisense strand), SS (sense strand), and DS (target double-stranded oligonucleotide) coexist can be observed. The present invention adjusts the type of mobile phase to avoid the denaturation of DS, thereby accurately detecting the purity of DS.

[0034] Compared with the prior art, the advantages of the present method are as follows: by adjusting the type of mobile phase, the double-stranded structure is maintained, and the inaccurate purity result caused by the denaturation of DS in the non-denaturing method is avoided; AS and DS or SS and DS can be detected simultaneously, providing more comprehensive purity information. The present method can be widely applied to fields such as nucleic acid drug research and development, gene therapy, and in vitro diagnosis, providing a reliable analysis means for the quality control of double-stranded oligonucleotides. Brief Description of the Drawings

[0035] The following further describes the present invention with reference to the drawings.

[0036] Figure 1 It is the chromatographic analysis result diagram in Example 1 of the present invention;

[0037] Figure 2 It is the chromatographic analysis result diagram in Example 2 of the present invention;

[0038] Figure 3 It is the chromatographic analysis result diagram in Example 3 of the present invention;

[0039] Figure 4 It is the chromatographic analysis result diagram in Comparative Example 1 of the present invention;

[0040] Figure 5It is the chromatogram analysis result diagram in Comparative Example 2 of the present invention. Detailed implementation manners

[0041] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0042] Obviously, the following description is only some examples or embodiments of the present application. For those of ordinary skill in the art, without making creative efforts, the present application can also be applied to other similar scenarios. In addition, it can also be understood that although the efforts made in this development process may be complex and lengthy, for those of ordinary skill in the art related to the content disclosed in the present application, some designs, manufacturing or production changes based on the technical content disclosed in the present application are only conventional technical means and should not be understood that the content disclosed in the present application is insufficient.

[0043] However, there will be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters are omitted and repeated descriptions of actually identical structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the following description is provided for those skilled in the art to fully understand the present application and is not intended to limit the subject matter recited in the claims.

[0044] If there is no special description, all the implementation manners and optional implementation manners of the present application can be combined with each other to form new technical solutions, and all the technical features and optional technical features of the present application can be combined with each other to form new technical solutions.

[0045] The following is a detailed description of a method for detecting the purity of a double-stranded oligonucleotide in an embodiment of the present application.

[0046] An embodiment of the present application provides a method for detecting the purity of a double-stranded oligonucleotide, including the following steps:

[0047] S1. Dissolve or dilute the double-stranded oligonucleotide sample to be tested with water to prepare a test solution.

[0048] S2. First, perform liquid chromatography detection on the test solution.

[0049] S3. According to the detection result of the liquid chromatography, perform quantitative analysis on the purity of the oligonucleotide compounds in the sample.

[0050] In some specific embodiments, the following contents need to be prepared in the experimental stage:

[0051] 1. Experimental materials

[0052] 1.1 Instrumentation:

[0053] Thermo Vanquish Flex or equivalent instrument; XPR205 analytical balance (minimum scale to 0.01 mg) or equivalent instrument; KH-500DB numerically controlled ultrasonic cleaner or equivalent instrument; Milli-Q water purifier or equivalent instrument; Class A volumetric flasks and pipettes.

[0054] 1.2 Reagents and samples:

[0055] Ultra-pure water (prepared by Milli-Q water purifier); methanol (CH3OH) (HPLC grade or equivalent grade); hexafluoroisopropanol (HFIP) (chromatographic grade or higher grade); N,N-diisopropylethylamine (DIPEA) (HPLC grade or equivalent grade); sodium chloride; oligonucleotide DS, AS, and SS samples.

[0056] 1.3 Preparation of solutions:

[0057] 1.3.1 Preparation of mobile phase:

[0058] Mobile phase A (5 - 50 mM NaCl + 100 mM HFIP + 20 mM DIPEA aqueous solution): Dissolve NaCl, HFIP, and DIPEA in water, make up the volume to a constant volume, mix well, filter through a 0.22 μm membrane filter, and degas by ultrasonic treatment.

[0059] Mobile phase B: Measure methanol and degas by ultrasonic treatment.

[0060] Note: If necessary, the preparation of the mobile phase can be scaled up or down proportionally while maintaining the concentration unchanged.

[0061] 1.3.2 Preparation of diluent:

[0062] The diluent is ultra-pure water.

[0063] 1.3.3 Preparation of blank solution:

[0064] Pipette approximately 1.0 mL of diluent into an injection vial, cap and seal it.

[0065] 1.3.4 Preparation of localization solution:

[0066] AS localization solution: Weigh accurately 5 - 10 mg of AS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, then make up the volume to the calibration line and shake well; label it as AS.

[0067] SS calibration solution: Accurately weigh 5-10 mg of SS chain, place it in a 10 mL volumetric flask, dissolve it with the diluent, then make up to the calibration line and shake well; label it as SS.

[0068] 1.3.5 Preparation of test solution:

[0069] Accurately weigh 5-10 mg of the sample, transfer it to a 10 mL volumetric flask, add the diluent to dissolve and dilute to the calibration line, shake well to obtain.

[0070] 2. Chromatographic conditions

[0071] 2.1 Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7μm, 2.1×150mm, P / N: 186009486 or equivalent chromatographic column; column temperature is 20 - 40°C; flow rate is 0.1 - 0.3 mL / min; injection temperature is 10 - 20°C;

[0072] 2.2 Gradient elution program is as follows:

[0073] At 0 min, mobile phase A is 70%, mobile phase B is 30%;

[0074] At 12 min, mobile phase A is 55%, mobile phase B is 45%;

[0075] At 14 min, mobile phase A is 55%, mobile phase B is 45%;

[0076] At 15 min, mobile phase A is 70%, mobile phase B is 30%;

[0077] At 20 min, mobile phase A is 70%, mobile phase B is 30%.

[0078] 2.3 Injection sequence:

[0079] Number of injection needles for blank solution ≥ 1 needle;

[0080] Number of injection needles for AS calibration solution is 1 needle;

[0081] Number of injection needles for SS calibration solution is 1 needle;

[0082] Number of injection needles for test solution is 1 needle.

[0083] The following is specifically described in conjunction with the examples.

[0084] Example 1

[0085] 1. Experimental materials

[0086] 1.1 Instrument and equipment:

[0087] Thermo Vanquish Flex; XPR205 analytical balance (minimum scale to 0.01 mg); KH-500DB numerically controlled ultrasonic cleaner; Milli-Q water purifier; Class A volumetric flasks and pipettes.

[0088] 1.2 Reagents and samples:

[0089] Ultra-pure water (prepared by Milli-Q water purifier); methanol (CH3OH) (HPLC grade); hexafluoroisopropanol (HFIP) (chromatographic grade); N,N-diisopropylethylamine (DIPEA) (HPLC grade); sodium chloride; oligonucleotide DS, AS, and SS samples.

[0090] 1.3 Preparation of solutions:

[0091] 1.3.1 Preparation of mobile phase:

[0092] Mobile phase A (50 mM NaCl + 100 mM HFIP + 20 mM DIPEA aqueous solution): Accurately measure 2925 mg of NaCl, 10.6 mL of HFIP, and 1.74 mL of DIPEA, add water to a volume of 1 L, mix well, filter through a 0.22 μm membrane filter, and degas by ultrasonic treatment.

[0093] Mobile phase B: Measure 1000 mL of methanol and degas by ultrasonic treatment.

[0094] 1.3.2 Preparation of diluent:

[0095] The diluent is ultra-pure water.

[0096] 1.3.3 Preparation of blank solution:

[0097] Pipette approximately 1.0 mL of diluent into an injection vial, cover and seal.

[0098] 1.3.4 Preparation of localization solution:

[0099] AS localization solution: Weigh accurately 10 mg of AS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the scale mark, shake well; label it as AS.

[0100] SS localization solution: Weigh accurately 10 mg of SS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the scale mark, shake well; label it as SS.

[0101] 1.3.5 Preparation of test solution:

[0102] Weigh accurately 10 mg of the sample, transfer it to a 10 mL volumetric flask, dissolve it with diluent and dilute to the scale mark, shake well to obtain.

[0103] 2. Chromatographic conditions

[0104] 2.1 Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7μm, 2.1×150mm, P / N: 186009486;

[0105] 2.2 Gradient elution program: as shown in Table 1 below:

[0106] Table 1

[0107]

[0108]

[0109] 2.3 Injection order: as shown in Table 2 below:

[0110] Table 2

[0111] Serial Number Sample Name Number of Injection Needles 1 Blank Solution ≥1 2 AS Positioning Solution 1 3 SS Positioning Solution 1 4 Test Solution 1

[0112] The results obtained by analyzing the sample using the above chromatographic conditions are as Figure 1 shown. It can be found through Figure 1 that there is only one SS single strand in the DS spectrum, indicating that adding sodium chloride to the mobile phase can enhance the stability of the double strand and prevent the double strand from unwinding during detection.

[0113] Example 2

[0114] This example is different from Example 1 in that the concentration of sodium chloride in mobile phase A is changed.

[0115] Specifically:

[0116] 1. Experimental materials

[0117] 1.1 Instrument and equipment:

[0118] Thermo Vanquish Flex; XPR205 analytical balance (minimum scale to 0.01 mg); KH-500DB numerically controlled ultrasonic cleaner; Milli-Q water purifier; A-grade volumetric flask and pipette.

[0119] 1.2 Reagents and samples:

[0120] Ultra-pure water (prepared by Milli-Q water purifier); methanol (CH3OH) (HPLC grade); hexafluoroisopropanol (HFIP) (chromatographic grade); N,N-diisopropylethylamine (DIPEA) (HPLC grade); sodium chloride; oligonucleotide DS, AS, and SS samples.

[0121] 1.3 Preparation of solutions:

[0122] 1.3.1 Preparation of mobile phase:

[0123] Mobile phase B: Measure 1000 mL of methanol and degas it by ultrasonic treatment.

[0124] Mobile phase A (aqueous solution of 25 mM NaCl + 100 mM HFIP + 20 mM DIPEA): Accurately weigh 1462.5 mg of NaCl, 10.6 mL of HFIP, and 1.74 mL of DIPEA, add water to make up to 1 L, mix well, filter through a 0.22 μm filter membrane, and degas by ultrasonic treatment.

[0125] 1.3.2 Preparation of diluent:

[0126] The diluent is ultrapure water.

[0127] 1.3.3 Preparation of blank solution:

[0128] Pipette approximately 1.0 mL of diluent into an injection vial, cover and seal it.

[0129] 1.3.4 Preparation of localization solution:

[0130] AS localization solution: Accurately weigh 10 mg of AS chain, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the scale line and shake well; label it as AS.

[0131] SS localization solution: Accurately weigh 10 mg of SS chain, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the scale line and shake well; label it as SS.

[0132] 1.3.5 Preparation of test solution:

[0133] Accurately weigh 10 mg of the sample, transfer it to a 10 mL volumetric flask, dissolve it with diluent and dilute to the scale line, shake well to obtain.

[0134] 2. Chromatographic conditions

[0135] 2.1 Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7 μm, 2.1×150 mm, P / N: 186009486;

[0136] 2.2 Gradient elution program: As shown in Table 3 below:

[0137] Table 3

[0138]

[0139] 2.3 Injection sequence: As shown in Table 4 below:

[0140] Table 4

[0141] Serial Number Sample Name Number of Injection Needles 1 Blank Solution ≥1 2 AS Positioning Solution 1 3 SS Positioning Solution 1 4 Test Solution 1

[0142] The results obtained by analyzing the sample using the above chromatographic conditions are as Figure 2 shown. When other chromatographic conditions remain unchanged, it can be found through Figure 2 that there is only one single-stranded SS in the DS spectrum, indicating that when the sodium chloride concentration in the mobile phase decreases from 50 mM to 25 mM, the binding ability of sodium chloride to double-stranded DNA does not change.

[0143] Example 3

[0144] This example is different from Example 1 in that the concentration of sodium chloride in mobile phase A is changed.

[0145] Specifically:

[0146] 1. Experimental materials

[0147] 1.1 Instrument and equipment:

[0148] Thermo Vanquish Flex; XPR205 analytical balance (minimum scale to 0.01 mg); KH-500DB numerical control ultrasonic cleaner; Milli-Q pure water instrument; Class A volumetric flask and pipette.

[0149] 1.2 Reagents and samples:

[0150] Ultra-pure water (prepared by Milli-Q pure water instrument); methanol (CH3OH) (HPLC grade); hexafluoroisopropanol (HFIP) (chromatographic grade); N,N-diisopropylethylamine (DIPEA) (HPLC grade); sodium chloride; oligonucleotide DS, AS and SS samples.

[0151] 1.3 Preparation of solutions:

[0152] 1.3.1 Preparation of mobile phase:

[0153] Mobile phase B: Measure 1000 mL of methanol and degas it by ultrasonic.

[0154] Mobile phase A (5 mM NaCl + 100 mM HFIP + 20 mM DIPEA aqueous solution): Accurately measure 292.5 mg of NaCl, 10.6 mL of HFIP, 1.74 mL of DIPEA, add water to make up to 1 L, mix well, filter with a 0.22 μm filter membrane, and degas by ultrasonic.

[0155] 1.3.2 Preparation of diluent:

[0156] The diluent is ultra-pure water.

[0157] 1.3.3 Preparation of blank solution:

[0158] Pipette approximately 1.0 mL of diluent into an injection vial, cover and seal it.

[0159] 1.3.4 Preparation of positioning solution:

[0160] AS positioning solution: Weigh accurately 10 mg of AS chain, place it in a 10 mL volumetric flask, dissolve it with diluent, then make up to the mark with diluent and shake well; label it as AS.

[0161] SS positioning solution: Weigh accurately 10 mg of SS chain, place it in a 10 mL volumetric flask, dissolve it with diluent, then make up to the mark with diluent and shake well; label it as SS.

[0162] 1.3.5 Preparation of test solution:

[0163] Weigh accurately 10 mg of the sample, transfer it to a 10 mL volumetric flask, dissolve it with diluent and dilute to the mark, then shake well to obtain the solution.

[0164] 2. Chromatographic conditions

[0165] 2.1 Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7μm, 2.1×150mm, P / N: 186009486;

[0166] 2.2 Gradient elution program: As shown in Table 5 below:

[0167] Table 5

[0168]

[0169]

[0170] 2.3 Injection sequence: As shown in Table 6 below:

[0171] Table 6

[0172] Serial Number Sample Name Number of Injection Needles 1 Blank Solution ≥1 2 AS Positioning Solution 1 3 SS Positioning Solution 1 4 Test Solution 1

[0173] The results obtained by analyzing the sample using the above chromatographic conditions are as Figure 3 shown. When other chromatographic conditions remain unchanged, by Figure 3 it can be found that there is only one SS single strand in the DS spectrum, indicating that when the sodium chloride concentration in the mobile phase drops from 25 mM to 5 mM, the binding ability of sodium chloride to the double strand has not changed.

[0174] Comparative Example 1

[0175] This comparative example is different from Example 1 in that the composition of mobile phase A is changed and sodium chloride is not added. Specifically:

[0176] 1. Experimental materials

[0177] 1.1 Instrumentation:

[0178] Thermo Vanquish Flex; XPR205 analytical balance (minimum scale to 0.01 mg); KH-500DB numerically controlled ultrasonic cleaner; Milli-Q water purifier; Class A volumetric flasks and pipettes.

[0179] 1.2 Reagents and samples:

[0180] Ultra-pure water (prepared by Milli-Q water purifier); methanol (CH3OH) (HPLC grade); hexafluoroisopropanol (HFIP) (chromatographic grade); N,N-diisopropylethylamine (DIPEA) (HPLC grade); oligonucleotide DS, AS, and SS samples.

[0181] 1.3 Preparation of solutions:

[0182] 1.3.1 Preparation of mobile phases:

[0183] Mobile phase C (100 mM HFIP + 20 mM DIPEA aqueous solution): Accurately measure 10.6 mL of HFIP and 1.74 mL of DIPEA, add water to make up to 1 L, mix well, filter through a 0.22 μm filter membrane, and degas by ultrasound.

[0184] Mobile phase B: Measure 1000 mL of methanol and degas by ultrasound.

[0185] 1.3.2 Preparation of diluent:

[0186] The diluent is ultra-pure water.

[0187] 1.3.3 Preparation of blank solution:

[0188] Pipette about 1.0 mL of diluent into an injection vial, cover and seal.

[0189] 1.3.4 Preparation of localization solution:

[0190] AS localization solution: Weigh accurately 10 mg of AS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the graduation mark and shake well; label it as AS.

[0191] SS localization solution: Weigh accurately 10 mg of SS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the graduation mark and shake well; label it as SS.

[0192] 1.3.5 Preparation of the test solution:

[0193] Accurately weigh 10 mg of the sample, transfer it to a 10-mL volumetric flask, dissolve it with the diluent and dilute to the mark, then shake well to obtain the solution.

[0194] 2. Chromatographic conditions

[0195] 2.1 Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7 μm, 2.1×150 mm, P / N: 186009486;

[0196] 2.3 Injection sequence: as shown in Table 7 below:

[0197] Table 7

[0198]

[0199]

[0200] 2.3 Injection sequence: as shown in Table 8 below:

[0201] Table 8

[0202] Serial Number Sample Name Number of Injection Needles 1 Blank Solution ≥1 2 AS Positioning Solution 1 3 SS Positioning Solution 1 4 Test Solution 1

[0203] The results obtained by analyzing the sample using the above chromatographic conditions are as Figure 4 shown. When analyzing the sample using the above chromatographic conditions, it was found that while DS was present, AS (5.308 min) and SS (6.773 min) were also present

[0204] Comparative Example 2

[0205] This comparative example is different from Example 1 in that the elution gradient program is changed. Specifically:

[0206] 1. Experimental materials

[0207] 1.1 Instrument and equipment:

[0208] Thermo Vanquish Flex; XPR205 analytical balance (minimum scale to 0.01 mg); KH-500DB numerically controlled ultrasonic cleaner; Milli-Q water purifier; Class A volumetric flasks and pipettes.

[0209] 1.2 Reagents and samples:

[0210] Ultra-pure water (prepared by Milli-Q water purification instrument); methanol (CH3OH) (HPLC grade); hexafluoroisopropanol (HFIP) (chromatographic grade); N,N-diisopropylethylamine (DIPEA) (HPLC grade); sodium chloride; oligonucleotide DS, AS, and SS samples.

[0211] 1.3 Preparation of solutions:

[0212] 1.3.1 Preparation of mobile phase:

[0213] Mobile phase B: Measure 1000 mL of methanol and degas it by sonication.

[0214] Mobile phase A (50 mM NaCl + 100 mM HFIP + 20 mM DIPEA aqueous solution): Accurately measure 2925 mg of NaCl, 10.6 mL of HFIP, and 1.74 mL of DIPEA, add water to make up to 1 L, mix well, filter through a 0.22 μm filter membrane, and degas by sonication.

[0215] 1.3.2 Preparation of diluent:

[0216] The diluent is ultra-pure water.

[0217] 1.3.3 Preparation of blank solution:

[0218] Transfer approximately 1.0 mL of diluent into an injection vial, cover and seal it.

[0219] 1.3.4 Preparation of localization solution:

[0220] AS localization solution: Weigh accurately 10 mg of AS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the scale mark and shake well; label it as AS.

[0221] SS localization solution: Weigh accurately 10 mg of SS strand, place it in a 10 mL volumetric flask, dissolve it with diluent, and then make up to the scale mark and shake well; label it as SS.

[0222] 1.3.5 Preparation of test solution:

[0223] Weigh accurately 10 mg of the sample, transfer it to a 10 mL volumetric flask, dissolve it with diluent and dilute to the scale mark, shake well to obtain.

[0224] 2. Chromatographic conditions

[0225] 2.1 Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7 μm, 2.1×150 mm, P / N: 186009486;

[0226] 2.3 Injection sequence: as shown in Table 9 below:

[0227] Table 9

[0228]

[0229] 2.3 Injection sequence: as shown in Table 10 below:

[0230] Table 10

[0231]

[0232]

[0233] The results obtained by analyzing the samples using the above chromatographic conditions are as Figure 5 shown. Compared with Example 1, it was found that by adjusting the AB elution gradient, the DS would not unwind. Compared with Comparative Example 1, it was found that adding sodium chloride to the mobile phase could enhance the stability of the double strand and prevent the double strand from unwinding during the detection process.

[0234] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0235] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the purity of a double-stranded oligonucleotide, characterized in that, It includes the following steps: S1. Dissolve or dilute the double-stranded oligonucleotide test sample with water to prepare a test solution. S2. First, perform liquid chromatography detection on the test solution. Composition of the mobile phase for chromatography detection: Mobile phase A is an aqueous solution of 5 - 50 mM NaCl + 100 mM HFIP + 20 mM DIPEA; Mobile phase B is methanol. S3. According to the results of the liquid chromatography detection, quantitatively analyze the purity of the oligonucleotide compounds in the sample.

2. The detection method for the purity of a double-stranded oligonucleotide according to claim 1, wherein Conditions for chromatography detection include: column temperature is 20 - 40 °C; flow rate is 0.1 - 0.3 mL / min; injection temperature is 10 - 20 °C.

3. The detection method for the purity of a double-stranded oligonucleotide according to claim 1, characterized in that, Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C18 1.7μm, 2.1×150mm, P / N: 186009486 or equivalent chromatographic column.

4. The detection method for the purity of a double-stranded oligonucleotide according to claim 1, wherein The gradient elution program for liquid chromatography detection is as follows: At 0 min, mobile phase A is 70%, and mobile phase B is 30%. At 12 min, mobile phase A is 55%, and mobile phase B is 45%. At 14 min, mobile phase A is 55%, and mobile phase B is 45%. At 15 min, mobile phase A is 70%, and mobile phase B is 30%. At 20 min, mobile phase A is 70%, and mobile phase B is 30%.

5. The detection method for the purity of a double-stranded oligonucleotide according to claim 1, wherein Injection sequence for liquid chromatography detection: Number of injection needles for the blank solution ≥ 1 needle. Number of injection needles for the AS positioning solution is 1 needle. Number of injection needles for the SS positioning solution is 1 needle. Number of injection needles for the test solution is 1 needle.

6. The detection method for the purity of a double-stranded oligonucleotide according to claim 1, characterized in that, The resolution of liquid chromatography detection is greater than 1.

5.

7. The detection method for the purity of a double-stranded oligonucleotide according to claim 1, characterized in that, The concentration of the test solution is 0.5 - 1 mg / L.

8. A method for detecting the purity of a double-stranded oligonucleotide according to claim 1, characterized in that, During the preparation of the mobile phase for liquid chromatography detection, filter with a 0.22 μm filter membrane and perform ultrasonic degassing; perform ultrasonic degassing during the preparation of mobile phase B.

9. The detection method for the purity of a double-stranded oligonucleotide according to claim 5, characterized in that, The concentration of the AS positioning solution is 0.5 - 1 mg / L; the concentration of the SS positioning solution is 0.5 - 1 mg / L; the blank solution is ultrapure water.

10. The detection method for the purity of a double-stranded oligonucleotide according to claim 5, wherein The preparation steps for the AS positioning solution are: dissolve and shake the AS strand with a diluent. The preparation steps for the SS positioning solution are: dissolve the SS strand with a diluent. The diluent is ultrapure water.

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