A method for detecting related substances in cationic lipid YK-009 and its application

Abstract

The present invention provides a detection method and application of related substances in cationic lipid YK-009, the detection method comprising the following steps: (1) mixing a sample to be tested with a diluent to obtain a test solution; (2) mixing a related substance standard with a diluent to obtain a reference solution; (3) subjecting the test solution and the reference solution to liquid chromatography detection, and calculating the content of the related substances according to the detection results; the mobile phase of the liquid chromatography detection comprises a mobile phase A and a mobile phase B, the mobile phase A is an aqueous solution of acetic acid-triethylamine, and the mobile phase B is an acetonitrile solution of acetic acid-triethylamine. The detection method provided by the present invention can accurately detect a variety of related substances in YK-009 at the same time, has high sensitivity, high accuracy, high reproducibility, good durability, and is convenient for accurate detection and quality control of related substances in YK-009.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to a detection method for related substances in cationic lipid YK-009 and application thereof. Background Art

[0002] Since the successful launch of the mRNA vaccine for novel coronavirus infection, lipid nanoparticles (LNP) have become the preferred delivery carrier for nucleic acid products such as mRNA and siRNA, and are widely used in mRNA vaccines and therapeutic drugs. mRNA has a large molecular weight and strong hydrophilicity. Its biological activity is thousands of times that of small molecule drugs, and its industrial production is relatively easy. However, its single-stranded structure makes it extremely unstable and easily degraded. It also carries a negative charge and has to pass through the cell membrane with the same negative charge on the surface, making its delivery difficult. LNP is a multi-component system that can encapsulate mRNA in the internal cavity of the structure. This structure can improve the stability of mRNA in vivo and protect it from degradation, thereby exerting its effect.

[0003] Lipid nanoparticles (LNP) are lipid vesicles with a uniform lipid core, which are widely used in the delivery of RNA vaccines and nucleic acid drugs. LNP encapsulation can protect mRNA from extracellular RNases and assist in the intracellular delivery of mRNA. LNP has become the most widely used non-viral vector due to its simple operation, high biosafety, good repeatability, high transfection efficiency and low cytotoxicity. The mRNA novel coronavirus infection vaccines of the three giants BioNTech, Moderna and CureVac all use LNP delivery technology.

[0004] The LNP delivery system is mainly composed of ionized cationic lipids (SM102 or ALC-0315 or YK-009), phospholipids (DSPC or DOPE), cholesterol and auxiliary lipids (DMG-PEG2000) to prepare lipid prescriptions. The four lipids usually have a molar ratio of 50:10:38.5:1.5, and the ratio of ionizable cationic lipids to RNA is maintained at 15:1 (w / w), so the amount of cationic lipids used in each unit preparation is very large. Ionizable lipids are protonated under acidic conditions, carry positive charges, and electrostatically bind to the negatively charged nucleic acid phosphate backbone, resulting in the RNA drug substance being encapsulated, thereby forming lipid nanoparticles to ensure the stability of mRNA. LNPs will be taken up by cells through receptor-mediated endocytosis. After being taken up by cells, LNPs will enter the endosomes. Ionizable lipids are protonated in the acidic environment of the endosomes and carry positive charges, which helps to destroy the endosomal membrane and enable LNPs to escape into the cytoplasm. After escaping from the endosomal structure, mRNA is released from the LNP and enters the cytoplasm, while a large number of ionizable cationic lipids remain in the cell. These fragments can interact with cellular components and cause various adverse effects in the cell: the positive charge of the cationic lipids can react with the negative charge of the cellular components, resulting in membrane porosity and calcium ion influx, which may lead to cell death; cationic lipids can affect hepatocytes (most LNPs are currently targeted to the liver) by interfering with amino acid metabolism, energy metabolism, lipid metabolism, and increasing oxidative stress; the interaction of cationic lipids with the negative charge of the mitochondrial membrane may destroy its integrity, leading to the release of reactive oxygen species (ROS) in the cell, which may trigger exponential cell damage; if the cationic lipids reach certain areas of the brain, they can cause serious neurological effects.

[0005] Since cationic lipids are used in large amounts and have a long residence time in the body, the presence of relatively small amounts of impurities will lead to an increase in the absolute number of impurities in the body. The structures of these impurities usually contain similar cationic structures and lipophilic genes that are not easily metabolized, which can cause many unpredictable cytotoxic effects. Therefore, it is very necessary and essential to strictly limit the impurity content in the cations and find scientific quality testing methods to strictly control the impurity limits. Unfortunately, in searching the existing literature, no very scientific quality control methods have been found for cationic lipids that have been on the market or have been made public.

[0006] YK-009(C 40 H 79NO5) is the third-generation cationic liposome independently developed by Yuekang Pharmaceutical Group Co., Ltd. Compared with the second-generation LNP made of DLin-MC3-DMA, the LNP made of YK-009 liposome has the advantages of smaller particle size, higher uniformity, higher in vitro cell transfection efficiency, lower cytotoxicity and high in vivo expression, but its synthesis process is relatively complicated, and a series of impurities with similar properties will be produced during the process, including intermediates, degradation impurities, by-products and by-product derivative impurities. Since these impurities have no obvious chromophore groups in the structure of YK-009 and their properties are very similar, it is difficult to separate YK-009 from its impurities (related substances), and no separation and detection methods for related substances in YK-009 or other similar cations have been retrieved.

[0007]

[0008] Therefore, how to provide a method that can effectively detect related substances in YK-009 has become an urgent problem to be solved. Summary of the invention

[0009] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting related substances in cationic lipid YK-009 and its application. The detection method provided by the present invention can accurately detect multiple related substances in YK-009 at the same time, with high sensitivity, high accuracy, high reproducibility, good durability, and is convenient for accurate detection and quality monitoring of related substances in YK-009.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In one aspect, the present invention provides a method for detecting related substances in cationic lipid YK-009, the detection method comprising the following steps:

[0012] (1) Mix the sample to be tested with the diluent to obtain a test solution;

[0013] (2) Mixing the standard substance of the relevant substance with the diluent to obtain a reference solution;

[0014] (3) The test solution and the reference solution are subjected to liquid chromatography detection, and the content of the related substances is calculated based on the detection results; the mobile phase of the liquid chromatography detection includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of acetic acid-triethylamine, and the mobile phase B is an acetonitrile solution of acetic acid-triethylamine.

[0015] Steps (1) and (2) are performed in no particular order.

[0016] The related substances include any one or a combination of at least two of impurities 1 to 10:

[0017] Impurity 1;

[0018] Impurity 2;

[0019] Impurity 3;

[0020] Impurity 4;

[0021] Impurity 5;

[0022] Impurity 6;

[0023] Impurity 7;

[0024] Impurity 8;

[0025] Impurity 9;

[0026] Impurity 10.

[0027] The above method can accurately detect multiple related substances in YK-009 at the same time, with high sensitivity, high accuracy, high reproducibility and good durability, which is convenient for accurate detection and quality monitoring of related substances in YK-009.

[0028] Preferably, the diluent comprises methanol and / or acetonitrile, preferably acetonitrile.

[0029] Preferably, the mobile phase for the liquid chromatography detection in step (3) comprises mobile phase A and mobile phase B;

[0030] The mobile phase A is an aqueous solution of acetic acid-triethylamine, and the mobile phase B is an acetonitrile solution of acetic acid-triethylamine.

[0031] Preferably, the mobile phase A is prepared by mixing an acetic acid-triethylamine aqueous solution with water, and the mobile phase B is prepared by mixing an acetic acid-triethylamine aqueous solution with acetonitrile;

[0032] In the acetic acid-triethylamine aqueous solution used in the preparation of mobile phase A and mobile phase B, the volume ratio of triethylamine to water is independently (1-3):(17-19), and the pH is independently 6.5-7.5, preferably 7.0, wherein the number of triethylamine parts can be 1, 1.5, 2, 2.5 or 3, etc., the number of water parts can be 17, 17.5, 18, 18.5 or 19, etc., and the pH can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0033] Preferably, the concentration of the acetic acid-triethylamine aqueous solution in the mobile phase A and the mobile phase B is independently 0.8-1.2%, for example 0.8%, 0.9%, 1%, 1.1%, 1.2%, etc., but is not limited to the above-listed values. Other values ​​not listed within the above numerical range are also applicable, preferably 1%.

[0034] Preferably, the liquid chromatography detection in step (3) is performed by gradient elution, and the specific elution gradient is as follows:

[0035] From 0 to 15 min, the volume fraction of mobile phase A was 23-27%, and the rest was mobile phase B;

[0036] From 15 to 30 minutes, the volume fraction of mobile phase A was changed from 23-27% to 4-6% at a constant rate, and the rest was mobile phase B;

[0037] At 30-40 min, the volume fraction of mobile phase A was 4-6%, and the rest was mobile phase B;

[0038] At 40-41 minutes, the volume fraction of mobile phase A changed from 4-6% to 0 at a constant rate, and the rest was mobile phase B;

[0039] From 41 to 45 min, the volume fraction of mobile phase A was 0, and the rest was mobile phase B;

[0040] From 45 to 45.1 min, the volume fraction of mobile phase A changed uniformly from 0 to 23-27%, and the rest was mobile phase B;

[0041] After 45.1 min, the volume fraction of mobile phase A was 23-27%, and the rest was mobile phase B.

[0042] Preferably, in the liquid chromatography detection in step (3), the stationary phase is hexyl-linked phenyl-bonded silica gel.

[0043] Preferably, in the liquid chromatography detection in step (3), the column temperature is 20-50°C, preferably 40°C, the mobile phase flow rate is 0.2-0.4 mL / min, preferably 0.2 mL / min, and the injection volume is 2-10 μL, preferably 5 μL; wherein the column temperature may be 20°C, 25°C, 30°C, 35°C, 40°C, etc., the mobile phase flow rate may be 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min or 0.4 mL / min, etc., and the injection volume may be 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL or 10 μL, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are equally applicable.

[0044] Preferably, the related substances include impurities 1 to 10.

[0045] The above-mentioned specific parameters and reagent raw materials can effectively improve the sensitivity and accuracy of detection.

[0046] On the other hand, the present invention also provides the use of the above-mentioned method for detecting related substances in cationic lipid YK-009 in the quality control of cationic lipid YK-009.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention provides a method for simultaneously separating and detecting multiple related substances in cationic lipid YK-009, wherein the separation degree between impurity 3 (by-product impurity) and impurity 6 (process impurity) and the main peak is 2.72, and the separation degree between the remaining impurities and the main peak is greater than 2.72, which is significantly better than the separation standard (>1.5), and the peak shape is obvious, and the separation of YK-009 and 10 impurities can be achieved with good separation degree. The detection method of related substances in YK-009 is not recorded in the prior art, but the method realizes the effective separation and detection of impurities in YK-009, which is beneficial to the quality control of YK-009.

[0049] By optimizing the conditions, the peak shapes of YK-009 and its impurities are improved, so that the sensitivity of each component detection is further improved. The method of the present invention uses a charged aerosol detector (CAD) for detection. Based on the characteristics of the detector itself, the main component self-control method is used to quantitatively calculate the relevant substances in YK-009. Through the recovery test, it is verified that the method has good accuracy, high reproducibility, and good durability, which is convenient for accurate detection and quality control of the relevant substances in YK-009. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a graph of the specificity detection results in Example 1;

[0051] Figure 2 is a graph showing the detection results when the chromatographic column in Example 2 is Waters ACQUITY UPLC Oligonucleotide BEH C18;

[0052] Figure 3 is a graph showing the detection results when the chromatographic column in Example 2 is Agilent Poroshell 120 EC-C18;

[0053] Figure 4 This is a graph showing the detection results when the solvent of mobile phase B is methanol in Example 3;

[0054] Figure 5 This is a graph showing the detection results when the amount of acetic acid-triethylamine solution added to mobile phases A and B in Example 4 is 0.5%;

[0055] Figure 6 This is a graph showing the detection results when the amount of acetic acid-triethylamine solution added to mobile phases A and B in Example 4 is 1.5%;

[0056] Figure 7 is a graph showing the detection results of elution gradient method 1 in Example 5;

[0057] Figure 8 is a graph showing the detection results of elution gradient method 2 in Example 5;

[0058] Fig. 9 This is a graph showing the detection results when the mobile phase additive in Example 6 is ammonium formate. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] Example 1

[0061] This example provides a method for detecting related substances in cationic lipid YK-009, which is as follows:

[0062] Solution preparation:

[0063] Diluent: acetonitrile;

[0064] Impurity 1 location solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0065] Impurity 2 location solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0066] Impurity 3 location solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0067] Impurity 4 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0068] Impurity 5 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0069] Impurity 6 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0070] Impurity 7 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0071] Impurity 8 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0072] Impurity 9 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0073] Impurity 10 localization solution: Take about 15 mg of the impurity reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake it well, and use it as the stock solution; accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, shake it well, and you have it.

[0074] Mixed impurity solution: Take about 60 mg of YK-009, weigh it accurately, put it in a 20 mL volumetric flask, accurately add 1.0 mL of each impurity stock solution mentioned above, dissolve it with acetonitrile and dilute it to the scale, shake well, and obtain.

[0075] Test solution: Take about 60 mg of YK-009, accurately weigh it, put it in a 20 mL volumetric flask, dissolve it in acetonitrile and dilute it to the scale, shake well, and you have it.

[0076] The test solution and each impurity location solution are subjected to liquid chromatography detection, and the types and contents of the relevant substances therein are determined based on the detection results.

[0077] The test conditions are as follows:

[0078] Instrument: High performance liquid chromatography-charged aerosol detector;

[0079] Chromatographic column: Phenyl-bonded silica gel connected with hexyl (6 carbons) as filler, ACQUITY UPLC CSH Phenyl-Hexyl Column 1.7 µm, 2.1 mm × 100 mm;

[0080] Mobile phase A: 1.0% acetic acid-triethylamine solution in water;

[0081] Mobile phase B: 1.0% acetic acid-triethylamine solution in acetonitrile;

[0082] The acetic acid-triethylamine solution was prepared as follows: 15 mL of triethylamine was transferred into 85 mL of ultrapure water, and the pH was adjusted to 7.0 with acetic acid.

[0083] Column temperature: 40°C;

[0084] Injection volume: 5 μL;

[0085] Detector: Charged aerosol detector;

[0086] Evaporation temperature: 50℃;

[0087] Flow rate: 0.2 mL / min;

[0088] Sample pan temperature: 5°C;

[0089] Elution method: gradient elution;

[0090] Table 1

[0091]

[0092] Elution time: 55min.

[0093] Exclusive properties:

[0094] Accurately measure 5 μL of the above-prepared blank solvent, each impurity location solution and mixed impurity reference solution, and inject them into the high performance liquid chromatograph respectively, record the chromatogram and results, and the results are shown in the table below.

[0095] Table 2

[0096]

[0097] Note: “ND” means not detected.

[0098] Table 3

[0099]

[0100] in conclusion:

[0101] The above samples were separated and tested by high performance liquid chromatography, and the test results show that:

[0102] The blank solvent / diluent had no interference with the detection of related substances of YK-009. In the mixed impurity solution, the separation degree between YK-009 and adjacent known impurity peaks was 2.72>1.2, and the minimum separation degree between known impurity peaks was 2.44>1.2. The theoretical plate number of each known impurity peak was greater than 1500, indicating that the method had good specificity. Figure 1 .

[0103] Linearity and Range:

[0104] Solution preparation:

[0105] Diluent: Acetonitrile

[0106] Linear solution: Accurately weigh appropriate amount of each impurity reference substance, add diluent to prepare each stock solution, accurately measure appropriate amount, dilute with diluent to form a series of linear solutions, shake well, and obtain.

[0107] Experimental steps and conclusions:

[0108] Accurately measure 5µl of each of the above linear solutions and inject them into the high performance liquid chromatograph respectively. Record the results. The results are shown in the table below.

[0109] Table 4

[0110]

[0111]

[0112] Conclusion: In the concentration range of impurity 1 from 1.803μg / mL to 72.115μg / mL, the linear equation is y = 0.0939x+0.4165, r=0.995>0.990, the linear regression is significant, and the linear relationship is good.

[0113] For impurity 2 in the concentration range of 3.423μg / mL~54.774μg / mL, the linear equation is y = 0.0708x+0.0843, r=0.996>0.990, the linear regression is significant, and the linear relationship is good.

[0114] For impurity 3 in the concentration range of 1.201μg / mL~48.044μg / mL, the linear equation is y = 0.1321x+0.295, r=0.996>0.990, the linear regression is significant, and the linear relationship is good.

[0115] For impurity 4 in the concentration range of 1.163μg / mL~46.508μg / mL, the linear equation is y = 0.1293x +0.3446, r=0.995>0.990, the linear regression is significant, and the linear relationship is good.

[0116] For impurity 5 in the concentration range of 1.157μg / mL~46.272μg / mL, the linear equation is y = 0.1213x +0.312, r=0.994>0.990, the linear regression is significant, and the linear relationship is good.

[0117] For impurity 6 in the concentration range of 1.297μg / mL~51.898μg / mL, the linear equation is y = 0.1299x +0.4053, r=0.995>0.990, the linear regression is significant, and the linear relationship is good.

[0118] For impurity 7 in the concentration range of 1.149μg / mL~45.968μg / mL, the linear equation is y = 0.1445x +0.3755, r=0.995>0.990, the linear regression is significant, and the linear relationship is good.

[0119] For impurity 8 in the concentration range of 1.161μg / mL~46.440μg / mL, the linear equation is y = 0.1405x+0.2586, r=0.997>0.990, the linear regression is significant, and the linear relationship is good.

[0120] For impurity 9 in the concentration range of 1.174μg / mL~46.950μg / mL, the linear equation is y = 0.1232x +0.3316, r=0.995>0.990, the linear regression is significant, and the linear relationship is good.

[0121] For impurity 10 in the concentration range of 3.006μg / mL~72.138μg / mL, the linear equation is y = 0.0794x+0.0887, r=0.998>0.990, the linear regression is significant, and the linear relationship is good.

[0122] In the concentration range of 1.176μg / mL~47.052μg / mL, the linear equation of YK-009 is y = 0.1299x +0.3621, r=0.994>0.990, the linear regression is significant, and the linear relationship is good.

[0123] Recovery rate:

[0124] Solution preparation:

[0125] (1) Recovery test 1 (impurity 1, impurity 2, impurity 10)

[0126] Diluent: Acetonitrile

[0127] Impurity reference solution: Take 30 mg of Impurity 10 reference, 40 mg of Impurity 1 reference, and 50 mg of Impurity 2 reference, accurately weigh, place in the same 50 mL volumetric flask, dissolve and dilute to the scale with acetonitrile, shake well, and use as the reference stock solution; accurately transfer 1 mL of the reference stock solution to a 20 mL volumetric flask, dissolve and dilute to the scale with acetonitrile, shake well, and obtain.

[0128] Impurity stock solution: Take 65 mg of impurity 10 reference substance, 45 mg of impurity 1 reference substance, and 90 mg of impurity 2 reference substance, accurately weigh, place in the same 200 mL volumetric flask, dissolve and dilute to the scale with acetonitrile, and shake well.

[0129] Background solution: Take 30 mg of YK-009 test sample, weigh accurately, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0130] Self-control solution: Accurately measure 1 mL of background solution, place it in a 100 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0131] R1-50% solution: Take 30 mg of YK-009 test sample, weigh accurately, and place in 10 mL volumetric flasks, accurately add 0.5 mL of impurity stock solution, add acetonitrile to dissolve and dilute to scale, shake well, and prepare 3 portions in parallel.

[0132] R2-100% solution: Take 30 mg of YK-009 test sample, weigh accurately, place in 10 mL volumetric flasks, accurately add 1.0 mL of impurity stock solution, add acetonitrile to dissolve and dilute to scale, shake well, and prepare 3 portions in parallel.

[0133] R3-150% solution: Take 30 mg of YK-009 test sample, weigh accurately, and place in 10 mL volumetric flasks, accurately add 1.5 mL of impurity stock solution, add acetonitrile to dissolve and dilute to scale, shake well, and prepare 3 portions in parallel.

[0134] (2) Recovery test 2 (impurity 3, impurity 8)

[0135] Diluent: Acetonitrile

[0136] Impurity reference solution: Take 15 mg of impurity 3 reference, accurately weigh it, place it in a 50 mL volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well, and use it as impurity stock solution ①. Take 15 mg of impurity 8 reference, accurately weigh it, place it in a 50 mL volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well, and use it as impurity stock solution ②. Accurately transfer 1 mL of impurity stock solution ① and 1 mL of impurity stock solution ②, place them in a 10 mL volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well, and you have it.

[0137] Background solution: Take 60 mg of YK-009 test sample, weigh accurately, place in a 20 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0138] Self-control solution: Accurately measure 1 mL of background solution, place it in a 100 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0139] R1-80% solution: Take 60 mg of YK-009 test sample, weigh accurately, and place in 20 mL volumetric flasks, accurately add 0.5 mL of impurity stock solution ① and 1.0 mL of impurity stock solution ②, add acetonitrile to dissolve and dilute to scale, shake well, and the solution is ready. Prepare 3 portions in parallel.

[0140] R2-100% solution: Take 60 mg of YK-009 test sample, weigh accurately, and place in 20 mL volumetric bottles, accurately add 1.0 mL of impurity stock solution ① and 1.5 mL of impurity stock solution ②, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready. Prepare 3 portions in parallel.

[0141] R3-120% solution: Take 60 mg of YK-009 test sample, weigh accurately, and place in 20 mL volumetric flasks, accurately add 1.5 mL of impurity stock solution ① and 2 mL of impurity stock solution ②, add acetonitrile to dissolve and dilute to scale, shake well, and the solution is ready. Prepare 3 portions in parallel.

[0142] (3) Recovery test 3 (impurity 4, impurity 5, impurity 6, impurity 7, impurity 9)

[0143] Diluent: Acetonitrile

[0144] Impurity reference solution: weigh 30 mg each of impurity 4, impurity 5, impurity 6, impurity 7 and impurity 9 reference substances, place them in the same 100 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, and use this as the reference stock solution. Take 1 mL of the reference stock solution into a 10 mL volumetric flask, dilute to the mark with acetonitrile, shake well, and you have the solution.

[0145] Background solution: Take 60 mg of YK-009 test sample, weigh accurately, place in a 20 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0146] Self-control solution: Accurately measure 1 mL of background solution, place it in a 100 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0147] R1-80% solution: Take 60 mg of the YK-009 test sample, weigh it accurately, and place it in a 20 mL volumetric bottle. Accurately add 0.5 mL of the reference stock solution, dissolve it in acetonitrile and dilute it to the mark, shake well, and the solution is ready. Prepare 3 portions in parallel.

[0148] R2-100% solution: Take 60 mg of YK-009 test sample, weigh accurately, and place in 20 mL volumetric bottles, accurately add 1.0 mL of reference stock solution, dissolve in acetonitrile and dilute to scale, shake well, and prepare 3 portions in parallel.

[0149] R3-120% solution: Take 60 mg of the YK-009 test sample, weigh it accurately, and place it in a 20 mL volumetric bottle. Add 1.5 mL of the reference stock solution accurately, dissolve it in acetonitrile and dilute it to the mark, shake well, and the solution is ready. Prepare 3 portions in parallel.

[0150] Experimental steps and conclusions:

[0151] Accurately measure 5µl of each of the above blank diluents, impurity reference solution, background solution, self-control solution and different levels of spiked solutions, inject them into the high performance liquid chromatograph respectively, record the chromatogram and results, calculate the recovery rate by the external standard method, and the results are shown in the table below.

[0152] Table 5

[0153]

[0154] Table 6

[0155]

[0156] Table 7

[0157]

[0158] Table 8

[0159]

[0160] Table 9

[0161]

[0162] Table 10

[0163]

[0164] Table 11

[0165]

[0166] Table 12

[0167]

[0168] Table 13

[0169]

[0170] Table 14

[0171]

[0172] Conclusion: At the three concentration levels of 50%, 100%, and 150%, the recovery rates of impurities 2 and 10 calculated by the external standard method were between 90% and 110%. The maximum recovery rate of impurity 10 was 105.4%, and the minimum was 91.5%; the maximum recovery rate of impurity 2 was 107.7%, and the minimum was 90.5%; the maximum recovery rate of impurity 1 was 95.5%, and the minimum was 80.0%; the maximum RSD was 6.3%, and the minimum was 5.6%, which was less than 10.0%, indicating that the accuracy of this method was good.

[0173] At the three concentration levels of 80%, 100%, and 120%, the recovery rates of impurities 3 and 8 were calculated by external standard method at each point and were between 90% and 110%. The maximum recovery rate of impurity 3 was 103.0%, and the minimum was 92.0%; the maximum recovery rate of impurity 8 was 99.6%, and the minimum was 91.8%; the RSD values ​​were 4.6% and 2.8%, respectively, less than 10.0%, indicating that the accuracy of this method was good.

[0174] At the three concentration levels of 80%, 100% and 120%, the recovery rates of impurities 4, 5, 6, 7 and 9 calculated by external standard method were all between 90% and 110%. The maximum recovery rate of impurity 4 was 106.5% and the minimum was 91.1%; the maximum recovery rate of impurity 5 was 103.3% and the minimum was 90.2%; the maximum recovery rate of impurity 6 was 107.5% and the minimum was 92.3%; the maximum recovery rate of impurity 7 was 107.3% and the minimum was 93.2%; the maximum recovery rate of impurity 9 was 107.0% and the minimum was 90.3%; the maximum RSD was 6.8% and the minimum was 4.7%, which was less than 10.0%, indicating that the accuracy of this method was good.

[0175] Durability:

[0176] Solution preparation:

[0177] Diluent: Acetonitrile

[0178] Test solution: Accurately weigh 30 mg of the product, place in a 10 mL volumetric flask, dissolve in acetonitrile and dilute to the mark, shake well, and the solution is ready.

[0179] Self-control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with acetonitrile, shake well, and obtain.

[0180] Experimental steps and conclusions:

[0181] Accurately measure 5 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0182] Table 15

[0183]

[0184] Table 16

[0185]

[0186] Conclusion: When the column temperature was adjusted within ±5℃ in the chromatographic conditions, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the separation of YK-009 and adjacent impurities was 2.0 and 1.9, respectively, both greater than 1.5, indicating that the system was in good condition. When the pH value of the acetic acid-triethylamine solution was 6.5, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the separation of YK-009 and adjacent impurities was 2.4. When the pH value of the acetic acid-triethylamine solution was 7.5, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the separation of YK-009 and adjacent impurities was 2.3, indicating that the system was in good condition. After adjusting the column temperature and the pH value of the acetic acid-triethylamine solution, there was no significant difference between the results of each impurity and the normal conditions. Based on the above results, the column temperature and the pH value of the acetic acid-triethylamine solution in the chromatographic conditions had good durability.

[0187] Example 2 Selection of chromatographic column

[0188] Based on the chromatographic conditions of Example 1, the type of chromatographic column was changed, and other chromatographic conditions remained unchanged, and YK-009 and its 10 known impurities were detected. Accurately measure 5 μL of the mixed impurity reference solution prepared in Example 1, inject it into the high performance liquid chromatograph respectively, and record the results. Figure 2-3 .

[0189] Table 17

[0190]

[0191] Conclusion: The separation of YK-009 and its impurities by HPLC analysis using the two different chromatographic columns mentioned above revealed that the chromatographic column used in method 1 (Waters ACQUITY UPLC Oligonucleotide BEH C18) yielded a spectrum ( Figure 2 ) has a wide peak shape for YK-009 and its impurities, of which the symmetry factor of YK-009 is 2.91, impurities 5 and 9 are not baseline separated, impurities 3 and 6 are wrapped by the main peak, and the separation degree between adjacent impurities and YK-009 is 0.51, indicating poor impurity separation. Using the ordinary C18 column (Agilent Poroshell 120 EC-C18) of method 2, the spectrum obtained is ( Figure 3 ) has a wider peak shape of YK-009, which is triangular and has a symmetry factor of 4.43. The peak shapes of impurities 5, 9 and 4 are also wider, which reduces the sensitivity of impurities. The tail of the main peak YK-009 can clearly be seen to contain impurities, and the impurity separation effect is not good. In contrast, the chromatographic column (Waters ACQUITY UPLC CSH Phenyl-Hexyl) in Example 1 is used to obtain the spectrum ( Figure 1) It can be clearly seen that the peak shapes of the 10 impurities and YK-009 are good, the impurity symmetry factors are between 0.9 and 1.1, the YK-009 symmetry factor is 0.74, and the separation degree between the main peak YK-009 and the adjacent impurities is 2.72, which achieves good separation of YK-009 and the 10 impurities and meets the detection requirements of related impurities in YK-009.

[0192] Example 3 Selection of mobile phase solvent

[0193] Based on the chromatographic conditions of Example 1, the mobile phase B solvent was changed, and other chromatographic conditions remained unchanged, and YK-009 and its 10 known impurities were detected. Accurately measure 5 μL of the test solution prepared in Example 1 and inject them into the high performance liquid chromatograph respectively, and record the results. Figure 4 .

[0194] Table 18

[0195]

[0196] Conclusion: The separation of YK-009 and its impurities by HPLC analysis using the two different solvents as mobile phases found that the spectrum obtained by using acetonitrile as the solvent in Example 1 ( Figure 1 ) has a good peak shape, the symmetry factors of each impurity are between 0.9 and 1.1, the symmetry factor of YK-009 is 0.74, all impurities can be well separated from YK-009, and the separation degree between adjacent impurities and YK-009 is greater than 1.5, which can achieve the separation and detection of 10 known impurities in YK-009. In contrast, when methanol in the comparative example is used as the solvent, the spectrum obtained ( Figure 4 ) The main peak YK-009 has a poor peak shape, severe broadening, and a symmetry factor of 3.61. The separation between the main peak YK-009 and the adjacent impurities is less than 1.0. There is one impurity peak before YK-009, which may be wrapped by the main peak, making it impossible to separate and detect the main peak and 10 impurities.

[0197] Example 4 Selection of the amount of acetic acid-triethylamine added in the mobile phase

[0198] Based on the chromatographic conditions of Example 1, the amount of ion pair reagent (acetic acid-triethylamine) added was changed, and other chromatographic conditions remained unchanged, and YK-009 and its 10 known impurities were detected. Accurately measure 5 μL of the test solution prepared in Example 1 and inject it into the high performance liquid chromatograph respectively, and record the results, see Figure 5 and Figure 6 .

[0199] Table 19

[0200]

[0201] Conclusion: The separation of YK-009 and its impurities was analyzed by HPLC using the mobile phases with different ion pair reagent additions. It was found that when 0.5% acetic acid-triethylamine was added in method 1, the spectrum obtained was ( Figure 5 ) has a poor peak shape, a symmetry factor of 2.14, and a separation degree of 2.5 between YK-009 and adjacent impurities. However, it can be clearly seen from the figure that there is a problem with the elution of the peak tail, and the impurity peak may be wrapped. When 0.7% of acetic acid-triethylamine is added, the peak shape of the main peak is significantly improved, and the separation degree of YK-009 and adjacent impurities is 1.2, which basically meets the separation requirements, but impurities 4 and 9 cannot be baseline separated, and the separation degree is only 0.85. When 1.5% of acetic acid-triethylamine is added, the spectrum obtained is ( Figure 6 ) in which the separation degree of YK-009 from adjacent impurities is greater than 1.5, but impurities 4 and 9 are inseparable, and the peak shape of YK-009 is not much different from that when the addition amount is 1.0%, and both are better than the peak shape when the addition amount is 0.5%. When 1.0% of acetic acid-triethylamine is added in Example 1, the spectrum obtained is ( Figure 1 ) The main peak YK-009 has a good peak shape, the symmetry factor of YK-009 is 0.74, and the separation between YK-009 and adjacent impurities is greater than 1.5, which can achieve the separation and detection of 10 known impurities in YK-009. At the same time, considering the influence of ion pair reagents on separation, instruments and chromatographic columns, a 1.0% addition amount can meet the needs.

[0202] Example 5 Selection of elution gradient

[0203] Based on the chromatographic conditions of Example 1, the elution gradient was changed, and other chromatographic conditions remained unchanged to detect YK-009 and its impurities. Accurately measure 5 μL of the test solution prepared in Example 1 and inject it into the high performance liquid chromatograph respectively, and record the results. Figure 7 and Figure 8 .

[0204] Table 20

[0205]

[0206] Conclusion: The separation of YK-009 and its impurities was analyzed by HPLC using the above different elution gradients. It was found that when the elution gradient of method 1 was used, the spectrum obtained ( Figure 7 ) has a separation degree of 0.49 between YK-009 and adjacent impurities. It can be clearly seen from the figure that the low-proportion organic phase has a poor elution effect on the main peak and impurities, resulting in no impurity peaks in the first 30 minutes of the chromatogram, and the main peak and impurities are concentrated in the back and are dense, with poor separation. When the elution gradient of method 2 is used, the spectrum obtained is ( Figure 8) It can be seen that the impurity separation is improved compared with method 1, but impurities 5 and 9 are encapsulated, and the separation degree between the main peak YK-009 and the subsequent impurities is 0.90, which needs further optimization. When the elution gradient in Example 1 is used, the spectrum obtained is ( Figure 1 ) The main peak YK-009 has a good peak shape, the symmetry factor of YK-009 is 0.74, and the separation degree between YK-009 and adjacent impurities is greater than 1.5, which can realize the separation and detection of 10 known impurities in YK-009.

[0207] Example 6 Selection of mobile phase additives

[0208] Based on the chromatographic conditions of Example 1, the mobile phase additives were changed to detect YK-009 and its impurities. Accurately measure 5 μL of the test solution prepared in Example 1 and inject them into the high performance liquid chromatograph respectively, and record the results. Fig. 9 .

[0209] Table 21

[0210]

[0211] Conclusion: The separation of YK-009 and its impurities by HPLC analysis with the mobile phases of the above-mentioned different additives revealed that the spectrum obtained by using ammonium formate in method 1 ( Fig. 9 ) has a wide main peak shape, a symmetry factor of 3.89, and a separation degree of 2.06 between YK-009 and adjacent impurities. However, it can be seen from the figure that the number of impurity peaks detected is small, and there may be impurity peaks that are wrapped. At the same time, the impurity peaks have a poor peak shape and a tailing phenomenon. When acetic acid-triethylamine in Example 1 is used, the spectrum obtained is ( Figure 1 ) The main peak YK-009 has a good peak shape, the symmetry factor of YK-009 is 0.74, and the separation degree between YK-009 and adjacent impurities is greater than 1.5, which can realize the separation and detection of 10 known impurities in YK-009.

[0212] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detection method and application of the related substances in the cationic lipid YK-009 of the present invention, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0213] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0214] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for detecting related substances in cationic lipid YK-009, characterized in that: The detection method comprises the following steps: (1) Mix the sample to be tested with the diluent to obtain a test solution; (2) Mixing the standard substance of the relevant substance with the diluent to obtain a reference solution; (3) subjecting the test solution and the reference solution to liquid chromatography detection, and calculating the content of the related substances according to the detection results; the mobile phase of the liquid chromatography detection comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution of acetic acid-triethylamine, and the mobile phase B is an acetonitrile solution of acetic acid-triethylamine; There is no order distinction between steps (1) and (2); The liquid chromatography detection in step (3) is carried out by gradient elution, and the specific elution gradient is as follows: From 0 to 15 min, the volume fraction of mobile phase A was 23-27%, and the rest was mobile phase B; From 15 to 30 minutes, the volume fraction of mobile phase A changes at a constant rate from 23-27% to 4-6%, and the rest is mobile phase B; At 30-40 min, the volume fraction of mobile phase A was 4-6%, and the rest was mobile phase B; At 40-41 min, the volume fraction of mobile phase A changed from 4-6% to 0 at a constant rate, and the rest was mobile phase B; From 41 to 45 min, the volume fraction of mobile phase A was 0, and the rest was mobile phase B; From 45 to 45.1 min, the volume fraction of mobile phase A changed from 0 to 23-27% at a constant rate, and the rest was mobile phase B; After 45.1 min, the volume fraction of mobile phase A was 23-27%, and the rest was mobile phase B; In the liquid chromatography detection of step (3), the stationary phase is hexyl-linked phenyl-bonded silica gel; The related substances include any one or a combination of at least two of impurities 1 to 10: Impurity 1; Impurity 2; Impurity 3; Impurity 4; Impurity 5; Impurity 6; Impurity 7; Impurity 8; Impurity 9; Impurity 10.

2. The method for detecting related substances in the cationic lipid YK-009 according to claim 1, characterized in that: The diluent includes methanol and / or acetonitrile.

3. The method for detecting related substances in the cationic lipid YK-009 according to claim 2, characterized in that: The mobile phase A is prepared by mixing an acetic acid-triethylamine aqueous solution with water, and the mobile phase B is prepared by mixing an acetic acid-triethylamine aqueous solution with acetonitrile.

4. The method for detecting related substances in the cationic lipid YK-009 according to claim 3, characterized in that: In the acetic acid-triethylamine aqueous solution used in the preparation of mobile phase A and mobile phase B, the volume ratio of triethylamine to water is independently (1-3):(17-19), and the pH is independently 6.5-7.

5.

5. The method for detecting related substances in the cationic lipid YK-009 according to claim 4, characterized in that: The concentration of the acetic acid-triethylamine aqueous solution in the mobile phase A and the mobile phase B is independently 0.8-1.2%.

6. The method for detecting related substances in the cationic lipid YK-009 according to claim 5, characterized in that: In the liquid chromatography detection of step (3), the column temperature is 20-50°C, the mobile phase flow rate is 0.2-0.4 mL / min, and the injection volume is 2-10 μL.

7. The method for detecting related substances in the cationic lipid YK-009 according to claim 6, characterized in that: The related substances include impurities 1 to 10.

8. Use of the method for detecting related substances in the cationic lipid YK-009 according to any one of claims 1 to 7 in the quality control of the cationic lipid YK-009.

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