Method for determining related substances in liposome adjuvant and application thereof

Abstract

The invention discloses a method for determining related substances in a liposome adjuvant and application of the method, and relates to the field of medicine detection. The method is a high performance liquid chromatography, and a mobile phase A comprises an aqueous solution containing trifluoroacetic acid; the mobile phase A comprises a mixed solution of methyl alcohol containing trifluoroacetic acid and acetonitrile, the mobile phase B comprises a mixed solution of methyl alcohol containing trifluoroacetic acid and acetonitrile, simultaneous detection of multiple target substances including oleic acid, ketocholesterol, two isomers of Lyso-DOTAP and two isomers of Lyso-DOPC in the liposome adjuvant is achieved through a specific elution procedure, and the method is good in specificity, high in sensitivity, good in separation degree and high in detection sensitivity. And a new way is provided for evaluating the effectiveness, safety and stability of the liposome adjuvant.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, in particular to a method for determining related substances in a liposome adjuvant and an application thereof. Background Art

[0002] Adjuvants are substances that enhance specific immune responses. They can be broadly categorized into two types: those inherently immunogenic, such as Bordetella pertussis, acid-fast bacilli (Mycobacterium tuberculosis), and Gram-negative bacilli; and those inherently non-immunogenic, such as aluminum hydroxide adjuvants, calcium phosphate, mineral oil emulsions, and surfactants. Freund's adjuvant, aluminum hydroxide adjuvant, and liposomes are the most commonly used. Liposomes are currently one of the most effective carriers for transporting various antigens into cells.

[0003] Liposomes used as vaccine adjuvant systems typically contain phospholipids, cholesterol, and immunostimulants. Liposomes are artificially synthesized lipid vesicles with a monolayer or multilayer membrane-like structure, acting as both adjuvants and carriers. As vaccine adjuvants, liposomes can enhance both humoral and cellular immunity. Phospholipids are a crucial component of liposomes and form the bilayer. Phospholipids used in vaccine adjuvant systems primarily include cationic phospholipids and neutral phospholipids. Commonly used cationic phospholipids include DOTAP (2,3-dioleoyl-trimethylammonium propane chloride) and DDA (Dioctadecyl Dimethyl Ammonium Chloride); commonly used neutral phospholipids include DOPC (Dioleoyl Phosphatidylcholine), DMPC (Dimyristoyl Phosphatidylcholine), and DMPG (Dimyristoyl Phosphatidylglycerol). Lysophospholipids (LPs) are intermediates in the synthesis of phospholipids and are also their major degradation products and metabolites. They are present in small amounts in cell membranes and tissues. Excessive levels in the body can cause red blood cells to rupture, leading to hemolysis or cell necrosis. Therefore, accurate measurement of lysophospholipid content is a key indicator for evaluating the quality, stability, and safety of liposomes. Ketocholesterol, among other substances, is a major degradation and metabolite of cholesterol. Therefore, accurate measurement of ketocholesterol content is also a key indicator for evaluating the quality, stability, and safety of liposomes.

[0004] As a universal detector, the ELSD detector has been widely used in the field of drug testing. Many studies have used the ELSD method to determine lysophosphatidylcholine in small-molecule liposome drugs. Currently, there are few literature reports on analytical methods for liposome adjuvant-related substances (lysophospholipids, oleic acid, ketocholesterol, etc.) in biological products such as vaccines. Detection is particularly difficult when the antigen and liposome adjuvant are in the same solution, and it is also possible to effectively separate lysophospholipid isomers. Existing methods cannot simultaneously determine the related substances of each component of the liposome adjuvant.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for determining related substances in liposome adjuvants and its application.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a method for determining a target substance in a liposome adjuvant, comprising: detecting the target substance in a sample to be tested using high performance liquid chromatography; the chromatography comprises gradient elution using mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution containing trifluoroacetic acid; and mobile phase B comprises a mixed solution of methanol containing trifluoroacetic acid and acetonitrile; the elution conditions of the chromatography are as follows: from 0 to 20 to 30 minutes, the volume percentage of the mobile phase A decreases from 15% to 25% to 0 to 10%, and the volume percentage of the mobile phase B increases from 75% to 85% to 90% to 100%; from 20 to 30 minutes to 30 to 40 minutes, the volume percentage of the mobile phase A is maintained at 0 to 10%, and the volume percentage of the mobile phase B is maintained at 90% to 100%; the target substance comprises: components of the liposome adjuvant and / or degradation products and / or metabolites of the components.

[0009] In a second aspect, an embodiment of the present invention provides the use of a reagent combination in preparing a product for determining a target substance in a liposome adjuvant, wherein the reagent combination includes reagents for implementing the method described in the aforementioned embodiment; the target substance includes: components of the liposome adjuvant and / or metabolites of the components.

[0010] In a third aspect, an embodiment of the present invention provides a product for determining a target substance in a liposome adjuvant, which includes: a reagent for implementing the method described in the above embodiment.

[0011] In a fourth aspect, embodiments of the present invention provide applications of the methods described in the preceding embodiments in evaluating the quality of liposome adjuvants or detecting the effectiveness, safety, and / or stability of liposome adjuvants.

[0012] The present invention has the following beneficial effects:

[0013] The present invention provides a method capable of simultaneously detecting target substances in a liposome adjuvant. The target substances include cationic phospholipid DOTAP, neutral phospholipid DOPC, cholesterol and their related substances. The related substances mainly include lysophospholipids (two isomers of Lyso-DOTAP and two isomers of Lyso-DOPC), oleic acid and ketocholesterol. The method has good separation effect and provides a new approach for evaluating the effectiveness, safety and stability of liposome adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 HPLC chromatogram of the control solution (Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketocholesterol: 20 μg / ml);

[0016] Figure 2 This is the HPLC chromatogram of test solution-1 (containing antigen protein, DOTAP, DOPC, cholesterol, etc.);

[0017] Figure 3 This is the HPLC chromatogram of test solution-2 (containing DOPC, cholesterol, etc.);

[0018] Figure 4 This is the HPLC chromatogram of test solution-3 (containing DOTAP, DOPC, cholesterol, etc.);

[0019] Figure 5 HPLC chromatogram of DOTAP acid degradation (containing two isomers of Lyso-DOTAP);

[0020] Figure 6 HPLC chromatogram of DOPC acid degradation (containing two isomers of Lyso-DOPC);

[0021] Figure 7 HPLC chromatogram of the control solution (Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketocholesterol, DOTAP, DOPC, cholesterol: 25 μg / ml);

[0022] Figure 8HPLC chromatogram of the test sample spiked solution (Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketocholesterol: 25 μg / ml);

[0023] Figure 9 HPLC chromatogram of the control solution (DOTAP, DOPC, cholesterol: 61, 298, 106 μg / ml);

[0024] Figure 10 HPLC chromatogram of the control solution (DOTAP, DOPC, cholesterol: 61, 298, 106 μg / ml);

[0025] Figure 11 HPLC chromatogram of control solution (DOTAP, DOPC, cholesterol: 61, 298, 106 μg / ml). DETAILED DESCRIPTION

[0026] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0027] To address the problems of the prior art, the present invention develops a novel detection process for detecting target substances in liposome adjuvants. This method enables the simultaneous detection of multiple target substances, such as lysophospholipids (Lyso-DOTAP, Lyso-DOPC), oleic acid, and ketocholesterol, and can effectively separate the two isomers of Lyso-DOTAP and Lyso-DOPC. This method has strong specificity, is simple to operate, has good resolution, and is highly sensitive, providing a pathway for testing the effectiveness, stability, and safety of liposome adjuvants.

[0028] In one aspect, an embodiment of the present invention provides a method for determining a target substance in a liposome adjuvant, comprising: detecting the target substance in a sample to be tested by high performance liquid chromatography;

[0029] The chromatography method includes gradient elution using mobile phase A and mobile phase B, wherein the mobile phase A includes an aqueous solution containing trifluoroacetic acid; the mobile phase B includes a mixed solution of methanol and acetonitrile containing trifluoroacetic acid; and the elution conditions of the chromatography are as follows:

[0030] From 0 to 20 to 30 minutes, the volume percentage of the mobile phase A is reduced from 15% to 25% to 0 to 10%, and the volume percentage of the mobile phase B is increased from 75% to 85% to 90% to 100%;

[0031] From 20 to 30 minutes to 30 to 40 minutes, the volume percentage of the mobile phase A is maintained at 0 to 10%, and the volume percentage of the mobile phase B is maintained at 90% to 100%.

[0032] In some embodiments, the elution conditions of the chromatography are as follows:

[0033] From 0 to 24-26 min, the volume percentage of the mobile phase A decreased from 18%-22% to 0-5%, and the volume percentage of the mobile phase B increased from 78%-82% to 95%-100%;

[0034] From 24 to 26 minutes to 34 to 36 minutes, the volume percentage of the mobile phase A is maintained at 0 to 5%, and the volume percentage of the mobile phase B is maintained at 95% to 100%.

[0035] In some embodiments, the volume fraction of trifluoroacetic acid in the mobile phase A in the aqueous solution is 0.05% to 0.15%, specifically any one of 0.05%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, 0.15%, or a range between any two of them.

[0036] In some embodiments, the volume fraction of trifluoroacetic acid in the mobile phase B in the mixed solution of methanol and acetonitrile is 0.05% to 0.15%, specifically any one of 0.05%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, and 0.15%, or a range between any two of them; the mixed volume ratio of methanol and acetonitrile is 1 to 5:1 to 5, specifically any one of 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:1, 3:2, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, and 5:4, or a range between any two of them.

[0037] In some embodiments, the flow rate of the gradient elution is 0.5 to 1.5 mL / min, specifically any one of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL / min or a range between any two thereof.

[0038] In some embodiments, the chromatographic column used in the chromatography comprises a reverse phase chromatography column, wherein the reverse phase chromatography column comprises any one of C8 and C18; the specifications of the chromatographic column are column length 50-250 mm x inner diameter 2.1-4.6 mm, and the filler particle size is 1.7-10 μm. Specifically, the column length can be any one of 50, 100, 150, 200, 250 mm, or a range between any two thereof; the inner diameter can be any one of 2.1, 3.0, 4.6 mm, or a range between any two thereof; and the filler particle size can be any one of 1.7, 2.6, 3, 3.5, 5, or 10 μm, or a range between any two thereof.

[0039] In some embodiments, the chromatographic conditions of the chromatography method include: a column temperature of 30-50° C. and an injection volume of 10-50 μL. Specifically, the column temperature can be any one of 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50° C., or a range between any two thereof; and the injection volume can be any one of 10, 15, 20, 25, 30, 35, 40, 45, and 50 μL, or a range between any two thereof.

[0040] In some embodiments, before the detection, the method further comprises: taking a liposome adjuvant sample solution, diluting it, and centrifuging it, and taking the supernatant as the sample to be tested.

[0041] In some embodiments, the liposome adjuvant sample can be an antigen-containing or antigen-free sample.

[0042] In some embodiments, the amount of the liposome adjuvant sample solution can be 0.2-2 mL, specifically any one of 0.2, 0.5, 1, 1.5, and 2 mL, or a range between any two of them.

[0043] In some embodiments, the diluent used for dilution includes any one or more combinations of methanol, ethanol, and acetonitrile.

[0044] In some embodiments, the dilution multiple is 5 to 10 times, specifically any one of 5, 6, 7, 8, 9, 10 times or a range between any two of them.

[0045] In some embodiments, the centrifugation conditions include: 3000-15000 rpm, 3-10 min. The speed can be any one of 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 1500 rpm, or a range between any two thereof; and the centrifugation time can be any one of 3, 4, 5, 6, 7, 8, 9, or 10 min, or a range between any two thereof.

[0046] In some embodiments, the target substance includes: components of the liposome adjuvant and / or degradation products and / or metabolites of the components. The detection method provided in the embodiments of the present invention can be applied to the simultaneous detection of components of the liposome adjuvant and their metabolites and / or degradation products.

[0047] In some embodiments, the component includes at least one of a cationic phospholipid DOTAP, a neutral phospholipid DOPC, and cholesterol.

[0048] In some embodiments, the degradation products and / or metabolites of the component include at least one of lysophospholipids, oleic acid, and ketocholesterol. The lysophospholipids include Lyso-DOTAP-1, Lyso-DOTAP-2, which is an isomer of the Lyso-DOTAP, Lyso-DOPC-1, and Lyso-DOOC-2, which is an isomer of the Lyso-DOPC.

[0049] The structural formula of the Lyso-DOTAP-1 is:

[0050]

[0051] The structural formula of the Lyso-DOTAP-2 is:

[0052]

[0053] The structural formula of the Lyso-DOPC-1 is:

[0054]

[0055] The structural formula of the Lyso-DOOC-2 is:

[0056]

[0057] In some embodiments, the method further includes: obtaining a control solution, performing chromatographic detection on the control solution, the detection conditions are the same as those described in any of the aforementioned embodiments, calculating a linear regression equation based on the logarithmic value of the control solution concentration and the logarithmic value of the corresponding peak area, and using the linear regression equation to calculate the content of each target substance in the test solution.

[0058] In some embodiments, the detector used for the detection comprises an ELSD detector.

[0059] In some embodiments, the ELSD detector is AllChrom 6000 or Agilent G7102A.

[0060] In some embodiments, the AllChrom 6000 is operated with a drift tube temperature of 40-80°C, a gas flow rate of 1.0-3 L / min, and an impactor position set to on. Alternatively, the drift tube temperature can be any one of 40, 45, 50, 55, 60, 65, 70, 75, or 80°C, or a range between any two thereof. The gas flow rate can be any one of 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.5, or 3 L / min, or a range between any two thereof.

[0061] On the other hand, an embodiment of the present invention provides the use of a reagent combination in preparing a product for determining a target substance in a liposome adjuvant, wherein the reagent combination includes a reagent for implementing the method described in any of the aforementioned embodiments; the target substance includes: a component of the liposome adjuvant and / or a metabolite of the component.

[0062] On the other hand, an embodiment of the present invention further provides a product for determining a target substance in a liposome adjuvant, which includes: a reagent for implementing the method described in any of the above embodiments.

[0063] In some embodiments, the types of products include: reagents and kits.

[0064] In some embodiments, the reagents for implementing the method described in any of the foregoing embodiments include: the mobile phase A described in any of the foregoing embodiments and the mobile phase B described in any of the foregoing embodiments.

[0065] In some embodiments, the reagent for implementing the method as described in any of the preceding embodiments further includes: the diluent described in any of the preceding embodiments.

[0066] In some embodiments, the reagents used to implement the method described in any of the foregoing embodiments further include: any one or more combinations of standard products, quality control products, reagents for configuring standard products, and reagents for configuring quality control products.

[0067] In addition, embodiments of the present invention also provide the use of the method described in any of the aforementioned embodiments in evaluating the quality of liposome adjuvants or detecting the effectiveness, safety and / or stability of liposome adjuvants.

[0068] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0069] Example 1

[0070] A method for determining a target substance in a liposome adjuvant is a high performance liquid chromatography method, specifically comprising the following steps.

[0071] 1.1 Chromatographic conditions

[0072] (1) Chromatographic column: Reversed-phase chromatography C18 column (Spolar C18), size 250 × 4.6 mm, filler particle size 5 μm; column temperature 40°C;

[0073] (2) Mobile phase: Mobile phase A is an aqueous solution containing 0.1 v / v% trifluoroacetic acid, and mobile phase B is a mixed solution of methanol and acetonitrile containing 0.1 v / v% trifluoroacetic acid (the volume ratio of methanol to acetonitrile is 1:1);

[0074] (3) Gradient elution program: elution time is 35 min; the specific gradient elution program is:

[0075] Table 1 Elution program

[0076] Time (minutes) Mobile phase A Mobile phase B 0 20 80 25 0 100 35 0 100

[0077] (4) Detector: AllChrom 6000 ELSD detector was used, with drift tube temperature of 45 °C, gas flow rate of 1.5 L / min, and impactor position on;

[0078] (5) The mobile phase flow rate was 1.0 mL / min and the injection volume was 30 μl.

[0079] 1.2 Solution Preparation

[0080] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol reference substances, dissolve them in anhydrous ethanol, and dilute them to make a solution containing 100 μg of each component per 1 ml.

[0081] Control solution: Take an appropriate amount of the control stock solution and add 80% ethanol to prepare solutions containing 5, 10, 20, 30, and 50 μg per 1 ml.

[0082] Test solution-1: Accurately measure 0.2 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, take the solution and centrifuge it at 5000 rpm for 5 minutes, take the supernatant to obtain the test solution-1.

[0083] Test solution-2: Accurately measure 0.2 ml of liposome adjuvant (including appropriate amounts of DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, take the solution and centrifuge it at 5000 rpm for 5 minutes, take the supernatant to obtain test solution-2.

[0084] Test solution-3: Accurately measure 0.2 ml of liposome adjuvant (including appropriate amounts of DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, take the solution and centrifuge at 5000 rpm for 5 minutes, take the supernatant to obtain test solution-3.

[0085] 1.3 Assay

[0086] Take 30 μL of control solution and test solution 1 to 3 respectively, and the results are as follows Figures 1 to 4 shown.

[0087] Example 2 Specificity

[0088] The specificity of the method of Example 1 was investigated using blank solution, control solution, test solution, and test solution spiked with the test solution.

[0089] 2.1 Chromatographic conditions were the same as in Example 1.

[0090] 2.2 Solution preparation:

[0091] Blank solution: Take 0.2 ml of purified water, add 0.8 ml of anhydrous ethanol to dissolve, shake well, take the solution and centrifuge at 5000 rpm for 5 minutes, take the supernatant to obtain the blank solution.

[0092] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketocholesterol, DOTAP, DOPC and cholesterol reference substances, dissolve them in anhydrous ethanol and dilute them to make a solution containing 100 μg of each component per 1 ml.

[0093] Control solution: Take an appropriate amount of the control stock solution and add 80% ethanol to prepare solutions containing 5, 15, 25, 35, and 50 μg per 1 ml.

[0094] DOTAP acid degradation solution: Take 0.5 ml of 4 mg / ml DOTAP control stock solution, add 0.4 ml of 1N hydrochloric acid solution, then add 1.1 ml of anhydrous ethanol, mix well, and let it stand at room temperature for 4 hours.

[0095] DOPC acid degradation solution: Take 0.5 ml of 4 mg / ml DOPC control stock solution, add 0.4 ml of 1N hydrochloric acid solution, then add 1.1 ml of anhydrous ethanol, mix well, and let it stand at room temperature for 4 hours.

[0096] Test solution: Accurately measure 0.2 ml of liposome adjuvant (including appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, take the solution and centrifuge at 5000 rpm for 5 minutes, take the supernatant to obtain the test solution;

[0097] Test sample spike solution: Accurately measure 2.0 ml of liposome adjuvant (including appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution, place them in the same 10 ml volumetric flask, make up to volume with diluent, and shake well; centrifuge the solution at 5000 rpm for 5 minutes and collect the supernatant;

[0098] 2.3 The results are shown in Table 2 and Figures 5 to 8 As shown in the figure, the other components did not interfere with the detection of the two isomers of Lyso-DOTAP, the two isomers of Lyso-DOPC, oleic acid and ketocholesterol, and the method was highly specific.

[0099] Table 2 Specificity results

[0100]

[0101] The results showed that the other components in the blank solution and the test solution did not interfere with the detection of the two isomers of Lyso-DOTAP, the two isomers of Lyso-DOPC, oleic acid and ketocholesterol, and could effectively separate the two isomers of Lyso-DOTAP and Lyso-DOPC ( Figures 5-6 ), showing strong specificity. The relative retention times (RRT) of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketocholesterol and DOPC are 0.28, 0.37, 0.62, 0.74 and 1.0, respectively. Figures 7-8 .

[0102] Example 3 Linear

[0103] The linear response of the method of Example 1 was tested over a concentration range of 5 μg / ml to 50 μg / ml for each target substance. The results showed that the logarithm of the response value (peak area) was proportional to the logarithm of the concentration of the analyte in the sample within the given range, as shown below.

[0104] 3.1 Chromatographic conditions were the same as in Example 1.

[0105] 3.2 Solution preparation:

[0106] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid and ketocholesterol reference substances, dissolve them in anhydrous ethanol and dilute them to make a solution containing 100 μg of each component per 1 ml.

[0107] Linear solution: Take an appropriate amount of the control stock solution and add 80% ethanol to prepare solutions containing 5, 10, 20, 25, and 50 μg per 1 ml.

[0108] 3.3 The test results are shown in Table 3:

[0109] Table 3 Linearity results

[0110]

[0111] The results showed that Lyso-DOTAP-1 had a good linear relationship in the range of 4.54μg / ml to 45.45μg / ml, Lyso-DOPC-1 had a good linear relationship in the range of 4.90μg / ml to 49.00μg / ml, oleic acid had a good linear relationship in the range of 5.32μg / ml to 53.22μg / ml, and ketone cholesterol had a good linear relationship in the range of 5.22μg / ml to 52.18μg / ml, meeting the acceptance criteria.

[0112] Example 4 Limit of Quantitation (LOQ) 4.1 Chromatographic conditions were the same as in Example 1.

[0113] 4.2 Solution preparation

[0114] Quantitation limit solution: Take a solution containing 5 μg per 1 ml of the linear solution in step 3.2 of Example 3.

[0115] 4.3 The test results are shown in Table 4:

[0116] Table 4 Quantitation limit results

[0117]

[0118]

[0119] The results showed that the quantification limits of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol were 4.54 μg / ml, 4.90 μg / ml, 5.32 μg / ml, and 5.22 μg / ml, respectively. The quantification limits of each component met the acceptance criteria of a signal-to-noise ratio ≥ 10.

[0120] Example 5 Accuracy

[0121] The method provided in Example 1 was used to determine the difference between the measurement results and the true values ​​when measuring the target substances Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol, so as to confirm whether the method can obtain accurate test results.

[0122] 5.1 Chromatographic conditions were the same as in Example 1.

[0123] 5.2 Solution preparation:

[0124] Control stock solution-1: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol reference substances, dissolve them in anhydrous ethanol, and dilute them to make a solution containing 25 μg of each component per 1 ml.

[0125] Reference stock solution-2: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol reference substances, dissolve them in anhydrous ethanol, and dilute them to make a solution containing 100 μg of each component per 1 ml.

[0126] Control stock solution-3: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol reference substances, dissolve them in anhydrous ethanol, and dilute them to make a solution containing 200 μg of each component per 1 ml.

[0127] Test solution: Accurately measure 0.2 ml of liposome adjuvant (including appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, take the solution and centrifuge at 5000 rpm for 5 minutes, take the supernatant to obtain the test solution;

[0128] Accuracy Solution 1 (20%, relative to 25 μg / ml): Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution-1, place them in the same 10 ml volumetric flask, make up to volume with anhydrous ethanol, and shake well; centrifuge the solution at 5000 rpm for 5 minutes, and collect the supernatant; prepare three replicates.

[0129] Accuracy Solution 2 (100%, relative to 25 μg / ml): Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution-2, place them in the same 10 ml volumetric flask, make up to volume with anhydrous ethanol, and shake well; centrifuge the solution at 5000 rpm for 5 minutes, and collect the supernatant; prepare three replicates.

[0130] Accuracy Solution 3 (200%, relative to 25 μg / ml): Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution-3, place them in the same 10 ml volumetric flask, make up to volume with anhydrous ethanol, and shake well; centrifuge the solution at 5000 rpm for 5 minutes, and collect the supernatant; prepare three replicates.

[0131] 5.3 The test results are shown in 5

[0132] Table 5 Accuracy results of each target substance

[0133]

[0134] The results showed that the accuracy of each target substance at three levels met the acceptance criteria of recovery between 70.0% and 130.0% and RSD ≤ 15.0%.

[0135] Example 6: Precision

[0136] Precision refers to the closeness of results obtained from multiple measurements of the same homogeneous sample under specified test conditions. Repeatability refers to the closeness of results obtained by the same analyst under the same conditions. Three replicates of the same concentration of the sample spiked with the sample are measured in parallel, and the mean and RSD of the spiked recovery are calculated.

[0137] 6.1 Chromatographic conditions were the same as in Example 1.

[0138] 6.2 Solution preparation

[0139] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketocholesterol reference substances, dissolve them in anhydrous ethanol, and dilute them to make a solution containing 100 μg of each component per 1 ml.

[0140] Test sample spike solution: Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution, place them in the same 10 ml volumetric flask, make up to volume with diluent, and shake well; centrifuge the solution at 5000 rpm for 5 min, and collect the supernatant; prepare three replicates.

[0141] 6.3 The test results are shown in Table 6:

[0142] Table 6 Repeatability results

[0143]

[0144] The results showed that in the three sample spiked solutions, the RSDs of the recoveries of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid and ketocholesterol were 1.8%, 1.1%, 1.3% and 1.4%, respectively, and the repeatability met the acceptance standard of RSD≤5.0%.

[0145] Example 7 Verification of different mobile phases

[0146] 7.1 Chromatographic conditions

[0147] The chromatographic conditions were substantially the same as those in Example 1, except that the mobile phase B was a methanol solution containing 0.1 v / v% trifluoroacetic acid.

[0148] 7.2 Solution Preparation

[0149] Control solution: Take appropriate amounts of DOTAP, DOPC, and cholesterol reference substances, dissolve them in anhydrous ethanol, and dilute them to make a solution containing 61 μg of DOTAP, 298 μg of DOPC, and 106 μg of cholesterol per 1 mL.

[0150] 7.3 Determination method

[0151] Take 30 μL of the reference solution and inject it. The result is as follows Figure 9 shown.

[0152] The results showed that when methanol was used as the organic phase, the elution times of DOPC and cholesterol were reversed, and the elution times of cholesterol and DOPC were close, resulting in a poor separation effect.

[0153] Example 8 Different elution gradients 1

[0154] 8.1 Chromatographic conditions

[0155] The chromatographic conditions were substantially the same as those in Example 1, with the only difference being the gradient elution procedure, which was as follows.

[0156] Table 7 Elution program

[0157] Time (minutes) Mobile phase A Mobile phase B 0 20 80 15 8 92 20 0 100 35 8 100 .

[0158] 8.2 Solution Preparation

[0159] Control solution: Take appropriate amounts of DOTAP, DOPC, and cholesterol reference substances, dissolve them in 80% ethanol, and dilute to make a solution containing 61 μg of DOTAP, 298 μg of DOPC, and 106 μg of cholesterol per 1 ml.

[0160] 8.3 Determination method

[0161] Take 30 μL of the reference solution and inject it. The result is as follows Figure 10 shown.

[0162] The results showed that after adjusting the elution gradient, DOPC and cholesterol co-eluted and failed to achieve effective separation.

[0163] Example 9 Different elution gradients 2

[0164] 8.1 Chromatographic conditions

[0165] The chromatographic conditions were substantially the same as those in Example 1, with the only difference being the gradient elution procedure, which was as follows.

[0166] Table 8 Elution program

[0167] Time (minutes) Mobile phase A Mobile phase B 0 20 80 15 8 92 30 0 100 35 8 100 .

[0168] 9.2 Solution Preparation

[0169] Control solution: Take appropriate amounts of DOTAP, DOPC, and cholesterol reference substances, dissolve them in 80% ethanol, and dilute to make a solution containing 61 μg of DOTAP, 298 μg of DOPC, and 106 μg of cholesterol per 1 ml.

[0170] 9.3 Determination method

[0171] Take 30 μL of the reference solution and inject it. The result is as follows Figure 11 shown.

[0172] The results showed that after adjusting the elution gradient, the elution time of DOPC and cholesterol was close, the baseline separation was not achieved, and the separation effect was poor.

[0173] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for determining a target substance in a liposome adjuvant, characterized in that: It includes: Detecting the target substance in the sample by high performance liquid chromatography; The chromatography method comprises gradient elution using mobile phase A and mobile phase B, wherein the mobile phase A comprises an aqueous solution containing trifluoroacetic acid; the mobile phase B comprises a mixed solution of methanol and acetonitrile containing trifluoroacetic acid; and the elution conditions of the chromatography are as follows: From 0 to 20 to 30 minutes, the volume percentage of the mobile phase A decreases from 15% to 25% to 0 to 10%, and the volume percentage of the mobile phase B increases from 75% to 85% to 90% to 100%; From 20 to 30 minutes to 30 to 40 minutes, the volume percentage of the mobile phase A is maintained at 0 to 10%, and the volume percentage of the mobile phase B is maintained at 90% to 100%; The target substance includes: components of the liposome adjuvant and / or degradation products and / or metabolites of the components.

2. The method according to claim 1, characterized in that The volume fraction of trifluoroacetic acid in the mobile phase A in the aqueous solution is 0.05% to 0.15%; The volume fraction of trifluoroacetic acid in the mobile phase B in the mixed solution of methanol and acetonitrile is 0.05% to 0.15%, and the mixed volume ratio of methanol to acetonitrile is 1 to 5:1 to 5.

3. The method according to claim 1, characterized in that The flow rate of the gradient elution is 0.5-1.5 mL / min.

4. The method according to claim 1, characterized in that: The chromatographic column used in the chromatography comprises a reverse phase chromatographic column, and the reverse phase chromatographic column comprises any one of C8 and C18; the specification of the chromatographic column is a column length of 50 to 250 mm×inner diameter of 2.1 to 4.6 mm, and the filler particle size is 1.7 to 10 μm; The chromatographic conditions of the chromatography method include: the column temperature of the chromatographic column is 30-50° C., and the injection volume is 10-50 μL.

5. The method according to claim 1, characterized in that Before the detection, the method further comprises: taking a liposome adjuvant sample solution, centrifuging after dilution, and taking the supernatant as a sample to be tested; The diluent used for the dilution includes any one or more combinations of methanol, ethanol and acetonitrile; The centrifugal conditions include: 3000-15000 rpm, 3-10 min.

6. The method according to claim 1, characterized in that The components include: at least one of cationic phospholipid DOTAP, neutral phospholipid DOPC and cholesterol; The degradation products and / or metabolites of the components include: at least one of lysophospholipids, oleic acid and ketocholesterol; The lysophospholipids include: Lyso-DOTAP-1, Lyso-DOTAP-2 which is an isomer of the Lyso-DOTAP-1, Lyso-DOPC-1, and Lyso-DOOC-2 which is an isomer of the Lyso-DOPC-1; The structural formula of the Lyso-DOTAP-1 is: The structural formula of the Lyso-DOTAP-2 is: The structural formula of the Lyso-DOPC-1 is: The structural formula of the Lyso-DOOC-2 is:

7. The method according to any one of claims 1 to 6, characterized in that: The detector used in the detection includes an ELSD detector.

8. Use of a reagent combination in preparing a product for determining a target substance in a liposome adjuvant, characterized in that: The reagent combination comprises reagents for implementing the method according to any one of claims 1 to 7; the target substance comprises: components of the liposome adjuvant and / or metabolites of the components.

9. Use of the method according to any one of claims 1 to 7 in the quality assessment of liposome adjuvants or in the detection of the effectiveness, safety and / or stability of liposome adjuvants.

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