A method for simultaneously detecting the contents of adjuvant main components in composite adjuvant / composite adjuvant-containing products and application thereof

Abstract

This invention discloses a method and its application for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants. The method includes the following steps: preparing a QS-21 calibration standard solution sample; preparing calibration standard solutions of dioleoylphosphatidylcholine and cholesterol; preparing a test sample; loading the sample into a high-performance liquid chromatograph (HPLC) and running the analytical method; and calculating the content of the major components of the compound adjuvant. This invention provides a method and its application for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants, solving the technical problem that existing technologies require two different methods for two separate detections of dioleoylphosphatidylcholine and cholesterol content, as well as the content of saponin QS-21. The high-performance liquid chromatography method disclosed in this invention requires only one sample loading and detection to detect the content of dioleoylphosphatidylcholine, cholesterol, and QS-21 in the product, greatly improving the detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method and its application for simultaneously detecting the content of the main adjuvant components in compound adjuvants / products containing compound adjuvants. Background Technology

[0002] Varicella-zoster virus (VZV), also known as human herpesvirus type 3, is a type of human alpha herpesvirus. The VZV genome is approximately 125 kb in size and encodes about 69 proteins, including eight glycoproteins: gB, gC, gE, gH, gI, gK, gL, and gM. Among these, glycoprotein E (gE) is the most abundant and immunogenic glycoprotein on the viral envelope and host cell membrane, capable of inducing both cellular and humoral immunity.

[0003] GlaxoSmithKline's recombinant protein shingles vaccine Approved by the FDA in 2017 for the prevention of shingles in adults aged 50 and older. The vaccine consists of two parts: a truncated VZV glycoprotein E (gE) expressed by CHO cells and the AS01B adjuvant system (which consists of the immune enhancer 3D-MPL, the immune enhancer saponin QS-21, and liposomes; the main components of the liposomes are dioleoylphosphatidylcholine (DOPC) and cholesterol).

[0004] Compared to the live attenuated vaccines from Merck and Brut, GSK's adjuvanted subunit vaccine... It offers higher protection, but more severe clinical side effects. Therefore, its immunogenicity is superior to... However, the clinical side effects are lower. The recombinant varicella-zoster vaccine is a promising candidate vaccine in clinical trials (immunogenicity data can be found in CN 116747298B and CN 117003896 B): This candidate vaccine contains the gE fusion protein (PADRE-gE-P2) antigen and the XA-401 complex adjuvant [the XA-401 complex adjuvant is composed of the immunostimulant saponin QS-21 and liposomes; the main components of the liposomes are dioleoylphosphatidylcholine (DOPC) and cholesterol]; with In contrast, this candidate vaccine removes the immune enhancer 3D-MPL (3-O-deacyl-4′-monophosphoryllipid A), which aggravates clinical side effects.

[0005] 3D-MPL is a Toll-like receptor (TLR)4 agonist. After binding to TLR4, it activates the primary response to myeloid differentiation 88 (MyD88) and downstream signaling pathways, leading to activation of nuclear factor-κB (NFκB) and activator protein (AP)-1, as well as the expression of pro-inflammatory cytokine genes. The production process of 3D-MPL is highly complex, and its production process and quality control are quite challenging. The production process of 3D-MPL powder includes: fermentation and cultivation of Salmonella Minnesota R595 strain, LPS extraction, LPS acid hydrolysis detoxification, LPS alkaline hydrolysis detoxification, chromatographic purification, protonation, salting, and freeze-drying. Among these, the LPS acid hydrolysis and alkaline hydrolysis detoxification steps are particularly difficult. Quality control of 3D-MPL is also challenging: it requires controlling the distribution ratios of 3D-MPL tetraacyl, pentacyl, hexaacyl, and heptaacyl isoforms in the 3D-MPL powder and AS01B adjuvant system.

[0006] QS-21 is an immunostimulant extracted from the bark of the Chilean soapberry tree, *Quillajasaponaria*. In aqueous solution, the QS-21A isomer partially converts to the QS-21B isomer, but the QS-21A isomer remains the predominant molecular form. Both the QS-21A and QS-21B isomers possess the same adjuvant activity.

[0007] To control vaccine quality, it is essential to test the content of the main adjuvant components in the vaccine. Therefore, it is necessary to establish a method for detecting the content of the main components of the XA-401 compound adjuvant [dioleoylphosphatidylcholine (DOPC), cholesterol, and saponin QS-21] in this candidate vaccine.

[0008] Currently, the recombinant zoster vaccine already on the market In quality control, two different liquid chromatography methods were used to detect the content of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol. In summary, the detection... The methods for determining the content of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccines are cumbersome, time-consuming, material-intensive, labor-intensive, and inefficient. Summary of the Invention

[0009] The purpose of this invention is to provide a method and application for simultaneously detecting the content of the main components of adjuvants in compound adjuvants / products containing compound adjuvants, so as to solve the technical problems of cumbersome, time-consuming, material-intensive, labor-intensive and inefficient methods for detecting components in adjuvants in the prior art.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] This invention provides a method for simultaneously detecting the content of the main adjuvant components in a compound adjuvant / product containing a compound adjuvant. The method employs liquid chromatography. The compound adjuvant is XA-401 compound adjuvant, which is composed of neutral liposomes and the immune enhancer saponin QS-21. The main components of the neutral liposomes are dioleoylphosphatidylcholine and cholesterol.

[0012] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra-high performance liquid chromatography.

[0013] Furthermore, the liquid chromatography method is reversed-phase liquid chromatography, wherein the chromatographic column contains alkyl or phenyl reversed-phase chromatographic packing material, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water.

[0014] Furthermore, the alkyl reversed-phase chromatography packing material can be selected from any of the following chromatographic packing materials, but is not limited to: butylsilane-bonded silica gel (C4), hexaalkylsilane-bonded silica gel (C6), octaalkylsilane-bonded silica gel (C8), hexadecylsilane-bonded silica gel (C16), and octadecylsilane-bonded silica gel (C18).

[0015] Furthermore, the liquid chromatography method is either the internal standard method or the external standard method.

[0016] Furthermore, the liquid chromatography external standard method includes the following steps:

[0017] S1. Prepare QS-21 calibration standard solution samples;

[0018] S2. Prepare dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples;

[0019] S3. Prepare the test sample;

[0020] S4. Load the sample into the liquid chromatograph and run the analytical method;

[0021] S5. Calculate the content of the main components of the compound adjuvant.

[0022] Furthermore, in step S1, the purity of the QS-21 standard solution sample used to prepare the QS-21 calibration standard solution sample is not less than 90%, and its preparation method can be selected from any of the following methods, but is not limited to the following methods: dissolving QS-21 powder (purity not less than 90%) in a buffer solution with a pH not less than 4 to prepare a QS-21 standard solution sample; and detecting the concentration of the QS-21 solution (purity not less than 90%) using liquid chromatography to prepare a QS-21 standard solution sample.

[0023] Furthermore, the liquid chromatography method for detecting the concentration of QS-21 solution uses an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector, a reversed-phase column, and acetonitrile as the organic phase in the mobile phase.

[0024] Furthermore, the concentration of QS-21 in the QS-21 calibration standard solution sample is 10 μg / ml to 100 μg / ml.

[0025] Furthermore, the QS-21 calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0026] Furthermore, in step S2, the preparation of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples can be selected from any one of the following two methods: 1. Preparing dioleoylphosphatidylcholine calibration standard solution samples and cholesterol calibration standard solution samples (prepared separately); 2. Preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples (prepared together).

[0027] Furthermore, the preparation of dioleoylphosphatidylcholine calibration standard solution samples and cholesterol calibration standard solution samples (prepared separately) are carried out using the methods described below, but not limited to the following methods: Dioleoylphosphatidylcholine (purity not less than 90%) is accurately weighed, then dissolved in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluted to the target volume and mixed thoroughly to prepare the dioleoylphosphatidylcholine standard solution sample; cholesterol (purity not less than 90%) is accurately weighed, then dissolved in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluted to the target volume and mixed thoroughly to prepare the cholesterol standard solution sample.

[0028] Further, a standard solution sample of dioleoylphosphatidylcholine and cholesterol (prepared by mixing) is prepared using the method described below, but not limited to: The concentrations of dioleoylphosphatidylcholine and cholesterol in a liposome solution (with a purity of not less than 90% in both liposomes) are determined by liquid chromatography to prepare a standard solution sample of dioleoylphosphatidylcholine and cholesterol; after accurately weighing dioleoylphosphatidylcholine and cholesterol (with a purity of not less than 90%), they are dissolved in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluted to the target volume and mixed to prepare the standard solution sample of dioleoylphosphatidylcholine and cholesterol.

[0029] Furthermore, the liquid chromatography method for detecting the concentration of liposome solution uses an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector, a reversed-phase column, and acetonitrile or methanol as the organic phase in the mobile phase.

[0030] Furthermore, the concentrations of dioleoylphosphatidylcholine and cholesterol in the calibration standard solution samples were 200 μg / ml to 2000 μg / ml and 50 μg / ml to 500 μg / ml, respectively.

[0031] Furthermore, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples may contain DMSO at a concentration of 0.5% to 5%.

[0032] Furthermore, the test sample prepared in S3 can be prepared using a vaccine or XA-401 compound adjuvant.

[0033] Furthermore, the vaccine used to prepare the test samples has a protein content of 10 μg / ml to 400 μg / ml, a dioleoylphosphatidylcholine content of 400 μg / ml to 4000 μg / ml, a cholesterol content of 100 μg / ml to 1000 μg / ml, and a QS-21 content of 20 μg / ml to 200 μg / ml.

[0034] Furthermore, the XA-401 compound adjuvant used in S3 to prepare the test sample has a dioleoylphosphatidylcholine content of 400 μg / ml to 4000 μg / ml, a cholesterol content of 100 μg / ml to 1000 μg / ml, and a QS-21 content of 20 μg / ml to 200 μg / ml.

[0035] Furthermore, the concentration of saponin QS-21 in the tested sample is 10 μg / ml to 100 μg / ml, the concentration of dioleoylphosphatidylcholine is 200 μg / ml to 2000 μg / ml, and the concentration of cholesterol is 50 μg / ml to 500 μg / ml.

[0036] Furthermore, the sample being tested may contain DMSO at a concentration of 0.5% to 5%.

[0037] Furthermore, the detector of the liquid chromatograph in S4 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0038] Furthermore, the analytical method in S4 includes a chromatographic column, liquid chromatograph parameters, and elution program.

[0039] Furthermore, the chromatographic column used in the analytical method in S4 is a reversed-phase chromatographic column.

[0040] Furthermore, the liquid chromatography parameters of the analytical method in S4 include column temperature, injection volume, flow rate, detector parameters, etc.

[0041] Furthermore, the column temperature is 40℃ to 60℃, such as 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃.

[0042] Furthermore, when the instrument is a high-performance liquid chromatograph, the injection volume is 30 μl to 100 μl, such as 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, 75 μl, 80 μl, 85 μl, 90 μl, 95 μl, or 100 μl.

[0043] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the injection volume is 5 μl to 30 μl, such as 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, or 30 μl.

[0044] Furthermore, when the instrument is a high-performance liquid chromatograph, the flow rate is 1.0 ml / min to 2.5 ml / min, such as 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, 1.6 ml / min, 1.7 ml / min, 1.8 ml / min, 1.9 ml / min, 2.0 ml / min, 2.1 ml / min, 2.2 ml / min, 2.3 ml / min, 2.4 ml / min, or 2.5 ml / min.

[0045] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the flow rate is 0.1 ml / min to 1.0 ml / min, such as 0.1 ml / min, 0.2 ml / min, 0.3 ml / min, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, or 1.0 ml / min.

[0046] Furthermore, the detector parameters are characterized as follows: when the detector is an ultraviolet detector, the detection wavelength is 200nm to 400nm, such as 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, or 400nm.

[0047] Furthermore, when the detector is a CAD detector, the atomization temperature is 35℃~70℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃.

[0048] Furthermore, when the detector is an evaporative photodetector, the atomization temperature is 35℃~70℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃.

[0049] Furthermore, the elution procedure of the analytical method in S4 shall include at least the following two elution methods: elution method for eluting the retained peak of saponin QS-21; elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0050] Furthermore, the elution method for the retained peak of saponin QS-21 can also be used to elute the retained peak of the fusion protein in recombinant varicella-zoster vaccine.

[0051] Furthermore, the acetonitrile concentration in the mobile phase for eluting saponin QS-21 is not higher than the acetonitrile concentration in the mobile phase for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0052] Furthermore, the elution method for the saponin QS-21 and protein retention peaks was gradient elution, with acetonitrile concentration gradients in the mobile phase of 5%–95%, such as 5%–95%, 10%–90%, 15%–85%, 20%–80%, 25%–75%, 30%–70%, 35%–65%, 40%–65%, 45%–65%, 45%–70%, 45%–75%, and 45%. ~80%, 45%~85%, 45%~90%, 45%~95%, 50%~65%, 50%~70%, 50%~75%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 55%~65%, 55%~70%, 55%~75%, 55%~80%, 55%~85%, 55%~90%, 55%~95%, 60% ~65%, 60%~70%, 60%~75%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, or 65%~75%, with gradient elution times of 2 min~1920 min, such as 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, 960 min, or 1920 min, and trifluoroacetic acid concentration in the mobile phase of 0.01%~0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0053] Furthermore, the elution method for the retained peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0054] Furthermore, the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution: the acetonitrile concentration in the mobile phase is 90%–100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and the isocratic elution time is 2 min–1920 min, such as 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 3 min, etc. The timeframes are 0 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, 960 min, or 1920 min, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0055] Furthermore, the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol is gradient elution: the acetonitrile concentration in the mobile phase is 90%–100%, such as 90%–100%, 91%–100%, 92%–100%, 93%–100%, 94%–100%, 95%–100%, 96%–100%, 97%–100%, 98%–100%, 99%–100%, 90%–99%, 91%–99%, 92 ... 99%–93%–99%, 94%–99%, 95%–99%, 96%–99%, 97%–99%, 98%–99%, 90%–98%, 91%–98%, 92%–98%, 93%–98%, 94%–98%, 95%–98%, 96%–98%, 97%–98%, 90%–97%, 91%–97%, 92%–97%, 93%–97%, 94%–97%, 95%–99% 7%, 96%–97%, 90%–96%, 91%–96%, 92%–96%, 93%–96%, 94%–96%, 95%–96%, 90%–95%, 91%–95%, 92%–95%, 93%–95%, or 94%–95%, with gradient elution times of 2 min–1920 min, such as 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min. The time intervals are 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, 960 min, or 1920 min, with the concentration of trifluoroacetic acid in the mobile phase being 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0056] Furthermore, in the elution procedure of the analytical method in S4, an isocratic elution method can be added between the elution method for eluting the retained peak of saponin QS-21 and the elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0057] Furthermore, an isocratic elution method is added between the elution method for the retention peak of saponin QS-21 and the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile concentration is less than the acetonitrile concentration in the mobile phase for eluting dioleoylphosphatidylcholine and cholesterol, and not less than the highest value of the acetonitrile concentration gradient in the mobile phase for eluting the retention peak of saponin QS-21.

[0058] Furthermore, an isocratic elution method is added between the elution method for the retention peak of saponin QS-21 and the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol. The mobile phase contains acetonitrile concentrations of 65%–94%, such as 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, and the isocratic elution time is 1 minute. The timeframe is from 1 min to 960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0059] Furthermore, in the elution procedure of the analytical method in S4, a gradient elution method can be added between the elution method for eluting the retained peaks of saponin QS-21 and protein and the elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0060] Furthermore, a gradient elution method is added between the elution method for eluting saponin QS-21 and the protein retention peak and the elution method for eluting the dioleoylphosphatidylcholine and cholesterol retention peak, wherein the maximum value of the acetonitrile concentration gradient is not higher than the acetonitrile concentration in the mobile phase for eluting dioleoylphosphatidylcholine and cholesterol, and the minimum value of the acetonitrile concentration gradient is not lower than the maximum value of the acetonitrile concentration gradient in the mobile phase for eluting saponin QS-21 and the protein retention peak.

[0061] Furthermore, a gradient elution method is added between the elution method for eluting saponin QS-21 and the protein retention peak and the elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks, wherein the acetonitrile concentration gradient in the mobile phase is 65%–95%, such as 65%–70%, 65%–75%, 65%–80%, 65%–85%, 65%–90%, 65%–95%, 70%–75%, 70%–80%, 70%–85%, 70%–90%, 70%–95%, 70%–95%, 75%–80%, 75%–85%, 75%–90%, 75%–95%, 80%–85%, 80%–90%, 80%–95%, 85%–90%. The concentration of trifluoroacetic acid in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%. The gradient elution time is 1 to 960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min.

[0062] Furthermore, in the elution procedure of the analytical method in S4, a pre-equilibration method and / or a pre-elution method can be added before the elution method for eluting the retained peak of saponin QS-21.

[0063] Furthermore, a pre-equilibration method is added before eluting the retention peak of saponin QS-21, wherein the concentration of acetonitrile in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient of the protein retention peak.

[0064] Furthermore, a pre-equilibration method is added before the elution method for the retention peak of saponin QS-21, wherein the concentration of acetonitrile in the mobile phase is 5%–60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, and the equilibration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, or 25 min. The timeframes are 1 minute, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or 960 minutes, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0065] Furthermore, a pre-elution method is added before the elution method for the retained peak of saponin QS-21, wherein the concentration of acetonitrile in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient of the retained protein peak.

[0066] Furthermore, a pre-elution method is added before the elution method for the retained peak of saponin QS-21, wherein the acetonitrile gradient concentration in the mobile phase is 5%–60%, such as 5%–10%, 5%–15%, 5%–20%, 5%–25%, 5%–30%, 5%–35%, 5%–40%, 5%–45%, 5%–50%, 5%–55%, 5%–60%, 10%–15%, 10%–20%, 10%–25%, 10%–30%, 10%–35%, 10%–40%, 10%–45%, 10%–50%, 1 ... 0%–55%, 10%–60%, 15%–20%, 15%–25%, 15%–30%, 15%–35%, 15%–40%, 15%–45%, 15%–50%, 15%–55%, 15%–60%, 20%–25%, 20%–30%, 20%–35%, 20%–40%, 20%–45%, 20%–50%, 20%–55%, 20%–60%, 25%–30%, 25%–35%, 25%–40%, 25%–45%, 25%–50%, 25%–55% 25%–60%, 30%–35%, 30%–40%, 30%–45%, 30%–50%, 30%–55%, 30%–60%, 35%–40%, 35%–45%, 35%–50%, 35%–55%, 35%–60%, 40%–45%, 40%–50%, 40%–55%, 40%–60%, 45%–50%, 45%–55%, 45%–60%, 50%–55%, 50%–60%, or 55%–60%, with gradient elution times of 1–960 min, such as 1 minute. The time intervals are 1 minute, 2 minutes, 4 minutes, 8 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or 960 minutes, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0067] Furthermore, in the elution procedure of the analytical method in S4, a column regeneration method and / or a post-equilibration method may be added after the elution method for eluting the retained peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0068] Furthermore, when both the column regeneration method and the post-equilibration method exist simultaneously, the column regeneration method should precede the post-equilibration method.

[0069] Furthermore, after eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol, a column regeneration method is added, wherein the acetonitrile concentration in the mobile phase is 98%–100%, such as 98%, 98.5%, 99%, 99.5%, or 100%, and the column regeneration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, or 30 min. The mobile phase may contain trifluoroacetic acid at concentrations of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%, for durations of 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min.

[0070] Furthermore, after eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, a post-equilibration method is added, wherein the concentration of acetonitrile in the mobile phase is not higher than the minimum value of the mobile phase concentration gradient for eluting the protein retention peaks.

[0071] Furthermore, after eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol, a post-equilibration method is added, wherein the acetonitrile concentration in the mobile phase is 5%–60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, and the equilibration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 2... The timeframes are 5 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0072] Furthermore, in step S5, the content of the main components of the compound adjuvant is calculated using two methods: Method 1 and Method 2.

[0073] Furthermore, the calculation method one is as follows: first, establish a calibration standard curve in which the concentrations of saponin QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area, and then calculate the content (concentration) of saponin QS-21, dioleoylphosphatidylcholine, and cholesterol.

[0074] Further, calculation method one: First, establish a calibration standard curve in which the concentrations of saponins QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area; then, substitute the peak area values ​​of saponins QS-21, dioleoylphosphatidylcholine, and cholesterol in the sample into the calibration standard curve formula of the corresponding component to calculate the concentration of the corresponding component in the sample; multiply the concentration of each component in the sample by the dilution factor to calculate the content (concentration) of each component [QS-21, dioleoylphosphatidylcholine, and cholesterol] in the sample.

[0075] Furthermore, the second calculation method is to directly calculate the content (concentration) of saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the sample.

[0076] Furthermore, the second calculation method is as follows: The calculation formula is: A content (concentration) in vaccine or XA-401 compound adjuvant = peak area of ​​A in the test sample * concentration of A calibration standard solution sample * dilution factor / peak area of ​​A calibration standard solution sample, where "A" represents "saponin QS-21" or "dioleoylphosphatidylcholine (DOPC)" or "cholesterol".

[0077] This invention provides a method and application for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants, wherein the products containing compound adjuvants include, but are not limited to, vaccines.

[0078] Furthermore, it is used to detect the content of saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol.

[0079] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0080] This invention provides a method and application for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants. It solves the technical problem that existing technologies require two different methods for two separate detections to determine the content of dioleoylphosphatidylcholine (DOPC) and cholesterol, as well as the content of saponin QS-21. The high-performance liquid chromatography method disclosed in this invention requires only one sample loading and detection to determine the content of DOPC, cholesterol, and QS-21 in the product, greatly improving the detection efficiency. Attached Figure Description

[0081] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0082] Figure 1 This is a liquid chromatogram of the main components of the vaccine under different mobile phase pH adjuster conditions in Example 1 of the present invention;

[0083] The left chromatogram represents acetic acid; the right chromatogram represents trifluoroacetic acid.

[0084] Figure 2 This is a liquid chromatogram of saponin QS-21 and PADRE-gE-P2 fusion protein in a vaccine under different elution conditions of saponin QS-21 in Example 2 of the present invention.

[0085] The chromatograms, from left to right, show gradient elutions of 5%–95% acetonitrile for 15 min, 50%–75% acetonitrile for 10 min, 50%–75% acetonitrile for 20 min, and 50%–75% acetonitrile for 30 min.

[0086] Figure 3 This is a liquid chromatography (LC) chromatogram of saponin QS-21 and PADRE-gE-P2 fusion protein in a vaccine under different column temperatures in Example 3 of this invention (from left to right: chromatograms at column temperatures of 30℃, 40℃, 50℃, and 60℃). At a column temperature of 60℃, the retention peaks of the QS-21A isomer and the PADRE-gE-P2 fusion protein could not be completely separated, which does not meet the requirements.

[0087] Figure 4 This is a comparison chart of the tailing factors of the retention peaks of the main components of the vaccine under different column temperatures in Example 3 of the present invention. At column temperatures of 30℃ and 40℃, the tailing factor of the DOPC retention peak is <0.8, which does not meet the requirements; at column temperatures of 30℃ and 40℃, the tailing factor of the cholesterol retention peak is >1.8, which does not meet the requirements; at a column temperature of 60℃, the software did not calculate the tailing factors of the QS-21B isomer, QS-21A isomer, and DOPC retention peaks, which does not meet the requirements; at a column temperature of 30℃, the software did not calculate the tailing factor of the QS-21B isomer retention peak, which does not meet the requirements.

[0088] Figure 5 This is a comparison chart of the tailing factors of the retained peaks of the main adjuvant component in the vaccine under different injection volumes in Example 4 of this invention. Figure description: When the injection volume is 50 μL, the tailing factor of the DOPC retained peak is >1.8, which does not meet the requirements.

[0089] Figure 6 This is a liquid chromatography (LC) chromatogram of saponin QS-21 and PADRE-gE-P2 fusion protein in a vaccine under different flow rate conditions in Example 5 of this invention (from left to right: chromatograms at flow rates of 1.0 ml / min, 1.5 ml / min, and 2.0 ml / min). At a flow rate of 1.0 ml / min, the retention peaks of the QS-21A isomer and the PADRE-gE-P2 fusion protein could not be completely separated, which does not meet the requirements.

[0090] Figure 7 This is a comparison chart of the tailing factors of the retained peaks of the main components of the vaccine under different flow rates in Example 5 of the present invention. When the flow rate is 1.0 ml / min, the tailing factor of the DOPC retained peak is <0.8; when the flow rate is 1.0 ml / min, the tailing factor of the cholesterol retained peak is >1.8, which does not meet the requirements.

[0091] Figure 8 This is a chromatogram of the retained peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol under different elution times in Example 6 of this invention. When the elution time for DOPC and cholesterol was 2 min, the baseline was not flat.

[0092] Figure 9 This is the standard curve of the concentration and peak area of ​​saponin QS-21 in Example 7 of the present invention.

[0093] Figure 10 This is a standard curve of the concentration and peak area of ​​dioleoylphosphatidylcholine (DOPC) in Example 7 of this invention.

[0094] Figure 11 This is the standard curve of cholesterol concentration versus peak area in Example 7 of the present invention.

[0095] Figure 12 This is a liquid chromatography chromatogram of Example 7 of the present invention, showing the simultaneous detection of the contents of three main adjuvant components [dioleoylphosphatidylcholine (DOPC), cholesterol, and saponin QS-21] in a recombinant varicella-zoster vaccine containing PADRE-gE-P2 fusion protein and XA-401 compound adjuvant. The retained peak of each substance is completely separated from the preceding peak, meeting the requirements.

[0096] Figure 13 This is a comparison chart of the contents of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples detected simultaneously (in one test) in Example 7 of the present invention and in two separate tests using existing technology. Detailed Implementation

[0097] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0098] It should be understood that the optimal liquid chromatography parameters and elution methods may differ when using reversed-phase columns containing different chromatographic packing materials to detect target substances (such as QS-21, DOPC, and cholesterol in vaccines or adjuvants), as is known to those skilled in the art.

[0099] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0100] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0101] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0102] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0103] Example 1: Study on the types of acidic pH adjusters in the mobile phase

[0104] 1) Preparation and concentration detection of PADRE-gE-P2 fusion protein

[0105] I. Codon Optimization and Whole Genome Synthesis of Fusion Proteins

[0106] See patent CN 117003896 B for details.

[0107] II. Construction of Fusion Protein Expression Plasmids

[0108] See patent CN 117003896 B for details.

[0109] III. Construction of Stable Cell Lines

[0110] See patent CN 117003896 B for details.

[0111] IV. Expression of the Target Product

[0112] See patent CN 117003896 B for details.

[0113] V. Fusion Protein Purification

[0114] See patent CN 117003896 B for details.

[0115] VI. Determination of Fusion Protein (Vaccine Stock Solution) Concentration [Lowry Method, Method I]

[0116] (1) Reagent preparation

[0117] Folin-Ciocalteu solution: Add 5.0 ml of phenol reagent to 75 ml of water and mix well; Solution A: Weigh 10.0 g of sodium hydroxide and 50.0 g of sodium carbonate into a plastic reagent bottle, add 400 ml of water to dissolve, and mix well; Potassium tartrate solution (0.5 g → 50 ml): Weigh 0.5 g of potassium tartrate into a reagent bottle, add 50 ml of water to dissolve, and mix well; Copper sulfate solution (0.25 g → 30 ml): Weigh 0.39 g of copper sulfate pentahydrate into a reagent bottle, add 30 ml of water to dissolve, and mix well; Solution B: Prepare according to the ratio of potassium tartrate solution (0.5 g → 50 ml): copper sulfate solution (0.25 g → 30 ml) = 5:3 (volume ratio), and mix well; Alkaline copper solution: Prepare according to the ratio of Solution A: Solution B: water = 40:8:2 (volume ratio), and mix well.

[0118] (2) Preparation of working solution for protein reference standard

[0119] 200 μg / ml protein reference solution (e.g., bovine serum albumin solution): Take one vial of protein reference standard, reconstitute it with purified water to prepare a 200 μg / ml protein reference solution, aliquot it into 5 ml plastic tubes, and store at -20℃ or below. When using, dilute the protein reference solution twice and mix well to obtain the working solution (100 μg / ml).

[0120] (3) Protein sample concentration detection

[0121] Test sample detection: Accurately transfer an appropriate amount of the sample solution to be tested, dilute with water until the protein content is within the standard curve range; accurately transfer 1.0 ml of the above solution into two test tubes, add 1.0 ml of alkaline copper solution, mix well, let stand at room temperature for 10 min, add 4.0 ml of Folin-Ciocalteu solution, mix immediately, let stand at room temperature for 30 min for color development, and then measure the absorbance at a wavelength of 650 nm using ultraviolet-visible spectrophotometry (if turbidity is found after color development, centrifuge at 3000 rpm / min for 15 min, and then take the supernatant for measurement).

[0122] Protein standard detection: Accurately transfer 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of 100 μg / ml protein standard working solution into test tubes, making duplicates. For solutions less than 1.0 ml, add water to bring the volume to 1.0 ml. Proceed with the same procedure starting from "Add 1.0 ml of alkaline copper solution". Separately, accurately transfer 1.0 ml of water and proceed with the same procedure starting from "Add 1.0 ml of alkaline copper solution" as a blank control.

[0123] (4) Protein sample concentration calculation

[0124] A linear regression was performed with the content of a series of protein reference standards as the X-axis and the corresponding mean absorbance as the Y-axis to obtain the linear regression equation. The mean absorbance of the test sample solution was then substituted into the linear regression equation to calculate the protein content of the test sample. The protein content of the sample (μg / ml) = A*n; where: A is the protein content of the test sample obtained by substituting the measured absorbance into the linear regression equation, in μg / ml; and n is the dilution factor of the test sample.

[0125] 2) Liposome preparation and detection of dioleoylphosphatidylcholine and cholesterol concentrations

[0126] I. Liposome preparation (theoretical concentrations of dioleoylphosphatidylcholine and cholesterol are 4 mg / ml and 1 mg / ml, respectively)

[0127] Weigh out 400 mg of dioleoylphosphatidylcholine (DOPC; Nippon Fine Chemicals Co., Ltd.) and 100 mg of cholesterol (Nippon Fine Chemicals Co., Ltd.); then completely dissolve DOPC and cholesterol in 10 ml of anhydrous ethanol and mix thoroughly to obtain the organic phase; inject 10 ml of the organic phase into 90 ml of 10 mM PBS buffer solution (pH 7.0) to prepare the liposome proemulsion; then use a high-pressure microfluidic homogenizer to granulate the liposomes to a particle size of about 100 nm; then remove ethanol by dialysis; finally, filter and sterilize using a 0.22 μm sterile filter to obtain the finished liposome product.

[0128] II. Detection of Dioleoylphosphatidylcholine (DOPC) and Cholesterol Concentration

[0129] (1) Preparation of mobile phase

[0130] Mobile phase A [methanol:water:trifluoroacetic acid (95:5:0.1)]: Measure 950 ml of methanol, add 50 ml of ultrapure water and 1 ml of trifluoroacetic acid, mix and shake well, and sonicate to degas for 10 min to obtain the mobile phase.

[0131] (2) Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol standard solution samples

[0132] Preparation of 90% ethanol solution: Accurately measure 45 ml of anhydrous ethanol, mix it with 5 ml of ultrapure water and shake well.

[0133] Preparation of 89.3% ethanol solution: Accurately measure 44.65 ml of anhydrous ethanol, mix with 5.35 ml of ultrapure water and shake well. Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol reference stock solution: Accurately weigh 37.5 mg of cholesterol (Nippon Fine Chemicals Co., Ltd.) and 150 mg of dioleoylphosphatidylcholine (DOPC; Nippon Fine Chemicals Co., Ltd.) into a 25 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, shake well.

[0134] Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol standard solutions: Transfer 1.667 ml of the reference stock solution to a 25 ml volumetric flask, dilute to 25 ml with 89.3% ethanol solution, and vortex to obtain a standard solution with a cholesterol concentration of 100 μg / ml and a DOPC concentration of 400 μg / ml. Transfer 7.5 ml, 5 ml, 2.5 ml, and 1.25 ml of the standard solution to 10 ml volumetric flasks, respectively, dilute to 10 ml with 90% ethanol solution, and vortex to obtain a standard solution. Standard solutions ④ [cholesterol concentration 75 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 300 μg / ml], ③ [cholesterol concentration 50 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 200 μg / ml], ② [cholesterol concentration 25 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 100 μg / ml], and ① [cholesterol concentration 12.5 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 50 μg / ml] were obtained.

[0135] (3) Preparation of test sample solution

[0136] Transfer 0.1 ml of the sample solution to be tested into a vial, add 1.9 ml of anhydrous ethanol, cap the vial, mix well, and the sample is ready for testing.

[0137] (4) Instrument parameters and elution program

[0138]

[0139] (5) Calculation of dioleoylphosphatidylcholine and cholesterol concentrations

[0140] Using the concentration of standard solutions of dioleoylphosphatidylcholine (DOPC) or cholesterol as the abscissa (X) and the peak area of ​​DOPC or cholesterol as the ordinate (Y), standard curves were plotted to show that the concentration of DOPC and cholesterol is directly proportional to the peak area. The peak area of ​​cholesterol or DOPC in the test sample solution was substituted into the standard curve for calculation, and then multiplied by the dilution factor used in the preparation of the test sample solution to obtain the concentration of DOPC and cholesterol in the test sample solution.

[0141] 3) Preparation and concentration detection of saponin QS-21 solution

[0142] I. Preparation of Saponin QS-21 Solution (Theoretical Concentration 4 mg / ml)

[0143] Weigh 40 mg of saponin QS-21 (Desert King), then dissolve it completely in 4 ml of phosphate solution (10 mM; pH 6.0), and then bring the volume up to 10 ml with phosphate solution (10 mM; pH 6.0). Mix well to obtain saponin QS-21 solution.

[0144] II. Detection of Saponin QS-21 Solution Concentration

[0145] (1) Preparation of mobile phase

[0146] Mobile phase A [water:acetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of acetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0147] Mobile phase B [acetonitrile:acetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of acetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0148] (2) Preparation of standard solution of saponin QS-21

[0149] Reference stock solution: Weigh 10 mg of QS-21 reference standard (Desert King) into a 2 ml volumetric flask, dissolve and dilute to the mark with phosphate solution (10 mM; pH 6.0), shake well, and use as the stock solution.

[0150] Standard solutions: Transfer 150 μl of the reference stock solution to a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, and mix well to obtain a solution with a concentration of 1 mg / ml; transfer 0.5 ml of the above solution to a 5 ml volumetric flask, dilute to the mark with 1% DMSO solution, and mix well to obtain a solution with a concentration of 100 μg / ml; then transfer 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution to different injection vials, and add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of 1% DMSO solution respectively, and mix well to obtain standard solutions with concentrations of 87.5 μg / ml, 75 μg / ml, 62.5 μg / ml, 50 μg / ml, 37.5 μg / ml, and 25 μg / ml respectively.

[0151] (3) Preparation of test sample solution

[0152] Transfer 0.125 ml of the sample solution to a 5 ml volumetric flask, then dilute to the mark with phosphate solution (10 mM; pH 6.0) and mix well to obtain a solution with a concentration of 100 μg / ml. Take 0.5 ml of the above 100 μg / ml solution into a vial, add 0.5 ml of 2% DMSO solution, and mix well to obtain the final solution.

[0153] (4) Instrument parameters and elution program

[0154]

[0155] (5) Calculation of saponin QS-21 concentration

[0156] Using the concentration of the standard solution of saponin QS-21 as the X-axis and the corresponding peak area as the Y-axis, a linear regression curve is plotted. The peak area of ​​the test sample solution is then substituted into the standard curve and multiplied by the dilution factor of the test sample solution to obtain the content of QS-21 in the test sample solution.

[0157] 4) Vaccine sample preparation

[0158] I. Preparation of Adjuvants

[0159] The concentrations of liposome solution were determined as follows: DOPC and cholesterol concentrations in liposomes were 4.20 and 1.05 mg / ml, respectively; the concentration of saponin QS-21 solution was determined as follows: the concentration of saponin QS-21 was 4.14 mg / ml; 4.76 ml of liposomes were taken, 0.24 ml of saponin QS-21 solution was added, and the mixture was stirred evenly to obtain the adjuvant.

[0160] II. Vaccine Preparation [10ml volume; target concentration of PADRE-gE-P2 fusion protein: 150μg / ml; target concentration of saponin QS-21: 100μg / ml; liposome components: target concentrations of dioleoylphosphatidylcholine and cholesterol: 2mg / ml and 0.5mg / ml, respectively]

[0161] The protein concentration of the PADRE-gE-P2 fusion protein solution was determined as follows: The concentration of the PADRE-gE-P2 fusion protein was 1.52 mg / ml. 0.99 ml of the PADRE-gE-P2 fusion protein solution was added to the adjuvant, and then the total volume was brought up to 10 ml using phosphate solution (10 mM; pH 6.0). The mixture was stirred thoroughly to obtain the PADRE-gE-P2 fusion protein adjuvant vaccine. The formula for calculating the volume (ml) of the added PADRE-gE-P2 fusion protein solution is: Mass of added PADRE-gE-P2 fusion protein (μg) / Concentration of PADRE-gE-P2 fusion protein (μg / ml).

[0162] 5) Investigate the effects of acidic pH adjusters (trifluoroacetic acid and acetic acid) in the mobile phase on the retention peaks of PADRE-gE-P2 fusion protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol.

[0163] I. Solution Preparation

[0164] (1) Preparation of mobile phase (0.05% trifluoroacetic acid)

[0165] Mobile phase A [water:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of trifluoroacetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0166] Mobile phase B [acetonitrile:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0167] (2) Preparation of mobile phase (0.05% acetic acid)

[0168] Mobile phase A [water:acetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of acetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0169] Mobile phase B [acetonitrile:acetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of acetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0170] (3) Preparation of sample dilution solution

[0171] 1% Dimethyl sulfoxide (DMSO) solution: Measure 99 ml of ultrapure water, add 1 ml of DMSO solution, mix well, and the solution is ready.

[0172] 1.25% Dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0173] 2% Dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0174] 3.5% Dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0175] II. Sample Preparation

[0176] Test sample: Transfer 0.5 ml of vaccine into a vial, add 0.5 ml of 2% DMSO solution, cap the vial, mix well, and the sample is ready.

[0177] III. Chromatographic column, instrument parameters, and elution program

[0178]

[0179] IV. Results Analysis

[0180] Liquid chromatography methods used to control drug quality have the following characteristics: the analyte must have a retained peak, the resolution of each retained peak must be >1.5, the tailing factor must be 0.8 to 1.8, and the baseline near the retained peak must be horizontal (parallel to the X-axis of the chromatogram).

[0181] (1) The acidic pH adjuster in the mobile phase is acetic acid.

[0182]

[0183]

[0184] Note: "NA" means "Not applicable"; "No peak" means "No retention peak of this substance on the chromatogram".

[0185] (2) The acidic pH adjuster in the mobile phase is trifluoroacetic acid.

[0186]

[0187] Note: "NA" means "Not applicable"; "No data output" means "The liquid chromatography analysis software did not calculate any data".

[0188] like Figure 1As shown, when acetic acid is used as the pH adjuster of the mobile phase, dioleoylphosphatidylcholine did not have a retention peak.

[0189] Example 2: Method for studying the elution of saponin QS-21 from saponins

[0190] 1) Preparation and protein concentration detection of PADRE-gE-P2 fusion protein - Same as in Example 1.

[0191] 2) Liposome preparation and detection of dioleoylphosphatidylcholine and cholesterol concentrations - same as in Example 1.

[0192] 3) Preparation and concentration detection of saponin QS-21 solution - same as in Example 1.

[0193] 4) Vaccine sample preparation - same as in Example 1.

[0194] 5) Methods for studying the elution of saponin QS-21 from soap trees

[0195] I. Solution Preparation

[0196] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0197] (2) Preparation of vaccine sample dilution solution - same as in Example 1.

[0198] II. Sample preparation for testing - Same as in Example 1.

[0199] III. Chromatographic column, instrument parameters, and elution program

[0200] (1) Chromatographic column and instrument parameters

[0201]

[0202] (2) Elution program 1: Gradient elution of acetonitrile concentration from 5% to 95% (5% mobile phase B to 95% mobile phase B) for 15 min to elute QS-21

[0203]

[0204] (3) Elution program 2: Acetonitrile concentration 50%-75% (50% mobile phase B-75% mobile phase B) gradient elution for 10 min to elute QS-21.

[0205]

[0206] (4) Elution program 3: Gradient elution of acetonitrile concentration 50%-75% (50% mobile phase B-75% mobile phase B) for 20 min to elute QS-21.

[0207]

[0208] (5) Elution program 4: Acetonitrile concentration 50%–75% (50% mobile phase B–75% mobile phase B) gradient elution for 30 min to elute QS-21.

[0209]

[0210] IV. Results Analysis

[0211] Liquid chromatography methods used to control drug quality have the following characteristics: the analyte must have a retained peak, the resolution of each retained peak must be >1.5, the tailing factor must be 0.8 to 1.8, and the baseline near the retained peak must be horizontal (parallel to the X-axis of the chromatogram).

[0212] (1) Elution program 1: Elute QS-21 with acetonitrile concentration gradient of 5% to 95% for 15 min.

[0213]

[0214] Note: "NA" means "Not applicable"; "No data output" means "The liquid chromatography analysis software did not calculate any data".

[0215] (2) Elution program 2: Elute QS-21 with acetonitrile concentration gradient of 50% to 75% for 10 min.

[0216]

[0217] Note: "NA" means "Not applicable".

[0218] (3) Elution program 3: Elution of QS-21 with acetonitrile concentration gradient of 50% to 75% for 20 min.

[0219]

[0220] Note: "NA" means "Not applicable".

[0221] (4) Elution program 4: Elution of QS-21 with acetonitrile concentration gradient of 50% to 75% for 30 min.

[0222]

[0223] Note: "NA" means "Not applicable".

[0224] like Figure 2 As shown, when eluting saponin QS-21 using a gradient of 5% to 95% acetonitrile for 15 min, the retention peaks of the QS-21A isomer and the PADRE-gE-P2 fusion protein could not be completely separated, which did not meet the requirements.

[0225] Example 3: Study on column temperature

[0226] 1) The preparation and protein concentration detection of PADRE-gE-P2 fusion protein are the same as in Example 1.

[0227] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0228] 3) The preparation and concentration detection of the saponin QS-21 solution were the same as in Example 1.

[0229] 4) The preparation of vaccine samples is the same as in Example 1.

[0230] 5) Study column temperature

[0231] I. Solution Preparation

[0232] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0233] (2) The preparation of the vaccine sample dilution solution is the same as in Example 1.

[0234] II. The preparation of the test samples is the same as in Example 1.

[0235] III. Chromatographic column, instrument parameters, and elution program

[0236]

[0237]

[0238] IV. Results Analysis

[0239] Liquid chromatography methods used to control drug quality have the following characteristics: the analyte must have a retained peak, the resolution of each retained peak must be >1.5, the tailing factor must be 0.8 to 1.8, and the baseline near the retained peak must be horizontal (parallel to the X-axis of the chromatogram).

[0240] (1) Column temperature 30℃

[0241]

[0242] Note: "NA" means "Not applicable"; "No data output" means "The liquid chromatography analysis software did not calculate any data".

[0243] (2) Column temperature 40℃

[0244]

[0245]

[0246] Note: "NA" means "Not applicable".

[0247] (3) Column temperature 50℃

[0248]

[0249] Note: "NA" means "Not applicable".

[0250] (4) Column temperature 60℃

[0251]

[0252] Note: "NA" means "Not applicable"; "No data output" means "The liquid chromatography analysis software did not calculate any data".

[0253] like Figure 3 As shown, at a column temperature of 60℃, the retention peaks of the QS-21A isomer and the PADRE-gE-P2 fusion protein cannot be completely separated, which does not meet the requirements.

[0254] like Figure 4 As shown, at column temperatures of 30℃ and 40℃, the tailing factor of the DOPC retention peak is <0.8, which does not meet the requirements; at column temperatures of 30℃ and 40℃, the tailing factor of the cholesterol retention peak is >1.8, which does not meet the requirements; at a column temperature of 60℃, the software did not calculate the tailing factors of the QS-21B isomer, QS-21A isomer, and DOPC retention peaks, which does not meet the requirements; at a column temperature of 30℃, the software did not calculate the tailing factor of the QS-21B isomer retention peak, which does not meet the requirements.

[0255] Example 4: Studying the injection volume

[0256] 1) The preparation and protein concentration detection of PADRE-gE-P2 fusion protein are the same as in Example 1.

[0257] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0258] 3) The preparation and concentration detection of the saponin QS-21 solution were the same as in Example 1.

[0259] 4) The preparation of vaccine samples is the same as in Example 1.

[0260] 5) Study the injection volume

[0261] I. Solution Preparation

[0262] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0263] (2) The preparation of the vaccine sample dilution solution is the same as in Example 1.

[0264] II. The preparation of the test samples is the same as in Example 1.

[0265] III. Chromatographic column, instrument parameters, and elution program

[0266]

[0267] IV. Results Analysis

[0268] Liquid chromatography methods used to control drug quality have the following characteristics: the analyte must have a retained peak, the resolution of each retained peak must be >1.5, the tailing factor must be 0.8 to 1.8, and the baseline near the retained peak must be horizontal (parallel to the X-axis of the chromatogram).

[0269] (1) Injection volume 50 μl

[0270]

[0271] Note: "NA" means "Not applicable".

[0272] (2) Injection volume 75 μl

[0273]

[0274] Note: "NA" means "Not applicable".

[0275] (3) Injection volume 100 μl

[0276]

[0277] Note: "NA" means "Not applicable".

[0278] like Figure 5 As shown, when the injection volume is 50 μL, the tailing factor of the DOPC retention peak is >1.8, which does not meet the requirements.

[0279] Example 5: Studying Flow Rate

[0280] 1) The preparation and protein concentration detection of PADRE-gE-P2 fusion protein are the same as in Example 1.

[0281] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0282] 3) The preparation and concentration detection of the saponin QS-21 solution were the same as in Example 1.

[0283] 4) The preparation of vaccine samples is the same as in Example 1.

[0284] 5) Study flow velocity

[0285] I. Solution Preparation

[0286] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0287] (2) The preparation of the vaccine sample dilution solution is the same as in Example 1.

[0288] II. The preparation of the test samples is the same as in Example 1.

[0289] III. Chromatographic column, instrument parameters, and elution program

[0290]

[0291] IV. Results Analysis

[0292] Liquid chromatography methods used to control drug quality have the following characteristics: the analyte must have a retained peak, the resolution of each retained peak must be >1.5, the tailing factor must be 0.8 to 1.8, and the baseline near the retained peak must be horizontal (parallel to the X-axis of the chromatogram).

[0293] (1) Flow rate 1.0 ml / min

[0294]

[0295] Note: "NA" means "Not applicable"; "No data output" means "The liquid chromatography analysis software did not calculate any data".

[0296] (2) Flow rate 1.5 ml / min

[0297]

[0298] Note: "NA" means "Not applicable".

[0299] (3) Flow rate 2.0 ml / min

[0300]

[0301] Note: "NA" means "Not applicable".

[0302] like Figure 6 As shown, at a flow rate of 1.0 ml / min, the retention peaks of the QS-21A isoform and the PADRE-gE-P2 fusion protein could not be completely separated, which did not meet the requirements.

[0303] like Figure 7 As shown, at a flow rate of 1.0 ml / min, the tailing factor of the DOPC retention peak is <0.8; at a flow rate of 1.0 ml / min, the tailing factor of the cholesterol retention peak is >1.8, which does not meet the requirements.

[0304] Example 6: Study on the duration of elution of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0305] 1) The preparation and protein concentration detection of PADRE-gE-P2 fusion protein are the same as in Example 1.

[0306] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0307] 3) The preparation and concentration detection of the saponin QS-21 solution were the same as in Example 1.

[0308] 4) The preparation of vaccine samples is the same as in Example 1.

[0309] 5) Study the duration of elution of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0310] I. Solution Preparation

[0311] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0312] (2) The preparation of the vaccine sample dilution solution is the same as in Example 1.

[0313] II. The preparation of the test samples is the same as in Example 1.

[0314] III. Chromatographic column, instrument parameters, and elution program

[0315] (1) Chromatographic column and instrument parameters

[0316]

[0317] (2) Elution program 1: Elution with 95% acetonitrile (95% mobile phase B) for 2 min to elute DOPC and cholesterol.

[0318]

[0319]

[0320] (3) Elution program 2: 95% acetonitrile concentration (95% mobile phase B) isocratic elution for 6 min to elute DOPC and cholesterol.

[0321]

[0322] (4) Elution program 3: Elution with 95% acetonitrile (95% mobile phase B) for 10 min to elute DOPC and cholesterol.

[0323]

[0324] IV. Results Analysis

[0325] Liquid chromatography methods used to control drug quality have the following characteristics: the analyte must have a retained peak, the resolution of each retained peak must be >1.5, the tailing factor must be 0.8 to 1.8, and the baseline near the retained peak must be horizontal (parallel to the X-axis of the chromatogram).

[0326] (1) Elution program 1: Elution with 95% acetonitrile for 2 min to elute DOPC and cholesterol.

[0327]

[0328] Note: "NA" means "Not applicable".

[0329] (2) Elution program 2: 95% acetonitrile isocratic elution for 6 min to elute DOPC and cholesterol.

[0330]

[0331] Note: "NA" means "Not applicable".

[0332] (3) Elution program 3: Elution with 95% acetonitrile for 10 min to remove DOPC and cholesterol.

[0333]

[0334]

[0335] Note: "NA" means "Not applicable".

[0336] like Figure 8 As shown, the baseline was uneven when the elution time for dioleoylphosphatidylcholine (DOPC) and cholesterol was 2 minutes.

[0337] Example 7: Simultaneous detection (single detection) of saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol content in vaccine samples.

[0338] 1) The preparation and protein concentration detection of PADRE-gE-P2 fusion protein are the same as in Example 1.

[0339] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0340] 3) The preparation and concentration detection of the saponin QS-21 solution were the same as in Example 1.

[0341] 4) The preparation of vaccine samples is the same as in Example 1.

[0342] 5) Detect the content of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples.

[0343] I. Solution Preparation

[0344] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0345] (2) The preparation of the vaccine sample dilution solution is the same as in Example 1.

[0346] II. Vaccine Sample Testing

[0347] Preparation of S1, Saponin QS-21 Calibration Standard Solution Sample

[0348] Transfer 150 μl of the QS-21 saponin reference standard stock solution (concentration of 5 mg / ml) to a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, and mix well to obtain a solution with a concentration of 1 mg / ml. Transfer 0.5 ml of the above solution to a 5 ml volumetric flask, dilute to the mark with 1% DMSO solution, and shake well to obtain a solution with a concentration of 100 μg / ml. Then transfer 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution to different injection vials, and add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of the solution respectively. Mix 1% DMSO solution, cap the bottle, and obtain calibration standard solutions with concentrations of 87.5 μg / ml, 75 μg / ml, 62.5 μg / ml, 50 μg / ml, 37.5 μg / ml, and 25 μg / ml.

[0349] Preparation of S2, dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples

[0350] Transfer 3 ml of the liposome reference solution (DOPC and cholesterol concentrations of 4.20 mg / ml and 1.05 mg / ml, respectively) to 1.2 ml of 3.5% DMSO, dissolve and mix, and shake well to obtain solutions with DOPC and cholesterol concentrations of 3 mg / ml and 0.75 mg / ml, respectively. Then, transfer 0.5 ml, 0.5 ml, 0.3 ml, and 0.2 ml of the above solution into different injection vials, and add 0.5 ml, 0.7 ml, 1.0 ml, 0.9 ml, and 1.0 ml of 1% DMSO solution, respectively. Cap the vials and mix well to obtain calibration standard solutions with DOPC and cholesterol concentrations of 1.5 mg / ml, 1.25 mg / ml, 1 mg / ml, 0.75 mg / ml, 0.5 mg / ml, 0.375 mg / ml, 0.3125 mg / ml, 0.25 mg / ml, 0.1875 mg / ml, and 0.125 mg / ml, respectively.

[0351] S3. Preparation of test samples

[0352] Transfer 0.5 ml of the vaccine sample solution to be tested into a vial, add 0.5 ml of 2% DMSO solution, cap the vial, mix well, and the solution is ready.

[0353] S4. Load the sample into the liquid chromatograph and run the analytical method.

[0354] The vials containing the QS-21 calibration standard solution of saponin, the vials containing the calibration standard solutions of dioleoylphosphatidylcholine (DOPC) and cholesterol, and the vial containing the test sample were loaded into the injection tray of the high performance liquid chromatograph, and the analytical method was run using the liquid chromatograph.

[0355]

[0356]

[0357] S5. Calculate the content of the main components of the compound adjuvant in the vaccine.

[0358] (1) Establishment of the standard curve

[0359] Calibration standard curves were established using the peak area values ​​of calibration standard solution samples of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol, respectively, to show that the concentrations of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol are proportional to the peak area.

[0360] The test results and standard curve of the QS-21 calibration standard solution of saponins from saponins are as follows: Figure 9 As shown

[0361]

[0362] Standard curve of peak area for saponin QS-21: y = 5781.4x - 371.7, R 2 =0.9995.

[0363] The test results and standard curves for dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solutions are as follows: Figure 10 , 11 As shown

[0364] Standard curve solution-1 (500 μg / ml) 840670 1.766 Standard curve solution-2 (750 μg / ml) 1255779 1.553 Standard curve solution - 3 (1000 μg / ml) 1710671 1.173 Standard curve solution-4 (1250 μg / ml) 2141153 0.962 Standard curve solution - 5 (1500 μg / ml) 2577165 0.994

[0365] DOPC peak area standard curve: y = 1743.3x - 38258, R 2 =0.9999.

[0366] Standard curve solution-1 (125 μg / ml) 1349974 1.550 Standard curve solution - 2 (187.5 μg / ml) 1980964 1.637 Standard curve solution - 3 (250 μg / ml) 2600131 1.700 Standard curve solution - 4 (312.5 μg / ml) 3198711 1.789 Standard curve solution - 5 (375 μg / ml) 3794897 1.753

[0367] Standard curve for cholesterol peak area: y = 9772.1x + 141898, R² = 0.9998.

[0368] (2) Content Calculation

[0369] The peak area values ​​of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine sample were substituted into the calibration standard curve formulas of the corresponding components, and then multiplied by the vaccine dilution factor (twice) to calculate the content of QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine.

[0370]

[0371] 6) Compare the differences in the content of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples detected simultaneously (one test) and using existing technology (two separate tests).

[0372] I. Vaccine Sample Testing (Prior Technology): A method for detecting the content of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples twice.

[0373] (1) Detection of saponin QS-21 content

[0374] The "concentration detection of saponin QS-21 solution" in "3) Preparation and concentration detection of saponin QS-21 solution" in Example 1 is the same as that in Example 1.

[0375] (2) Detection of dioleoylphosphatidylcholine (DOPC) and cholesterol levels

[0376] The procedure is the same as the "2) Preparation of liposomes and detection of dioleoylphosphatidylcholine (DOPC) and cholesterol concentration" in Example 1.

[0377] II. Comparison of the differences in the content of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples by simultaneous detection (1 test) and existing technology detection (2 separate tests), such as Figure 12 , 13 As shown, the retained peak of each substance is completely separated from the front peak, which meets the requirements.

[0378]

[0379] Finally, it should be noted that:

[0380] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants, characterized in that, Includes the following steps: S1. Prepare a standard solution sample of saponin QS21 from saponins; S2. Prepare dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples; S3. Prepare the test sample; S4. Load the sample into the liquid chromatograph and run the analytical method; S5. Calculate the content of the main components of the compound adjuvant; The compound adjuvant is XA401 compound adjuvant, which is composed of neutral liposomes and the immune enhancer saponin QS21. The main components of the neutral liposomes are dioleoylphosphatidylcholine and cholesterol. When detecting the content of PADREgEP2 fusion protein, saponin QS21, dioleoylphosphatidylcholine, and cholesterol in a compound adjuvant sample, the elution procedure uses mobile phase A and mobile phase B. The mobile phase A consists of water and trifluoroacetic acid in a volume ratio of 100:0.05; the mobile phase B consists of acetonitrile and trifluoroacetic acid in a volume ratio of 100:0.05; the specific analytical methods in step S4 are shown in the table.

2. The method for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants according to claim 1, characterized in that, The liquid chromatograph parameters of the analytical method in S4 include, but are not limited to: column temperature, injection volume, flow rate, and detector parameters; the column temperature is 40℃~60℃; the injection volume is 5μl~100μl; the flow rate is 0.1ml / min~2.5ml / min; and when the detector is an ultraviolet detector, the detection wavelength is 200~400nm.

3. The method for simultaneously detecting the content of major adjuvant components in compound adjuvants / products containing compound adjuvants according to claim 1, characterized in that, The calculation of the content of the main components of the compound adjuvant in S5 includes two calculation methods. Calculation Method 1: First, establish a calibration standard curve showing that the concentrations of saponin QS21, dioleoylphosphatidylcholine, and cholesterol are directly proportional to their peak areas. Then, substitute the peak area values ​​of saponin QS21, dioleoylphosphatidylcholine, and cholesterol into the corresponding calibration standard curve formulas to calculate the concentrations of the corresponding components in the test sample. Finally, multiply the concentrations of each component in the test sample by the dilution factor to calculate the concentration of each component. Calculation Method 2: The calculation formula is: A concentration = peak area of ​​sample A * concentration of calibration standard solution A * dilution factor / peak area of ​​calibration standard solution A, where A represents saponin QS21, dioleoylphosphatidylcholine, or cholesterol.

4. The application of the method for simultaneously detecting the content of the main adjuvant component in a compound adjuvant / product containing a compound adjuvant according to claim 1, characterized in that, The products containing compound adjuvants include, but are not limited to, vaccines.

5. The application of the method for simultaneously detecting the content of the main adjuvant component in a compound adjuvant / product containing a compound adjuvant according to claim 4, characterized in that, It is used to detect the content of saponin QS21 and / or dioleoylphosphatidylcholine and / or cholesterol.

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