Method for simultaneously detecting contents of saponin immunopotentiators and lipoids in composite adjuvant / product containing composite adjuvant and application of method for simultaneously detecting contents of saponin immunopotentiators and lipoids in composite adjuvant / product containing composite adjuvant

Abstract

The invention discloses a method for simultaneously detecting the contents of saponin immunopotentiators and lipoids in a composite adjuvant / product containing the composite adjuvant and application. The method comprises the following steps: preparing a saponin immunopotentiator calibration standard solution sample; preparing a lipoid calibration standard solution sample; preparing a detection sample; loading the sample into a liquid chromatograph, and operating an analysis method; and calculating the content of the saponin immunopotentiator and the lipoid. The invention provides a method for simultaneously detecting the contents of saponin immunopotentiators and lipoids in a composite adjuvant or a product containing the composite adjuvant and application of the method. The technical problem that in the prior art, the contents of the saponin immunopotentiators and the lipoids in the composite adjuvant need to be detected for at least two times through at least two different methods is solved. According to the high performance liquid chromatography disclosed by the invention, the contents of the saponin immunopotentiators and the lipoids in the product can be detected only by one-time sample loading detection, so that the detection efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method and application for simultaneously detecting the contents of saponin immunopotentiators and lipids in a composite adjuvant or a product containing the composite adjuvant. Background Art

[0002] Quillaja saponaria saponin QS-21 is an immunopotentiator extracted from the bark of the Chilean soap tree, Quillaja saponaria. QS-21 stimulates Th2 and Th1 immune responses by acting on antigen-presenting cells (APCs) and T cells. QS-21 also activates the NLRP3 inflammasome, leading to the release of caspase-1-dependent cytokines, IL-1β, and IL-18. In aqueous solution, the QS-21 A isomer partially converts to the QS-21 B isomer, but the QS-21 A isomer remains the predominant molecular form. Both the QS-21 A and QS-21 B isomers exhibit equivalent adjuvant activity.

[0003] Quillaja saponin QS-7 is also an immune enhancer extracted from the bark of the Chilean soap tree, Quillaja saponaria. Its content in the bark is lower than that of Quillaja saponin QS-21. Quillaja saponin QS-7 has similar immune-enhancing effects to Quillaja saponin QS-21, but is less toxic. As saponin component A, Quillaja saponin QS-7 has been used in Matrix-M TM Composite adjuvant.

[0004] Lipids include phospholipids, sphingolipids, glycolipids, steroids and sterols, lipoproteins and other compounds. Liposomes containing lipids phospholipids and sterols have been widely used in drugs and vaccines.

[0005] QS-21, MPL and neutral liposomes are AS01 ® The key components of the adjuvant system; QS-21, QS-7 and neutral liposomes are the key components of Matrix-M TM Neutral liposomes (containing lipid dioleoylphosphatidylcholine and lipid cholesterol) are used together with saponin immunopotentiators QS-21 and QS-7 in vaccines. This is primarily to eliminate the hemolytic activity of QS-21 and QS-7 and reduce clinical side effects of the vaccine. The combination of saponin immunopotentiators and neutral liposomes has been used in a number of approved vaccines, such as GSK's Shingrix. ® Arexvy ® 、Mosquirix(RTS,S / AS01 ®) vaccines, Novavax's NVX-CoV2373 vaccine, and the R21 / Matrix-M developed and produced by the University of Oxford and the Serum Institute of India. TM vaccine.

[0006] In addition, more vaccines containing saponin immunopotentiators QS-21 and / or QS-7 and neutral liposomes / cationic liposomes are in clinical trial and non-clinical trial studies: clinical trial studies see NCT00470574, NCT00000809, NCT00001044, NCT00006387, NCT05841550, NCT05270265, NCT03293498, CTR20233995, CTR20241986, CTR20243742, CTR20243851, CTR20244652, etc.; non-clinical trial studies see CN116350770B, CN114767844A, CN115894707A, CN116747298B and CN117003896 B, etc.

[0007] In order to control the quality of vaccines, it is necessary to test the content of the main adjuvant components in the vaccine. ® In the quality control of Shingrix, two different liquid chromatography methods were used to detect the content of saponin immunopotentiator (QS-21) and lipids (dioleoylphosphatidylcholine and cholesterol). ® The methods for determining the content of saponin immunopotentiators (QS-21) and lipids (dioleoylphosphatidylcholine and cholesterol) in vaccines are cumbersome, time-consuming, material-consuming, labor-intensive, and inefficient. Summary of the Invention

[0008] The purpose of the present invention is to provide a method and application for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / product containing a composite adjuvant, so as to solve the technical problems in the prior art of detecting the content of saponin immunopotentiators and lipids, which are cumbersome, time-consuming, material-consuming, labor-consuming and inefficient.

[0009] To achieve the above objectives, the present invention provides the following technical solutions: The present invention provides a method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing the composite adjuvant. The method adopts a liquid chromatography method. The immunopotentiators include but are not limited to QS-21 and QS-7, and the lipids include but are not limited to dioleoylphosphatidylcholine and cholesterol.

[0010] Furthermore, the composite adjuvant may also contain immunopotentiators such as monophosphoryl lipid A (MPL) and / or unmethylated cytosine guanine dinucleotide (CPG) and / or polyinosinic acid (polyinosinic acid:polycytidylic acid; Poly I:C), and the lipids may also contain cationic lipids such as dioleoyl-trimethylammonium chloride (DOTAP) and / or ALC-0315 and / or D-Lin-MC3-DMA and / or SM102, and / or anionic lipids such as phosphatidylinositol (PI) and / or phosphatidylserine (PS).

[0011] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra high performance liquid chromatography.

[0012] Furthermore, the liquid chromatography method is a reverse phase liquid chromatography method, wherein the chromatographic column contains an alkyl or phenyl reverse phase chromatography filler, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water, and the mobile phase may also contain methanol.

[0013] Furthermore, the alkyl reverse phase chromatography filler can be selected from any one of the following chromatography fillers, but is not limited to the following: butylsilane bonded silica gel (C4), hexadecylsilane bonded silica gel (C6), octadecylsilane bonded silica gel (C8), hexadecylsilane bonded silica gel (C16), and octadecylsilane bonded silica gel (C18).

[0014] Furthermore, the liquid chromatography method is a liquid chromatography internal standard method or a liquid chromatography external standard method.

[0015] Furthermore, the liquid chromatography external standard method comprises the following steps: S1. Prepare saponin immunopotentiator calibration standard solution sample; S2, preparing lipid calibration standard solution samples; S3, preparing test samples; S4, loading the sample into the liquid chromatograph and running the analytical method; S5. Calculate the content of immunopotentiators and lipids.

[0016] Furthermore, the saponin immunopotentiator calibration standard solution samples are prepared in S1, including but not limited to QS-21 and / or QS-7 calibration standard solution samples.

[0017] Furthermore, the concentration of QS-21 and / or QS-7 in the QS-21 and / or QS-7 calibration standard solution sample is 10 μg / ml to 100 μg / ml.

[0018] Furthermore, the QS-21 and / or QS-7 calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0019] Furthermore, the lipid calibration standard solution samples prepared in S2 include but are not limited to the preparation of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples, and the preparation method is selected from any one of the following two preparation methods: 1. Preparing dioleoylphosphatidylcholine calibration standard solution samples and preparing cholesterol calibration standard solution samples (prepared separately); 2. Preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples (mixed preparation).

[0020] Furthermore, a dioleoylphosphatidylcholine calibration standard solution sample and a cholesterol calibration standard solution sample (prepared separately) are prepared by the following method, but are not limited to the following method: after accurately weighing dioleoylphosphatidylcholine (purity not less than 90%), dissolving it in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), finally diluting to the target volume and mixing, to prepare a dioleoylphosphatidylcholine standard solution sample; after accurately weighing cholesterol (purity not less than 90%), dissolving it in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), finally diluting to the target volume and mixing, to prepare a cholesterol standard solution sample.

[0021] Furthermore, a dioleoylphosphatidylcholine and cholesterol calibration standard solution sample (mixed preparation) is prepared, and the preparation method is the following method, but not limited to the following method: using liquid chromatography to detect the concentration of dioleoylphosphatidylcholine and cholesterol in the liposome solution [the purity of dioleoylphosphatidylcholine and cholesterol in the liposome is not less than 90%], and preparing a dioleoylphosphatidylcholine and cholesterol standard solution sample; accurately weighing dioleoylphosphatidylcholine and cholesterol [the purity of dioleoylphosphatidylcholine and cholesterol is not less than 90%], and then dissolving them in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluting to the target volume and mixing to prepare a dioleoylphosphatidylcholine and cholesterol standard solution sample.

[0022] Furthermore, the liquid chromatography method for detecting the concentration of the liposome solution has a detector that is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector, a chromatographic column that is a reversed-phase column, and an organic phase in the mobile phase that is acetonitrile or methanol.

[0023] Furthermore, the dioleoylphosphatidylcholine and cholesterol concentrations of the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples are 200 μg / ml to 2000 μg / ml and 50 μg / ml to 500 μg / ml, respectively.

[0024] Furthermore, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples may contain DMSO at a concentration of 0.5% to 5%.

[0025] Furthermore, the test sample prepared in S3 can be prepared using a product containing a composite adjuvant or a composite adjuvant.

[0026] Furthermore, a product containing a composite adjuvant is prepared for use in preparing test samples, wherein the protein or nucleic acid content of the product containing the composite adjuvant is 10 μg / ml to 400 μg / ml, the phospholipid content is 400 μg / ml to 8000 μg / ml, the sterol content is 100 μg / ml to 2000 μg / ml, and the immunopotentiator content is 20 μg / ml to 7000 μg / ml.

[0027] Furthermore, the protein includes but is not limited to varicella-zoster virus glycoprotein E (gE) or gE fusion protein, respiratory syncytial virus fusion protein (F protein), metapneumovirus fusion protein (F protein), parainfluenza virus hemagglutinin neuraminidase (HN) glycoprotein and fusion protein (F protein), Plasmodium circumsporozoite protein (CSP) or its fusion protein (such as: a fusion protein expressed by fusion of a partial sequence of the circumsporozoite protein with the hepatitis B virus surface antigen), a fusion protein expressed by fusion of Mycobacterium tuberculosis MTB32A and MTB39A (M72 fusion protein), influenza A and B virus hemagglutinin protein (HA), herpes simplex virus glycoprotein D (gD) or gD fusion protein, novel coronavirus spike glycoprotein (S protein) or its fusion protein, human immunodeficiency virus gp120 protein, rabies virus glycoprotein (G protein) or its fusion protein.

[0028] Furthermore, the phospholipids contain dioleoylphosphatidylcholine (DOPC) in an amount of 400 μg / ml to 4000 μg / ml; the phospholipids may contain distearoylphosphatidylcholine (DSPC) in an amount of 400 μg / ml to 2000 μg / ml; the phospholipids may contain cationic lipids, including but not limited to dioleoyl-trimethylammonium chloride (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), ALC-0315, D-Lin-MC3-DMA, and SM102, in an amount of 400 μg / ml to 2000 μg / ml; the phospholipids may also contain anionic lipids, including but not limited to phosphatidylinositol (PI) and phosphatidylserine (PS), in an amount of 400 μg / ml to 2000 μg / ml.

[0029] Furthermore, the sterol includes but is not limited to cholesterol, and the content is 100 μg / ml to 2000 μg / ml.

[0030] Furthermore, the immunopotentiator contains saponin QS-21 and / or QS-7, with a content of 20 μg / ml to 1000 μg / ml; the immunopotentiator may also contain MPL (monophosphoryl lipid A) and / or CPG ODN (artificially synthesized oligodeoxynucleotide sequence containing non-methylated cytosine guanine dinucleotide) and / or poly I:C (polyinosinic acid), with a content of 10 μg / ml to 6000 μg / ml.

[0031] Furthermore, the MPL includes but is not limited to any one or more combinations of the following: MPL (triacyl; monophosphoryl lipid A), MPL (tetraacyl; monophosphoryl lipid A), MPL (pentaacyl; monophosphoryl lipid A), MPL (hexaacyl; monophosphoryl lipid A), MPL (heptaacyl; monophosphoryl lipid A), 3D-MPL (triacyl; 3-O-deacyl-4´-monophosphoryl lipid A), 3D-MPL (tetraacyl; 3-O-deacyl-4´-monophosphoryl lipid A), 3D-MPL (pentaacyl; 3-O-deacyl-4´-monophosphoryl lipid A), 3D-MPL (hexaacyl; 3-O-deacyl-4´-monophosphoryl lipid A).

[0032] Furthermore, the CpG ODN includes but is not limited to the following three categories: Class A, Class B, and Class C; the Class A CpG ODN: a CpG dinucleotide palindromic sequence as the core, poly G tails at both ends, a phosphodiester bond backbone that is partially thiolated, and a higher-order structure formed by the palindromic sequence and poly G, which can activate plasmacytoid dendritic cells to induce a large amount of type I interferon, but has weak activity on B cells, such as: CpG 2216; the Class B CpG ODN is characterized by: a fully thiolated linear CpG ODN that has strong immunostimulatory activity on B cells but cannot activate plasmacytoid dendritic cells, such as: CpG 1018, CpG 2006, and CpG 1826; the Class C CpG ODN is characterized by: a fully thiolated CpG ODN that can form dimers through a palindromic sequence and has both type A and type B CpG The activity of ODN can activate both plasmacytoid dendritic cells and B cells, such as CpG2395.

[0033] Furthermore, the poly I:C (polyinosinic-polycytidylic acid) includes but is not limited to any one or more combinations of the following: poly I:C (without stabilizer), poly I:C (stabilizer is polylysine), and poly I:C (stabilizer is kanamycin).

[0034] Furthermore, the composite adjuvant used to prepare the test sample in S3 has a phospholipid content of 400 μg / ml to 8000 μg / ml, a sterol content of 100 μg / ml to 2000 μg / ml, and an immunopotentiator content of 20 μg / ml to 7000 μg / ml.

[0035] Furthermore, the phospholipids contain dioleoylphosphatidylcholine (DOPC) in an amount of 400 μg / ml to 4000 μg / ml; the phospholipids may contain distearoylphosphatidylcholine (DSPC) in an amount of 400 μg / ml to 2000 μg / ml; the phospholipids may contain cationic lipids, including but not limited to dioleoyl-trimethylammonium chloride (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), ALC-0315, D-Lin-MC3-DMA, and SM102, in an amount of 400 μg / ml to 2000 μg / ml; the phospholipids may also contain anionic lipids, including but not limited to phosphatidylinositol (PI) and phosphatidylserine (PS), in an amount of 400 μg / ml to 2000 μg / ml.

[0036] Furthermore, the sterol includes but is not limited to cholesterol, and the content is 100 μg / ml to 2000 μg / ml.

[0037] Furthermore, the immunopotentiator contains saponin QS-21 and / or QS-7 in an amount of 20 μg / ml to 1000 μg / ml; the immunopotentiator may also contain MPL and / or CPG ODN and / or poly I:C in an amount of 10 μg / ml to 6000 μg / ml.

[0038] Furthermore, in the test sample in S3, the concentration of the immunopotentiator is 10 μg / ml to 3500 μg / ml, the concentration of phospholipid is 200 μg / ml to 4000 μg / ml, and the concentration of sterol is 50 μg / ml to 1000 μg / ml.

[0039] Furthermore, in the test sample in S3, the concentration of the saponin immunopotentiator is 10 μg / ml to 100 μg / ml, the concentration of dioleoylphosphatidylcholine (DOPC) is 200 μg / ml to 2000 μg / ml, and the concentration of sterol is 50 μg / ml to 500 μg / ml.

[0040] Furthermore, the phospholipids contain dioleoylphosphatidylcholine (DOPC) in an amount of 200 μg / ml to 2000 μg / ml; the phospholipids may contain distearoylphosphatidylcholine (DSPC) in an amount of 200 μg / ml to 1000 μg / ml; the phospholipids may contain cationic lipids, including but not limited to dioleoyl-trimethylammonium chloride (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), ALC-0315, D-Lin-MC3-DMA, and SM102, in an amount of 200 μg / ml to 1000 μg / ml; the phospholipids may also contain anionic lipids, including but not limited to phosphatidylinositol (PI) and phosphatidylserine (PS), in an amount of 200 μg / ml to 1000 μg / ml.

[0041] Furthermore, the sterol includes but is not limited to cholesterol, and the content is 50 μg / ml to 1000 μg / ml.

[0042] Furthermore, the immunopotentiator contains saponin QS-21 and / or QS-7 in an amount of 10 μg / ml to 500 μg / ml; the immunopotentiator may also contain MPL and / or CPG ODN and / or poly I:C in an amount of 5 μg / ml to 3000 μg / ml.

[0043] Furthermore, the test sample in S3 may contain DMSO at a concentration of 0.5% to 5%.

[0044] Furthermore, the detector of the liquid chromatograph in S4 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0045] Furthermore, the analysis method in S4 includes a chromatographic column, liquid chromatography parameters, and an elution procedure.

[0046] Furthermore, the chromatographic column used in the analysis method in S4 is a reverse phase chromatographic column.

[0047] Furthermore, the liquid chromatography parameters of the analysis method in S4 include column temperature, injection volume, flow rate, detector parameters, etc.

[0048] Further, the column temperature is 40°C to 60°C, such as 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0049] 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.

[0050] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the injection volume is 1 μl to 30 μl, such as 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, or 30 μl.

[0051] Further, 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.

[0052] Furthermore, when the instrument is an ultra 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.

[0053] Further, the detector parameters, when the detector is an ultraviolet detector, the detection wavelength is 200nm~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.

[0054] Furthermore, when the detector is a CAD detector, the atomization temperature is 35°C to 70°C, such as 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C.

[0055] Furthermore, when the detector is an evaporative light detector, the atomization temperature is 35°C to 70°C, such as 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C.

[0056] Furthermore, the elution procedure of the analysis method in S4 includes at least the following two elution methods: an elution method for eluting the retained peak of the immunopotentiator; and an elution method for eluting the retained peak of the lipid.

[0057] Furthermore, the elution method for eluting the retained peak of the immunopotentiator is gradient elution and / or isocratic elution.

[0058] Furthermore, the elution method for eluting the retained peak of the immunopotentiator contains a sub-elution method for eluting the retained peak of Quillaja saponin QS-21 and / or QS-7; the elution method for eluting the retained peak of the immunopotentiator may also contain a sub-elution method for eluting MPL and / or CPGODN and / or poly I:C.

[0059] Furthermore, the sub-elution method for eluting the retained peak of Quillaja saponin QS-21 and / or QS-7 can elute the retained peak of the protein in the recombinant varicella-zoster vaccine.

[0060] Further, the sub-elution method for eluting the retained peak of Quillaja saponin QS-21 and / or QS-7 is gradient elution, and the acetonitrile concentration gradient in the mobile phase is 5% to 95%, such as 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 65%, 45% to 65%, 45% to 70%, 45% to 75%, 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%, 6 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 2 min to 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 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%.

[0061] Furthermore, the elution method for eluting the lipid retention peak is gradient elution and / or isocratic elution, and the mobile phase system includes but is not limited to an acetonitrile system or a methanol system.

[0062] Furthermore, the elution method for eluting the lipid retention peak contains a sub-elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks, and the sub-elution method can also elute some cationic lipids (such as DOTAP) and / or anionic lipids; the elution method for eluting the lipid retention peak may also contain a sub-elution method for eluting cationic lipids and / or anionic lipids and / or distearoylphosphatidylcholine.

[0063] Furthermore, the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0064] Furthermore, the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution: the concentration of acetonitrile or methanol in the mobile phase is 90% to 100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and the isocratic elution time is 2 min to 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 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%.

[0065] Furthermore, the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is gradient elution: the concentration of acetonitrile or methanol in the mobile phase is 90% to 100%, such as 90% to 100%, 91% to 100%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 90% to 99%, 91% to 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% ~97%, 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%, and the gradient elution time is 2min~1920min, such as 2min, 4min, 8min, 10min, 15min, 20min, 25min, 3min, 45min, 5min, 6min, 7min, 8min, 9min, 10min, 15min, 20min, 25min, 3min, 4 ... The mobile phase is stirred for 10 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 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%.

[0066] Furthermore, in the elution procedure of the analysis method in S4, a pre-equilibration method may be added before the sub-elution method for eluting the retained peaks of Quillaja saponin QS-21 and / or QS-7.

[0067] Furthermore, a pre-equilibration method is added before the sub-elution method for eluting the retention peak of Quillaja saponin QS-21 and / or QS-7, wherein the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient for eluting the retention peak of Quillaja saponin QS-21 and / or QS-7.

[0068] Further, a pre-equilibrium method is added before the elution method of eluting the retained peak of Quillaja saponin QS-21 and / or QS-7, the acetonitrile concentration in the mobile phase is 5% to 60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, the equilibration 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, and the trifluoroacetic acid concentration 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%.

[0069] Furthermore, in the elution procedure of the analysis method in S4, a chromatographic column regeneration method and / or a post-equilibration method may be added after the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0070] Furthermore, when the chromatographic column regeneration method and the post-equilibrium method exist at the same time, the chromatographic column regeneration method should be performed before the post-equilibrium method.

[0071] Further, a chromatographic column regeneration method is added after the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile concentration in the mobile phase is 98% to 100%, such as 98%, 98.5%, 99%, 99.5% or 100%, and the chromatographic column regeneration 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, and the mobile phase may also contain trifluoroacetic acid at a concentration 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, a post-equilibrium method is added after the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, and the acetonitrile concentration in the mobile phase is not higher than the minimum value of the mobile phase concentration gradient for eluting the retention peaks of Quillaja saponin QS-21 and / or QS-7.

[0073] Further, a post-equilibrium method is added after the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile concentration in the mobile phase is 5% to 60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, the equilibrium 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, and the trifluoroacetic acid concentration 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%.

[0074] Furthermore, the calculation of the saponin immunopotentiator and lipid content in S5 includes calculation method one and calculation method two.

[0075] Furthermore, the calculation method 1 is: first establish a calibration standard curve in which the concentration of the saponin immunopotentiator and the lipid is proportional to the peak area, and then calculate the content (concentration) of the saponin immunopotentiator and the lipid.

[0076] Furthermore, calculation method 1: first establish a calibration standard curve in which the concentration of saponin immunopotentiators and lipids is proportional to the peak area; then substitute the peak area values of saponin immunopotentiators and lipids in the sample into the calibration standard curve formula of the corresponding components, and calculate the concentration of the corresponding components in the test sample; multiply the concentration of each component of the test sample by the dilution multiple, and calculate the content (concentration) of saponin immunopotentiators and lipids in the sample.

[0077] Furthermore, the second calculation method is to directly calculate the content (concentration) of saponin immunopotentiators and lipids in the sample.

[0078] Furthermore, the second calculation method is: the calculation formula is A content (concentration) in the product or compound adjuvant = A peak area of the test sample * A calibration standard solution sample concentration * dilution factor / A calibration standard solution sample peak area, "A" represents "saponin immunopotentiator" or "lipid".

[0079] The present invention also provides an application for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant or a product containing the composite adjuvant, wherein the product containing the composite adjuvant includes but is not limited to vaccines.

[0080] Furthermore, the method is used to detect the content of Quillaja saponin QS-21 and / or Quillaja saponin QS-7 and / or dioleoylphosphatidylcholine and / or cholesterol.

[0081] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: The present invention provides a method and application for simultaneously detecting the content of saponin immunopotentiators and lipids in composite adjuvants / products containing composite adjuvants. This method overcomes the technical problem of existing techniques requiring at least two separate methods for detecting saponin immunopotentiators and lipids. The high-performance liquid chromatography method disclosed in the present invention can detect the content of saponin immunopotentiators and lipids in a product with only a single sample loading, significantly improving assay efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0083] Figure 1 This is a liquid chromatogram of the main components of the vaccine under different mobile phase pH adjusters in Example 1 of the present invention; The left chromatogram is acetic acid; the right chromatogram is trifluoroacetic acid.

[0084] Figure 2 This is a liquid chromatogram of Quillaja saponin QS-21 and PADRE-gE-P2 fusion protein in the vaccine under different elution conditions of the immunopotentiator Quillaja saponin QS-21 according to Example 2 of the present invention; From left to right are the chromatograms of 5% to 95% acetonitrile gradient elution for 15 minutes, 50% to 75% acetonitrile gradient elution for 10 minutes, 50% to 75% acetonitrile gradient elution for 20 minutes, and 50% to 75% acetonitrile gradient elution for 30 minutes.

[0085] Figure 3 This is the liquid chromatogram of the immune enhancer Quillaja saponin QS-21 and PADRE-gE-P2 fusion protein in the vaccine under different column temperature conditions in Example 3 of the present invention (from left to right are the chromatograms at column temperatures of 30°C, 40°C, 50°C, and 60°C).

[0086] 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 temperature conditions in Example 3 of the present invention.

[0087] Figure 5This is a comparison chart of the tailing factors of the retained peaks of the main adjuvant components in the vaccine under different injection volume conditions in Example 4 of the present invention. Figure 6 These are liquid chromatograms of the immunopotentiator Quillaja saponin QS-21 and the PADRE-gE-P2 fusion protein in the vaccine under different flow rate conditions in Example 5 of the present invention (from left to right are the chromatograms at flow rates of 1.0 ml / min, 1.5 ml / min, and 2.0 ml / min, respectively). Figure 7 This is a comparison chart of the tailing factors of the retention peaks of the main components of the vaccine under different flow rate conditions in Example 5 of the present invention.

[0088] Figure 8 6 is a chromatogram of the retention peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol under different elution time conditions of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol in Example 6 of the present invention.

[0089] Figure 9 This is a standard curve of concentration and peak area of the immunopotentiator Quillaja saponin QS-21 of Example 7 of the present invention.

[0090] Figure 10 This is the standard curve of lipid dioleoylphosphatidylcholine (DOPC) concentration and peak area in Example 7 of the present invention.

[0091] Figure 11 This is the standard curve of lipid cholesterol concentration and peak area in Example 7 of the present invention.

[0092] Figure 12 This is a liquid chromatogram of Example 7 of the present invention for simultaneously detecting the contents of immunopotentiators and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in a recombinant varicella-zoster vaccine containing PADRE-gE-P2 fusion protein and XA-401 composite adjuvant.

[0093] Figure 13 This is a comparison chart of the content of the immune enhancer Quillaja saponin QS-21, the lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine sample detected simultaneously (one time) by Example 7 of the present invention and detected by the prior art (two separate detections).

[0094] Figure 14 This is a liquid chromatogram of Example 8 of the present invention for simultaneously detecting the contents of immunopotentiators and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in the XA-401 composite adjuvant.

[0095] The retention peak and front of each substance are completely separated, meeting the requirements.

[0096] Figure 15This is a comparison chart of Example 8 of the present invention for simultaneously detecting the contents of immunopotentiators and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in the XA-401 composite adjuvant.

[0097] Figure 16 This is a liquid chromatogram of Example 9 of the present invention for simultaneously detecting the contents of the immunopotentiator and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in the composite adjuvant 1.

[0098] Figure 17 This is a comparison chart of Example 9 of the present invention for simultaneously detecting the contents of the immunopotentiator and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 1.

[0099] Figure 18 This is a liquid chromatogram of Example 10 of the present invention for simultaneously detecting the contents of the immunopotentiator and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 2.

[0100] Figure 19 This is a comparison chart of the simultaneous detection of the contents of the immunopotentiator and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 2 according to Example 10 of the present invention.

[0101] Figure 20 This is a liquid chromatogram of Example 11 of the present invention for simultaneously detecting the contents of the immunopotentiator and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 3.

[0102] Figure 21 This is a comparison chart of Example 11 of the present invention for simultaneously detecting the contents of the immunopotentiator and lipids [immunopotentiator Quillaja saponin QS-21; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 3.

[0103] Figure 22 This is a standard curve of concentration and peak area of the immunopotentiator Quillaja saponin QS-21 according to Example 12 of the present invention.

[0104] Figure 23 This is the standard curve of lipid dioleoylphosphatidylcholine (DOPC) concentration and peak area in Example 12 of the present invention.

[0105] Figure 24 This is the standard curve of lipid cholesterol concentration and peak area in Example 12 of the present invention.

[0106] Figure 25This is a liquid chromatogram of Example 12 of the present invention for simultaneously detecting the contents of immunopotentiators and lipids [immunopotentiators Quillaja saponins QS-21 and QS-7; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 4.

[0107] Figure 26 This is a comparison chart of Example 12 of the present invention for simultaneously detecting the contents of immunopotentiators and lipids [immunopotentiators Quillaja saponins QS-21 and QS-7; lipids dioleoylphosphatidylcholine (DOPC), cholesterol] in composite adjuvant 4. DETAILED DESCRIPTION

[0108] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0109] It should be understood that the optimal liquid chromatography parameters and elution methods when using reversed-phase chromatography columns containing different chromatographic fillers to detect target substances (such as the immune enhancers Quillaja saponins QS-21 and QS-7, lipid dioleoylphosphatidylcholine, and cholesterol) may be different, which is known to those skilled in the art.

[0110] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0111] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0112] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0113] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0114] Example 1: Study on the Type of Acidic pH Regulator in the Mobile Phase for Eluting the Immunopotentiator Quillaja Saponin QS-21, Lipid Dioleoylphosphatidylcholine, and Cholesterol 1) Preparation of PADRE-gE-P2 fusion protein and protein concentration detection 1. Fusion protein codon optimization and whole gene synthesis For details, see patent CN 117003896 B.

[0115] 2. Construction of fusion protein expression plasmid For details, see patent CN 117003896 B.

[0116] 3. Construction of Stable Cell Lines For details, see patent CN 117003896 B.

[0117] 4. Expression of target product For details, see patent CN 117003896 B.

[0118] 5. Fusion Protein Purification For details, see patent CN 117003896 B.

[0119] VI. Fusion protein (vaccine stock solution) concentration detection (Lowry method) The first method.

[0120] 2) Liposome preparation and determination of dioleoylphosphatidylcholine and cholesterol concentrations 1. Liposome Preparation (Theoretical Concentrations of Dioleoylphosphatidylcholine and Cholesterol are 4 mg / ml and 1 mg / ml, respectively) 400 mg of dioleoylphosphatidylcholine (DOPC; Japan Fine Chemicals Co., Ltd.) and 100 mg of cholesterol (Japan Fine Chemicals Co., Ltd.) were weighed separately. DOPC and cholesterol were then completely dissolved in 10 ml of anhydrous ethanol and mixed evenly to form the organic phase. 10 ml of the organic phase was injected into 90 ml of 10 mM PBS buffer solution (pH 7.0) to produce liposome colostrum. The liposomes were then granulated using a high-pressure microfluidizer to a particle size of approximately 100 nm. Ethanol was then removed by dialysis. Finally, the liposomes were sterilized by filtration using a 0.22 μm sterilizing filter to obtain the finished liposome product.

[0121] 2. Dioleoylphosphatidylcholine (DOPC) and cholesterol concentration detection (1) Preparation of mobile phase 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 well, and degas by ultrasonication for 10 minutes.

[0122] (2) Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol standard solution sample Preparation of 90% ethanol solution: Accurately measure 45 ml of anhydrous ethanol, mix with 5 ml of ultrapure water and shake well.

[0123] Preparation of 89.3% ethanol solution: Accurately measure 44.65 ml of anhydrous ethanol, mix it with 5.35 ml of ultrapure water and shake well.

[0124] Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol reference stock solution: Accurately weigh 37.5 mg of cholesterol (Japan Fine Chemicals Co., Ltd.) and 150 mg of dioleoylphosphatidylcholine (DOPC; Japan Fine Chemicals Co., Ltd.) into a 25 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, shake well, and obtain the solution.

[0125] Mix dioleoylphosphatidylcholine (DOPC) and cholesterol standard solution sample preparation: Pipette 1.667 ml of the reference stock solution into a 25 ml volumetric flask, dilute to 25 ml with 89.3% ethanol solution, and shake to obtain standard solution ⑤ with a cholesterol concentration of 100 μg / ml and a dioleoylphosphatidylcholine (DOPC) concentration of 400 μg / ml; Pipette 7.5 ml, 5 ml, 2.5 ml and 1.25 ml of standard solution ⑤ into a 10 ml volumetric flask, dilute to 10 ml with 90% ethanol solution, and shake to obtain standard solution ④ [cholesterol concentration of 75 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration of 300 μg / ml] and standard solution ③ [cholesterol concentration of 50 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration of 200 μg / ml], standard solution ② [cholesterol concentration: 25 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration: 100 μg / ml)], and standard solution ① [cholesterol concentration: 12.5 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration: 50 μg / ml].

[0126] (3) Preparation of test sample solution Pipette 0.1 ml of the sample solution to be tested into a sampling vial, add 1.9 ml of anhydrous ethanol, cover the vial cap, and mix well to obtain the test sample.

[0127] (4) Instrument parameters and elution procedures (5) Calculation of dioleoylphosphatidylcholine and cholesterol concentrations With the concentration of the standard solution of dioleoylphosphatidylcholine (DOPC) or cholesterol as the abscissa (X) and the peak area of dioleoylphosphatidylcholine (DOPC) or cholesterol as the ordinate (Y), draw standard curves in which the concentration of dioleoylphosphatidylcholine (DOPC) and cholesterol are proportional to the peak area. Substitute the peak area of cholesterol or dioleoylphosphatidylcholine (DOPC) in the test sample solution into the standard curve for calculation, and then multiply it by the dilution factor used in the preparation of the test sample solution to obtain the concentration of dioleoylphosphatidylcholine (DOPC) and cholesterol in the sample solution to be tested.

[0128] 3) Preparation and concentration determination of Quillaja saponin QS-21 solution 1. Preparation of Quillaja saponin QS-21 solution (theoretical concentration 4 mg / ml) Weigh 40 mg of Quillaja saponin QS-21 (Desert King), dissolve it completely in 4 ml of 10 mM phosphate solution (pH 6.0), dilute the volume to 10 ml with 10 mM phosphate solution (pH 6.0), and mix well to obtain the Quillaja saponin QS-21 solution.

[0129] 2. Concentration detection of Quillaja saponin QS-21 solution (1) Preparation of mobile phase 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 degas by ultrasonication for 5 minutes.

[0130] 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 ultrasonically degas for 5 minutes.

[0131] (2) Preparation of standard solution sample of Quillaja saponin QS-21 Reference substance stock solution: Weigh 10 mg of QS-21 reference substance (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.

[0132] Standard solution: Pipette 150 μl of the reference substance stock solution into a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, and mix to obtain a solution with a concentration of 1 mg / ml; Pipette 0.5 ml of the above solution into a 5 ml volumetric flask, dilute to the scale with 1% DMSO solution, and mix to obtain a solution with a concentration of 100 μg / ml; then pipette 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution into different injection vials, and then add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of 1% DMSO solution, respectively, and mix 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.

[0133] (3) Preparation of test sample solution Pipette 0.125 ml of the sample solution to be tested into a 5 ml volumetric flask. Dilute to the mark with 10 mM phosphate solution (pH 6.0) and mix thoroughly to obtain a 100 μg / ml solution. Transfer 0.5 ml of the 100 μg / ml solution to a vial, add 0.5 ml of 2% DMSO solution, and mix thoroughly.

[0134] (4) Instrument parameters and elution procedures (5) Calculation of concentration of Quillaja saponin QS-21 A linear regression curve was drawn with the concentration of the standard solution of Quillaja saponin QS-21 as the X-axis and the corresponding peak area as the Y-axis. The peak area of the test sample solution was substituted into the standard curve and then multiplied by the dilution factor of the test sample solution to obtain the content of QS-21 in the test sample solution.

[0135] 4) Vaccine sample preparation 1. Preparation of adjuvant The concentration of the liposome solution was detected: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively. The concentration of the Quillaja saponin QS-21 solution was detected: the concentration of the Quillaja saponin QS-21 was 4.14 mg / ml. 4.76 ml of liposomes was added with 0.24 ml of the Quillaja saponin QS-21 solution and stirred evenly to obtain the adjuvant.

[0136] II. Vaccine Preparation [10 ml volume; target concentration of PADRE-gE-P2 fusion protein: 150 μg / ml; target concentration of Quillaja saponin QS-21: 100 μg / ml; liposome components: target concentrations of dioleoylphosphatidylcholine and cholesterol: 2 mg / ml and 0.5 mg / ml, respectively] Detect the protein concentration of the PADRE-gE-P2 fusion protein solution: the concentration of the PADRE-gE-P2 fusion protein is 1.52 mg / ml; add 0.99 ml of the PADRE-gE-P2 fusion protein solution to the adjuvant, and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir evenly to obtain the PADRE-gE-P2 fusion protein adjuvant vaccine; the volume (ml) of the added PADRE-gE-P2 fusion protein solution is calculated as follows: the mass of the added PADRE-gE-P2 fusion protein (μg) / the concentration of the PADRE-gE-P2 fusion protein (μg / ml).

[0137] 5) Study the effect of acidic pH regulators (trifluoroacetic acid and acetic acid) in the mobile phase on the retention peaks of Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol 1. Solution preparation (1) Preparation of mobile phase (0.05% trifluoroacetic acid) 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 ultrasonically degas for 5 minutes.

[0138] 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 degas by ultrasonication for 5 minutes.

[0139] (2) Preparation of mobile phase (0.05% acetic acid) 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 degas by ultrasonication for 5 minutes.

[0140] 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 ultrasonically degas for 5 minutes.

[0141] (3) Preparation of sample dilution solution 1% dimethyl sulfoxide (DMSO) solution: Measure 99 ml of ultrapure water, add 1 ml of dimethyl sulfoxide solution, and mix well.

[0142] 1.25% dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, and mix well.

[0143] 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix well.

[0144] 3.5% dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, and mix well.

[0145] 2. Sample Preparation Test sample: Pipette 0.5 ml of vaccine into a sample vial, add 0.5 ml of 2% DMSO solution, cover the vial cap, and mix well.

[0146] 3. Chromatographic column, instrument parameters and elution procedure 4. Results Analysis The liquid chromatography method used to control the quality of drugs has the following characteristics: the substance to be tested must have a retention peak, and the separation degree of the retention peaks of each substance must be greater than 1.5, the tailing factor must be 0.8-1.8, and the baseline near the retention peak must be horizontal (parallel to the X-axis of the spectrum).

[0147] (1) The acidic pH regulator in the mobile phase is acetic acid Note: “NA” means “not applicable”; “no peak” means “no retention peak of the substance on the chromatogram”.

[0148] (2) The acidic pH regulator in the mobile phase is trifluoroacetic acid substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.140 5846 NA 1.433 flat QS-21 A isomer 9.536 258313 2.289 No data released flat DOPC 21.072 1496668 15.356 0.716 Unfair cholesterol 23.849 2533631 3.887 3.215 flat Note: “NA” means “not applicable”; “data not available” means “data not calculated by the liquid chromatography analysis software”.

[0149] like Figure 1 As shown in the figure, when acetic acid was used as the mobile phase pH adjuster, there was no retention peak for dioleoylphosphatidylcholine.

[0150] Example 2: Study on the method of eluting the immunopotentiator Quillaja saponin QS-21 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0151] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0152] 3) The preparation of Quillaja saponin QS-21 solution and the concentration determination were the same as in Example 1.

[0153] 4) The vaccine sample preparation was the same as in Example 1.

[0154] 5) Study on the method of eluting Quillaja saponin QS-21 1. Solution preparation (1) Preparation of mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0155] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0156] 2. Preparation of test samples is the same as in Example 1.

[0157] 3. Chromatographic column, instrument parameters and elution procedure (1) Chromatographic column and instrument parameters (2) Elution procedure 1: Gradient elution of acetonitrile concentration 5% to 95% (5% mobile phase B-95% mobile phase B) for 15 minutes to elute QS-21 (3) Elution procedure 2: 50% to 75% acetonitrile concentration (50% mobile phase B-75% mobile phase B) gradient elution for 10 min to elute QS-21 (4) Elution procedure 3: Gradient elution of acetonitrile concentration 50% to 75% (50% mobile phase B to 75% mobile phase B) for 20 min to elute QS-21 (5) Elution procedure 4: 50% to 75% acetonitrile concentration (50% mobile phase B-75% mobile phase B) gradient elution for 30 min to elute QS-21 4. Results Analysis The liquid chromatography method used to control the quality of drugs has the following characteristics: the substance to be tested must have a retention peak, and the separation degree of the retention peaks of each substance must be greater than 1.5, the tailing factor must be 0.8-1.8, and the baseline near the retention peak must be horizontal (parallel to the X-axis of the spectrum).

[0158] (1) Elution procedure 1: gradient elution with acetonitrile concentration of 5% to 95% for 15 minutes to elute QS-21 substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.140 3452 NA 1.430 flat QS-21 A isomer 9.538 262898 2.284 No data released flat DOPC 21.086 1445088 14.109 0.642 Unfair cholesterol 23.843 2452095 3.409 3.050 flat Note: “NA” means “not applicable”; “data not available” means “data not calculated by the liquid chromatography analysis software”.

[0159] (2) Elution procedure 2: gradient elution with acetonitrile concentration of 50% to 75% for 10 minutes to elute QS-21 substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.600 4594 NA 1.692 flat QS-21 A isomer 10.035 265124 2.276 1.342 flat DOPC 28.098 1457212 16.459 0.634 Unfair cholesterol 30.548 2464920 3.033 3.083 flat Note: “NA” means “not applicable”.

[0160] (3) Elution procedure 3: gradient elution with acetonitrile concentration of 50% to 75% for 20 minutes to elute QS-21 substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.604 4228 NA 1.156 flat QS-21 A isomer 10.048 266858 2.194 1.390 flat DOPC 38.020 1464766 20.647 0.638 Unfair cholesterol 40.221 2481466 2.797 2.944 flat Note: “NA” means “not applicable”.

[0161] (4) Elution procedure 4: gradient elution with acetonitrile concentration of 50% to 75% for 30 minutes to elute QS-21 substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.615 5312 NA 1.835 flat QS-21 A isomer 10.068 267136 2.124 1.342 flat DOPC 48.076 1434968 22.957 0.635 Unfair cholesterol 49.988 2481402 2.413 2.877 flat Note: “NA” means “not applicable”.

[0162] like Figure 2 As shown, when the method for eluting Quillaja saponin QS-21 was 5% to 95% acetonitrile gradient elution for 15 min, the retention peak of QS-21 A isomer and the retention peak of PADRE-gE-P2 fusion protein could not be completely separated, which did not meet the requirements.

[0163] Example 3: Study on column temperature 1) Preparation of PADRE-gE-P2 fusion protein and detection of protein concentration were the same as in Example 1.

[0164] 2) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0165] 3) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0166] 4) Vaccine sample preparation is the same as in Example 1.

[0167] 5) Study column temperature 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0168] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0169] 2. The preparation of test samples is the same as that in Example 1.

[0170] 3. Chromatographic column, instrument parameters and elution procedure 4. Results Analysis The liquid chromatography method used to control the quality of drugs has the following characteristics: the substance to be tested must have a retention peak, and the separation degree of the retention peaks of each substance must be greater than 1.5, the tailing factor must be 0.8-1.8, and the baseline near the retention peak must be horizontal (parallel to the X-axis of the spectrum).

[0171] (1) Column temperature 30°C substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.525 6993 NA No data released flat QS-21 A isomer 9.938 264531 1.802 1.350 flat DOPC 39.859 1626068 14.951 0.584 Unfair cholesterol 45.854 2489880 3.438 4.622 flat Note: “NA” means “not applicable”; “data not available” means “data not calculated by the liquid chromatography analysis software”.

[0172] (2) Column temperature 40°C substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.603 4413 NA 1.186 flat QS-21 A isomer 10.046 269066 2.289 1.379 flat DOPC 37.956 1550917 20.306 0.642 Unfair cholesterol 40.187 2504344 2.813 3.027 flat Note: “NA” means “not applicable”.

[0173] (3) Column temperature 50°C substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.624 4882 NA 1.228 flat QS-21 A isomer 10.069 279205 2.294 1.356 flat DOPC 36.556 1323866 24.669 0.918 Unfair cholesterol 37.576 2397250 2.493 1.797 Unfair Note: “NA” means “not applicable”.

[0174] (4) Column temperature 60°C substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.57 6035 NA No data released flat QS-21 A isomer 9.998 304782 2.189 No data released flat DOPC 35.127 1566384 26.490 No data released Unfair cholesterol 35.717 2420564 1.439 1.334 Unfair Note: “NA” means “not applicable”; “data not available” means “data not calculated by the liquid chromatography analysis software”.

[0175] like Figure 3 As shown in the figure, when the column temperature is 60℃, the QS-21 A isomer retention peak and the PADRE-gE-P2 fusion protein retention peak cannot be completely separated, which does not meet the requirements.

[0176] like Figure 4 As shown in the figure, when the column temperature is 30℃ and 40℃, the tailing factor of the DOPC retention peak is less than 0.8, which does not meet the requirements; when the column temperature is 30℃ and 40℃, the tailing factor of the cholesterol retention peak is greater than 1.8, which does not meet the requirements; when the column temperature is 60℃, the software does not calculate the tailing factors of the QS-21 B isomer, QS-21 A isomer and DOPC retention peaks, which does not meet the requirements; when the column temperature is 30℃, the software does not calculate the tailing factor of the QS-21 B isomer retention peak, which does not meet the requirements.

[0177] Example 4: Study of injection volume 1) Preparation of PADRE-gE-P2 fusion protein and detection of protein concentration were the same as in Example 1.

[0178] 2) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0179] 3) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0180] 4) Vaccine sample preparation is the same as in Example 1.

[0181] 5) Study the injection volume 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0182] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0183] 2. Preparation of test samples is the same as in Example 1.

[0184] 3. Chromatographic column, instrument parameters and elution procedure 4. Results Analysis The liquid chromatography method used to control the quality of drugs has the following characteristics: the substance to be tested must have a retention peak, and the separation degree of the retention peaks of each substance must be greater than 1.5, the tailing factor must be 0.8-1.8, and the baseline near the retention peak must be horizontal (parallel to the X-axis of the spectrum).

[0185] (1) Injection volume 50 μl substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.603 2968 NA 1.207 flat QS-21 A isomer 10.043 134910 2.444 1.425 flat DOPC 36.155 664372 21.909 2.225 Unfair cholesterol 37.531 1215828 2.360 1.615 Unfair Note: “NA” means “not applicable”.

[0186] (2) Injection volume 75 μl substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.602 4616 NA 1.281 flat QS-21 A isomer 10.043 199087 2.418 1.401 flat DOPC 36.286 1022350 26.755 1.253 Unfair cholesterol 37.513 1789017 3.285 1.732 Unfair Note: “NA” means “not applicable”.

[0187] (3) Injection volume 100 μl substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.600 6029 NA 1.198 flat QS-21 A isomer 10.041 270223 2.404 1.368 flat DOPC 36.442 1386046 25.023 0.911 Unfair cholesterol 37.531 2347518 2.489 1.778 Unfair Note: “NA” means “not applicable”.

[0188] like Figure 5 As shown, when the injection volume is 50 μL, the tailing factor of the DOPC retention peak is greater than 1.8, which does not meet the requirements.

[0189] Example 5: Study flow rate 1) Preparation of PADRE-gE-P2 fusion protein and detection of protein concentration were the same as in Example 1.

[0190] 2) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0191] 3) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0192] 4) Vaccine sample preparation is the same as in Example 1.

[0193] 5) Study flow rate 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0194] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0195] 2. Preparation of test samples is the same as in Example 1.

[0196] 3. Chromatographic column, instrument parameters and elution procedure 4. Results Analysis The liquid chromatography method used to control the quality of drugs has the following characteristics: the substance to be tested must have a retention peak, and the separation degree of the retention peaks of each substance must be greater than 1.5, the tailing factor must be 0.8-1.8, and the baseline near the retention peak must be horizontal (parallel to the X-axis of the spectrum).

[0197] (1) Flow rate 1.0 ml / min substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 10.797 5660 NA 1.239 flat QS-21 A isomer 11.362 409708 2.725 No data released flat DOPC 38.691 2431044 19.674 0.660 Unfair cholesterol 40.504 3567625 2.320 2.210 flat Note: “NA” means “not applicable”; “data not available” means “data not calculated by the liquid chromatography analysis software”.

[0198] (2) Flow rate 1.5 ml / min substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.611 4898 NA 1.309 flat QS-21 A isomer 10.060 275281 2.441 1.390 flat DOPC 36.484 1380402 25.400 0.916 Unfair cholesterol 37.555 2373898 2.661 1.759 Unfair Note: “NA” means “not applicable”.

[0199] (3) Flow rate 2.0 ml / min substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 8.977 3302 NA 1.049 flat QS-21 A isomer 9.395 197910 2.587 1.290 flat DOPC 35.187 1061516 25.622 1.185 flat cholesterol 36.011 1735962 2.199 1.494 Unfair Note: “NA” means “not applicable”.

[0200] like Figure 6 As shown in the figure, when the flow rate is 1.0 ml / min, the QS-21 A isomer retention peak and the PADRE-gE-P2 fusion protein retention peak cannot be completely separated, which does not meet the requirements.

[0201] like Figure 7 As shown, when the flow rate is 1.0 ml / min, the tailing factor of the DOPC retention peak is less than 0.8; when the flow rate is 1.0 ml / min, the tailing factor of the cholesterol retention peak is greater than 1.8, which does not meet the requirements.

[0202] Example 6: Study on the duration of elution of lipid dioleoylphosphatidylcholine and cholesterol 1) Preparation of PADRE-gE-P2 fusion protein and detection of protein concentration were the same as in Example 1.

[0203] 2) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0204] 3) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0205] 4) Vaccine sample preparation is the same as in Example 1.

[0206] 5) Study the elution time of dioleoylphosphatidylcholine (DOPC) and cholesterol 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0207] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0208] 2. Preparation of test samples is the same as in Example 1.

[0209] 3. Chromatographic column, instrument parameters and elution procedure (1) Chromatographic column and instrument parameters (2) Elution procedure 1: 95% acetonitrile concentration (95% mobile phase B) isocratic elution for 2 minutes to elute DOPC and cholesterol (3) Elution procedure 2: 95% acetonitrile concentration (95% mobile phase B) isocratic elution for 6 minutes to elute DOPC and cholesterol (4) Elution procedure 3: 95% acetonitrile concentration (95% mobile phase B) isocratic elution for 10 min to elute DOPC and cholesterol 4. Results Analysis The liquid chromatography method used to control the quality of drugs has the following characteristics: the substance to be tested must have a retention peak, and the separation degree of the retention peaks of each substance must be greater than 1.5, the tailing factor must be 0.8-1.8, and the baseline near the retention peak must be horizontal (parallel to the X-axis of the spectrum).

[0210] (1) Elution procedure 1: Isocratic elution at 95% acetonitrile for 2 minutes to elute DOPC and cholesterol substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.617 5949 NA 1.364 flat QS-21 A isomer 10.067 271947 2.488 1.303 flat DOPC 36.461 1472846 23.913 0.890 Unfair cholesterol 37.565 2391725 2.486 1.782 Unfair Note: “NA” means “not applicable”.

[0211] (2) Elution procedure 2: Isocratic elution at 95% acetonitrile for 6 minutes to elute DOPC and cholesterol substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.590 4829 NA 1.247 flat QS-21 A isomer 10.035 278522 2.315 1.362 flat DOPC 36.389 1559581 24.288 1.272 flat cholesterol 37.976 2451320 3.040 1.724 flat Note: “NA” means “not applicable”.

[0212] (3) Elution procedure 3: Isocratic elution at 95% acetonitrile for 10 min to elute DOPC and cholesterol substance Retention time (min) Peak area (μAU*min) Separation (from front peak) tailing factor Peak baseline QS-21 B isomer 9.604 4228 NA 1.156 flat QS-21 A isomer 10.048 266858 2.194 1.390 flat DOPC 38.020 1464766 20.647 0.638 Unfair cholesterol 40.221 2481466 2.797 2.944 flat Note: “NA” means “not applicable”.

[0213] like Figure 8 As shown, when the elution time of dioleoylphosphatidylcholine (DOPC) and cholesterol is 2 minutes, the baseline is not flat.

[0214] Example 7: Simultaneous Detection (One-Stop Detection) of the Contents of the Immunopotentiator Quillaja Saponin QS-21, the Lipid Dioleoylphosphatidylcholine (DOPC), and Cholesterol in Vaccine Samples 1) Preparation of PADRE-gE-P2 fusion protein and detection of protein concentration were the same as in Example 1.

[0215] 2) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0216] 3) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0217] 4) Vaccine sample preparation is the same as in Example 1.

[0218] 5) Detection of the content of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccine samples 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0219] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0220] 2. Sample Testing S1. Preparation of calibration standard solution sample of the immunopotentiator Quillaja saponin QS-21 Pipette 150 μl of the reference stock solution of QS-21 (5 mg / ml) into a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, and mix to obtain a solution with a concentration of 1 mg / ml; pipette 0.5 ml of the above solution into a 5 ml volumetric flask, dilute to the mark with 1% DMSO solution, and shake to obtain a solution with a concentration of 100 μg / ml; then pipette 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution into different injection vials, and then add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of 1% DMSO solution, cover the vials, and mix to obtain concentrations of 87.5 μg / ml, 750 μl, 625 μl, 375 μl, and 250 μl, respectively. Calibration standard solutions of 1 μg / ml, 75 μg / ml, 62.5 μg / ml, 50 μg / ml, 37.5 μg / ml, and 25 μg / ml.

[0221] S2. Preparation of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples Pipette 3 ml of liposome reference solution (DOPC and cholesterol concentrations of 4.20 mg / ml and 1.05 mg / ml, respectively) and dissolve it in 1.2 ml of 3.5% DMSO, mix well, and shake well to obtain solutions with DOPC and cholesterol concentrations of 3 mg / ml and 0.75 mg / ml; then pipette 0.5 ml, 0.5 ml, 0.5 ml, 0.3 ml, and 0.2 ml of the above solution into different injection vials, and then add 0.5 ml, 0.7 ml, 1.0 ml, 0.9 ml, and 1.0 ml of 1% DMSO solution, cover 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, and 0.375 mg / ml, 0.3125 mg / ml, 0.25 mg / ml, 0.1875 mg / ml, and 0.125 mg / ml, respectively.

[0222] S3. Preparation of test samples Pipette 0.5 ml of the vaccine sample solution to be tested into a sampling vial, add 0.5 ml of 2% DMSO solution, cover the vial cap, and mix well.

[0223] S4. Load the sample into the liquid chromatograph and run the analysis method The injection vials containing the calibration standard solution of Quillaja saponin QS-21, the injection vials containing the calibration standard solution of dioleoylphosphatidylcholine (DOPC) and cholesterol, and the injection vials containing the test samples were loaded onto the injection tray of the high performance liquid chromatography, and the analytical method was run using the liquid chromatography. S5. Calculate the content of the immune enhancer Quillaja saponin QS-21, the lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine 1. Establishment of the standard curve The peak area values of the calibration standard solutions of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol were used to establish calibration standard curves in which the concentration of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol were proportional to the peak area.

[0224] (1) Sample test results and standard curve of the calibration standard solution of Quillaja saponin QS-21, such as Figure 9 shown QS-21 Calibration Standard Solution Peak area (A+B isomer) (μAU*min) Tailing factor (A isomer) Tailing factor (B isomer) Calibration standard solution-1 (25 μg / ml) 145662 1.500 1.267 Calibration standard solution-2 (37.5 μg / ml) 218382 1.420 1.224 Calibration standard solution-3 (50 μg / ml) 283212 1.427 1.231 Calibration standard solution-4 (62.5 μg / ml) 362991 1.450 1.275 Calibration standard solution-5 (75 μg / ml) 433930 1.438 1.274 Calibration standard solution-6 (87.5 μg / ml) 506752 1.414 1.291 Standard curve of peak area of Quillaja saponin QS-21: y=5781.4x-371.7, R 2 =0.9995.

[0225] (2) Sample test results and standard curves of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solutions, such as Figure 10 、 11 shown DOPC calibration standard solution Peak area (μAU*min) tailing factor 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 DOPC peak area standard curve: y = 1743.3x - 38258, R 2 =0.9999. Cholesterol calibration standard solution Peak area (μAU*min) tailing factor 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 Cholesterol peak area standard curve: y=9772.1x+141898, R2=0.9998.

[0226] 2. Content Calculation Substitute the peak area values of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the vaccine sample into the calibration standard curve formula of the corresponding components, and then multiply them by the dilution factor of the vaccine (two times) to calculate the content of QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the vaccine. The chromatogram of the vaccine sample is shown in Figure 2. Figure 12 As shown in the figure, the retention peak and the front of each substance are completely separated, which meets the requirements. 6) Comparison of the differences in the content of the immunopotentiator Quillaja saponin QS-21, the lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples detected by simultaneous testing (single test) and existing technology testing (two separate tests) 1. Vaccine Sample Testing (Existing Technology) A method for testing the contents of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccine samples twice.

[0227] (1) Detection of the content of Quillaja saponin QS-21 The same as the “Concentration detection of Quillaja saponin QS-21 solution” in the item “3) Preparation and concentration detection of Quillaja saponin QS-21 solution” in Example 1.

[0228] (2) Detection of dioleoylphosphatidylcholine (DOPC) and cholesterol content The same as the "Detection of Dioleoylphosphatidylcholine (DOPC) and Cholesterol Concentrations" in the item "2) Liposome Preparation and Detection of Dioleoylphosphatidylcholine (DOPC) and Cholesterol Concentrations" in Example 1.

[0229] Second, the differences in the contents of the immune enhancer Quillaja saponin QS-21, lipid dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccine samples were compared between simultaneous detection (one detection) and prior art detection (two separate detections). Figure 13 shown. Example 8. Simultaneous Detection (1 Test) of the Contents of the Immunopotentiator Quillaja Saponin QS-21, the Lipid Dioleoylphosphatidylcholine (DOPC), and Cholesterol in the XA-401 Composite Adjuvant (Containing the Immunopotentiator Quillaja Saponin QS-21) 1) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0230] 2) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0231] 3) Preparation of XA-401 composite adjuvant samples (QS-21 concentration: 100 μg / ml; dioleoylphosphatidylcholine and cholesterol concentrations: 2 mg / ml and 0.5 mg / ml, respectively) Detection of liposome solution concentration: The concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; Detection of Quillaja saponin QS-21 solution concentration: The concentration of Quillaja saponin QS-21 was 4.14 mg / ml; Take 4.76 ml of liposomes, add 0.24 ml of Quillaja saponin QS-21 solution, and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir evenly to obtain XA-401 adjuvant.

[0232] 4) Detection of the contents of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in XA-401 composite adjuvant samples 1. Solution preparation (1) Preparation of mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0233] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0234] 2. Sample Testing S1. Preparation of calibration standard solution sample of the immunopotentiator Quillaja saponin QS-21 Same as Implementation Case 7.

[0235] S2. Preparation of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples Same as Implementation Case 7.

[0236] S3. Preparation of test samples Pipette 0.5 ml of the sample solution to be tested into a sampling vial, add 0.5 ml of 2% DMSO solution, cover the vial cap, and mix well.

[0237] S4. Load the sample into the liquid chromatograph and run the analysis method Same as Implementation Case 7.

[0238] S5. Calculate the content of the immunopotentiator Quillaja saponin QS-21, the lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in the XA-401 composite adjuvant. (1) Establishment of standard curve Same as Implementation Case 7.

[0239] (2) Content calculation The peak area values of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the sample were respectively substituted into the calibration standard curve formula of the corresponding components, and then multiplied by the dilution factor of the sample (twice) to calculate the contents of QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the XA-401 composite adjuvant. like Figure 14 , the retention peak and the front of each substance are completely separated, which meets the requirements; Figure 15 , the detection value of each substance is close to the theoretical value and meets the requirements.

[0240] Example 9: Simultaneous Detection (1 Test) of the Contents of the Immunopotentiator Quillaja Saponin QS-21, the Lipid Dioleoylphosphatidylcholine (DOPC), and Cholesterol in Composite Adjuvant 1 (Containing the Immunopotentiators Quillaja Saponin QS-21 and CPG1018) 1) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0241] 2) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0242] 3) Preparation of composite adjuvant 1 sample (QS-21 concentration: 100 μg / ml; CPG1018 concentration: 6 mg / ml; dioleoylphosphatidylcholine and cholesterol concentrations: 2 mg / ml and 0.5 mg / ml, respectively) Detection of liposome solution concentration: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; detection of Quillaja saponin QS-21 solution concentration: the concentration of Quillaja saponin QS-21 was 4.14 mg / ml; take 4.76 ml of liposomes, add 0.24 ml of Quillaja saponin QS-21 solution, then add 1.97 ml of CPG1018 solution (the detection CPG1018 concentration was 30.50 mg / ml), and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir evenly to obtain composite adjuvant 1.

[0243] 4) Detection of the contents of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in composite adjuvant 1 samples 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0244] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0245] 2. Sample Testing S1. Preparation of calibration standard solution sample of the immunopotentiator Quillaja saponin QS-21 Same as Implementation Case 7.

[0246] S2. Preparation of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples Same as Implementation Case 7.

[0247] S3. Preparation of test samples Same as Implementation Case 8.

[0248] S4. Load the sample into the liquid chromatograph and run the analysis method Same as Implementation Case 7.

[0249] S5. Calculate the content of the immunopotentiator Quillaja saponin QS-21, lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in composite adjuvant 1 (1) Establishment of standard curve Same as Implementation Case 7.

[0250] (2) Content calculation The peak area values of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the sample were respectively substituted into the calibration standard curve formula of the corresponding components, and then multiplied by the dilution factor of the sample (twice), thereby calculating the contents of QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in composite adjuvant 1. like Figure 16 , the retention peak and the front of each substance are completely separated, which meets the requirements; Figure 17 , the detection value of each substance is close to the theoretical value and meets the requirements.

[0251] Example 10: Simultaneous Detection (1 Test) of the Contents of the Immunopotentiator Quillaja Saponin QS-21, the Lipid Dioleoylphosphatidylcholine (DOPC), and Cholesterol in Composite Adjuvant 2 (Containing the Immunopotentiator Quillaja Saponin QS-21 and MPL) 1) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0252] 2) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0253] 3) Preparation of composite adjuvant 2 sample (QS-21 concentration: 100 μg / ml; MPL concentration: 100 μg / ml; dioleoylphosphatidylcholine and cholesterol concentrations: 2 mg / ml and 0.5 mg / ml, respectively) Detection of liposome solution concentration: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; detection of Quillaja saponin QS-21 solution concentration: the concentration of Quillaja saponin QS-21 was 4.14 mg / ml; take 4.76 ml of liposomes, add 0.24 ml of Quillaja saponin QS-21 solution, then add 1 ml of MPL solution (concentration of 1 mg / ml), and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir evenly to obtain composite adjuvant 2.

[0254] 4) Detection of the contents of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in composite adjuvant 2 samples 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0255] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0256] 2. Sample Testing S1. Preparation of calibration standard solution sample of the immunopotentiator Quillaja saponin QS-21 Same as Implementation Case 7.

[0257] S2. Preparation of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples Same as Implementation Case 7.

[0258] S3. Preparation of test samples Same as Implementation Case 8.

[0259] S4. Load the sample into the liquid chromatograph and run the analysis method Same as Implementation Case 7.

[0260] S5. Calculate the content of the immunopotentiator Quillaja saponin QS-21, the lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in composite adjuvant 2. (1) Establishment of standard curve Same as Implementation Case 7.

[0261] (2) Content calculation The peak area values of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the sample were respectively substituted into the calibration standard curve formula of the corresponding components, and then multiplied by the dilution factor of the sample (twice), thereby calculating the contents of QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in composite adjuvant 2. like Figure 18 , the retention peak and the front of each substance are completely separated, which meets the requirements; Figure 19 , the detection value of each substance is close to the theoretical value and meets the requirements.

[0262] Example 11: Simultaneous Detection (1 Test) of the Contents of the Immunopotentiator Quillaja Saponin QS-21, the Lipid Dioleoylphosphatidylcholine (DOPC), and Cholesterol in Composite Adjuvant 3 (Containing the Immunopotentiator Quillaja Saponin QS-21 and Poly I: C) 1) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0263] 2) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0264] 3) Preparation of Composite Adjuvant 3 Sample (QS-21 concentration: 100 μg / ml; Poly I:C concentration: 800 μg / ml; Dioleoylphosphatidylcholine and Cholesterol concentrations: 2 mg / ml and 0.5 mg / ml, respectively) Detection of liposome solution concentration: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; detection of Quillaja saponin QS-21 solution concentration: the concentration of Quillaja saponin QS-21 was 4.14 mg / ml; take 4.76 ml of liposomes, add 0.24 ml of Quillaja saponin QS-21 solution, then add 1.6 ml of Poly I:C solution (concentration of 5 mg / ml; stabilizer: kanamycin), and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml. Stir well to obtain composite adjuvant 3.

[0265] 4) Detection of the contents of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in composite adjuvant 3 samples 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0266] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0267] 2. Sample Testing S1. Preparation of calibration standard solution sample of the immunopotentiator Quillaja saponin QS-21 Same as Implementation Case 7.

[0268] S2. Preparation of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples Same as Implementation Case 7.

[0269] S3. Preparation of test samples Same as Implementation Case 8.

[0270] S4. Load the sample into the liquid chromatograph and run the analysis method Same as Implementation Case 7.

[0271] S5. Calculate the content of the immunopotentiator Quillaja saponin QS-21, lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in composite adjuvant 3 (1) Establishment of standard curve Same as Implementation Case 7.

[0272] (2) Content calculation The peak area values of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the sample were respectively substituted into the calibration standard curve formula of the corresponding components, and then multiplied by the dilution factor of the sample (twice), thereby calculating the contents of QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in composite adjuvant 3. like Figure 20 , the retention peak and the front of each substance are completely separated, which meets the requirements; Figure 21 , the detection value of each substance is close to the theoretical value and meets the requirements.

[0273] Example 12: Simultaneous Detection (1 Test) of the Contents of the Immunopotentiators Quillaja Saponins QS-7 and QS-21, the Lipid Dioleoylphosphatidylcholine (DOPC), and Cholesterol in Composite Adjuvant 4 (Containing the Immunopotentiators Quillaja Saponins QS-7 and QS-21) 1) Liposome preparation and dioleoylphosphatidylcholine and cholesterol concentration detection were the same as in Example 1.

[0274] 2) Preparation of Quillaja saponin QS-21 solution and concentration detection were the same as in Example 1.

[0275] 3) Preparation of composite adjuvant 4 sample (QS-21 concentration: 25 μg / ml; QS-7 concentration: 75 μg / ml; dioleoylphosphatidylcholine and cholesterol concentrations: 1 mg / ml and 0.25 mg / ml, respectively) Detection of liposome solution concentration: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; detection of Quillaja saponin QS-21 solution concentration: the concentration of Quillaja saponin QS-21 was 4.14 mg / ml; take 2.38 ml of liposomes, add 0.06 ml of Quillaja saponin QS-21 solution, then add 0.75 ml of QS-7 solution (1 mg / ml), and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir evenly to obtain composite adjuvant 4.

[0276] 4) Detection of the contents of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in composite adjuvant 4 samples 1. Solution preparation (1) Preparation of mobile phase (trifluoroacetic acid) 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 ultrasonically degas for 5 minutes.

[0277] 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 degas by ultrasonication for 5 minutes.

[0278] Mobile phase C [methanol:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of methanol, add 0.5 ml of trifluoroacetic acid solution, shake well, and degas by ultrasonication for 5 minutes.

[0279] (2) The sample dilution solution was prepared in the same manner as in Example 1.

[0280] 2. Sample Testing S1. Preparation of calibration standard solution sample of the immunopotentiator Quillaja saponin QS-21 Same as Implementation Case 7.

[0281] S2. Preparation of lipid dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples Same as Implementation Case 7.

[0282] S3. Preparation of test samples Same as Implementation Case 8.

[0283] S4. Load the sample into the liquid chromatograph and run the analysis method The injection vials containing the calibration standard solution of Quillaja saponin QS-21, the injection vials containing the calibration standard solution of dioleoylphosphatidylcholine (DOPC) and cholesterol, and the injection vials containing the test samples were loaded onto the injection tray of the high performance liquid chromatography, and the analytical method was run using the liquid chromatography. S5. Calculate the content of the immunopotentiators Quillaja saponin QS-21, Quillaja saponin QS-7, lipid dioleoylphosphatidylcholine (DOPC), and cholesterol in composite adjuvant 4. (1) Establishment of standard curve The peak area values of the calibration standard solutions of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol were used to establish calibration standard curves in which the concentration of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol were proportional to the peak area.

[0284] (1) Sample test results and standard curve of the calibration standard solution of Quillaja saponin QS-21, such as Figure 22 shown QS-21 Calibration Standard Solution Peak area (A+B isomer) (μAU*min) Tailing factor (A isomer) Tailing factor (B isomer) Calibration standard solution-1 (25 μg / ml) 139433 1.295 1.318 Calibration standard solution-2 (37.5 μg / ml) 211179 1.278 1.274 Calibration standard solution-3 (50 μg / ml) 282908 1.273 1.221 Calibration standard solution-4 (62.5 μg / ml) 351289 1.262 1.309 Calibration standard solution-5 (75 μg / ml) 424265 1.255 1.282 Calibration standard solution-6 (87.5 μg / ml) 493667 1.247 1.244 Standard curve of peak area of Quillaja saponin QS-21: y=5665.8x-1580.5, R 2 =0.9999.

[0285] (2) Sample test results and standard curves of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solutions, such as Figure 23 、 24 shown DOPC calibration standard solution Peak area (μAU*min) tailing factor Standard curve solution-1 (500 μg / ml) 550039 1.759 Standard curve solution-2 (750 μg / ml) 820342 1.767 Standard curve solution-3 (1000 μg / ml) 1086222 1.789 Standard curve solution-4 (1250 μg / ml) 1346325 1.788 Standard curve solution-5 (1500 μg / ml) 1615030 1.798 DOPC peak area standard curve: y = 1062.4x + 21206, R 2 =1. Cholesterol calibration standard solution Peak area (μAU*min) tailing factor Standard curve solution-1 (125 μg / ml) 970572 1.107 Standard curve solution-2 (187.5 μg / ml) 1426879 1.090 Standard curve solution-3 (250 μg / ml) 1858278 1.069 Standard curve solution-4 (312.5 μg / ml) 2266198 1.052 Standard curve solution-5 (375 μg / ml) 2679431 1.034 Cholesterol peak area standard curve: y=6811.3x+137457, R 2 =0.9995.

[0286] (2) Content calculation The peak area values of Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the sample were respectively substituted into the calibration standard curve formula of the corresponding components, and then multiplied by the dilution factor of the sample (twice), thereby calculating the contents of QS-21, Quillaja saponin QS-7, dioleoylphosphatidylcholine (DOPC) and cholesterol in composite adjuvant 4. like Figure 25 , the retention peak and the front of each substance are completely separated, which meets the requirements; Figure 26 , the detection value of each substance is close to the theoretical value and meets the requirements.

[0287] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant, characterized in that: The following steps are involved: S1. Prepare saponin immunopotentiator calibration standard solution sample; S2, preparing lipid calibration standard solution samples; S3, preparing test samples; S4, loading the sample into the liquid chromatograph and running the analytical method; S5. Calculate the content of saponin immunopotentiators and lipids.

2. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The composite adjuvant contains an immunopotentiator and liposomes; the immunopotentiator contains a saponin immunopotentiator or contains a saponin immunopotentiator, MPL and / or CPG ODN and / or poly I:C immunopotentiator; the saponin immunopotentiator is Quillaja saponin QS-21 and / or Quillaja saponin QS-7; the liposomes include but are not limited to dioleoylphosphatidylcholine and cholesterol; the product containing the composite adjuvant includes but is not limited to vaccines.

3. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The preparation of the saponin immunopotentiator calibration standard solution sample in S1 includes preparing a QS-21 and / or QS-7 calibration standard solution sample; the concentration of QS-21 and / or QS-7 in the QS-21 and / or QS-7 calibration standard solution sample is 10 μg / ml to 100 μg / ml.

4. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The preparation of lipid calibration standard solution samples in S2 includes preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples; the concentrations of the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples are 200μg / ml to 2000μg / ml and 50μg / ml to 500μg / ml, respectively.

5. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: In the test sample in S3, the concentration of QS-21 and / or QS-7 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.

6. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The chromatographic column of the analysis method in S4 contains an alkyl or phenyl reverse phase chromatographic filler, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water, and the mobile phase may contain methanol; the liquid chromatograph parameters of the analysis method in S4 include but are not limited to: column temperature, injection volume, flow rate, and detector parameters; the column temperature is 40°C to 60°C; the injection volume is 1μl to 100μl; the flow rate is 0.1 ml / min to 2.5 ml / min; when the detector is a UV detector, the detection wavelength is 200 to 400nm.

7. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The elution procedure of the analytical method in S4 at least includes: an elution method for eluting the saponin immunopotentiator retention peak and an elution method for eluting the lipid retention peak; the elution method for eluting the saponin immunopotentiator retention peak is isocratic elution or gradient elution, the acetonitrile concentration gradient in the mobile phase is 5% to 95%, the gradient elution time is 2 minutes to 1920 minutes, and the trifluoroacetic acid concentration in the mobile phase is 0.01% to 0.10%.

8. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 7, characterized in that: The elution method for eluting the lipid retention peak is isocratic elution or gradient elution, the mobile phase contains acetonitrile or methanol with a concentration of 90% to 100%, the elution time is 2 minutes to 1920 minutes, and the concentration of trifluoroacetic acid in the mobile phase is 0.01% to 0.10%.

9. The method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The calculation of the content of saponin immunopotentiators and lipids in S5 includes two calculation methods: Calculation method 1: First, establish a calibration standard curve in which the concentration of saponin immunopotentiators and lipids is proportional to the peak area. Then, substitute the values of the peak area of saponin immunopotentiators and lipids into the corresponding calibration standard curve formula to calculate the concentration of saponin immunopotentiators and lipids in the test sample. Then, multiply the concentration of saponin immunopotentiators and lipids by the dilution factor to calculate the concentration of saponin immunopotentiators and lipids in the composite adjuvant / product containing the composite adjuvant. Calculation method 2: The calculation formula is A concentration = test sample A peak area * A calibration standard solution sample concentration * dilution factor / A calibration standard solution sample peak area, where A represents "saponin immune enhancer" or "lipid".

10. Use of the method for simultaneously detecting the content of saponin immunopotentiators and lipids in a composite adjuvant / a product containing a composite adjuvant according to any one of claims 1 to 9 in detecting the content of Quillaja saponin QS-21 and / or Quillaja saponin QS-7 and / or the lipid dioleoylphosphatidylcholine and / or the lipid cholesterol.

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