Method for simultaneously detecting content of main components of adjuvant in composite adjuvant / product containing composite adjuvant and application

Abstract

The invention discloses a method for simultaneously detecting the content of main components of an adjuvant in a composite adjuvant / a product containing the composite adjuvant and application of the method. The method comprises the following steps: preparing a saponin QS-21 and / or saponin QS-7 calibration standard solution sample; preparing a dioleoyl phosphatidylcholine and cholesterol 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 main components of the adjuvant. The method solves the technical problem that at least two different methods are needed for detecting the content of main components of the adjuvant in the composite adjuvant for at least two times in the prior art. According to the high performance liquid chromatography disclosed by the invention, the contents of the saponin QS-21 and / or the saponin QS-7, the dioleoyl phosphatidylcholine and the cholesterol 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 content of main adjuvant components in a composite adjuvant / a product containing the composite adjuvant. Background Art

[0002] Quillaja saponaria (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. It 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 similar 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 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 the lipid dioleoylphosphatidylcholine and lipid cholesterol) are used together with saponin immunopotentiators QS-21 and / or QS-7 in vaccines, primarily to eliminate the hemolytic activity of QS-21 and / or 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 Shingrix, which contains the saponin immunopotentiator QS-21. ® Recombinant shingles vaccine (GSK), Arexvy ® Recombinant respiratory syncytial virus vaccine (GSK), Mosquirix (RTS,S / AS01 ®) recombinant malaria vaccine (GSK), NVX-CoV2373 recombinant COVID-19 vaccine containing saponin immunopotentiators QS-21 and QS-7 (Novavax), R21 / Matrix-M TM Recombinant malaria vaccine (Oxford University and Serum Institute of India).

[0006] In addition, more vaccines containing saponin immunopotentiators QS-21 and / or QS-7 in combination with 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 CN117003896B, 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 the main components of adjuvants in a composite adjuvant / a product containing a composite adjuvant, so as to solve the technical problems in the prior art of detecting the content of the main components of adjuvants, such as being 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 main adjuvant components in a composite adjuvant / a product containing the composite adjuvant, wherein the method adopts a liquid chromatography method. The composite adjuvant contains a saponin immunopotentiator and a lipid; the saponin immunopotentiator includes Quillaja saponin QS-21 and / or Quillaja saponin QS-7, and the lipid includes dioleoylphosphatidylcholine and cholesterol.

[0010] The saponin immunopotentiator may also contain saponin components with immunopotentiating effects, such as Quillaja saponin QS-17 and / or Quillaja saponin QS-18.

[0011] The composite adjuvant may also contain immunopotentiators such as MPL (monophosphoryl lipid A) and / or CPG ODN (artificially synthesized oligodeoxynucleotide sequence containing non-methylated cytosine guanine dinucleotide) and / or poly I:C (polyinosinic-polycytidylic acid).

[0012] The lipids may also contain lipid substances such as distearoylphosphatidylcholine (DSPC), dioleoyl-trimethylammonium chloride (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), ALC-0315, D-Lin-MC3-DMA, SM102, phosphatidylinositol (PI) and phosphatidylserine (PS).

[0013] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra high performance liquid chromatography.

[0014] 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.

[0015] 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).

[0016] Furthermore, the liquid chromatography method is a liquid chromatography internal standard method or a liquid chromatography external standard method.

[0017] Furthermore, the liquid chromatography external standard method comprises the following steps: S1. preparing a calibration standard solution sample of Quillaja saponin QS-21 and / or Quillaja saponin QS-7; S2. preparing dioleoylphosphatidylcholine and cholesterol 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 the main components of the adjuvant.

[0018] 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.

[0019] Furthermore, the QS-21 and / or QS-7 calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0020] Furthermore, the preparation of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples in S2 is characterized in that the preparation method is selected from any one of the following two 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).

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Furthermore, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples may contain DMSO at a concentration of 0.5% to 5%.

[0026] Furthermore, the test sample prepared in S3 can be prepared using a product containing a composite adjuvant or a composite adjuvant.

[0027] Furthermore, a product containing a composite adjuvant is used to prepare the test sample, wherein the protein or nucleic acid content is 10 μg / ml to 400 μg / ml, the dioleoylphosphatidylcholine content is 400 μg / ml to 8000 μg / ml, the cholesterol content is 100 μg / ml to 2000 μg / ml, and the immunopotentiator content is 20 μg / ml to 7000 μg / ml.

[0028] 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.

[0029] Furthermore, the composite adjuvant may contain distearoylphosphatidylcholine (DSPC) in an amount of 400 μg / ml to 2000 μg / ml; the composite adjuvant 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 composite adjuvant 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.

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

[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 is characterized in that: a palindromic sequence of CpG dinucleotides is the core, with poly G tails at both ends, and the phosphodiester bond backbone is partially thiolated, and a higher-order structure is 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 in that: a fully thiolated linear CpG ODN has strong immunostimulatory activity on B cells, but cannot activate plasmacytoid dendritic cells, such as: CpG 1018, CpG2006, and CpG 1826; the Class C CpG ODN is characterized in that: a fully thiolated CpG ODN can form a dimer 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 CpG 2395.

[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 dioleoylphosphatidylcholine content of 400 μg / ml to 8000 μg / ml, a cholesterol content of 100 μg / ml to 2000 μg / ml, and an immunopotentiator content of 20 μg / ml to 7000 μg / ml.

[0035] Furthermore, the composite adjuvant may contain distearoylphosphatidylcholine (DSPC) in an amount of 400 μg / ml to 2000 μg / ml; the composite adjuvant 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 composite adjuvant 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 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 3D-MPL and / or CPG ODN and / or poly I:C in an amount of 10 μg / ml to 6000 μg / ml.

[0037] Furthermore, in the test sample in S3, the concentration of the immunopotentiator is 10 μg / ml to 3500 μg / ml, the concentration of dioleoylphosphatidylcholine is 200 μg / ml to 4000 μg / ml, and the concentration of cholesterol is 50 μg / ml to 1000 μg / ml.

[0038] Furthermore, the test sample may contain distearoylphosphatidylcholine (DSPC) in an amount of 200 μg / ml to 1000 μg / ml; the test sample 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 test sample 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.

[0039] 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 3D-MPL and / or CPG ODN and / or poly I:C in an amount of 5 μg / ml to 3000 μg / ml.

[0040] Furthermore, the test sample in S3 may contain DMSO at a concentration of 0.5% to 5%.

[0041] Furthermore, the detector of the liquid chromatograph in S4 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0042] Furthermore, the analysis method in S4 includes a chromatographic column, liquid chromatography parameters, and an elution procedure.

[0043] Furthermore, the chromatographic column used in the analysis method in S4 is a reverse phase chromatographic column.

[0044] Furthermore, the liquid chromatography parameters of the analysis method in S4 include column temperature, injection volume, flow rate, detector parameters, etc.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Furthermore, the elution method for eluting the retained peak of the immunopotentiator is gradient elution and / or isocratic elution.

[0055] Furthermore, the elution method for eluting the lipid retention peak is gradient elution and / or isocratic elution.

[0056] 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 3D-MPL and / or CPGODN and / or poly I:C.

[0057] Furthermore, the sub-elution method for eluting the retained peak of Quillaja saponin QS-21 and / or QS-7 may also be able to elute the retained peak of proteins in the composite adjuvant product.

[0058] 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%.

[0059] Furthermore, the elution method for eluting the lipid retention peak contains at least sub-elution methods for eluting the dioleoylphosphatidylcholine and cholesterol retention peaks.

[0060] Furthermore, the sub-elution method for eluting the dioleoylphosphatidylcholine and cholesterol retention peaks 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.

[0061] Furthermore, subelution methods that elute the retained peaks of dioleoylphosphatidylcholine and cholesterol may also elute cationic lipids (e.g., DOTAP) and / or anionic lipids and / or distearoylphosphatidylcholine.

[0062] Furthermore, the sub-elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0063] 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%.

[0064] 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%.

[0065] 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.

[0066] 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.

[0067] Further, a pre-equilibrium method is added before the sub-elution method for 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%.

[0068] 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.

[0069] 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.

[0070] 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%.

[0071] 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.

[0072] Further, a post-equilibrium 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 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%.

[0073] Furthermore, the calculation of the content of the main component of the adjuvant in S5 includes calculation method one and calculation method two.

[0074] Furthermore, the calculation method 1 is: first establish a calibration standard curve in which the concentration of Quillaja saponin QS-21 and / or QS-7, dioleoylphosphatidylcholine and cholesterol are proportional to the peak area, and then calculate the content (concentration) of the main component of the adjuvant.

[0075] Furthermore, calculation method 1: first establish a calibration standard curve in which the concentration of Quillaja saponin QS-21 and / or QS-7, dioleoylphosphatidylcholine and cholesterol is proportional to the peak area; then substitute the peak area values of the main adjuvant components of the sample into the calibration standard curve formula of the corresponding components to calculate the concentration of the corresponding components in the test sample; multiply the concentration of each component of the test sample by the dilution factor to calculate the content (concentration) of the main adjuvant component in the sample.

[0076] Furthermore, the second calculation method is to directly calculate the content (concentration) of the main components of the adjuvant in the sample.

[0077] 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 "Quillaja saponin QS-21" or "Quillaja saponin QS-7" or "dioleoylphosphatidylcholine" or "cholesterol".

[0078] The present invention provides a method and application for simultaneously detecting the content of main adjuvant components in a composite adjuvant / a product containing the composite adjuvant, wherein the product containing the composite adjuvant includes but is not limited to a vaccine.

[0079] 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.

[0080] 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 the main adjuvant components in a composite adjuvant or a product containing the composite adjuvant. This method addresses the technical problem of existing techniques requiring at least two separate methods to detect the content of saponin immunopotentiators and lipids. The high-performance liquid chromatography method disclosed in the present invention can detect the content of both saponin immunopotentiators and lipids in a product with only a single sample loading, significantly improving assay efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] 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.

[0082] Figure 1 This is a liquid chromatogram of Quillaja saponin QS-21 calibration standard solution-1 (25 μg / ml) in Example 1 of the present invention.

[0083] Figure 2 This is a liquid chromatogram of Quillaja saponin QS-21 calibration standard solution-1 (37.5 μg / ml) in Example 1 of the present invention.

[0084] Figure 3 This is a liquid chromatogram of Quillaja saponin QS-21 calibration standard solution-1 (50 μg / ml) in Example 1 of the present invention.

[0085] Figure 4 This is a liquid chromatogram of Quillaja saponin QS-21 calibration standard solution-1 (62.5 μg / ml) in Example 1 of the present invention.

[0086] Figure 5 This is a liquid chromatogram of Quillaja saponin QS-21 calibration standard solution-1 (75 μg / ml) in Example 1 of the present invention.

[0087] Figure 6 This is a liquid chromatogram of Quillaja saponin QS-21 calibration standard solution-1 (87.5 μg / ml) in Example 1 of the present invention.

[0088] Figure 7 This is a standard curve diagram of the concentration and peak area of Quillaja saponin QS-21 in Example 1 of the present invention.

[0089] Figure 8 This is a liquid chromatogram of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution-1 (the concentrations of dioleoylphosphatidylcholine and cholesterol are 500 μg / ml and 125 μg / ml, respectively) in Example 1 of the present invention.

[0090] Figure 9 This is a liquid chromatogram of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution-2 (the concentrations of dioleoylphosphatidylcholine and cholesterol are 750 μg / ml and 187.5 μg / ml, respectively) in Example 1 of the present invention.

[0091] Figure 10 This is a liquid chromatogram of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution-3 (the concentrations of dioleoylphosphatidylcholine and cholesterol are 1000 μg / ml and 250 μg / ml, respectively) in Example 1 of the present invention.

[0092] Figure 11 This is a liquid chromatogram of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution-4 (the concentrations of dioleoylphosphatidylcholine and cholesterol are 1250 μg / ml and 312.5 μg / ml, respectively) in Example 1 of the present invention.

[0093] Figure 12 This is a liquid chromatogram of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution-5 (the concentrations of dioleoylphosphatidylcholine and cholesterol are 1500 μg / ml and 375 μg / ml, respectively) in Example 1 of the present invention.

[0094] Figure 13 This is a standard curve diagram of dioleoylphosphatidylcholine (DOPC) concentration and peak area in Example 1 of the present invention.

[0095] Figure 14 This is a standard curve diagram of cholesterol concentration and peak area in Example 1 of the present invention.

[0096] Figure 15 This is a liquid chromatogram for simultaneously detecting the contents of Quillaja saponin QS-7, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the composite adjuvant according to Example 1 of the present invention.

[0097] Figure 16 This is an analysis chart of the results of simultaneous detection of the contents of Quillaja saponin QS-7, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the composite adjuvant in Example 1 of the present invention. DETAILED DESCRIPTION

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0104] Example 1. Simultaneous Detection (One-Stop Detection) of the Contents of Quillaja Saponin QS-7, Quillaja Saponin QS-21, Dioleoylphosphatidylcholine (DOPC), and Cholesterol in the Composite Adjuvant 1) 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.

[0105] 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.

[0106] (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.

[0107] 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.

[0108] 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.

[0109] 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].

[0110] (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.

[0111] (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.

[0112] 2) 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.

[0113] 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.

[0114] 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.

[0115] (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.

[0116] 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; 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.

[0117] (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.

[0118] (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.

[0119] 3) Preparation of composite adjuvant samples (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) Detect the concentration of the liposome solution: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; detect the concentration of the Quillaja saponin QS-21 solution: the concentration of the Quillaja saponin QS-21 was 4.14 mg / ml; take 2.38 ml of liposomes, add 0.06 ml of the Quillaja saponin QS-21 solution, then add 0.75 ml of the 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 the composite adjuvant.

[0120] 4) Detection of the contents of Quillaja saponin QS-21, Quillaja saponin QS-7, dioleoylphosphatidylcholine (DOPC) and cholesterol in the composite adjuvant 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.

[0121] 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.

[0122] 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.

[0123] (2) 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.

[0124] 1.25% dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, and mix well.

[0125] 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix well.

[0126] 3.5% dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, and mix well.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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 the composite adjuvant. (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.

[0131] The test results and standard curve of the sample of the calibration standard solution of Quillaja saponin QS-21 are as follows: Figures 1-6 As shown in the standard curve Figure 7 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.

[0132] The test results and standard curve of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples are shown in the liquid chromatogram. Figures 8-12 As shown in the standard curve Figure 13-14 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.

[0133] (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), so as to calculate the contents of QS-21, Quillaja saponin QS-7, dioleoylphosphatidylcholine (DOPC) and cholesterol in the composite adjuvant. like Figure 15 As shown in the figure, the retention peak and the front of each substance are completely separated, which meets the requirements.

[0134] like Figure 16 As shown, the content of each substance meets the quality control requirements.

[0135] 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 the main components of adjuvants in a composite adjuvant / a product containing a composite adjuvant, characterized in that: The following steps are involved: S1. preparing a calibration standard solution sample of Quillaja saponin QS-21 and / or Quillaja saponin QS-7; S2. preparing dioleoylphosphatidylcholine and cholesterol 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 the main components of the adjuvant.

2. The method for simultaneously detecting the content of the main components of the adjuvant in the composite adjuvant / composite adjuvant-containing product according to claim 1, characterized in that: The composite adjuvant contains a saponin immunopotentiator and liposomes; the saponin immunopotentiator is Quillaja saponin QS-21 and / or Quillaja saponin QS-7; the liposomes are composed of lipids, and the lipids are dioleoylphosphatidylcholine and cholesterol.

3. The method for simultaneously detecting the content of the main components of the adjuvant in the composite adjuvant / composite adjuvant-containing product 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.

4. The method for simultaneously detecting the content of the main components of the adjuvant in the composite adjuvant / composite adjuvant-containing product 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 retention peak of Quillaja saponin QS-21 and / or Quillaja saponin QS-7; an elution method for eluting the retention peak of dioleoylphosphatidylcholine and cholesterol; the elution method for eluting the retention peak of Quillaja saponin QS-21 and / or Quillaja saponin QS-7 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%.

5. The method for simultaneously detecting the content of the main components of adjuvants in a composite adjuvant / a product containing a composite adjuvant according to claim 4, characterized in that: The elution method for eluting dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution, the mobile phase contains acetonitrile or methanol at 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%.

6. The method for simultaneously detecting the content of the main components of adjuvants in a composite adjuvant / a product containing a composite adjuvant according to claim 1, characterized in that: The calculation of the content of the main components of the adjuvant in S5 includes two calculation methods: Calculation method 1: First, establish a calibration standard curve in which the concentration of Quillaja saponin QS-21 and / or Quillaja saponin QS-7, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area. Then, substitute the peak area values of Quillaja saponin QS-21 and / or Quillaja saponin QS-7, dioleoylphosphatidylcholine, and cholesterol into the corresponding calibration standard curve formula to calculate the concentration of the main adjuvant component in the test sample. Then, multiply the concentration of the main adjuvant component by the dilution factor to calculate the content (concentration) of the main adjuvant component 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 "Quillaja saponin QS-21" or "Quillaja saponin QS-7" or "dioleoylphosphatidylcholine" or "cholesterol".

7. Application of a method for simultaneously detecting the content of main adjuvant components in a composite adjuvant / a product containing a composite adjuvant, characterized in that: The products containing composite adjuvants include but are not limited to vaccines.

8. The use of the method for simultaneously detecting the content of the main components of adjuvants in composite adjuvants / composite adjuvant-containing products according to claim 7, characterized in that: It is used to detect the content of Quillaja saponin QS-21 and / or Quillaja saponin QS-7 and / or dioleoylphosphatidylcholine and / or cholesterol.

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