A method for detecting the content of protein and main components of complex adjuvant in recombinant varicella-zoster vaccine and application thereof

Abstract

The application discloses a method for detecting the content of protein and main components of composite adjuvant in recombinant varicella-zoster vaccine and application. The method comprises the following steps: preparing a protein calibration standard solution sample; preparing a QS-21 calibration standard solution sample; preparing a dioleoylphosphatidylcholine and cholesterol calibration standard solution sample; preparing a detection sample; loading the sample into a liquid chromatograph and running an analysis method; and calculating the content of protein and main components of adjuvant in the vaccine. The application is a high performance liquid chromatography method which can simultaneously detect the content of protein, dioleoylphosphatidylcholine, cholesterol and quillaja saponin QS-21 in the vaccine. The content of protein, dioleoylphosphatidylcholine, cholesterol and quillaja saponin QS-21 in the vaccine can be accurately detected only once, and the efficiency of vaccine detection is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method and application for detecting the content of protein and main components of compound adjuvant in recombinant varicella-zoster vaccine. Background Technology

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

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

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

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

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

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

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

[0009] The purpose of this invention is to provide a method and application for detecting the content of protein and adjuvant main components in recombinant varicella-zoster vaccine, so as to solve the problems of cumbersome, time-consuming, material-intensive, labor-intensive and inefficient methods for detecting components in vaccines in the prior art.

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

[0011] This invention provides a method for detecting the content of protein and compound adjuvant main components in recombinant varicella-zoster vaccine, wherein the method employs liquid chromatography.

[0012] The protein is a PADRE-gE-P2 fusion protein, comprising the extracellular region of varicella-zoster virus (VZV) glycoprotein E (gE), the universal DR Th epitope peptide PADRE (PADRE), and the amino acid sequence of tetanus toxin Th epitope P2 (P2). The gE, PADRE, and P2 are linked by a linker peptide, which is a GGS and / or GGGS and / or GGGGS and / or GGSSG linker peptide.

[0013] Furthermore, an optional molecular structure of the PADRE-gE-P2 fusion protein is (from N-terminus to C-terminus): PADRE-GSGSG (linker peptide)-gE(AA31-AA544)-GGS (linker peptide)-P2, and the amino acid sequence of the PADRE-gE-P2 fusion protein is shown in SEQ ID NO.1.

[0014] Furthermore, the compound adjuvant is XA-401 compound adjuvant, which is composed of neutral liposomes and the immune enhancer saponin QS-21. The main components of the neutral liposomes are dioleoylphosphatidylcholine and cholesterol.

[0015] Furthermore, the recombinant varicella-zoster vaccine contains 10 μg / ml to 400 μg / ml of protein, 400 μg / ml to 4000 μg / ml of dioleoylphosphatidylcholine, 100 μg / ml to 1000 μg / ml of cholesterol, and 20 μg / ml to 200 μg / ml of QS-21.

[0016] Furthermore, the recombinant varicella-zoster vaccine contains buffering components to maintain the stability of the vaccine's pH value and components to regulate the vaccine's osmotic pressure.

[0017] Furthermore, the buffering component for maintaining the stability of the vaccine's pH value is selected from any one or more of the following substances, but is not limited to: disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, histidine, histidine hydrochloride, glycine, hydrochloric acid, sodium hydroxide, sodium carbonate, potassium carbonate, sodium citrate, citric acid, succinic acid, sodium succinate, acetic acid, sodium acetate, tris(hydroxymethyl)aminomethane, and 4-hydroxyethylpiperazine ethanesulfonic acid.

[0018] Furthermore, the component for regulating vaccine osmotic pressure may be selected from any one or more of the following substances, but is not limited to: sodium chloride, sucrose, trehalose, glucose, mannitol, and sorbitol.

[0019] Furthermore, the recombinant varicella-zoster vaccine may also contain components that maintain the stability of proteins in the vaccine.

[0020] Furthermore, the component that maintains protein stability in the vaccine may be selected from any one or more of the following substances, but is not limited to: polysorbate 80, polysorbate 20, and poloxamer 188.

[0021] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra-high performance liquid chromatography.

[0022] Furthermore, the liquid chromatography method is reversed-phase liquid chromatography, wherein the chromatographic column contains alkyl or phenyl reversed-phase chromatographic packing material, and the mobile phase contains acetonitrile, methanol, trifluoroacetic acid, and water.

[0023] Furthermore, the alkyl reversed-phase chromatography packing material can be selected from any of the following chromatographic packing materials, but is not limited to: butylsilane-bonded silica gel (C4), hexaalkylsilane-bonded silica gel (C6), octaalkylsilane-bonded silica gel (C8), hexadecylsilane-bonded silica gel (C16), and octadecylsilane-bonded silica gel (C18).

[0024] Furthermore, the liquid chromatography method is either the internal standard method or the external standard method.

[0025] Furthermore, the liquid chromatography external standard method includes the following steps:

[0026] S1. Prepare protein calibration standard solution samples;

[0027] S2. Prepare QS-21 calibration standard solution samples;

[0028] S3. Prepare calibration standard solution samples of dioleoylphosphatidylcholine and cholesterol;

[0029] S4. Prepare the test sample;

[0030] S5. Load the sample into the liquid chromatograph and run the analytical method;

[0031] S6. Calculate the content of protein, QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine.

[0032] Furthermore, in S1, the protein calibration standard solution sample is prepared, and the protein used can be selected from any of the following, but is not limited to: bovine serum albumin (BSA), bovine gamma-globulin (BGG), varicella-zoster virus (VZV) glycoprotein E (gE) extracellular segment (gE), and PADRE-gE-P2 fusion protein.

[0033] Furthermore, the protein calibration standard solution sample in S1 has a protein concentration of 5 μg / ml to 200 μg / ml.

[0034] Furthermore, the protein calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0035] Furthermore, in step S2, the purity of the QS-21 standard solution sample used to prepare the QS-21 calibration standard solution sample is not less than 90%, and its preparation method can be selected from any of the following methods, but is not limited to the following methods: dissolving QS-21 powder (purity not less than 90%) in a buffer solution with a pH not less than 4 to prepare a QS-21 standard solution sample; and detecting the concentration of the QS-21 solution (purity not less than 90%) using liquid chromatography to prepare a QS-21 standard solution sample.

[0036] Furthermore, the liquid chromatography method for detecting the concentration of QS-21 solution uses an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector, a reversed-phase column, and acetonitrile as the organic phase in the mobile phase.

[0037] Furthermore, the concentration of QS-21 in the QS-21 calibration standard solution sample is 10 μg / ml to 100 μg / ml.

[0038] Furthermore, the QS-21 calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0039] Furthermore, in step S3, the preparation of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples can be selected from any one of the following two methods: 1. Preparing dioleoylphosphatidylcholine calibration standard solution samples and cholesterol calibration standard solution samples (prepared separately); 2. Preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples (prepared together).

[0040] Furthermore, the preparation of dioleoylphosphatidylcholine calibration standard solution samples and cholesterol calibration standard solution samples (prepared separately) are carried out using the methods described below, but not limited to the following methods: Dioleoylphosphatidylcholine (purity not less than 90%) is accurately weighed, then dissolved in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluted to the target volume and mixed thoroughly to prepare the dioleoylphosphatidylcholine standard solution sample; cholesterol (purity not less than 90%) is accurately weighed, then dissolved in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluted to the target volume and mixed thoroughly to prepare the cholesterol standard solution sample.

[0041] Further, a standard solution sample of dioleoylphosphatidylcholine and cholesterol (prepared by mixing) is prepared using the method described below, but not limited to: The concentrations of dioleoylphosphatidylcholine and cholesterol in a liposome solution (with a purity of not less than 90% in both liposomes) are determined by liquid chromatography to prepare a standard solution sample of dioleoylphosphatidylcholine and cholesterol; after accurately weighing dioleoylphosphatidylcholine and cholesterol (with a purity of not less than 90%), they are dissolved in a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.), and finally diluted to the target volume and mixed to prepare the standard solution sample of dioleoylphosphatidylcholine and cholesterol.

[0042] Furthermore, the liquid chromatography method for detecting the concentration of liposome solution uses an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector, a reversed-phase column, and acetonitrile or methanol as the organic phase in the mobile phase.

[0043] Furthermore, the concentrations of dioleoylphosphatidylcholine and cholesterol in the calibration standard solution samples were 200 μg / ml to 2000 μg / ml and 50 μg / ml to 500 μg / ml, respectively.

[0044] Furthermore, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples may contain DMSO at a concentration of 0.5% to 5%.

[0045] Furthermore, the vaccine used in S4 to prepare the test sample has a protein content of 10 μg / ml to 400 μg / ml, a dioleoylphosphatidylcholine content of 400 μg / ml to 4000 μg / ml, a cholesterol content of 100 μg / ml to 1000 μg / ml, and a QS-21 content of 20 μg / ml to 200 μg / ml.

[0046] Furthermore, the sample tested in S4 has a protein concentration of 5 μg / ml to 200 μg / ml, a saponin QS-21 concentration of 10 μg / ml to 100 μg / ml, a dioleoylphosphatidylcholine concentration of 200 μg / ml to 2000 μg / ml, and a cholesterol concentration of 50 μg / ml to 500 μg / ml.

[0047] Furthermore, the sample being tested may contain DMSO at a concentration of 0.5% to 5%.

[0048] Furthermore, the detector of the liquid chromatograph in S5 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0049] Furthermore, the analytical method in S5 includes a chromatographic column, liquid chromatograph parameters, and elution program.

[0050] Furthermore, the chromatographic column used in the analytical method in S5 is a reversed-phase chromatographic column.

[0051] Furthermore, the liquid chromatography parameters of the analytical method in S5 include column temperature, injection volume, flow rate, detector parameters, etc.

[0052] Furthermore, the column temperature is 40℃ to 60℃, such as 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃.

[0053] 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.

[0054] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the injection volume is 5 μl to 30 μl, such as 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, or 30 μl.

[0055] Furthermore, when the instrument is a high-performance liquid chromatograph, the flow rate is 1.0 ml / min to 2.5 ml / min, such as 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, 1.6 ml / min, 1.7 ml / min, 1.8 ml / min, 1.9 ml / min, 2.0 ml / min, 2.1 ml / min, 2.2 ml / min, 2.3 ml / min, 2.4 ml / min, or 2.5 ml / min.

[0056] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the flow rate is 0.1 ml / min to 1.0 ml / min, such as 0.1 ml / min, 0.2 ml / min, 0.3 ml / min, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, or 1.0 ml / min.

[0057] Furthermore, the detector parameters are characterized as follows: when the detector is an ultraviolet detector, the detection wavelength is 200nm to 400nm, such as 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, or 400nm.

[0058] Furthermore, when the detector is a CAD detector, the atomization temperature is 35℃~70℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃.

[0059] Furthermore, when the detector is an evaporative photodetector, the atomization temperature is 35℃~70℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃.

[0060] Furthermore, the elution procedure of the analytical method in S5 shall include at least the following two elution methods: elution method for eluting saponin QS-21 and protein retention peaks; elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks.

[0061] Furthermore, the elution method for the saponin QS-21 and protein retention peaks was gradient elution, with acetonitrile concentration gradients in the mobile phase of 5%–95%, such as 5%–95%, 10%–90%, 15%–85%, 20%–80%, 25%–75%, 30%–70%, 35%–65%, 40%–65%, 45%–65%, 45%–70%, 45%–75%, and 45%. ~80%, 45%~85%, 45%~90%, 45%~95%, 50%~65%, 50%~70%, 50%~75%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 55%~65%, 55%~70%, 55%~75%, 55%~80%, 55%~85%, 55%~90%, 55%~95%, 60% ~65%, 60%~70%, 60%~75%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, or 65%~75%, with gradient elution times of 2 min~1920 min, such as 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, 960 min, or 1920 min, and trifluoroacetic acid concentration in the mobile phase of 0.01%~0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0062] Furthermore, the elution method for the retained peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0063] Furthermore, the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution: the methanol concentration in the mobile phase is 90%–100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and the isocratic elution time is 2 min–1920 min, such as 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 3 min, etc. The timeframes are 0 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, 960 min, or 1920 min, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0064] Furthermore, the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol was gradient elution: the methanol concentration in the mobile phase was 90%–100%, such as 90%–100%, 91%–100%, 92%–100%, 93%–100%, 94%–100%, 95%–100%, 96%–100%, 97%–100%, 98%–100%, 99%–100%, 90%–99%, 91%–99%, 92 ... 99%–93%–99%, 94%–99%, 95%–99%, 96%–99%, 97%–99%, 98%–99%, 90%–98%, 91%–98%, 92%–98%, 93%–98%, 94%–98%, 95%–98%, 96%–98%, 97%–98%, 90%–97%, 91%–97%, 92%–97%, 93%–97%, 94%–97%, 95%–99% 7%, 96%–97%, 90%–96%, 91%–96%, 92%–96%, 93%–96%, 94%–96%, 95%–96%, 90%–95%, 91%–95%, 92%–95%, 93%–95%, or 94%–95%, with gradient elution times of 2 min–1920 min, such as 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min. The time intervals are 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, 960 min, or 1920 min, with the concentration of trifluoroacetic acid in the mobile phase being 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0065] Furthermore, in the elution procedure of the analytical method in S5, an isocratic elution method can be added between the elution method for eluting the retained peaks of saponin QS-21 and protein and the elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0066] Furthermore, an isocratic elution method is added between the elution method for eluting saponin QS-21 and the protein retention peak and the elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks, wherein the methanol concentration is lower than the methanol concentration in the mobile phase used for eluting dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0067] Furthermore, an isocratic elution method is added between the elution method for eluting saponin QS-21 and the protein retention peak and the elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks. The mobile phase contains methanol at a concentration of 65%–94%, such as 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, and the isocratic elution time is [not specified]. The 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 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%.

[0068] Furthermore, in the elution procedure of the analytical method in S5, a gradient elution method can be added between the elution method for eluting the retained peaks of saponin QS-21 and protein and the elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0069] Furthermore, a gradient elution method is added between the elution method for eluting saponin QS-21 and the protein retention peak and the elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks, wherein the maximum value of the methanol concentration gradient is not higher than the methanol concentration in the mobile phase for eluting dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0070] Furthermore, a gradient elution method is added between the elution method for eluting saponin QS-21 and the protein retention peak and the elution method for eluting dioleoylphosphatidylcholine and cholesterol retention peaks, wherein the methanol concentration gradient in the mobile phase is 65%–95%, such as 65%–70%, 65%–75%, 65%–80%, 65%–85%, 65%–90%, 65%–95%, 70%–75%, 70%–80%, 70%–85%, 70%–90%, 70%–95%, 70%–95%, 75%–80%, 75%–85%, 75%–90%, 75%–95%, 80%–85%, 80%–90%, 80%–95%, 85%–90%. The concentration of trifluoroacetic acid in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%. The gradient elution time is 1 to 960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min.

[0071] Furthermore, in the elution procedure of the analytical method in S5, a pre-equilibration method and / or pre-elution method can be added before the elution method for eluting saponin QS-21 and protein retention peaks.

[0072] Furthermore, a pre-equilibration method is added before the elution of saponin QS-21 and the protein retention peak, wherein the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient for eluting saponin QS-21 and the protein retention peak.

[0073] Furthermore, a pre-equilibration method is added before the elution of saponin QS-21 and the protein retention peak. The acetonitrile concentration in the mobile phase is 5%–60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, and the equilibration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, etc. The timeframes are 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0074] Furthermore, a pre-elution method is added before the elution method for saponin QS-21 and the protein retention peak, wherein the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient for eluting saponin QS-21 and the protein retention peak.

[0075] Furthermore, a pre-elution method is added before the elution of saponin QS-21 and the protein retention peak, wherein the acetonitrile gradient concentration in the mobile phase is 5%–60%, such as 5%–10%, 5%–15%, 5%–20%, 5%–25%, 5%–30%, 5%–35%, 5%–40%, 5%–45%, 5%–50%, 5%–55%, 5%–60%, 10%–15%, 10%–20%, 10%–25%, 10%–30%, 10%–35%, 10%–40%, 10%–45%, and 10%–50%. 10%–55%, 10%–60%, 15%–20%, 15%–25%, 15%–30%, 15%–35%, 15%–40%, 15%–45%, 15%–50%, 15%–55%, 15%–60%, 20%–25%, 20%–30%, 20%–35%, 20%–40%, 20%–45%, 20%–50%, 20%–55%, 20%–60%, 25%–30%, 25%–35%, 25%–40%, 25%–45%, 25%–50%, 25%–55% %, 25%–60%, 30%–35%, 30%–40%, 30%–45%, 30%–50%, 30%–55%, 30%–60%, 35%–40%, 35%–45%, 35%–50%, 35%–55%, 35%–60%, 40%–45%, 40%–50%, 40%–55%, 40%–60%, 45%–50%, 45%–55%, 45%–60%, 50%–55%, 50%–60%, or 55%–60%, with gradient elution times of 1–960 min, such as 1 m The time intervals are 1 minute, 2 minutes, 4 minutes, 8 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or 960 minutes, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0076] Furthermore, in the elution procedure of the analytical method in S5, a column regeneration method and / or a post-equilibration method may be added after the elution method for eluting the retained peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0077] Furthermore, when both the column regeneration method and the post-equilibration method exist simultaneously, the column regeneration method should precede the post-equilibration method.

[0078] Furthermore, after eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol, a column regeneration method is added, wherein the acetonitrile concentration in the mobile phase is 98%–100%, such as 98%, 98.5%, 99%, 99.5%, or 100%, and the column regeneration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, or 30 min. The mobile phase may contain trifluoroacetic acid at concentrations of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%, for durations of 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min.

[0079] Furthermore, after the elution method for the retention peaks of dioleoylphosphatidylcholine and cholesterol, a post-equilibration method is added, wherein the concentration of acetonitrile in the mobile phase is not higher than the minimum value of the mobile phase concentration gradient for eluting the retention peaks of saponin QS-21 and protein.

[0080] Furthermore, after eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol, a post-equilibration method is added, wherein the acetonitrile concentration in the mobile phase is 5%–60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, and the equilibration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 2... The timeframes are 5 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0081] Furthermore, in S6, the content of protein, QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine is calculated, including calculation method one and calculation method two.

[0082] Furthermore, the calculation method one is as follows: first, establish a calibration standard curve in which the concentrations of protein, saponin QS-21, dioleoylphosphatidylcholine and cholesterol are proportional to the peak area, and then calculate the content (concentration) of protein, saponin QS-21, dioleoylphosphatidylcholine and cholesterol.

[0083] Further, calculation method one: First, establish a calibration standard curve in which the concentrations of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area; then, substitute the peak area values ​​of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the test sample into the calibration standard curve formula of the corresponding component to calculate the concentration of the corresponding component in the test sample; multiply the concentration of each component in the test sample by the dilution factor to calculate the content (concentration) of each component [protein, QS-21, dioleoylphosphatidylcholine, and cholesterol] in the vaccine.

[0084] Furthermore, the second calculation method is to directly calculate the content (concentration) of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine.

[0085] Furthermore, the second calculation method is as follows: The calculation formula is: Vaccine A content (concentration) = Peak area of ​​test sample A * Concentration of A calibration standard solution sample * Dilution factor / Peak area of ​​A calibration standard solution sample, where "A" represents "protein" or "saponin QS-21" or "dioleoylphosphatidylcholine (DOPC)" or "cholesterol".

[0086] This invention also provides a method for detecting the content of proteins and major components of a compound adjuvant in a recombinant varicella-zoster vaccine, applicable to the simultaneous detection of components in the recombinant varicella-zoster vaccine. The proteins in the recombinant varicella-zoster vaccine include, but are not limited to, the PADRE-gE-P2 fusion protein. The PADRE-gE-P2 fusion protein comprises the extracellular segment gE of varicella-zoster virus glycoprotein E, the universal DR Th epitope peptide PADRE, and the amino acid sequence of the tetanus toxin Th epitope P2. gE, PADRE, and P2 are linked by a linker peptide, which is a GGS and / or GGGS and / or GGGGS and / or GGSSG linker peptide. The adjuvant in the recombinant varicella-zoster vaccine is a compound adjuvant containing neutral liposomes and the immunostimulant QS-21.

[0087] Furthermore, the method for detecting the content of major components of protein and adjuvant in recombinant varicella-zoster vaccine is applied to the simultaneous detection of components in recombinant varicella-zoster vaccine:

[0088] 1. Simultaneously detect the content of protein and saponin QS-21; 2. Simultaneously detect the content of protein and dioleoylphosphatidylcholine; 3. Simultaneously detect the content of protein and cholesterol; 4. Simultaneously detect the content of protein, saponin QS-21, and cholesterol; 5. Simultaneously detect the content of protein, saponin QS-21, and dioleoylphosphatidylcholine; 6. Simultaneously detect the content of protein, dioleoylphosphatidylcholine, and cholesterol; 7. Simultaneously detect the content of saponin QS-21, dioleoylphosphatidylcholine, and cholesterol; 8. Simultaneously detect the content of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol.

[0089] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0090] This invention provides a method and application for detecting the content of protein and major components of compound adjuvant in recombinant varicella-zoster vaccine. It not only solves the technical problem of existing technologies requiring three different methods and three separate tests to detect protein, dioleoylphosphatidylcholine (DOPC), cholesterol, and saponin QS-21 content in vaccines, but also addresses the limitation of existing technologies in quantitative detection of protein in vaccines, which cannot provide qualitative analysis. The high-performance liquid chromatography method disclosed in this invention requires only a single sample loading and detection to determine the content of protein, DOPC, cholesterol, and QS-21 in vaccines, significantly improving vaccine detection efficiency.

[0091] This invention provides a method and application for detecting the content of protein and main components of compound adjuvant in recombinant varicella-zoster vaccine, which can simultaneously detect the content of protein and / or saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol in recombinant varicella-zoster vaccine.

[0092] This invention provides a method and application for detecting the content of protein and main components of compound adjuvant in recombinant varicella-zoster vaccine. It can also be used in conjunction with liquid chromatography for qualitative detection of protein in recombinant varicella-zoster vaccine, enabling both qualitative and quantitative detection of protein in recombinant varicella-zoster vaccine. Attached Figure Description

[0093] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0094] Figure 1 This is a standard curve of the concentration and peak area of ​​saponin QS-21 in Example 1 of the present invention.

[0095] Figure 2 This is a standard curve of dioleoylphosphatidylcholine concentration versus peak area in Example 1 of this invention.

[0096] Figure 3 This is the standard curve of cholesterol concentration versus peak area in Example 1 of the present invention.

[0097] Figure 4 This is a standard curve of the concentration and peak area of ​​the PADRE-gE-P2 fusion protein in Example 1 of this invention.

[0098] Figure 5 This is a liquid chromatogram of the protein and the content of three main adjuvant components in the XA-401 compound adjuvant in the recombinant varicella-zoster vaccine containing PADRE-gE-P2 fusion protein and XA-401 compound adjuvant, as shown in Example 1 of the present invention.

[0099] Figure 6 This is a comparison chart of the protein, saponin QS-21, dioleoylphosphatidylcholine and cholesterol content in vaccine samples detected in Example 1 of this invention (one simultaneous detection; PADRE-gE-P2 fusion protein as protein control) and the prior art detection (three separate detections).

[0100] Figure 7 This is the standard curve of BSA concentration versus peak area in Example 2 of this invention.

[0101] Figure 8 This is a comparison chart of the protein, saponin QS-21, dioleoylphosphatidylcholine and cholesterol content in vaccine samples detected in Example 2 of this invention (one simultaneous detection; BSA as protein control) and the prior art detection (three separate detections). Detailed Implementation

[0102] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0103] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0104] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0105] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0106] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0107] A liquid chromatography method for detecting the content of protein and adjuvant main components [dioleoylphosphatidylcholine, cholesterol, and saponin QS-21] in a recombinant varicella-zoster vaccine containing PADRE-gE-P2 fusion protein and XA-401 complex adjuvant.

[0108] An optional molecular structure of the PADRE-gE-P2 fusion protein (from N-terminus to C-terminus) is: PADRE—GSGSG (linker peptide)-gE(AA31-AA544)-GGS (linker peptide)-P2, the nucleic acid sequence of which is shown in CN 117003896 B and the amino acid sequence of which is shown in SEQ ID NO.1.

[0109] After codon optimization and full-gene synthesis of the nucleic acid sequence of the PADRE-gE-P2 fusion protein, a fusion protein expression plasmid was constructed, followed by the construction of a stable cell line, expression of the target product (PADRE-gE-P2 fusion protein), and finally purification to obtain the PADRE-gE-P2 fusion protein. The protein concentration of the fusion protein solution was then detected.

[0110] Liposomes were prepared, and the concentrations of dioleoylphosphatidylcholine and cholesterol in the liposomes were detected; a saponin QS-21 solution was prepared, and the concentration of saponin QS-21 was detected; the PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine was formulated using the PADRE-gE-P2 fusion protein, liposomes, and saponin QS-21 solution.

[0111] The PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine showed superior immunogenicity in mice compared to GSK's recombinant herpes zoster vaccine Shingrix, as detailed in CN 116747298 B and CN 117003896 B.

[0112] The contents of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the PADRE-gE-P2 fusion protein vaccine were determined using liquid chromatography (LC), and the results were compared with those obtained using existing techniques. The comparison results show that both LC and existing techniques can be used to detect the contents of protein and the main adjuvant components (dioleoylphosphatidylcholine, cholesterol, and saponin QS-21) in vaccines, and LC can detect protein content more accurately. Furthermore, the optimal LC parameters and elution methods for detecting target substances (such as PADRE-gE-P2 fusion protein, QS-21, DOPC, and cholesterol in vaccines) may differ when using reversed-phase columns with different chromatographic packing materials, as is known to those skilled in the art.

[0113] Example 1: Detection (1 test) of protein, saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol content in vaccine samples (PADRE-gE-P2 fusion protein was used as a protein standard).

[0114] 1) Preparation and concentration detection of PADRE-gE-P2 fusion protein

[0115] I. Codon Optimization and Whole Genome Synthesis of Fusion Proteins

[0116] See patent CN 117003896 B for details.

[0117] II. Construction of Fusion Protein Expression Plasmids

[0118] See patent CN 117003896 B for details.

[0119] III. Construction of Stable Cell Lines

[0120] See patent CN 117003896 B for details.

[0121] IV. Expression of the Target Product

[0122] See patent CN 117003896 B for details.

[0123] V. Fusion Protein Purification

[0124] See patent CN 117003896 B for details.

[0125] VI. Determination of Fusion Protein (Vaccine Stock Solution) Concentration [Lowry Method, Method I]

[0126] (1) Reagent preparation

[0127] Folin-Ciocalteu solution: Add 5.0 ml of phenol reagent to 75 ml of water and mix well; Solution A: Weigh 10.0 g of sodium hydroxide and 50.0 g of sodium carbonate into a plastic reagent bottle, add 400 ml of water to dissolve, and mix well; Potassium tartrate solution (0.5 g → 50 ml): Weigh 0.5 g of potassium tartrate into a reagent bottle, add 50 ml of water to dissolve, and mix well; Copper sulfate solution (0.25 g → 30 ml): Weigh 0.39 g of copper sulfate pentahydrate into a reagent bottle, add 30 ml of water to dissolve, and mix well; Solution B: Prepare according to the ratio of potassium tartrate solution (0.5 g → 50 ml): copper sulfate solution (0.25 g → 30 ml) = 5:3 (volume ratio), and mix well; Alkaline copper solution: Prepare according to the ratio of Solution A: Solution B: water = 40:8:2 (volume ratio), and mix well.

[0128] (2) Preparation of working solution for protein reference standard

[0129] 200 μg / ml Protein Reference Solution (Bovine Serum Albumin Solution): Take one vial of protein reference standard, reconstitute it with purified water to prepare a 200 μg / ml protein reference solution, and dispense it into 5 ml plastic tubes; store at -20℃ or below. Before use, dilute twice and mix well to obtain the working solution (100 μg / ml).

[0130] (3) Protein sample concentration detection

[0131] Preparation and detection of test sample: Accurately transfer an appropriate amount of the sample to be tested, dilute with water until the protein content is within the standard curve range; accurately transfer 1.0 ml of the above solution into two test tubes, add 1.0 ml of alkaline copper solution, mix well, let stand at room temperature for 10 min, add 4.0 ml of Folin-Ciocalteu solution, mix immediately, let stand at room temperature for 30 min for color development, and then measure the absorbance at a wavelength of 650 nm using ultraviolet-visible spectrophotometry (if turbidity is found after color development, centrifuge at 3000 rpm / min for 15 min, and then take the supernatant for measurement).

[0132] Protein standard test: Accurately transfer 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0 ml of 100 μg / ml protein standard solution into test tubes, making two copies each; for solutions less than 1 ml, add water to 1.0 ml. Starting from "add 1.0 ml of alkaline copper solution", proceed in the same manner.

[0133] (4) Protein sample concentration calculation

[0134] A linear regression was performed with the content of a series of protein reference standards as the X-axis and the corresponding mean absorbance as the Y-axis to obtain the linear regression equation. The mean absorbance of the test sample solution was then substituted into the linear regression equation to calculate the protein content of the test sample. The protein content of the sample (μg / ml) = A*n; where: A is the protein content of the test sample obtained by substituting the measured absorbance into the linear regression equation, in μg / ml; and n is the dilution factor of the test sample.

[0135] 2) Liposome preparation and detection of dioleoylphosphatidylcholine and cholesterol concentrations

[0136] I. Liposome preparation (theoretical concentrations of dioleoylphosphatidylcholine and cholesterol are 4 mg / ml and 1 mg / ml, respectively)

[0137] Weigh out 400 mg of dioleoylphosphatidylcholine (DOPC; Nippon Fine Chemicals Co., Ltd.) and 100 mg of cholesterol (Nippon Fine Chemicals Co., Ltd.); then completely dissolve DOPC and cholesterol in 10 ml of anhydrous ethanol and mix thoroughly to obtain the organic phase; inject 10 ml of the organic phase into 90 ml of 10 mM PBS buffer solution (pH 7.0) to prepare the liposome proemulsion; then use a high-pressure microfluidic homogenizer to granulate the liposomes to a particle size of about 100 nm; then remove ethanol by dialysis; finally, filter and sterilize using a 0.22 μm sterile filter to obtain the finished liposome product.

[0138] II. Detection of Dioleoylphosphatidylcholine (DOPC) and Cholesterol Concentrations

[0139] (1) Preparation of mobile phase

[0140] Mobile phase A [methanol:water:trifluoroacetic acid (95:5:0.1)]: Measure 950 ml of methanol, add 50 ml of ultrapure water and 1 ml of trifluoroacetic acid, mix and shake well, and sonicate to degas for 10 min to obtain the mobile phase.

[0141] (2) Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol standard solution samples

[0142] Preparation of 90% ethanol solution: Accurately measure 45 ml of anhydrous ethanol, mix it with 5 ml of ultrapure water and shake well.

[0143] 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.

[0144] Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol reference stock solution: Accurately weigh 37.5 mg of cholesterol (Nippon Fine Chemicals Co., Ltd.) and 150 mg of dioleoylphosphatidylcholine (DOPC; Nippon Fine Chemicals Co., Ltd.) into a 25 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, and shake well to obtain the solution.

[0145] Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol standard solutions: Transfer 1.667 ml of the reference stock solution to a 25 ml volumetric flask, dilute to 25 ml with 89.3% ethanol solution, and vortex to obtain a standard solution with a cholesterol concentration of 100 μg / ml and a DOPC concentration of 400 μg / ml. Transfer 7.5 ml, 5 ml, 2.5 ml, and 1.25 ml of standard solution ⑤ to 10 ml volumetric flasks, dilute to 10 ml with 90% ethanol solution, and vortex to obtain... The following standard solutions were prepared: ④ [cholesterol concentration 75 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 300 μg / ml], ③ [cholesterol concentration 50 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 200 μg / ml], ② [cholesterol concentration 25 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 100 μg / ml], and ① [cholesterol concentration 12.5 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration 50 μg / ml].

[0146] (3) Preparation of test sample solution

[0147] Transfer 0.1 ml of the sample solution to be tested into a vial, add 1.9 ml of anhydrous ethanol, cap the vial, mix well, and the sample is ready for testing.

[0148] (4) Instrument parameters and elution program

[0149]

[0150]

[0151] (5) Calculation of dioleoylphosphatidylcholine and cholesterol concentrations

[0152] Using the concentration of standard solutions of dioleoylphosphatidylcholine or cholesterol as the abscissa (X) and the peak area of ​​dioleoylphosphatidylcholine or cholesterol as the ordinate (Y), standard curves were plotted to show that the concentration of dioleoylphosphatidylcholine and cholesterol are directly proportional to the peak area. The peak area of ​​cholesterol or dioleoylphosphatidylcholine in the test sample solution was substituted into the standard curve for calculation, and then multiplied by the dilution factor used in the preparation of the test sample solution to obtain the concentration of dioleoylphosphatidylcholine and cholesterol in the test sample solution.

[0153] 3) Preparation and concentration detection of saponin QS-21 solution

[0154] I. Preparation of Saponin QS-21 Solution (Theoretical Concentration 4 mg / ml)

[0155] Weigh 40 mg of saponin QS-21 (Desert King), then dissolve it completely in 4 ml of phosphate solution (10 mM; pH 6.0), and then bring the volume up to 10 ml with phosphate solution (10 mM; pH 6.0). Mix well to obtain saponin QS-21 solution.

[0156] II. Detection of Saponin QS-21 Solution Concentration

[0157] (1) Preparation of mobile phase

[0158] Mobile phase A [water:acetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of acetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0159] Mobile phase B [acetonitrile:acetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of acetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0160] (2) Preparation of standard solution of saponin QS-21

[0161] Reference stock solution: Weigh 10 mg of QS-21 reference standard (Desert King) into a 2 ml volumetric flask, dissolve and dilute to the mark with phosphate solution (10 mM; pH 6.0), shake well, and use as the stock solution.

[0162] Standard solutions: Transfer 150 μl of the reference stock solution to a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, mix well, and obtain a solution with a concentration of 1 mg / ml; transfer 0.5 ml of the above solution to a 5 ml volumetric flask, dilute to the mark with 1% DMSO solution, mix well, and obtain a solution with a concentration of 100 μg / ml; transfer 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution to different injection vials, and then add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of 1% DMSO solution respectively, mix well, and 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.

[0163] (3) Preparation of test sample solution

[0164] Transfer 0.125 ml of the sample solution to a 5 ml volumetric flask, then dilute to the mark with phosphate solution (10 mM; pH 6.0) and mix well to obtain a solution with a concentration of 100 μg / ml. Take 0.5 ml of the above 100 μg / ml solution into a vial, add 0.5 ml of 2% DMSO solution, and mix well to obtain the final solution.

[0165] (4) Instrument parameters and elution program

[0166]

[0167] (5) Calculation of saponin QS-21 concentration

[0168] Using the concentration of the standard solution of saponin QS-21 as the X-axis and the corresponding peak area as the Y-axis, a linear regression curve is plotted. The peak area of ​​the test sample solution is then substituted into the standard curve and multiplied by the dilution factor of the test sample solution to obtain the content of QS-21 in the test sample solution.

[0169] 4) Vaccine sample preparation

[0170] I. Preparation of Adjuvants

[0171] The concentrations of liposome solution were determined as follows: DOPC and cholesterol concentrations in liposomes were 4.20 and 1.05 mg / ml, respectively; the concentration of saponin QS-21 solution was determined as follows: the concentration of saponin QS-21 was 4.14 mg / ml; 4.76 ml of liposomes were taken, 0.24 ml of saponin QS-21 solution was added, and the mixture was stirred evenly to obtain the adjuvant.

[0172] II. Vaccine Preparation [10ml volume; target concentration of PADRE-gE-P2 fusion protein: 150μg / ml; target concentration of saponin QS-21: 100μg / ml; liposome components: target concentrations of dioleoylphosphatidylcholine and cholesterol: 2mg / ml and 0.5mg / ml, respectively]

[0173] The protein concentration of the PADRE-gE-P2 fusion protein solution was determined as follows: The concentration of the PADRE-gE-P2 fusion protein was 1.52 mg / ml. 0.99 ml of the PADRE-gE-P2 fusion protein solution was added to the adjuvant, and then the total volume was brought up to 10 ml using phosphate solution (10 mM; pH 6.0). The mixture was stirred thoroughly to obtain the PADRE-gE-P2 fusion protein adjuvant vaccine. The formula for calculating the volume (ml) of the added PADRE-gE-P2 fusion protein solution is: Mass of added PADRE-gE-P2 fusion protein (μg) / Concentration of PADRE-gE-P2 fusion protein (μg / ml).

[0174] 5) Detect (1 test) the protein, saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol content in the vaccine sample (PADRE-gE-P2 fusion protein is a protein standard).

[0175] I. Solution Preparation

[0176] (1) Preparation of mobile phase (0.05% trifluoroacetic acid)

[0177] Mobile phase A [water:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of trifluoroacetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0178] Mobile phase B [acetonitrile:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0179] 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 sonicate to degas for 5 min to obtain the mobile phase.

[0180] (2) Preparation of sample dilution solution

[0181] 1% Dimethyl sulfoxide (DMSO) solution: Measure 99 ml of ultrapure water, add 1 ml of DMSO solution, mix well, and the solution is ready.

[0182] 1.25% Dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0183] 2% Dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0184] 3.5% Dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0185] II. Vaccine Sample Testing

[0186] S1. Preparation of protein calibration standard solution samples

[0187] 658 μl of PADRE-gE-P2 fusion protein control solution (concentration 1.52 mg / ml) was placed in a 5 ml volumetric flask and diluted to the mark with phosphate solution (10 mM; pH 6.0). The solution was shaken well to obtain a concentration of 200 μg / ml. Then, 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution were transferred to different centrifuge tubes, and 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0) were added respectively. The solutions were mixed well to obtain concentrations of 175 μg / ml, 150 μg / ml, 125 μg / ml, and 100 μg / ml, respectively. Take 0.5 ml of each of the five concentration solutions and place them in different vials. Then add 0.5 ml of 2% DMSO solution to each vial and mix well to obtain standard solutions with protein concentrations of 100 μg / ml, 87.5 μg / ml, 75 μg / ml, 62.5 μg / ml and 50 μg / ml, respectively.

[0188] Preparation of S2, Saponin QS-21 Calibration Standard Solution Sample

[0189] Transfer 150 μl of the QS-21 saponin reference standard stock solution (concentration of 5 mg / ml) to a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, and mix well to obtain a solution with a concentration of 1 mg / ml. Transfer 0.5 ml of the above solution to a 5 ml volumetric flask, dilute to the mark with 1% DMSO solution, and shake well to obtain a solution with a concentration of 100 μg / ml. Then transfer 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution to different injection vials, and add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of the solution respectively. Mix 1% DMSO solution, cap the bottle, and obtain 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.

[0190] Preparation of S3, dioleoylphosphatidylcholine and cholesterol calibration standard solution samples

[0191] 3 ml of liposome reference solution (DOPC and cholesterol concentrations of 4.20 mg / ml and 1.05 mg / ml, respectively) was transferred to 1.2 ml of 3.5% DMSO solution and mixed well to obtain solutions with DOPC and cholesterol concentrations of 3 mg / ml and 0.75 mg / ml, respectively. Then, 0.5 ml, 0.5 ml, 0.3 ml, and 0.2 ml of the above solution were transferred to different vials, and 0.5 ml, 0.7 ml, 1.0 ml, 0.9 ml, and 1.0 ml of 1% DMSO solution were added respectively. The vials were capped and mixed well to obtain standard solutions with DOPC and cholesterol concentrations of 1.5 mg / ml, 1.25 mg / ml, 1 mg / ml, 0.75 mg / ml, 0.5 mg / ml, 0.375 mg / ml, 0.3125 mg / ml, 0.25 mg / ml, 0.1875 mg / ml, and 0.125 mg / ml, respectively.

[0192] S4. Preparation of test samples

[0193] Transfer 0.5 ml of the sample solution to be tested into a vial, add 0.5 ml of 2% DMSO solution, cap the vial, mix well, and the solution is ready.

[0194] S5. Load the sample into the liquid chromatograph and run the analytical method.

[0195] The vials containing protein calibration standard solution, saponin QS-21 calibration standard solution, dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution, and the vial containing the test sample were loaded into the injection tray of the high performance liquid chromatograph, and the analytical method was run using the liquid chromatograph.

[0196] Chromatogram as shown Figure 5 As shown, the retained peak of each substance is completely separated from the front peak, which meets the requirements.

[0197]

[0198] S6. Calculate the content of protein, saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine (1) Establishment of standard curve

[0199] Calibration standard curves were established using the peak area values ​​of calibration standard solution samples of PADRE-gE-P2 fusion protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol, respectively, to show that the concentration of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol is proportional to the peak area.

[0200] The test results and standard curve of the PADRE-gE-P2 fusion protein calibration standard solution sample are as follows: Figure 4 As shown

[0201] Calibration standard solution-1 (50 μg / ml) 11.759 2973317 1.092 Calibration standard solution - 2 (62.5 μg / ml) 11.753 3767740 1.093 Calibration standard solution - 3 (75 μg / ml) 11.755 4491807 1.090 Calibration standard solution-4 (87.5 μg / ml) 11.758 5241079 1.096 Calibration standard solution - 5 (100 μg / ml) 11.748 6015649 1.096

[0202] Standard curve of peak area of ​​PADRE-gE-P2 fusion protein: y = 60464x - 36883, R 2 =0.9998.

[0203] The test results and standard curve of the QS-21 calibration standard solution of saponins from saponins are as follows: Figure 1 As shown

[0204]

[0205] Standard curve of peak area for saponin QS-21: y = 5665.8x - 1580.5, R 2 =0.9999.

[0206] The test results and standard curves for the calibration standard solutions of dioleoylphosphatidylcholine and cholesterol are as follows: Figure 2 , 3 As shown

[0207]

[0208]

[0209] DOPC peak area standard curve: y = 1062.4x + 21206, R 2 =1.

[0210] 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

[0211] Standard curve for cholesterol peak area: y = 6811.3x + 137457, R 2 =0.9995.

[0212] (2) Content Calculation

[0213] The peak area values ​​of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine sample were substituted into the calibration standard curve formulas of the corresponding components, and then multiplied by the dilution factor of the vaccine (twice) to calculate the content of protein, QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine.

[0214]

[0215] Note: "NA" means "Not applicable".

[0216] 6) Compare the differences in the content of PADRE-gE-P2 fusion protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in vaccine samples using a comparative test (single simultaneous test; PADRE-gE-P2 fusion protein as a protein standard) and existing technology tests (three separate tests). Figure 6 As shown

[0217] I. Vaccine Sample Testing (Prior Technology): A method for detecting the content of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in vaccine samples three times.

[0218] (1) Detection of protein content

[0219] Since the components of XA-401 adjuvant, such as saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol, can interfere with the Lowry method for detecting vaccine protein content, it is necessary to remove the influence of XA-401 adjuvant during the detection process to reduce detection errors.

[0220] (A) Reagent preparation

[0221] Folin-Ciocalteu solution: Add 5.0 ml of phenol reagent to 75 ml of water and mix well; Solution A: Weigh 10.0 g of sodium hydroxide and 50.0 g of sodium carbonate into a plastic reagent bottle, add 400 ml of water to dissolve, and mix well; Potassium tartrate solution (0.5 g → 50 ml): Weigh 0.5 g of potassium tartrate into a reagent bottle, add 50 ml of water to dissolve, and mix well; Copper sulfate solution (0.25 g → 30 ml): Weigh 0.39 g of copper sulfate pentahydrate into a reagent bottle, add 30 ml of water to dissolve, and mix well; Solution B: Prepare according to the ratio of potassium tartrate solution (0.5 g → 50 ml): copper sulfate solution (0.25 g → 30 ml) = 5:3 (volume ratio), and mix well; Alkaline copper solution: Prepare according to the ratio of Solution A: Solution B: water = 40:8:2 (volume ratio), and mix well.

[0222] (B) Preparation of working solution for protein reference standard

[0223] 200 μg / ml protein reference solution (bovine serum albumin solution): Take one vial of protein reference standard, reconstitute it with purified water to prepare a 200 μg / ml protein reference solution, and dispense it into 5 ml plastic tubes; store at -20℃ or below. Before use, dilute twice and mix well to obtain the working solution (100 μg / ml).

[0224] (C)XA-401 Adjuvant Control Preparation

[0225] The concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively; the concentration of saponin QS-21 was 4.14 mg / ml. Take 4.76 ml of liposomes, add 0.24 ml of saponin QS-21 solution, and finally make up to 10 ml with phosphate solution (10 mM; pH 6.0), and stir well to obtain the final product.

[0226] (D) Protein concentration detection

[0227] Preparation and detection of test sample: Accurately transfer an appropriate amount of vaccine sample and dilute with water until the protein content is within the standard curve range; accurately transfer 1.0 ml of the above solution into two test tubes, add 1.0 ml of alkaline copper solution, mix well, and let stand at room temperature for 10 min. Add 4.0 ml of Folin-Ciocalteu solution, mix immediately, and let stand at room temperature for 30 min for color development. Then, measure the absorbance at a wavelength of 650 nm using ultraviolet-visible spectrophotometry (if turbidity is found after color development, centrifuge at 3000 rpm / min for 15 min and take the supernatant for measurement).

[0228] Protein standard test: Accurately transfer 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0 ml of 100 μg / ml protein standard solution into test tubes, making two copies each; for solutions less than 1 ml, add water to 1.0 ml. Starting from "add 1.0 ml of alkaline copper solution", proceed in the same manner.

[0229] XA-401 Adjuvant Control Test: Accurately transfer an appropriate amount of XA-401 adjuvant sample and dilute it with water (the dilution factor is the same as that of the vaccine sample); accurately transfer 1.0 ml of the above solution into a test tube, make two copies, and operate in the same way starting from "add 1.0 ml of alkaline copper solution", as the adjuvant control.

[0230] (E) Calculation of protein concentration in vaccines

[0231] A linear regression was performed with the protein content of a series of protein reference standards as the X-axis and the corresponding mean absorbance as the Y-axis to obtain the linear regression equation. The mean absorbance of the adjuvant control solution was subtracted from the mean absorbance of the test solution to obtain the protein absorbance value of the test solution. This protein absorbance value was then substituted into the linear regression equation to calculate the protein content of the test sample. The protein content of the vaccine sample (μg / ml) = A*n; where: A is the protein content of the test sample obtained by substituting the protein absorbance value of the test solution into the linear regression equation, in μg / ml; and n is the dilution factor of the vaccine sample.

[0232] (2) Detection of saponin QS-21 content

[0233] Same as "Detection of concentration of saponin QS-21 solution" in item "3)".

[0234] (3) Detection of dioleoylphosphatidylcholine and cholesterol content

[0235] Same as "Detection of dioleoylphosphatidylcholine and cholesterol concentrations" in item "2)".

[0236] II. Comparative testing (single simultaneous test; PADRE-gE-P2 fusion protein as protein standard) and existing technology testing (three separate tests) to compare the differences in protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol content in vaccine samples.

[0237]

[0238]

[0239] Example 2: Detection (simultaneous detection) of protein, saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples (BSA as protein standard).

[0240] 1) The preparation and protein concentration detection of PADRE-gE-P2 fusion protein are the same as in Example 1.

[0241] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0242] 3) The preparation and concentration detection of the saponin QS-21 solution were the same as in Example 1.

[0243] 4) The preparation of vaccine samples is the same as in Example 1.

[0244] 5) Detect the content of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in vaccine samples (BSA is the protein standard).

[0245] I. Solution Preparation

[0246] (1) The preparation of the mobile phase (trifluoroacetic acid) is the same as in Example 1.

[0247] (2) The preparation of the vaccine sample dilution solution is the same as in Example 1.

[0248] II. Vaccine Sample Testing

[0249] S1. Preparation of protein calibration standard solution samples

[0250] Preparation of BSA reference stock solution: Weigh 21.9 mg of BSA (China National Institutes for Food and Drug Control) into a 10 ml volumetric flask, dissolve it in 6 ml of phosphate solution (10 mM; pH 6.0); then dilute to the mark with phosphate solution (10 mM; pH 6.0) and mix well; finally, dispense into 500 μl tubes to obtain the BSA reference stock solution (concentration 2.19 mg / ml). 457 μl of BSA reference stock solution (concentration 2.19 mg / ml) was placed in a 5 ml volumetric flask and diluted to the mark with phosphate solution (10 mM; pH 6.0). The solution was shaken well to obtain a concentration of 200 μg / ml. Then, 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution were transferred to different centrifuge tubes, and 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0) were added respectively. The solutions were mixed well to obtain concentrations of 175 μg / ml, 150 μg / ml, 125 μg / ml, and 100 μg / ml, respectively. Take 0.5 ml of each of the five concentration solutions and place them in different vials. Then add 0.5 ml of 2% DMSO solution to each vial and mix well to obtain calibration standard solutions with protein concentrations of 100 μg / ml, 87.5 μg / ml, 75 μg / ml, 62.5 μg / ml and 50 μg / ml, respectively.

[0251] Preparation of S2, Saponin QS-21 Calibration Standard Solution Sample

[0252] The same as the "S2, Preparation of Saponin QS-21 Calibration Standard Solution Sample" item in Example 1.

[0253] Preparation of S3, dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples

[0254] The procedure is the same as that in Example 1, “Preparation of S3, dioleoylphosphatidylcholine and cholesterol calibration standard solution sample”.

[0255] S4. Preparation of test samples

[0256] Same as "S4, Preparation of test sample" in Example 1.

[0257] S5. Load the sample into the liquid chromatograph and run the analytical method.

[0258] The procedure is the same as "S5, Load the sample into the liquid chromatograph and run the analytical method" in Example 1.

[0259] S6. Calculate the content of protein, saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in the vaccine (1) Establishment of standard curve

[0260] Calibration standard curves were established using the peak area values ​​of calibration standard solution samples of BSA, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol, respectively, to show that the concentrations of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area.

[0261] BSA protein calibration standard solution sample test results and standard curve, such as Figure 7 As shown

[0262] Calibration standard solution-1 (50 μg / ml) 7.683 2714156 1.793 Calibration standard solution - 2 (62.5 μg / ml) 7.679 3443813 1.782 Calibration standard solution - 3 (75 μg / ml) 7.676 4162984 1.760 Calibration standard solution-4 (87.5 μg / ml) 7.676 4785582 1.787 Calibration standard solution - 5 (100 μg / ml) 7.670 5527499 1.720

[0263] BSA protein peak area standard curve: y = 55701x - 54266, R 2 =0.9993.

[0264] Test results and standard curve of QS-21 calibration standard solution of saponins

[0265] The results are the same as those in item “S6” of Example 1, “(2) Detection results and standard curve of saponin QS-21 calibration standard solution sample”.

[0266] Results and standard curves of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solutions.

[0267] The results are the same as those in "S6" of Example 1, "(3) Detection results and standard curves of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples".

[0268] (2) Content Calculation

[0269] The peak area values ​​of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine sample were substituted into the calibration standard curve formulas of the corresponding components, and then multiplied by the dilution factor of the vaccine (twice) to calculate the content of protein, QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine.

[0270]

[0271] 6) Comparative testing (one simultaneous test; BSA as protein standard) and existing technology testing (three separate tests) to identify differences in protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol content in vaccine samples, such as... Figure 8 As shown

[0272] I. Vaccine Sample Testing (Prior Technology): A method for detecting the content of protein, saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples three times.

[0273] (1) Detection of protein content

[0274] The same as "(1) Detection of protein content" in item "6)" of Example 1, "I. Detection of vaccine sample (prior art)".

[0275] (2) Detection of saponin QS-21 content

[0276] The "concentration detection of saponin QS-21 solution" in "3) Preparation and concentration detection of saponin QS-21 solution" in Example 1 is the same as that in Example 1.

[0277] (3) Detection of dioleoylphosphatidylcholine (DOPC) and cholesterol levels

[0278] The procedure is the same as the "2) Preparation of liposomes and detection of dioleoylphosphatidylcholine (DOPC) and cholesterol concentration" in Example 1.

[0279] II. Comparative testing (single simultaneous test; BSA as protein standard) and existing technology testing (three separate tests) to the differences in protein, saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples.

[0280]

[0281]

[0282] Finally, it should be noted that:

[0283] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of major components of protein and compound adjuvant in recombinant varicella-zoster vaccine, characterized in that, Includes the following steps: S1. Prepare protein calibration standard solution samples; S2. Prepare QS-21 calibration standard solution samples; S3. Prepare calibration standard solution samples of dioleoylphosphatidylcholine and cholesterol; S4. Prepare the test sample; S5. Load the sample into the liquid chromatograph and run the analytical method; S6. Calculate the content of the main protein and adjuvant components in the vaccine; The recombinant varicella-zoster vaccine is composed of a PADRE-gE-P2 fusion protein and a complex adjuvant. An optional molecular structure of the PADRE-gE-P2 fusion protein from the N-terminus to the C-terminus is PADRE-GSGSG-gE-GGS-P2, and the amino acid sequence of the PADRE-gE-P2 fusion protein is shown in SEQ ID NO.

1. The complex adjuvant is XA-401, whose main components are dioleoylphosphatidylcholine, cholesterol, and saponin QS-21. When detecting the concentrations of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol, the elution program uses mobile phases A, B, and C. Mobile phase A consists of water and trifluoroacetic acid in a volume ratio of 100:0.05; mobile phase B consists of acetonitrile and trifluoroacetic acid in a volume ratio of 100:0.05; and mobile phase C consists of methanol and trifluoroacetic acid in a volume ratio of 100:0.

05. The specific analytical methods in step S5 are shown in the table. 。 2. The method for detecting the content of protein and compound adjuvant main components in recombinant varicella-zoster vaccine according to claim 1, characterized in that, The protein concentration of the protein calibration standard solution sample in S1 is 5 μg / ml to 200 μg / ml.

3. The method for detecting the content of major components of protein and adjuvant in recombinant varicella-zoster vaccine according to claim 1, characterized in that, The concentration of the saponin QS-21 calibration standard solution sample in S2 is 10 μg / ml to 100 μg / ml, and the concentrations of the dioleoylphosphatidylcholine calibration standard solution sample and the cholesterol calibration standard solution sample in S3 are 200 μg / ml to 2000 μg / ml and 50 μg / ml to 500 μg / ml, respectively.

4. The method for detecting the content of protein and adjuvant main components in recombinant varicella-zoster vaccine according to claim 1, characterized in that, The liquid chromatograph parameters for the analytical method in S5 include: column temperature, injection volume, flow rate, and detector parameters; the column temperature is 40℃~60℃; the injection volume is 5μl~100μl; the flow rate is 0.1ml / min~2.5ml / min; and when the detector is an ultraviolet detector, the detection wavelength is 200~400nm.

5. The method for detecting the content of protein and compound adjuvant main components in recombinant varicella-zoster vaccine according to claim 1, characterized in that, The calculation of the content of the main protein and adjuvant components in the vaccine in S6 includes two calculation methods: Calculation Method 1: First, establish a calibration standard curve in which the concentrations of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area. Then, substitute the peak area values ​​of protein, saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the test sample into the corresponding calibration standard curve formulas to calculate the concentration of the corresponding component in the test sample. Finally, multiply the concentration of each component in the test sample by the dilution factor to calculate the concentration of each component in the vaccine. Calculation Method 2: The calculation formula is: Vaccine A concentration = Peak area of ​​test sample A * Concentration of A calibration standard solution sample * Dilution factor / Peak area of ​​A calibration standard solution sample, where A represents protein, saponin QS-21, dioleoylphosphatidylcholine, or cholesterol.

6. The method for detecting the content of major components of protein and adjuvant in recombinant varicella-zoster vaccine according to any one of claims 1-5, applied to the simultaneous detection of components in recombinant varicella-zoster vaccine, characterized in that, The recombinant varicella-zoster vaccine contains proteins including the extracellular segment gE of varicella-zoster virus glycoprotein E, the universal DRTh epitope peptide PADRE, and the amino acid sequence of tetanus toxin Th epitope P2; gE, PADRE, and P2 are linked by a linker peptide, which is GGS and / or GGGS and / or GGGGS and / or GGSSG linker peptide; the recombinant varicella-zoster vaccine contains adjuvant... The agent is a compound adjuvant containing neutral liposomes and the immune enhancer QS-21.

7. The method for detecting the content of protein and adjuvant main components in recombinant varicella-zoster vaccine according to claim 5, applied to the simultaneous detection of components in recombinant varicella-zoster vaccine, is characterized in that... It is used to detect the content of protein and / or saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol in recombinant varicella-zoster vaccine.

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