Method for simultaneously qualitatively detecting gE in recombinant varicella-zoster vaccine and quantitatively detecting gE, saponin QS-21, dioleoylphosphatidylcholine and cholesterol in vaccine and application

Abstract

The invention discloses a method for simultaneously qualitatively detecting gE in a recombinant chicken pox-herpes zoster vaccine and quantitatively detecting gE, saponin QS-21, dioleoyl phosphatidylcholine and cholesterol in the vaccine and application of the gE, the saponin QS-21, the dioleoyl phosphatidylcholine and the cholesterol. The invention solves the technical problem that in the prior art, four different methods are needed for four times of detection for qualitative detection of gE in recombinant varicella-zoster vaccines, quantitative detection of gE in vaccines, quantitative detection of saponin QS-21 in vaccines, and quantitative detection of dioleoyl phosphatidylcholine and cholesterol in vaccines. According to the method, the contents of gE, saponin QS-21, dioleoyl phosphatidylcholine and cholesterol in the vaccine can be accurately detected only through one-time detection, meanwhile, the gE in the vaccine can be qualitatively detected, and the vaccine verification 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 qualitatively detecting gE in a recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine. Background Art

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

[0003] GlaxoSmithKline's recombinant varicella-zoster vaccine Shingrix ® Shingrix ® ) was approved by the FDA in 2017 for the prevention of herpes zoster in adults 50 years and older. The vaccine consists of two components: the extracellular domain of VZV glycoprotein E (gE) expressed in CHO cells and the AS01B composite adjuvant / adjuvant system (the AS01B adjuvant system consists of the immunopotentiators monophosphoryl lipid A (MPL), the immunopotentiator Quillaja saponin QS-21, and liposomes; the main components of the liposomes are dioleoylphosphatidylcholine (DOPC) and cholesterol).

[0004] Compared with the live attenuated vaccines of Merck and Biopharma, GSK's recombinant varicella-zoster vaccine Shingrix ® The protection rate is higher. ® The protection rate of the vaccine is 69.8% among people aged 50 to 59 years old, and 51% among people over 60 years old. The protection rate gradually decreases with age, and the protection rate is only 18% among people aged 80 years and above. In terms of preventing PHN, the protection rate of the vaccine is 39% for people aged 60 years and above. ®Protection rate: Among people aged 40-49, the protection rate of the vaccine is 37.41%, among people aged ≥40, the protection rate is 57.62%, among people aged ≥50, the protection rate is 58.72%, among people aged ≥60, and the protection rate is 55.64% among people aged ≥70, and 18.63%. Shingrix® protection rate: The overall protection rate of the vaccine against HZ in subjects aged ≥50 years was 97.16%. When analyzed by stratification of 50-59 years, 60-69 years, and ≥70 years, the protection rate of the vaccine against HZ was comparable in all age groups, with protection rates of 96.57% for those aged 50-59 years, 97.36% for those aged 60-69 years, and 97.93% for those aged ≥70 years. In terms of preventing PHN, Shingrix® can reduce the incidence of PHN by 91.2% in the normal population aged ≥50 years; and by 88.8% in the normal population aged ≥70 years. Continuous research on the vaccine's effectiveness has shown that the vaccine's effectiveness can last for more than 10 years.

[0005] In order to control the quality of vaccines, it is necessary to qualitatively detect the protein in the vaccine and quantitatively detect the content of the main components of the protein and adjuvant in the vaccine. ® In the quality control of Shingrix, ELISA was used to qualitatively detect gE in the vaccine, and three different methods were used to detect gE content, Quillaja saponin QS-21 content, dioleoylphosphatidylcholine (DOPC) and cholesterol content. ® Quantitative detection of gE in vaccines, Shingrix ® The methods for producing gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccines are cumbersome, time-consuming, material-consuming, labor-intensive and inefficient. Summary of the Invention

[0006] The purpose of the present invention is to provide a method and application for simultaneously qualitatively detecting gE in recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine, so as to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solutions: The present invention provides a method for simultaneously qualitatively detecting gE in a recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine. The method adopts a liquid chromatography method, and the vaccine is a recombinant varicella-zoster vaccine.

[0008] The recombinant varicella-zoster vaccine consists of gE and a composite adjuvant; the gE is the extracellular segment of the varicella-zoster virus glycoprotein E, an optional molecular structure from the N-terminus to the C-terminus of which is: AA31-AA544, and its amino acid sequence is shown in SEQ ID NO.1; the composite adjuvant has dioleoylphosphatidylcholine, cholesterol, monophosphoryl lipid A and Quillaja saponin QS-21 as its main components.

[0009] Furthermore, the gE is a recombinant protein prepared using a eukaryotic expression system.

[0010] Furthermore, the eukaryotic expression system includes but is not limited to a mammalian expression system, a yeast expression system, an insect cell-baculovirus expression system, etc.; the mammalian expression system includes but is not limited to Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, etc.; the yeast expression system includes but is not limited to Pichia pastoris, etc.; the insect cells include but are not limited to SF9 cells, SF21 cells, HI5 cells, etc.

[0011] Furthermore, the optional molecular structure of gE may also be one of the following: AA31-AA547, AA31-AA546, AA31-AA545, AA31-AA543, AA31-AA542, AA31-AA541, AA31-AA540, AA31-AA539, AA31-AA538, AA31-AA537, AA31-AA536, AA31-AA535, AA31-AA547, AA31-AA548, AA31-AA549, AA31-AA550, AA31-AA551, AA31-AA552, AA31-AA553, AA31-AA554, AA31-AA555 1-AA534, AA31-AA533, AA31-AA532, AA31-AA531, AA31-AA530, AA31-AA529, AA31-AA528, AA31-AA 527, AA31-AA526, AA31-AA525, AA31-AA524, AA31-AA523, AA31-AA522, AA31-AA521, AA31-AA520.

[0012] Furthermore, the composite adjuvant is composed of neutral liposomes and immunopotentiators monophosphoryl lipid A and Quillaja saponin QS-21, and the main components of the neutral liposomes are dioleoylphosphatidylcholine and cholesterol.

[0013] Furthermore, the gE content in the recombinant varicella-zoster vaccine is 10 μg / ml to 400 μg / ml, the dioleoylphosphatidylcholine content is 400 μg / ml to 4000 μg / ml, the cholesterol content is 100 μg / ml to 1000 μg / ml, the Quillaja saponin QS-21 content is 20 μg / ml to 200 μg / ml, and the monophosphoryl lipid A content is 20 μg / ml to 200 μg / ml.

[0014] Furthermore, the source of the monophosphoryl lipid A includes but is not limited to any one or more combinations of the following: Salmonella extraction, Escherichia coli extraction, and chemical synthesis; the Salmonella includes but is not limited to Minnesota Salmonella R595 strain.

[0015] Furthermore, the monophosphoryl lipid A includes 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPL) and 3-O-acyl-4'-monophosphoryl lipid A (MPL); the monophosphoryl lipid A may be a mixture of 3-O-deacylated-4'-monophosphoryl lipid A and 3-O-acyl-4'-monophosphoryl lipid A (MPL).

[0016] Furthermore, the 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPL) can be a monomer or a mixture of various monomers, and the acyl chains of the monomer are 3, 4, 5, or 6; the 3-O-acyl-4'-monophosphoryl lipid A (MPL) can be a monomer or a mixture of various monomers, and the acyl chains of the monomer are 3, 4, 5, 6, or 7.

[0017] Furthermore, the recombinant varicella-zoster vaccine contains a buffer component for maintaining the pH value of the vaccine stable and a component for regulating the osmotic pressure of the vaccine.

[0018] Furthermore, the buffer component that maintains the stable pH value of the vaccine is selected from any one or more of the following substances, but is not limited to the following substances: 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, trishydroxymethylaminomethane, and 4-hydroxyethylpiperazineethanesulfonic acid.

[0019] Furthermore, the component for regulating the osmotic pressure of the vaccine can be selected from any one or more of the following substances, but is not limited to the following substances: sodium chloride, sucrose, trehalose, glucose, mannitol, and sorbitol.

[0020] Furthermore, the recombinant varicella-zoster vaccine may also contain components for maintaining protein stability in the vaccine.

[0021] Furthermore, the component that maintains protein stability in the vaccine can be selected from any one or more of the following substances, but is not limited to the following substances: polysorbate 80, polysorbate 20, and poloxamer 188.

[0022] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra high performance liquid chromatography.

[0023] 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 contain methanol.

[0024] 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).

[0025] Furthermore, the liquid chromatography method is a liquid chromatography internal standard method or a liquid chromatography external standard method.

[0026] Furthermore, the liquid chromatography external standard method comprises the following steps: S1. Prepare gE calibration standard solution samples (for qualitative and / or quantitative detection of gE in vaccines); S2. Prepare BSA calibration standard solution samples (for quantitative detection of gE in vaccines); S3, preparing a sample of a calibration standard solution of Quillaja saponin QS-21; S4, preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples; S5. preparing test samples; S6. Load the sample into the liquid chromatograph and run the analysis method; S7. Calculate the relative deviation of protein retention time in the vaccine; S8. Calculate the content of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine.

[0027] Furthermore, the protein concentration of the gE calibration standard solution sample in S1 is 5 μg / ml to 200 μg / ml.

[0028] Furthermore, the gE calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0029] Furthermore, the protein concentration of the BSA calibration standard solution sample in S2 is 5 μg / ml to 200 μg / ml.

[0030] Furthermore, the BSA calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0031] Furthermore, the purity of the QS-21 standard solution sample for preparing the quillaja saponin QS-21 calibration standard solution sample in S3 is not less than 90%, and its preparation method can be selected from any one of the following methods, but is not limited to the following methods: using a buffer solution with a pH of not less than 4 to dissolve QS-21 powder (purity not less than 90%) to form a QS-21 solution, and preparing a QS-21 standard solution sample; using liquid chromatography to detect the concentration of the QS-21 solution (purity not less than 90%), and preparing a QS-21 standard solution sample.

[0032] Furthermore, the concentration of the QS-21 solution is detected by liquid chromatography, wherein the detector is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector, the chromatographic column is a reverse phase chromatographic column, and the organic phase in the mobile phase is acetonitrile.

[0033] Furthermore, the concentration of QS-21 in the QS-21 calibration standard solution sample is 10 μg / ml to 100 μg / ml.

[0034] Furthermore, the QS-21 calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0035] Furthermore, in S4, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples are prepared by any one of the following two preparation methods: 1. Preparing a dioleoylphosphatidylcholine calibration standard solution sample and a cholesterol calibration standard solution sample (separate preparation); 2. Preparing a dioleoylphosphatidylcholine and cholesterol calibration standard solution sample (mixed preparation).

[0036] 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.

[0037] 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 using a suitable organic solvent (including but not limited to ethanol, isopropanol, etc.) to dissolve, and finally dilute to the target volume and mix to prepare a dioleoylphosphatidylcholine and cholesterol standard solution sample.

[0038] 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.

[0039] 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.

[0040] Furthermore, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples may contain DMSO at a concentration of 0.5% to 5%.

[0041] Furthermore, the vaccine used to prepare the test sample in S5 has a protein concentration of 10μg / ml to 400μg / ml, a dioleoylphosphatidylcholine concentration of 400μg / ml to 4000μg / ml, a cholesterol concentration of 100μg / ml to 1000μg / ml, a QS-21 concentration of 20μg / ml to 200μg / ml, and a monophosphoryl lipid A concentration of 20μg / ml to 200μg / ml.

[0042] Furthermore, the test sample in S5 has a protein concentration of 5 μg / ml to 200 μg / ml, a concentration of Quillaja saponin QS-21 of 10 μg / ml to 100 μg / ml, a concentration of monophosphoryl lipid A of 10 μg / ml to 100 μg / ml, a concentration of dioleoylphosphatidylcholine of 200 μg / ml to 2000 μg / ml, and a cholesterol concentration of 50 μg / ml to 500 μg / ml.

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

[0044] Furthermore, the detector of the liquid chromatograph in S6 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0045] Furthermore, the analysis method in S6 includes a chromatographic column, liquid chromatography parameters, and an elution procedure.

[0046] Furthermore, the chromatographic column used in the analysis method in S6 is a reverse phase chromatographic column.

[0047] Furthermore, the liquid chromatography parameters of the analysis method in S6 include column temperature, injection volume, flow rate, detector parameters, etc.

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

[0049] Furthermore, when the instrument is a high performance liquid chromatograph, the injection volume is 30 μl to 100 μl, such as 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, 75 μl, 80 μl, 85 μl, 90 μl, 95 μl, or 100 μl.

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

[0051] Further, when the instrument is a high performance liquid chromatograph, the flow rate is 1.0 ml / min to 2.5 ml / min, such as 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, 1.6 ml / min, 1.7 ml / min, 1.8 ml / min, 1.9 ml / min, 2.0 ml / min, 2.1 ml / min, 2.2 ml / min, 2.3 ml / min, 2.4 ml / min or 2.5 ml / min.

[0052] Furthermore, when the instrument is an ultra performance liquid chromatograph, the flow rate is 0.1 ml / min to 1.0 ml / min, such as 0.1 ml / min, 0.2 ml / min, 0.3 ml / min, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, or 1.0 ml / min.

[0053] Further, the detector parameters, when the detector is an ultraviolet detector, the detection wavelength is 200nm~400nm, such as 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm or 400nm.

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

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

[0056] Furthermore, the elution procedure of the analysis method in S6 includes at least the following two elution methods: an elution method for eluting the retained peaks of Quillaja saponin QS-21 and gE; and an elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0057] Furthermore, the acetonitrile concentration in the mobile phase for eluting the retention peaks of Quillaja saponin QS-21 and gE is not higher than the acetonitrile or methanol concentration in the mobile phase for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol.

[0058] Furthermore, the elution method for eluting the retained peaks of Quillaja saponin QS-21 and gE 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%, 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% 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 retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0060] Furthermore, the 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%.

[0061] Furthermore, the 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%.

[0062] Furthermore, in the elution procedure of the analysis method in S6, an isocratic elution method can be added between the elution method for eluting the retained peaks of Quillaja saponin QS-21 and gE and the elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0063] Furthermore, an isocratic elution method is added between the elution method for eluting the retention peaks of Quillaja saponin QS-21 and gE and the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile or methanol concentration is less than the acetonitrile or methanol concentration in the mobile phase for eluting dioleoylphosphatidylcholine and cholesterol, and is not lower than the maximum value of the acetonitrile concentration gradient in the mobile phase for eluting the retention peaks of Quillaja saponin QS-21 and gE.

[0064] Furthermore, an isocratic elution method is added between the elution method for eluting the retention peaks of Quillaja saponin QS-21 and gE and the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the concentration of acetonitrile or methanol in the mobile phase is 65% to 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 during isocratic elution The length 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%.

[0065] Furthermore, in the elution procedure of the analysis method in S6, a gradient elution method can be added between the elution method for eluting the retained peaks of Quillaja saponin QS-21 and gE and the elution method for eluting the retained peaks of dioleoylphosphatidylcholine and cholesterol.

[0066] Furthermore, a gradient elution method is added between the elution method for eluting the retention peaks of Quillaja saponin QS-21 and gE and the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the maximum value of the acetonitrile or methanol concentration gradient is not higher than the acetonitrile or methanol concentration in the mobile phase for eluting dioleoylphosphatidylcholine and cholesterol, and the minimum value of the acetonitrile or methanol concentration gradient is not lower than the maximum value of the acetonitrile concentration gradient in the mobile phase for eluting the retention peaks of Quillaja saponin QS-21 and protein.

[0067] Furthermore, a gradient elution method is added between the elution method for eluting the retention peaks of Quillaja saponin QS-21 and gE and the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile or methanol concentration gradient in the mobile phase is 65% to 95%, such as 65% to 70%, 65% to 75%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 70% to 75%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 80% to 85%, 80% to 90%, 80% to 95%, 85% to 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 analysis method in S6, a pre-equilibration method and / or a pre-elution method may be added before the elution method for eluting the retained peaks of Quillaja saponin QS-21 and gE.

[0069] Furthermore, a pre-equilibration method is added before the elution method for eluting the retention peaks of Quillaja saponin QS-21 and gE, wherein the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient for eluting the retention peaks of Quillaja saponin QS-21 and protein.

[0070] Furthermore, a pre-equilibrium method is added before the elution method of eluting the retention peaks of Quillaja saponin QS-21 and gE, 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%.

[0071] Furthermore, a pre-elution method is added before the elution method for eluting the retention peaks of Quillaja saponin QS-21 and gE, and the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient for eluting the retention peaks of Quillaja saponin QS-21 and gE.

[0072] Further, a pre-elution method is added before the elution method of eluting the retention peaks of Quillaja saponin QS-21 and gE, and the acetonitrile gradient concentration in the mobile phase is 5% to 60%, such as 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to 45%, 5% to 50%, 5% to 55%, 5% to 60%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 10% to 45%, 10% to 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 a gradient elution time of 1-960 min, such as 1 m In some embodiments, the mobile phase may be a column or column, and the mobile phase may be a column or column, and the column may be a column or column column, and the column may be a column or column column, and the column may be a column or column column, and the column may be a column or column column, and the column may be a column or column column, and the column may be a column or column column, and the column may be a column or column column, and the column may be a column or column, and the column may be a column or column, and the column may be a column or column, and the column may be a column or column, and the column may be a column or column, and the column may be a column or column, and the column may be a column or column, and the column may be

[0073] Furthermore, in the elution procedure of the analysis method in S6, a chromatographic column regeneration method and / or a post-equilibration method may be added after the elution method for eluting the retention peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0074] 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.

[0075] Furthermore, a chromatographic column regeneration method is added after the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile concentration in the mobile phase is 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%.

[0076] Furthermore, a post-equilibrium method is added after the 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 gE and QS-21.

[0077] Further, a post-equilibrium method is added after the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, wherein the acetonitrile concentration in the mobile phase is 5% to 60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, the equilibrium time is 1 to 960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, and the trifluoroacetic acid concentration in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0078] Furthermore, the calculation formula for the relative deviation of the retention time of the protein in the vaccine in S7 = [|retention time of the vaccine protein retention peak - average value of the retention time| / average value of the retention time] × 100%. Note: The average value of the retention time is the average value of the retention time of the vaccine protein retention peak and the retention time of the gE calibration standard solution sample retention peak.

[0079] Furthermore, the criterion for determining whether the protein in the vaccine is gE is that the relative deviation of the protein retention time in the vaccine is no more than 2%.

[0080] Furthermore, the calculation of the gE, QS-21, dioleoylphosphatidylcholine and cholesterol contents in the vaccine in S8 includes calculation method one and calculation method two.

[0081] Furthermore, the calculation method 1 is: first establish a calibration standard curve in which the concentration of gE or BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol is proportional to the peak area, and then calculate the content (concentration) of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol.

[0082] Furthermore, calculation method 1: first establish a calibration standard curve in which the concentration of gE or BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol is proportional to the peak area; then substitute the peak area values of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol of the test sample into the calibration standard curve formula of the corresponding component, and calculate the concentration of the corresponding component in the test sample; multiply the concentration of each component of the test sample by the dilution multiple, and calculate the content (concentration) of each component [gE, QS-21, dioleoylphosphatidylcholine and cholesterol] in the vaccine.

[0083] Furthermore, the second calculation method is to directly calculate the content (concentration) of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine.

[0084] Furthermore, the second calculation method is: the calculation formula is vaccine A content (concentration) = test sample A peak area * A calibration standard solution sample concentration * dilution factor / A calibration standard solution sample peak area, "A" represents "gE" or "Quillaja saponin QS-21" or "dioleoylphosphatidylcholine (DOPC)" or "cholesterol".

[0085] The present invention also provides an application of a method for simultaneously qualitatively detecting gE in a recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine. The gE in the recombinant varicella-zoster vaccine is an extracellular segment of varicella-zoster virus glycoprotein E prepared using a eukaryotic expression system; and the adjuvant in the recombinant varicella-zoster vaccine is a composite adjuvant containing neutral liposomes and immunopotentiators monophosphoryl lipid A and Quillaja saponin QS-21.

[0086] Further, the application of the method for simultaneously qualitatively detecting gE in recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine: 1. Simultaneous detection of gE and Quillaja saponin QS-21 content; 2. Simultaneous detection of gE and dioleoylphosphatidylcholine content; 3. Simultaneous detection of gE and cholesterol content; 4. Simultaneous detection of gE, Quillaja saponin QS-21, and cholesterol content; 5. Simultaneous detection of gE, Quillaja saponin QS-21, and dioleoylphosphatidylcholine content; 6. Simultaneous detection of gE, dioleoylphosphatidylcholine and cholesterol content; 7. Simultaneous detection of Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol content; 8. Simultaneous detection of gE, Quillaja saponin QS -21, dioleoylphosphatidylcholine and cholesterol content; 9. Simultaneous qualitative and quantitative detection of gE; 10. Simultaneous qualitative detection of gE and quantitative detection of Quillaja saponin QS-21; 11. Simultaneous qualitative detection of gE and quantitative detection of dioleoylphosphatidylcholine; 12. Simultaneous qualitative detection of gE and quantitative detection of cholesterol; 13. Simultaneous qualitative detection of gE and quantitative detection of gE and Quillaja saponin QS-21; 14. Simultaneous qualitative detection of gE and quantitative detection of gE and dioleoylphosphatidylcholine; 15. Simultaneous qualitative detection of gE and quantitative detection of gE and cholesterol; 16. Simultaneous qualitative detection of gE, quantitative detection of dioleoylphosphatidylcholine and cholesterol; 17. Simultaneous qualitative detection of gE, quantitative detection of gE, Quillaja saponin QS-21 and dioleoylphosphatidylcholine; 18. Simultaneous qualitative detection of gE, quantitative detection of gE, Quillaja saponin QS-21 and cholesterol; 19. Simultaneous qualitative detection of gE, quantitative detection of gE, dioleoylphosphatidylcholine and cholesterol; 20. Simultaneous qualitative detection of gE, quantitative detection of Quillaja saponin QS-21 and dioleoylphosphatidylcholine; 21. Simultaneous qualitative detection of gE, quantitative detection of Quillaja saponin QS-21 and cholesterol; 22. Simultaneous qualitative detection of gE, quantitative detection of Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol; 23. Simultaneous quantitative Detection of gE and Quillaja saponin QS-21; 24. Simultaneous quantitative detection of gE and dioleoylphosphatidylcholine; 25. Simultaneous quantitative detection of gE and cholesterol; 26. Simultaneous quantitative detection of gE, Quillaja saponin QS-21 and dioleoylphosphatidylcholine; 27. Simultaneous quantitative detection of gE, Quillaja saponin QS-21 and cholesterol; 28. Simultaneous quantitative detection of gE, dioleoylphosphatidylcholine and cholesterol; 29. Simultaneous quantitative detection of Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol; 30. Simultaneous quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol; 31. Simultaneous qualitative detection of gE and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol.

[0087] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: The present invention provides methods and applications for simultaneously qualitatively detecting gE and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in recombinant varicella-zoster vaccine. These methods address the technical problem of prior art techniques requiring four separate methods for qualitatively detecting gE, quantitatively detecting gE, quantitatively detecting QS-21, and quantitatively detecting dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccines. The high-performance liquid chromatography method disclosed in the present invention enables qualitative detection of gE and quantitative detection of gE, QS-21, dioleoylphosphatidylcholine, and cholesterol in recombinant varicella-zoster vaccine with only a single sample loading, significantly improving vaccine testing efficiency.

[0088] The present invention provides a method and application for simultaneously qualitatively detecting gE in a recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine. The method can quantitatively detect gE and / or Quillaja saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol in the vaccine while qualitatively detecting gE in the recombinant varicella-zoster vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] 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.

[0090] Figure 1 This is a liquid chromatogram of a sample of the gE calibration standard solution (75 μg / ml) according to Example 1 of the present invention.

[0091] Figure 2 This is the standard curve of BSA concentration and peak area in Example 1 of the present invention.

[0092] Figure 3 This is the standard curve of concentration and peak area of Quillaja saponin QS-21 in Example 1 of the present invention.

[0093] Figure 4 This is the standard curve of dioleoylphosphatidylcholine concentration and peak area in Example 1 of the present invention.

[0094] Figure 5 This is the standard curve of cholesterol concentration and peak area in Example 1 of the present invention.

[0095] Figure 6This is a liquid chromatogram of Example 1 of the present invention for detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the recombinant varicella-zoster vaccine.

[0096] Figure 7 It is a comparison chart of the contents of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in vaccine samples detected simultaneously (one detection) by Example 1 of the present invention and detected by the prior art (three separate detections). DETAILED DESCRIPTION

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0102] The invention relates to a method and application for simultaneously qualitatively detecting gE in a recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine. The recombinant varicella-zoster vaccine is composed of gE and a composite adjuvant. The gE is the extracellular segment of varicella-zoster virus glycoprotein E. The main components of the composite adjuvant are dioleoylphosphatidylcholine, cholesterol, monophosphoryl lipid A, and Quillaja saponin QS-21.

[0103] An optional molecular structure of the gE is (from N-terminus to C-terminus): AA31-AA544, and its amino acid sequence is shown in SEQ ID NO.1.

[0104] SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVF KGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLK VLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTF TSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGG After the nucleic acid sequence of the gE is codon-optimized and fully synthesized, a fusion protein expression plasmid is constructed, followed by a stable cell line, and then the target product is expressed. Finally, the gE is obtained by purification, and the protein concentration is detected.

[0105] Liposomes are prepared, and the concentrations of dioleoylphosphatidylcholine and cholesterol in the liposomes are detected; a Quillaja saponin QS-21 solution is prepared, and the concentration of Quillaja saponin QS-21 is detected; and the recombinant varicella-zoster vaccine is prepared using gE, liposomes, 3D-MPL, and the Quillaja saponin QS-21 solution.

[0106] The optimal liquid chromatography parameters and elution methods when detecting target substances (such as gE, QS-21, DOPC and cholesterol in vaccines) using reversed-phase chromatography columns containing different chromatographic fillers may be different, which is known to those skilled in the art.

[0107] The content of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the recombinant varicella-zoster vaccine was determined using a liquid chromatography method that met the requirements, and the results were compared with those obtained using existing techniques. The comparison results demonstrated that both the liquid chromatography method and existing techniques were suitable for determining the content of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine, and that the selected liquid chromatography method was also capable of qualitatively detecting gE in the vaccine.

[0108] Example 1. Simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine 1) Preparation of gE solution (stock solution) and protein content detection 1. Protein codon optimization and whole gene synthesis Codon optimization of the gE gene was performed to avoid commonly used restriction enzyme cleavage sites. Based on the codon preference of CHO cells, low-frequency synonymous codons were replaced with high-frequency codons to control rare codon usage. The GC content of the sequence was controlled to 40%-60% to improve mRNA transcription efficiency while preventing high GC content from affecting mRNA secondary structure and, in turn, translation efficiency. A gene sequence encoding a signal peptide was added to the front of the optimized gene sequence. A Hind III restriction site was introduced upstream, and a stop codon and BamH I restriction site were added downstream. The nucleotide sequence was then fully synthesized.

[0109] 2. Construction of protein expression plasmid The cloning vector containing the fully synthesized gene sequence was transformed into DH5α competent bacteria and then amplified. After plasmid extraction, the cloning vector was double-digested with the restriction endonucleases Hind III and Bam HI. Simultaneously, the expression vector pXNM3.0 was double-digested with the restriction endonucleases Hind III and Bam HI. The fusion protein gene was recovered from the double-digested cloning vector by gel excision, while the backbone was recovered from the expression vector by gel excision. The two fragments were ligated with T4 enzyme and transformed into DH5α competent bacteria. Plates containing ampicillin resistance were then plated for screening. Positive colonies were plaque-pick amplified and plasmids were extracted. The plasmids were then double-digested with Hind III and Bam HI for identification. The correct recombinant expression vector was verified by sequencing.

[0110] 3. Construction of Stable Cell Lines Identify the correct recombinant expression vector, enrich the cells, and extract the plasmid in large quantities. Use Pvμ I to digest the recombinant expression vector. Recover the linearized vector by gel excision and filter sterilize before use. After CHO-K1 cells have recovered, passage them twice or more, and the cell viability should be greater than 95%. In a clean bench, add 0.6 ml of cell suspension (approximately 1 × 10 7 The cells were electroporated at 300 V and 900 μF with 200 μl of the linearized recombinant expression vector. The cells were then transferred to a shaker containing 30 ml of CD CHO medium and cultured at 37°C, 5% CO2, and 125 rpm for 24 hours. The electroporated cell suspension was centrifuged at 100 g for 10 minutes, the supernatant discarded, and the cells were resuspended in CD CHO medium supplemented with 25 μM MSX and 200 μg / ml bleomycin. The cells were then seeded into 24-well plates. After 3 weeks of culture, expression levels were determined by ELISA. The three wells with the highest expression levels were pooled and seeded into 96-well plates by limiting dilution for single-clone screening. Single-cell imaging was used to image the cells on days 0, 1, 2, 3, 7, and 15. After 15 days, expression levels were determined by ELISA, and the three cell lines with the highest expression levels were cryopreserved. After stability studies, the cell line used for vaccine production was determined, and a two-tier cell bank was established.

[0111] 4. Expression of target product A frozen working seed was revived in OPM-CHO CDP9 medium, scaled up in shake flasks, and finally transferred into a 5 L bioreactor for culture at an inoculation density of 0.8 × 10 6 The culture parameters were set at 37°C, pH 7.0, 150 rpm, and 40% dissolved oxygen. Cell viability, density, lactate content, and glucose content were measured daily. On the third day of culture, the viable cell density reached 3×10 6 When the cell viability reaches 70% cells / ml, add feed medium CDF18 and CDF26, inoculating 250 ml and 25 ml, respectively. Continue adding the same volume of feed medium every other day. Maintain the glucose content in the culture medium above 2 g / L. If it falls below this concentration, add glucose to 4 g / L. Terminate the culture after approximately 15 days, when the cell viability drops to 70%. Remove cells and cell debris through a depth filter, and collect the cell culture supernatant.

[0112] 5. Protein Purification Deep filter the cell culture supernatant using a filter with a 0.2-2 μm cutoff range. Adjust the filtered cell culture supernatant to pH 7.5. Equilibrate the anion exchange column Capto Q with 20 mM PB buffer (pH 7.5) to the UV absorbance baseline. Once the pH stabilizes, pass the cell supernatant through the column and equilibrate to the UV absorbance baseline with the same buffer. Elute the column linearly with 20 mM PB elution buffer (pH 7.5) containing 1 M sodium chloride to collect the target product. Low pH inactivation (pH 3.0-4.0, 18-25°C for 60 minutes) is performed to purify the anion-purified product. Ammonium sulfate (final concentration 1 M) was added to the inactivated product, and the pH was adjusted to 7.5. A hydrophobic chromatography column, Capto PhenylImpRes, was equilibrated with 50 mM PB buffer (pH 7.5) + 1 M ammonium sulfate buffer until the UV absorbance reached baseline and the pH remained stable. The pH-adjusted inactivated solution was then passed through the column, and the column was equilibrated with the same buffer. Finally, the target product was linearly eluted with 50 mM PB buffer (pH 7.5). The hydrophobic purification product was purified by Sephacryl S-300 High Resolution molecular sieve chromatography, and the buffer was exchanged to obtain the purified protein. The purified protein was then nanofiltered through a 15 nm filter and sterilized by filtration through a 0.22 μm filter to obtain the gE solution (stock solution).

[0113] 6. Detection of protein content in gE solution (stock solution) (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.

[0114] 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 ultrasonically degas for 5 minutes.

[0115] (2) Preparation of 2% DMSO solution 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix well.

[0116] (3) gE solution (stock solution) sample testing = 1 \* GB3① Preparation of protein calibration standard solution samples Preparation of BSA reference stock solution: Weigh 21.9 mg of BSA (China Food and Drug Inspection Institute) into a 10 ml volumetric flask and dissolve in 6 ml of 10 mM phosphate solution (pH 6.0). Then, dilute to the mark with 10 mM phosphate solution (pH 6.0) and mix thoroughly. Finally, aliquot into 500 μl / tubes to obtain the BSA reference stock solution (concentration: 2.19 mg / ml). Place 457 μl of BSA reference stock solution (concentration: 2.19 mg / ml) in a 5 ml volumetric flask and dilute to the mark with phosphate solution (10 mM; pH 6.0). Shake well to obtain a solution with a concentration of 200 μg / ml. Then, pipette 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution into different centrifuge tubes. Then, add 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0), respectively, and mix well to obtain solutions with concentrations of 175 μg / ml, 150 μg / ml, 125 μg / ml, and 100 μg / ml, respectively. Pipette 0.5 ml of each of the above five concentration solutions into different injection vials, then add 0.5 ml of 2% DMSO solution 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.

[0117] = 2 \* GB3② Preparation of test samples Dilute 1 ml of the sample solution in two steps until the maximum dilution is 2. 8 times (256 times). 1 , 2 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 7 , 2 8 Take 0.5 ml of the sample solution and put it into 8 injection vials, then add 0.5 ml of 2% DMSO solution to each vial, cover the vials and mix well to obtain the dilution multiple of 2. 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 7 , 2 8 , 2 9 of test samples.

[0118] = 3 \* GB3③ Load the sample into the liquid chromatograph and run the analysis method The injection vials containing the protein calibration standard solution and the injection vials of the test samples were loaded into the injection tray of the high performance liquid chromatograph, and the analytical method was run using the liquid chromatograph.

[0119]

[0120] = 4 \* GB3④ calculates protein content Establishment of standard curve Use the peak area values of the BSA calibration standard solution samples to establish a calibration standard curve in which protein concentration is proportional to peak area. Content calculation Select the test sample with a protein peak area within the range of the standard curve as the target test sample, then substitute the target test sample protein peak area value into the calibration standard curve formula, and then multiply it by the target test sample dilution factor to calculate the protein content in the gE solution (stock solution).

[0121] 2) Liposome preparation and determination of dioleoylphosphatidylcholine and cholesterol concentrations 1. Liposome Preparation (Theoretical Concentrations of Dioleoylphosphatidylcholine and Cholesterol are 4 mg / ml and 1 mg / ml, respectively) 400 mg of dioleoylphosphatidylcholine (DOPC; Japan Fine Chemicals Co., Ltd.) and 100 mg of cholesterol (Japan Fine Chemicals Co., Ltd.) were weighed separately. DOPC and cholesterol were then completely dissolved in 10 ml of anhydrous ethanol and mixed evenly to form the organic phase. 10 ml of the organic phase was injected into 90 ml of 10 mM PBS buffer solution (pH 7.0) to produce liposome colostrum. The liposomes were then granulated using a high-pressure microfluidizer to a particle size of approximately 100 nm. Ethanol was then removed by dialysis. Finally, the liposomes were sterilized by filtration using a 0.22 μm sterilizing filter to obtain the finished liposome product.

[0122] 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.

[0123] (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.

[0124] 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.

[0125] 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.

[0126] 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].

[0127] (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.

[0128] (4) Instrument parameters and elution procedures

[0129] (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.

[0130] 3) Preparation and concentration determination of Quillaja saponin QS-21 solution 1. Preparation of Quillaja saponin QS-21 solution (theoretical concentration 4 mg / ml) Weigh 40 mg of Quillaja saponin QS-21 (Desert King), dissolve it completely in 4 ml of 10 mM phosphate solution (pH 6.0), dilute the volume to 10 ml with 10 mM phosphate solution (pH 6.0), and mix well to obtain the Quillaja saponin QS-21 solution.

[0131] 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.

[0132] 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.

[0133] (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.

[0134] 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.

[0135] (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.

[0136] (4) Instrument parameters and elution procedures

[0137] (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.

[0138] 4) Vaccine Sample Preparation [10 ml volume; target concentration of gE: 100 μg / ml; target concentration of Quillaja saponin QS-21: 100 μg / ml; target concentration of 3D-MPL: 100 μg / ml; liposome composition: target concentrations of dioleoylphosphatidylcholine and cholesterol: 2 mg / ml and 0.5 mg / ml, respectively] Detect the concentration of the liposome solution: the concentrations of DOPC and cholesterol in the liposomes are 4.20 and 1.05 mg / ml, respectively; detect the concentration of the Quillaja saponin QS-21 solution: the concentration of Quillaja saponin QS-21 is 4.14 mg / ml; take 4.76 ml of liposomes, add 0.24 ml of Quillaja saponin QS-21 solution and 1 ml of 3D-MPL solution (concentration is 1 mg / ml), and stir evenly to obtain the adjuvant.

[0139] Detect the protein concentration of the gE solution / stock solution: the protein concentration is 5.063 mg / ml; add 198 μl of the gE solution / stock solution to the adjuvant, and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir well to obtain the vaccine; the volume of the gE solution / stock solution added (ml) is calculated as: mass of gE added (μg) / concentration of the gE solution / stock solution (μg / ml).

[0140] 5) Simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine, and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine 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.

[0141] 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 ultrasonically degas for 5 minutes.

[0142] (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.

[0143] 1.25% dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, and mix well.

[0144] 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix well.

[0145] 3.5% dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, and mix well.

[0146] 2. Vaccine Sample Testing S1. Prepare gE calibration standard solution sample Place 284 μl of the gE reference solution (protein concentration: 3.52 mg / ml) in a 5 ml volumetric flask and dilute to the mark with 10 mM phosphate solution (pH 6.0). Shake well to obtain a 200 μg / ml solution. Pipette 1 ml of this 200 μg / ml solution into a centrifuge tube, add 0.33 ml of 10 mM phosphate solution (pH 6.0), and mix well to obtain a 150 μg / ml solution. Pipette 0.5 ml of this solution into a sample vial, add 0.5 ml of 2% DMSO solution, cap the vial, and mix well to obtain a gE calibration standard solution with a protein concentration of 75 μg / ml.

[0147] S2. Prepare BSA calibration standard solution sample Place 457 μl of BSA reference stock solution (concentration: 2.19 mg / ml) in a 5 ml volumetric flask and dilute to the mark with phosphate solution (10 mM; pH 6.0). Shake well to obtain a solution with a concentration of 200 μg / ml. Then, pipette 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution into different centrifuge tubes. Then, add 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0), respectively, and mix well to obtain solutions with concentrations of 175 μg / ml, 150 μg / ml, 125 μg / ml, and 100 μg / ml, respectively. Pipette 0.5 ml of each of the above five concentration solutions into different injection vials, then add 0.5 ml of 2% DMSO solution and mix well to obtain BSA 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.

[0148] S3. Preparation of QS-21 calibration standard solution sample 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.

[0149] S4. Preparation of 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.

[0150] S5. Prepare test samples Pipette 0.5 ml of the vaccine sample solution to be tested into a sampling vial, add 0.5 ml of 2% DMSO solution, cover the vial cap, and mix well.

[0151] S6. Load the sample into the liquid chromatograph and run the analysis method The injection vials containing the gE calibration standard solution, the BSA calibration standard solution, the Quillaja saponin QS-21 calibration standard solution, the dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solutions, and the test sample vials were loaded onto the injection tray of the high performance liquid chromatography (HPLC) instrument, and the analytical method was run using the HPLC instrument.

[0152]

[0153] S7. Calculate the relative deviation of protein retention time in vaccines Calculate the relative deviation of the retention time of the vaccine protein peak and the gE calibration standard solution peak using the formula: Relative deviation = [|Vaccine protein peak retention time - average retention time| / average retention time] × 100%. Note: The average retention time is the average of the retention time of the vaccine protein peak and the retention time of the qualitative standard (gE calibration standard solution sample). Criteria: If the relative deviation is no greater than 2%, the protein antigen in the vaccine sample can be determined to be gE. The liquid chromatogram of the gE calibration standard solution (75 μg / ml) sample is as follows: Figure 1 shown.

[0154]

[0155] S8. Calculate the content of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine (1) Establishment of standard curve The peak area values of the calibration standard solutions of BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol were used to establish calibration standard curves in which the concentrations of BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol were proportional to the peak area.

[0156] BSA calibration standard solution sample test results and standard curve, such as Figure 2 shown Protein calibration standard solution Retention time (min) Peak area (mAU*min) tailing factor Calibration standard solution-1 (50 μg / ml) 7.708 2814156 1.769 Calibration standard solution-2 (62.5 μg / ml) 7.703 3543813 1.797 Calibration standard solution-3 (75 μg / ml) 7.698 4312984 1.738 Calibration standard solution-4 (87.5 μg / ml) 7.706 5085582 1.764 Calibration standard solution-5 (100 μg / ml) 7.697 5827499 1.754 BSA peak area standard curve: y = 60497x - 224266, R 2 =0.9999.

[0157] The test results and standard curve of the sample of Quillaja saponin QS-21 calibration standard solution are as follows: Figure 3 shown QS-21 Calibration Standard Solution Peak area (A+B isomer) (μAU*min) Tailing factor (A isomer) Tailing factor (B isomer) Calibration standard solution-1 (25 μg / ml) 145662 1.500 1.267 Calibration standard solution-2 (37.5 μg / ml) 218382 1.420 1.224 Calibration standard solution-3 (50 μg / ml) 283212 1.427 1.231 Calibration standard solution-4 (62.5 μg / ml) 362991 1.450 1.275 Calibration standard solution-5 (75 μg / ml) 433930 1.438 1.274 Calibration standard solution-6 (87.5 μg / ml) 506752 1.414 1.291 Standard curve of peak area of Quillaja saponin QS-21: y=5781.4x-371.7, R 2 =0.9995.

[0158] Dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution sample test results and standard curve, such as Figure 4 and Figure 5 shown DOPC calibration standard solution Peak area (μAU*min) tailing factor Standard curve solution-1 (500 μg / ml) 840670 1.766 Standard curve solution-2 (750 μg / ml) 1255779 1.553 Standard curve solution-3 (1000 μg / ml) 1710671 1.173 Standard curve solution-4 (1250 μg / ml) 2141153 0.962 Standard curve solution-5 (1500 μg / ml) 2577165 0.994 DOPC peak area standard curve: y = 1743.3x - 38258, R 2 =0.9999.

[0159] Cholesterol calibration standard solution Peak area (μAU*min) tailing factor Standard curve solution-1 (125 μg / ml) 1349974 1.550 Standard curve solution-2 (187.5 μg / ml) 1980964 1.637 Standard curve solution-3 (250 μg / ml) 2600131 1.700 Standard curve solution-4 (312.5 μg / ml) 3198711 1.789 Standard curve solution-5 (375 μg / ml) 3794897 1.753 Cholesterol peak area standard curve: y=9772.1x+141898, R2=0.9998.

[0160] (2) Content calculation The peak area values of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine sample were respectively substituted into the calibration standard curve formula of the corresponding components, and then multiplied by the dilution factor of the vaccine (two times) to calculate the content of gE, QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in the vaccine. The retained peaks of each component of the vaccine were completely separated, such as Figure 6 shown.

[0161]

[0162] 6) Comparison of the differences in protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples detected by simultaneous testing and existing technology (three separate tests) 1. Vaccine Sample Testing (Existing Technology) A method for testing the content of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccine samples three times respectively.

[0163] (1) Detection of gE content Since components in the composite adjuvant, such as Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol, can interfere with the Lowry method for detecting vaccine gE content, it is necessary to remove the influence of the composite adjuvant during detection to reduce detection errors.

[0164] (A) Reagent preparation Folin-phenol test solution: Add 5.0 ml of phenol reagent to 75 ml of water and mix thoroughly. 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 thoroughly. 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 thoroughly. 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 thoroughly. Solution B: Prepare potassium tartrate solution (0.5 g → 50 ml): copper sulfate solution (0.25 g → 30 ml) in a volume ratio of 5:3 (volume ratio) and mix thoroughly. Alkaline copper solution: Prepare solution A: solution B: water in a volume ratio of 40:8:2 (volume ratio) and mix thoroughly.

[0165] (B) Preparation of protein reference working solution 200 μg / ml Protein Reference Solution (Bovine Serum Albumin Solution): Dissolve one vial of Protein Reference Solution in purified water to prepare a 200 μg / ml protein reference solution. Aliquot into 5 ml plastic tubes and store at -20°C or below. Dilute twice before use and mix thoroughly to obtain the reference working solution (100 μg / ml).

[0166] (C) Composite adjuvant control preparation The concentrations of DOPC and cholesterol in the liposomes are 4.20 and 1.05 mg / ml, respectively; the concentration of Quillaja saponin QS-21 is 4.14 mg / ml; take 4.76 ml of liposomes, add 0.24 ml of Quillaja saponin QS-21 solution, then add 1 ml of 3D-MPL solution (concentration is 1 mg / ml), and finally add phosphate solution (10 mM; pH 6.0) to 10 ml, and stir evenly to obtain the product.

[0167] (D) gE concentration detection Preparation and testing of test samples: Accurately pipette an appropriate amount of vaccine sample and dilute with water until the protein content is within the standard curve range; accurately pipette 1.0 ml of the above solution into a test tube in duplicate, add 1.0 ml of alkaline copper solution, mix well, and let it stand at room temperature for 10 minutes. Add 4.0 ml of folin phenol test solution, mix immediately, let it stand at room temperature for 30 minutes, and after color development, measure the absorbance at a wavelength of 650 nm by UV-visible spectrophotometry (after color development, if turbidity is found, centrifuge at 3000 rpm / min for 15 minutes, and then take the supernatant for determination).

[0168] Protein control test: Accurately pipette 0.2ml, 0.4ml, 0.6ml, 0.8ml, and 1.0ml of 100μg / ml protein control solution into test tubes in duplicate; if the solution is less than 1ml, add water to 1.0ml and proceed in the same manner starting from "add 1.0ml of alkaline copper solution".

[0169] Test of composite adjuvant reference substance: Accurately pipette an appropriate amount of composite adjuvant sample and dilute it with water (the dilution factor is the same as that of the vaccine sample); accurately pipette 1.0 ml of the above solution into a test tube, perform two replicates, and proceed in the same manner starting from "add 1.0 ml of alkaline copper solution" to serve as the adjuvant control.

[0170] (E) Calculation of gE concentration in vaccines A linear regression was performed using the protein control content as the X-axis and the corresponding mean absorbance as the Y-axis to determine the linear regression equation. The protein absorbance of the test solution was calculated by subtracting the mean absorbance of the adjuvant control solution from the mean absorbance of the test solution. Substituting the protein absorbance of the test solution into the linear regression equation, the protein content of the test sample was calculated. The gE content of the vaccine sample (μg / ml) is calculated as A*n. Where: A is the protein content of the test sample (μg / ml) obtained by substituting the protein absorbance of the test solution into the linear regression equation; n is the dilution factor of the vaccine sample.

[0171] (2) Detection of the content of Quillaja saponin QS-21 The same as the “Concentration detection of Quillaja saponin QS-21 solution” in the item “3) Preparation and concentration detection of Quillaja saponin QS-21 solution” in Example 1.

[0172] (3) Detection of dioleoylphosphatidylcholine (DOPC) and cholesterol content The same as the "Detection of Dioleoylphosphatidylcholine (DOPC) and Cholesterol Concentrations" in the item "2) Liposome Preparation and Detection of Dioleoylphosphatidylcholine (DOPC) and Cholesterol Concentrations" in Example 1.

[0173] Second, the differences in the contents of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol in vaccine samples were compared between simultaneous testing (one test) and prior art testing (three separate tests). Figure 7 shown

[0174] 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 qualitatively detecting gE in a recombinant varicella-zoster vaccine and quantitatively detecting gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine, characterized in that: The following steps are involved: S1. Prepare gE calibration standard solution samples (for qualitative and / or quantitative detection of gE in vaccines); S2. Prepare BSA calibration standard solution samples (for quantitative detection of gE in vaccines); S3, preparing a sample of a calibration standard solution of Quillaja saponin QS-21; S4, preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples; S5. preparing test samples; S6. Load the sample into the liquid chromatograph and run the analysis method; S7. Calculate the relative deviation of protein retention time in the vaccine; S8. Calculate the content of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine.

2. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The recombinant varicella-zoster vaccine consists of gE and a composite adjuvant; the gE is the extracellular segment of the varicella-zoster virus glycoprotein E, an optional molecular structure from the N-terminus to the C-terminus of which is: AA31-AA544, and its amino acid sequence is shown in SEQ ID NO.1; the composite adjuvant has dioleoylphosphatidylcholine, cholesterol, monophosphoryl lipid A and Quillaja saponin QS-21 as its main components.

3. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The protein concentration of the gE calibration standard solution sample in S1 is 5 μg / ml to 200 μg / ml, and the protein concentration of the BSA calibration standard solution sample in S2 is 5 μg / ml to 200 μg / ml.

4. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The concentration of the Quillaja saponin QS-21 calibration standard solution sample in S3 is 10 μg / ml to 100 μg / ml, and the concentrations of dioleoylphosphatidylcholine and cholesterol in the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples in S4 are 200 μg / ml to 2000 μg / ml and 50 μg / ml to 500 μg / ml, respectively.

5. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The concentrations of gE, Quillaja saponin QS-21, monophosphoryl lipid A, dioleoylphosphatidylcholine and cholesterol in the test sample in S5 are 5μg / ml~200μg / ml, 10μg / ml~100μg / ml, 10μg / ml~100μg / ml, 200μg / ml~2000μg / ml and 50μg / ml~500μg / ml, respectively.

6. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The chromatographic column of the analysis method in S6 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 S6 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 an ultraviolet detector, the detection wavelength is 200 to 400nm.

7. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The elution procedure of the analytical method in S6 includes: an elution method for eluting the retention peaks of quillaja saponin QS-21 and gE; an elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol; the elution method for eluting the retention peaks of quillaja saponin QS-21 and gE is 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%; the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution, the acetonitrile or methanol concentration in the mobile phase is 90% to 100%, the elution time is 2 minutes to 1920 minutes, and the trifluoroacetic acid concentration in the mobile phase is 0.01% to 0.10%.

8. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The calculation formula for the relative deviation of the retention time of the protein in the vaccine in S7 is as follows = [|retention time of the vaccine protein retention peak - average retention time| / average retention time] × 100%. Note: the average retention time is the average retention time of the vaccine protein retention peak and the retention time of the gE calibration standard solution sample retention peak; the criterion for determining whether the protein in the vaccine sample is gE: the relative deviation is not higher than 2%.

9. The method for simultaneous qualitative detection of gE in recombinant varicella-zoster vaccine and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the vaccine according to claim 1, characterized in that: The calculation of the contents of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine in S8 includes two calculation methods: Calculation method 1: First, establish a calibration standard curve in which the concentration of gE or BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to the peak area. Then, substitute the peak area values of gE, Quillaja 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; then, 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 = test sample A peak area * A calibration standard solution sample concentration * dilution factor / A calibration standard solution sample peak area, where A represents "gE" or "Quillaja saponin QS-21" or "dioleoylphosphatidylcholine" or "cholesterol".

10. Use of the method for simultaneous qualitative detection of gE and quantitative detection of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in recombinant varicella-zoster vaccine according to any one of claims 1 to 9 in simultaneous detection of components in recombinant varicella-zoster vaccine, characterized in that: The recombinant varicella-zoster vaccine includes gE and a composite adjuvant; the gE is the extracellular segment of the varicella-zoster virus glycoprotein E, and the composite adjuvant is a composite adjuvant containing neutral liposomes, the immunopotentiator monophosphoryl lipid A, and the immunopotentiator Quillaja saponin QS-21; and is used for qualitative detection of gE in the recombinant varicella-zoster vaccine, as well as for detecting the content of gE and / or Quillaja saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol in the recombinant varicella-zoster vaccine.

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