Method for detecting contents of main components of protein and composite adjuvant in recombinant protein vaccine and application

Abstract

The invention discloses a method for detecting the contents of main components of protein and a composite adjuvant in a recombinant protein 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 dioleoyl phosphatidylcholine and cholesterol calibration standard solution sample; preparing a detection sample; loading the sample into a liquid chromatograph, and operating an analysis method; and calculating the contents of main components of proteins and adjuvants in the vaccine. The method is a high performance liquid chromatography method capable of simultaneously detecting the contents of protein, dioleoyl phosphatidylcholine, cholesterol and saponin QS-21 in the recombinant protein vaccine. According to the method, the content of protein, dioleoyl phosphatidylcholine, cholesterol and saponin QS-21 in the recombinant protein vaccine can be accurately detected only through one-time detection, and the verification efficiency of the recombinant protein vaccine is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method and application for detecting the content of main components of protein and composite adjuvant in a recombinant protein vaccine. Background Art

[0002] Herpesviruses are large, enveloped DNA viruses with complex structures. They belong to the family Herpesviridae and are divided into three subfamilies: α, β, and γ. These viruses have a broad host range, infecting humans and other vertebrates. Herpesviruses that infect humans include varicella-zoster virus (VZV), herpes simplex virus (HSV) type 1 and type 2, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and Kaposi's sarcoma-associated herpesvirus (KSHV). Herpesviruses that infect animals include pseudorabies virus (PRV) in pigs and Marek's disease virus (MDV) in chickens. Herpesviruses primarily invade the skin, mucous membranes, and nervous system, potentially causing diseases such as chickenpox, viral rashes, herpes simplex, lymphadenopathy, and genital herpes. Some herpesviruses may also invade the central nervous system, causing diseases such as viral encephalitis. Glycoprotein E is one of the major glycoproteins of herpesviruses and is highly immunogenic.

[0003] Varicella zoster virus (VZV), also known as human herpesvirus type 3, is a human alphaherpesvirus that causes diseases such as chickenpox and herpes zoster. The VZV genome is approximately 125 kb in size and encodes approximately 69 proteins, including eight glycoproteins: glycoprotein B, glycoprotein C, glycoprotein E, glycoprotein H, glycoprotein I, glycoprotein K, glycoprotein L, and glycoprotein M. Glycoprotein E is the most abundant and highly immunogenic glycoprotein on the viral envelope and host cell membranes, inducing both cellular and humoral immunity.

[0004] GlaxoSmithKline's recombinant protein herpes 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: a truncated VZV glycoprotein E (gE) expressed in CHO cells and the AS01B adjuvant system [the AS01B adjuvant system consists of the immunopotentiators 3D-MPL, the immunopotentiator QS-21, and liposomes; the main components of the liposomes are dioleoylphosphatidylcholine (DOPC) and cholesterol].

[0005] Compared with the live attenuated vaccines of Merck and Biopharma, GSK's adjuvanted subunit vaccine Shingrix® The protection rate is higher, but the clinical side effects are more severe. Therefore, the immunogenicity is better than Shingrix ® , but the clinical side effects may be lower than Shingrix ® The recombinant varicella-zoster vaccine is a promising candidate vaccine in clinical trials (immunogenicity data are available in CN 116747298 B and CN 117003896 B). The candidate vaccine contains a gE fusion protein (PADRE-gE-P2) antigen and an XA-401 compound adjuvant [the XA-401 compound adjuvant consists of the immunopotentiator Quillaja saponin QS-21 and liposomes; the main components of the liposomes are dioleoylphosphatidylcholine (DOPC) and cholesterol]. ® In contrast, the candidate vaccine removes the immunopotentiator 3D-MPL (3-O-deacyl-4´-monophosphoryl lipid A), which exacerbates clinical side effects.

[0006] To control vaccine quality, it is essential to test the content of protein and the main adjuvant components in the vaccine. Therefore, it is necessary to establish a method for detecting the content of protein and the main components of the composite adjuvant [dioleoylphosphatidylcholine (DOPC), cholesterol, and Quillaja saponin QS-21] in the vaccine.

[0007] Currently, the recombinant herpes zoster vaccine Shingrix is on the market. ® In the quality control of Shingrix, three different methods were used to detect protein content, Quillaja saponin QS-21 content, dioleoylphosphatidylcholine (DOPC) and cholesterol content. ® The methods for determining the content of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol in vaccines are cumbersome, time-consuming, material-consuming, labor-intensive and inefficient. Summary of the Invention

[0008] The purpose of the present invention is to provide a method and application for detecting the content of the main components of protein and composite adjuvant in recombinant protein vaccines, so as to solve the technical problems in the existing technology of detecting components in vaccines that are cumbersome, time-consuming, material-consuming, labor-consuming and inefficient.

[0009] To achieve the above objectives, the present invention provides the following technical solutions: The present invention provides a method for detecting the content of main components of protein and composite adjuvant in recombinant protein vaccine, which adopts liquid chromatography method.

[0010] Furthermore, the recombinant protein is a herpes virus glycoprotein E extracellular region or a fusion protein thereof prepared using a eukaryotic expression system; the herpes virus includes varicella-zoster virus (VZV), herpes simplex virus (HSV) type I, herpes simplex virus (HSV) type II, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus (KSHV), pseudorabies virus (PRV), and Marek's disease virus (MDV); an optional molecular structure of the varicella-zoster virus (VZV) glycoprotein E extracellular region (hereinafter referred to as gE) from N-terminus to C-terminus is AA31-AA544, and the amino acid sequence is as shown in SEQ ID As shown in NO.1; an optional protein of the varicella-zoster virus (VZV) glycoprotein E extracellular region fusion protein is PADRE-gE-P2, and an optional molecular structure from N-terminus to C-terminus is: PADRE-GSGSG-gE-GGS-P2, and the amino acid sequence is shown in SEQ ID NO.2.

[0011] 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.

[0012] Furthermore, the optional molecular structure of the glycoprotein E extracellular region of varicella-zoster virus (VZV) 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-AA534, AA31-AA533, AA31-AA532, AA31-AA531, AA31-AA530, AA31-AA529, AA31-AA528, AA 31-AA527, AA31-AA526, AA31-AA525, AA31-AA524, AA31-AA523, AA31-AA522, AA31-AA521, AA31-AA520.

[0013] Furthermore, the optional protein of the varicella-zoster virus (VZV) glycoprotein E extracellular region fusion protein may also be one of the following: gE-Fc fusion protein, gE-Fc (mutant) fusion protein, human interleukin-gE fusion protein, interferon-gE fusion protein, immune enhancing peptide-gE fusion protein, human interleukin-gE-Fc fusion protein, interferon-gE-Fc fusion protein, immune enhancing peptide-gE-Fc fusion protein, human interleukin-gE-Fc (mutant) fusion protein, interferon-gE-Fc (mutant) fusion protein, immune enhancing peptide-gE-Fc (mutant) fusion protein.

[0014] Furthermore, the Fc is the Fc segment (Fc) of human immunoglobulin gamma (IgG), characterized in that IgG includes IgG1, IgG2, IgG3 and IgG4; the amino acids mutated in the Fc (mutant) to enhance the antibody-dependent cell-mediated phagocytosis (ADCP) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) of Fc include but are not limited to S239D and / or A330L and / or I332E and / or G236A; the immune enhancing peptides include but are not limited to universal DR Th epitope peptide PADRE and / or tetanus toxin Th epitope peptide P2; the human interleukins include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11) L-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 ( IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), interleukin-36 (IL-36), interleukin-37 (IL-37), interleukin-38 (IL-38); the human interferon includes interferon α (IFN-α), interferon β (IFN-β), and interferon γ (IFN-γ); the "-" in the fusion protein (such as: gE-Fc fusion protein) represents a "connecting peptide", characterized in that the "connecting peptide" includes but is not limited to the following: GGS, GGGS, GGGGS, GSGSG.

[0015] Furthermore, the compound adjuvant is XA-401 compound adjuvant, which is composed of neutral liposomes and the immunopotentiator Quillaja saponin QS-21. The main components of the neutral liposomes are dioleoylphosphatidylcholine and cholesterol.

[0016] Furthermore, the protein content of the recombinant protein 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, and the QS-21 content is 20 μg / ml to 200 μg / ml.

[0017] Furthermore, the recombinant protein vaccine may also contain other immunopotentiators, including but not limited to 3D-MPL (3-O-deacyl-4´-monophosphoryl lipid A) and / or saponin QS-7 and / or CpG ODN (artificially synthesized oligodeoxynucleotide sequence containing non-methylated cytosine guanine dinucleotide) and / or Poly I:C (polyinosinic-polycytidylic acid).

[0018] Furthermore, the CpG ODN includes but is not limited to the following three categories: Class A, Class B, and Class C; the Class A CpG ODN is characterized in that: a CpG dinucleotide palindromic sequence is a core, two poly G tails are at both ends, the phosphodiester bond backbone is partially thiolated, and a higher-order structure is formed by the palindromic sequence and poly G, which can activate plasmacytoid dendritic cells to induce a large amount of type I interferon, but has weak activity on B cells, such as: CpG 2216; the Class B CpG ODN is characterized in that: a fully thiolated linear CpG ODN has strong immunostimulatory activity on B cells, but cannot activate plasmacytoid dendritic cells, such as: CpG1018, CpG2006, and CpG 1826; the Class C CpG ODN is characterized in that: a fully thiolated CpG ODN can form dimers through palindromic sequences and has the activity of both type A and type B CpGODN. It can activate both plasmacytoid dendritic cells and B cells, such as CpG 2395.

[0019] Furthermore, the recombinant protein vaccine may also contain cationic lipids, including but not limited to the following: dioleoyl-trimethylammonium chloride (DOTAP), ALC-0315, D-Lin-MC3-DMA, and SM102.

[0020] Furthermore, the recombinant protein 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.

[0021] Furthermore, the buffer component that maintains the pH value of the vaccine stable 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.

[0022] 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.

[0023] Furthermore, the recombinant protein vaccine may also contain components that maintain the stability of the protein in the vaccine.

[0024] 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.

[0025] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra high performance liquid chromatography.

[0026] 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.

[0027] Furthermore, the mobile phase may also contain methanol.

[0028] 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).

[0029] Furthermore, the liquid chromatography method is a liquid chromatography internal standard method or a liquid chromatography external standard method.

[0030] Furthermore, the liquid chromatography external standard method comprises the following steps: S1. Prepare protein calibration standard solution sample; S2. Prepare QS-21 calibration standard solution sample; S3, preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples; S4, preparing test samples; S5. Load the sample into the liquid chromatograph and run the analysis method; S6. Calculate the protein, QS-21, dioleoylphosphatidylcholine, and cholesterol contents in the vaccine.

[0031] Furthermore, in the preparation of the protein calibration standard solution sample in S1, the protein used can be selected from any one of the following, but is not limited to the following: bovine serum albumin (BSA), bovine gamma-globulin (BGG), varicella-zoster virus (VZV) glycoprotein E (gE) extracellular segment (gE), PADRE-gE-P2 fusion protein.

[0032] Furthermore, the protein calibration standard solution sample in S1 has a protein concentration of 5 μg / ml to 200 μg / ml.

[0033] Furthermore, the protein calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0034] Furthermore, the purity of the QS-21 standard solution sample used to prepare the QS-21 calibration standard solution sample in S2 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, to prepare a QS-21 standard solution sample; using liquid chromatography to detect the concentration of the QS-21 solution (purity not less than 90%), to prepare a QS-21 standard solution sample.

[0035] 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.

[0036] Furthermore, the concentration of QS-21 in the QS-21 calibration standard solution sample is 10 μg / ml to 100 μg / ml.

[0037] Furthermore, the QS-21 calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0038] Furthermore, in S3, 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 (prepared separately); 2. Preparing a dioleoylphosphatidylcholine and cholesterol calibration standard solution sample (mixed preparation).

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Furthermore, the dioleoylphosphatidylcholine and cholesterol calibration standard solution samples may contain DMSO at a concentration of 0.5% to 5%.

[0044] Furthermore, the vaccine used to prepare the test sample in S4 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.

[0045] Furthermore, the sample tested in S4 has a protein concentration of 5 μg / ml to 200 μg / ml, a Quillaja 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.

[0046] Furthermore, the test sample may contain DMSO at a concentration of 0.5% to 5%.

[0047] Furthermore, the detector of the liquid chromatograph in S5 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0048] Furthermore, the analysis method in S5 includes a chromatographic column, liquid chromatography parameters, and an elution procedure.

[0049] Furthermore, the chromatographic column used in the analysis method in S5 is a reverse phase chromatographic column.

[0050] Furthermore, the liquid chromatography parameters of the analysis method in S5 include column temperature, injection volume, flow rate, detector parameters, etc.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] Furthermore, the elution procedure of the analysis method in S5 includes at least the following two elution methods: an elution method for eluting the retention peaks of Quillaja saponin QS-21 and protein; and an elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol.

[0060] Furthermore, the elution method for eluting the saponin QS-21 and the protein retention peak is gradient elution, and the acetonitrile concentration gradient in the mobile phase is 5% to 95%, such as 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 65%, 45% to 65%, 45% to 70%, 45% to 75%, 45% to 75%. ~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%.

[0061] Furthermore, the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0062] 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%.

[0063] 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%.

[0064] Furthermore, in the elution procedure of the analysis method in S5, an isocratic elution method can be added between the elution method for eluting the retention peak of Quillaja saponin QS-21 and protein and the elution method for eluting the retention peak of dioleoylphosphatidylcholine and cholesterol.

[0065] Furthermore, an isocratic elution method was added between the elution method for eluting the retention peak of Quillaja saponin QS-21 and protein and the elution method for eluting the retention peak of dioleoylphosphatidylcholine and cholesterol, wherein the concentration of acetonitrile or methanol was lower than the concentration of acetonitrile or methanol in the mobile phase for eluting dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0066] Furthermore, an isocratic elution method is added between the elution method for eluting the retention peak of Quillaja saponin QS-21 and protein and the elution method for eluting the retention peak 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%.

[0067] Furthermore, in the elution procedure of the analysis method in S5, a gradient elution method can be added between the elution method for eluting the retention peak of Quillaja saponin QS-21 and protein and the elution method for eluting the retention peak of dioleoylphosphatidylcholine and cholesterol.

[0068] Furthermore, a gradient elution method is added between the elution method for eluting the retention peak of Quillaja saponin QS-21 and protein and the elution method for eluting the retention peak 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 (DOPC) and cholesterol.

[0069] Furthermore, a gradient elution method is added between the elution method for eluting the retention peak of Quillaja saponin QS-21 and protein and the elution method for eluting the retention peak 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%.

[0070] Furthermore, in the elution procedure of the analysis method in S5, a pre-equilibration method and / or a pre-elution method may be added before the elution method for eluting Quillaja saponin QS-21 and the protein retention peak.

[0071] Furthermore, a pre-equilibration method is added before the elution method for eluting the Quillaja 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 the Quillaja saponin QS-21 and the protein retention peak.

[0072] Furthermore, a pre-equilibrium method is added before the elution method of eluting Quillaja saponin QS-21 and the protein retention peak, 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%.

[0073] Furthermore, a pre-elution method is added before the elution method for eluting Quillaja saponin QS-21 and the protein retention peak, and the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient for eluting Quillaja saponin QS-21 and the protein retention peak.

[0074] Further, a pre-elution method is added before the elution method of eluting the saponin QS-21 and the protein retention peak, 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

[0075] Furthermore, in the elution procedure of the analysis method in S5, 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.

[0076] 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.

[0077] 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%.

[0078] 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 Quillaja saponin QS-21 and protein.

[0079] 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%.

[0080] Furthermore, the calculation of the protein, QS-21, dioleoylphosphatidylcholine and cholesterol contents in the vaccine in S6 includes calculation method one and calculation method two.

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

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

[0083] Furthermore, the second calculation method is to directly calculate the content (concentration) of protein, 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 "protein" or "Quillaja saponin QS-21" or "dioleoylphosphatidylcholine (DOPC)" or "cholesterol".

[0085] The present invention also provides an application of a method for detecting the content of proteins and main components of a composite adjuvant in a recombinant protein vaccine to simultaneously detect components in the recombinant protein vaccine, wherein the proteins in the recombinant protein vaccine include the extracellular region of herpes virus glycoprotein E or a fusion protein thereof prepared using a eukaryotic expression system; the herpes viruses include varicella-zoster virus (VZV), herpes simplex virus (HSV) type I, herpes simplex virus (HSV) type II, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus (KSHV), pseudorabies virus (PRV), and Marek's disease virus (MDV); the composite adjuvant is XA-401, whose main components are dioleoylphosphatidylcholine, cholesterol, and Quillaja saponin QS-21.

[0086] Furthermore, the method for detecting the content of the main components of protein and composite adjuvant in recombinant protein vaccine is used to simultaneously detect the components in recombinant varicella-zoster vaccine: 1. Simultaneously detect the content of protein and Quillaja 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, Quillaja saponin QS-21, and cholesterol; 5. Simultaneously detect the content of protein, Quillaja saponin QS-21, and dioleoylphosphatidylcholine; 6. Simultaneously detect the content of protein, dioleoylphosphatidylcholine and cholesterol; 7. Simultaneously detect the content of Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol; 8. Simultaneously detect the content of protein, 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 a method and application for detecting the content of protein and the main components of a composite adjuvant in a recombinant protein vaccine. This method not only addresses the technical problem of existing techniques requiring three separate tests for detecting protein, dioleoylphosphatidylcholine (DOPC), cholesterol, and Quillaja saponin QS-21 in vaccines, but also addresses the technical problem of existing techniques being limited to quantitative, but not qualitative, detection of protein in vaccines. The high-performance liquid chromatography method disclosed in the present invention can detect the contents of protein, dioleoylphosphatidylcholine (DOPC), cholesterol, and QS-21 in vaccines with only a single sample loading, significantly improving vaccine testing efficiency.

[0088] The present invention provides a method and application for detecting the content of protein and main components of composite adjuvant in recombinant protein vaccine, which can simultaneously detect the content of protein and / or Quillaja saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol in recombinant varicella-zoster vaccine.

[0089] The present invention provides a method and application for detecting the content of main components of proteins and composite adjuvants in recombinant protein vaccines, which can also be used together with liquid chromatography for qualitatively detecting proteins in recombinant protein vaccines to simultaneously qualitatively and quantitatively detect proteins in recombinant protein vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] 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.

[0091] Figure 1 This is the standard curve of concentration and peak area of Quillaja saponin QS-21 in Example 1 of the present invention.

[0092] Figure 2 This is the standard curve of dioleoylphosphatidylcholine concentration and peak area in Example 1 of the present invention.

[0093] Figure 3 This is the standard curve of cholesterol concentration and peak area in Example 1 of the present invention.

[0094] Figure 4 This is the standard curve of PADRE-gE-P2 fusion protein concentration and peak area in Example 1 of the present invention.

[0095] Figure 5This is a liquid chromatogram of Example 1 of the present invention for detecting the contents of proteins in the recombinant varicella-zoster vaccine containing PADRE-gE-P2 fusion protein and XA-401 composite adjuvant and the three main adjuvant components in the XA-401 composite adjuvant.

[0096] Figure 6 It is a comparison chart of the protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol contents in the vaccine samples detected by Example 1 of the present invention (one simultaneous detection; PADRE-gE-P2 fusion protein is a protein reference) and the prior art detection (three separate detections).

[0097] Figure 7 This is the standard curve of BSA concentration and peak area in Example 2 of the present invention.

[0098] Figure 8 It is a comparison chart of the protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol contents in the vaccine samples detected by Example 2 of the present invention (one simultaneous detection; BSA is a protein reference substance) and the prior art detection (three separate detections).

[0099] Figure 9 This is the standard curve of BSA concentration and peak area in Example 3 of the present invention.

[0100] Figure 10 This is the standard curve of concentration and peak area of Quillaja saponin QS-21 in Example 3 of the present invention.

[0101] Figure 11 This is the standard curve of dioleoylphosphatidylcholine concentration and peak area in Example 3 of the present invention.

[0102] Figure 12 This is the standard curve of cholesterol concentration and peak area in Example 3 of the present invention.

[0103] Figure 13 This is a liquid chromatogram of Example 3 of the present invention for detecting the contents of protein in the recombinant varicella-zoster vaccine containing gE and a composite adjuvant and Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in the composite adjuvant.

[0104] Figure 14 This is a comparison chart of the gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol contents in the vaccine sample detected in Example 3 of the present invention and the theoretical contents of each component. DETAILED DESCRIPTION

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0110] A method and application for detecting the content of main components of protein and composite adjuvant in a recombinant protein vaccine.

[0111] The recombinant protein may be gE or gE fusion protein.

[0112] An optional molecular structure of gE is (from N-terminus to C-terminus): AA31-AA544, the nucleic acid sequence of which is shown in "GenBank: DQ008354.1", and the amino acid sequence of which is shown in SEQ ID NO.1.

[0113] SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVF KGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLK VLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTF TSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGG An optional molecular structure of the gE fusion protein PADRE-gE-P2 is (from N-terminus to C-terminus): PADRE—GSGSG (connecting peptide)-gE (AA31-AA544)-GGS (connecting peptide)-P2, whose nucleic acid sequence is shown in CN 117003896 B, and its amino acid sequence is shown in SEQ ID NO.2.

[0114] AKFVAAWTLKAAAGSGSGSVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNV DQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVL KVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQR VASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGGGSQYIKANSKFIGITE After the nucleic acid sequence of the PADRE-gE-P2 fusion protein is codon-optimized and fully synthesized, a fusion protein expression plasmid is constructed, followed by a stable cell line construction, and then the target product (PADRE-gE-P2 fusion protein) is expressed. Finally, the PADRE-gE-P2 fusion protein is obtained by purification, and the protein concentration of the fusion protein solution is detected.

[0115] Liposomes were prepared, and the concentrations of dioleoylphosphatidylcholine and cholesterol in the liposomes were detected; a Quillaja saponin QS-21 solution was prepared, and the concentration of Quillaja saponin QS-21 was detected; and the PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine was prepared using the PADRE-gE-P2 fusion protein, liposomes, and the Quillaja saponin QS-21 solution.

[0116] The PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine has better immunogenicity in mice than GSK's recombinant herpes zoster vaccine Shingrix, see CN 116747298 B and CN 117003896 B for details.

[0117] Liquid chromatography was used to determine the protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol contents in the PADRE-gE-P2 fusion protein vaccine, and the results were compared with those obtained using existing techniques. The comparison demonstrated that both the liquid chromatography method and existing techniques can be used to determine the protein and major adjuvant components (dioleoylphosphatidylcholine, cholesterol, and Quillaja saponin QS-21) in the vaccine, with the liquid chromatography method providing a more accurate measurement of protein content. Furthermore, it is known to those skilled in the art that optimal liquid chromatography parameters and elution methods may vary when using reversed-phase chromatography columns containing different chromatographic fillers to determine target substances (e.g., PADRE-gE-P2 fusion protein, QS-21, DOPC, and cholesterol in the vaccine).

[0118] Example 1. Detection (1 test) of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples (PADRE-gE-P2 fusion protein as a protein standard) 1) Preparation of PADRE-gE-P2 fusion protein and protein concentration detection 1. Fusion protein codon optimization and whole gene synthesis For details, see patent CN 117003896 B.

[0119] 2. Construction of fusion protein expression plasmid For details, see patent CN 117003896 B.

[0120] 3. Construction of Stable Cell Lines For details, see patent CN 117003896 B.

[0121] 4. Expression of target product For details, see patent CN 117003896 B.

[0122] 5. Fusion Protein Purification For details, see patent CN 117003896 B.

[0123] VI. Fusion protein (vaccine stock solution) concentration detection [(Lowry method) first method] (1) 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.

[0124] (2) 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 make 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).

[0125] (3) Protein sample concentration detection Preparation and testing of test samples: Accurately pipette an appropriate amount of the sample to be tested 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 well 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).

[0126] 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".

[0127] (4) Calculation of protein sample concentration Perform a linear regression using the protein control concentration as the X-axis and the corresponding mean absorbance as the Y-axis to determine the linear regression equation. Substitute the mean absorbance of the test solution into the linear regression equation to determine the protein content of the test sample. The protein content of the test sample (μg / ml) = A*n; where A is the protein content of the test sample (μg / ml) obtained by substituting the measured absorbance of the test sample into the linear regression equation; n is the dilution factor of the test sample.

[0128] 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.

[0129] 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.

[0130] (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.

[0131] 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.

[0132] 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.

[0133] Mix dioleoylphosphatidylcholine (DOPC) and cholesterol standard solutions: 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].

[0134] (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.

[0135] (4) Instrument parameters and elution procedures (5) Calculation of dioleoylphosphatidylcholine and cholesterol concentrations With the concentration of the standard solution of dioleoylphosphatidylcholine or cholesterol as the abscissa (X) and the peak area of dioleoylphosphatidylcholine or cholesterol as the ordinate (Y), draw standard curves in which the concentration of dioleoylphosphatidylcholine and cholesterol are proportional to the peak area. Substitute the peak area of cholesterol or dioleoylphosphatidylcholine 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 and cholesterol in the sample solution to be tested.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] (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.

[0140] 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, 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.

[0141] (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.

[0142] (4) Instrument parameters and elution procedures (5) Calculation of concentration of Quillaja saponin QS-21 A linear regression curve was drawn with the concentration of the standard solution of Quillaja saponin QS-21 as the X-axis and the corresponding peak area as the Y-axis. The peak area of the test sample solution was substituted into the standard curve and then multiplied by the dilution factor of the test sample solution to obtain the content of QS-21 in the test sample solution.

[0143] 4) Vaccine sample preparation 1. Preparation of adjuvant The concentration of the liposome solution was detected: the concentrations of DOPC and cholesterol in the liposomes were 4.20 and 1.05 mg / ml, respectively. The concentration of the Quillaja saponin QS-21 solution was detected: the concentration of the Quillaja saponin QS-21 was 4.14 mg / ml. 4.76 ml of liposomes was added with 0.24 ml of the Quillaja saponin QS-21 solution and stirred evenly to obtain the adjuvant.

[0144] II. Vaccine Preparation [10 ml volume; target concentration of PADRE-gE-P2 fusion protein: 150 μg / ml; target concentration of Quillaja saponin QS-21: 100 μg / ml; liposome components: target concentrations of dioleoylphosphatidylcholine and cholesterol: 2 mg / ml and 0.5 mg / ml, respectively] Detect the protein concentration of the PADRE-gE-P2 fusion protein solution: the concentration of the PADRE-gE-P2 fusion protein is 1.52 mg / ml; add 0.99 ml of the PADRE-gE-P2 fusion protein solution to the adjuvant, and then use phosphate solution (10 mM; pH 6.0) to make up the total volume to 10 ml, and stir evenly to obtain the PADRE-gE-P2 fusion protein adjuvant vaccine; the volume (ml) of the added PADRE-gE-P2 fusion protein solution is calculated as follows: the mass of the added PADRE-gE-P2 fusion protein (μg) / the concentration of the PADRE-gE-P2 fusion protein (μg / ml).

[0145] 5) Detection (1 test) of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples (PADRE-gE-P2 fusion protein is used as the protein standard) 1. Solution preparation (1) Preparation of mobile phase (0.05% trifluoroacetic acid) Mobile phase A [water: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of trifluoroacetic acid solution, shake well, and ultrasonically degas for 5 minutes.

[0146] Mobile phase B [acetonitrile: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and degas by ultrasonication for 5 minutes.

[0147] Mobile phase C [methanol:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of methanol, add 0.5 ml of trifluoroacetic acid solution, shake well, and degas by ultrasonication for 5 minutes.

[0148] (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.

[0149] 1.25% dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, and mix well.

[0150] 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix well.

[0151] 3.5% dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, and mix well.

[0152] 2. Vaccine Sample Testing S1. Preparation of protein calibration standard solution samples Place 658 μl of the PADRE-gE-P2 fusion protein control solution (concentration of 1.52 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 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 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.

[0153] S2. 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. Standard solutions of μg / ml, 75μg / ml, 62.5μg / ml, 50μg / ml, 37.5μg / ml, and 25μg / ml.

[0154] S3. Preparation of dioleoylphosphatidylcholine 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 mix with 1.2 ml of 3.5% DMSO solution, 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 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.

[0155] S4. Preparation of test samples Pipette 0.5 ml of the sample solution to be tested into a sampling vial, add 0.5 ml of 2% DMSO solution, cover the vial cap, and mix well.

[0156] S5. Load the sample into the liquid chromatograph and run the analysis method The injection vials containing protein calibration standard solution, Quillaja saponin QS-21 calibration standard solution, dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution, and the injection vials of the test sample were loaded onto the injection tray of the high performance liquid chromatography (HPLC) instrument, and the analytical method was run using the HPLC instrument.

[0157] Chromatogram Figure 5 As shown in the figure, the retention peak and the front peak of each substance are completely separated, which meets the requirements. S6. Calculate the content of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine (1) Establishment of standard curve The peak area values of the calibration standard solution samples of PADRE-gE-P2 fusion protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol were used to establish calibration standard curves in which the concentration of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol were proportional to the peak area.

[0158] PADRE-gE-P2 fusion protein calibration standard solution sample test results and standard curve, such as Figure 4 shown Protein calibration standard solution Retention time (min) Peak area (μAU*min) tailing factor 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 PADRE-gE-P2 fusion protein peak area standard curve: y = 60464x - 36883, R 2 =0.9998.

[0159] The test results and standard curve of the sample of Quillaja saponin QS-21 calibration standard solution are as follows: Figure 1 shown QS-21 Calibration Standard Solution Peak area (A+B isomer) (μAU*min) Tailing factor (A isomer) Tailing factor (B isomer) Calibration standard solution-1 (25 μg / ml) 139433 1.295 1.318 Calibration standard solution-2 (37.5 μg / ml) 211179 1.278 1.274 Calibration standard solution-3 (50 μg / ml) 282908 1.273 1.221 Calibration standard solution-4 (62.5 μg / ml) 351289 1.262 1.309 Calibration standard solution-5 (75 μg / ml) 424265 1.255 1.282 Calibration standard solution-6 (87.5 μg / ml) 493667 1.247 1.244 Standard curve of peak area of Quillaja saponin QS-21: y=5665.8x-1580.5, R 2 =0.9999.

[0160] Dioleoylphosphatidylcholine and cholesterol calibration standard solution sample test results and standard curve, such as Figure 2 、 3 shown DOPC calibration standard solution Peak area (μAU*min) tailing factor Standard curve solution-1 (500 μg / ml) 550039 1.759 Standard curve solution-2 (750 μg / ml) 820342 1.767 Standard curve solution-3 (1000 μg / ml) 1086222 1.789 Standard curve solution-4 (1250 μg / ml) 1346325 1.788 Standard curve solution-5 (1500 μg / ml) 1615030 1.798 DOPC peak area standard curve: y = 1062.4x + 21206, R 2 =1. Cholesterol calibration standard solution Peak area (μAU*min) tailing factor Standard curve solution-1 (125 μg / ml) 970572 1.107 Standard curve solution-2 (187.5 μg / ml) 1426879 1.090 Standard curve solution-3 (250 μg / ml) 1858278 1.069 Standard curve solution-4 (312.5 μg / ml) 2266198 1.052 Standard curve solution-5 (375 μg / ml) 2679431 1.034 Cholesterol peak area standard curve: y=6811.3x+137457, R 2 =0.9995.

[0161] (2) Content calculation Substitute the peak area values of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol of the vaccine sample into the calibration standard curve formula of the corresponding components, and then multiply them by the dilution factor of the vaccine (two times) to calculate the protein, QS-21, dioleoylphosphatidylcholine and cholesterol content in the vaccine. Figure 5 As shown in the figure, the retention peak and the front of each substance are completely separated, which meets the requirements. Note: “NA” means “not applicable”.

[0162] 6) Compare the differences in the contents of PADRE-gE-P2 fusion protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in vaccine samples between the comparative test (one simultaneous test; PADRE-gE-P2 fusion protein as the protein standard) and the existing technology test (three separate tests), e.g. Figure 6 shown 1. Vaccine Sample Testing (Existing Technology) A method for testing the protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol content in a vaccine sample three times.

[0163] (1) Detection of protein content Since the components of the XA-401 adjuvant, such as Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol, can interfere with the Lowry method for detecting vaccine protein content, the influence of the XA-401 adjuvant needs to be removed during the test 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) XA-401 adjuvant control formulation 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, and finally make up to 10 ml with phosphate solution (10 mM; pH 6.0), and stir evenly to obtain the product.

[0167] (D) Protein 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 well 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] XA-401 adjuvant control test: Accurately pipette an appropriate amount of XA-401 adjuvant sample and dilute it with water (the dilution factor is the same as that for 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 protein concentration in vaccine Perform a linear regression using the protein control content as the X-axis and the corresponding mean absorbance as the Y-axis to determine the linear regression equation. Subtract the mean absorbance of the adjuvant control solution from the mean absorbance of the test solution to determine the protein absorbance of the test solution. Substitute the protein absorbance of the test solution into the linear regression equation to determine the protein content of the test sample. The protein 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 item “3)”.

[0172] (3) Detection of dioleoylphosphatidylcholine and cholesterol content Same as "Detection of dioleoylphosphatidylcholine and cholesterol concentration" in item "2)".

[0173] 2. Comparative testing (one simultaneous test; PADRE-gE-P2 fusion protein as the protein standard) and existing technology testing (three separate tests) to determine the differences in protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol content in vaccine samples Example 2: Detection (1 simultaneous detection) of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples (BSA as a protein standard) 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0174] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0175] 3) The preparation of Quillaja saponin QS-21 solution and the concentration determination were the same as in Example 1.

[0176] 4) The vaccine sample preparation was the same as in Example 1.

[0177] 5) Detection of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol content in vaccine samples (BSA is a protein standard) 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0178] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0179] 2. Vaccine Sample Testing S1. 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.

[0180] S2. Preparation of QS-21 calibration standard solution sample The same as the item “S2, preparation of Quillaja saponin QS-21 calibration standard solution sample” in Example 1.

[0181] S3. Preparation of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples The same as the item "S3, preparation of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples" in Example 1.

[0182] S4. Preparation of test samples The same as the item "S4. Preparation of test samples" in Example 1.

[0183] S5. Load the sample into the liquid chromatograph and run the analysis method The same as the item “S5. Load the sample into the liquid chromatograph and run the analysis method” in Example 1.

[0184] S6. Calculation of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in the vaccine (1) Establishment of standard curve The peak area values of the calibration standard solution samples of BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol were used to establish calibration standard curves in which the concentrations of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol were proportional to the peak area.

[0185] BSA protein calibration standard solution sample test results and standard curve, such as Figure 7 shown Protein calibration standard solution Retention time (min) Peak area (μAU*min) tailing factor 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 BSA protein peak area standard curve: y = 55701x - 54266, R 2 =0.9993.

[0186] Test results and standard curve of Quillaja saponin QS-21 calibration standard solution samples The same as "(2) Detection results and standard curve of sample of Quillaja saponin QS-21 calibration standard solution" in item "S6" of Example 1.

[0187] Dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution sample test results and standard curve The same as "(3) Sample test results and standard curve of dioleoylphosphatidylcholine and cholesterol calibration standard solution" in "S6" of Example 1.

[0188] (2) Content calculation The peak area values of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine 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 multiple of the vaccine (twice) to calculate the protein, QS-21, dioleoylphosphatidylcholine and cholesterol contents in the vaccine. 6) Comparative testing (1 simultaneous test; BSA as protein standard) and existing technology testing (3 separate tests) to determine the differences in the content of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol in vaccine samples, e.g. Figure 8 shown 1. Vaccine Sample Testing (Existing Technology) A method for testing the protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol content in vaccine samples three times.

[0189] (1) Detection of protein content The same as “(1) Detection of protein content” in “I. Vaccine sample detection (existing technology)” of “6)” in Example 1.

[0190] (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.

[0191] (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.

[0192] 2. Comparative testing (one simultaneous test; BSA as a protein standard) and existing technology testing (three separate tests) to determine the differences in protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples Example 3. Detection (1 simultaneous detection) of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in vaccine samples (BSA as a protein standard) 1) gE preparation and protein concentration detection 1. Protein codon optimization and whole gene synthesis Codon optimization of the gE gene was performed: common restriction enzyme cleavage sites were avoided; based on codon preference in 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 enzyme cleavage site was introduced upstream, and a stop codon and BamHI restriction enzyme cleavage site were added downstream. Full gene synthesis of the nucleotide sequence was then performed.

[0193] 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.

[0194] 3. Construction of Stable Cell Lines Identify the correct recombinant expression vector, enrich the cells, and extract the plasmid in large quantities. Digest the recombinant expression vector with Pvμ I, then recover the linearized vector from the gel and filter-sterilize it for later use. Recover CHO-K1 cells and passage them twice or more, ensuring a cell viability greater than 95%. In a clean bench, add 0.6 ml of the cell suspension (approximately 1 × 107 cells) and 200 μl of the linearized recombinant expression vector (approximately 50 μg) to a 4 mm electroporation cuvette. Electroporation should be performed at a voltage of 300 V and a capacitance of 900 μF. After electroporation, transfer the cells to a shake flask containing 30 ml of CD CHO medium and incubate at 37°C, 5% CO2, and 125 rpm for 24 hours. After electroporation, the cell suspension was centrifuged at 100g for 10 minutes, the supernatant discarded, and the cells resuspended in CD CHO medium supplemented with 25μM MSX and 200μg / ml bleomycin. The cells were then seeded into 24-well plates. After 3 weeks of culture, expression levels were determined by ELISA. Three wells with the highest expression levels were pooled and seeded into 96-well plates using limiting dilution for single-clone screening. Images were taken using a single-cell imaging device 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 preparation was determined, and a two-stage cell bank was established.

[0195] 4. Expression of target product A frozen working seed was revived in OPM-CHO CDP9 medium, scaled up in shake flasks, and finally transferred to a 5-L bioreactor for cultivation at an inoculum density of 0.8 × 10⁶ cells / mL. Culture parameters were set at 37°C, pH 7.0, a rotational speed of 150 rpm, and a dissolved oxygen concentration of 40%. Cell viability, density, lactate content, and glucose content were measured daily. On day 3 of culture, when the viable cell density reached 3 × 10⁶ cells / mL, feed media (250 mL) of CDF18 and 25 mL of CDF26 were added, respectively. Equal volumes of feed media were then added every other day. The glucose concentration in the culture medium was maintained above 2 g / L. If it fell below this level, glucose was supplemented to 4 g / L. After approximately 15 days of culture, when the cell viability dropped to 70%, the culture was terminated. Cells and cell debris were removed by depth filtration, and the cell culture supernatant was collected.

[0196] 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 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 anion-purify the 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 stabilized. 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.

[0197] 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.

[0198] Mobile phase B [acetonitrile: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and degas by ultrasonication for 5 minutes.

[0199] (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.

[0200] (3) Protein concentration detection of gE solution / stock solution sample S1. Preparation of protein calibration standard solution samples The same as the item “S2, preparation of Quillaja saponin QS-21 calibration standard solution sample” in Example 1.

[0201] S2. Preparation of test samples 1 ml of the sample solution to be tested is diluted in two-fold gradients until the maximum dilution is 28 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.

[0202] S3. 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. S4. Calculate protein content Establishment of standard curve The peak area values of the BSA calibration standard solution samples were used to establish a calibration standard curve in which protein concentration was proportional to peak area.

[0203] 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.

[0204] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0205] 3) The preparation of Quillaja saponin QS-21 solution and the concentration determination were the same as in Example 1.

[0206] 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.

[0207] 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).

[0208] 5) Detection of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol content in vaccine samples (BSA is used as a protein standard) 1. Solution preparation (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0209] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0210] 2. Vaccine Sample Testing S1. Preparation of protein calibration standard solution samples The same as the item “S1, preparation of protein calibration standard solution sample” in Example 2.

[0211] S2. Preparation of QS-21 calibration standard solution sample The same as the item “S2, preparation of Quillaja saponin QS-21 calibration standard solution sample” in Example 1.

[0212] S3. Preparation of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples The same as the item "S3, preparation of dioleoylphosphatidylcholine and cholesterol calibration standard solution samples" in Example 1.

[0213] S4. Preparation of test samples The same as the item "S4. Preparation of test samples" in Example 1.

[0214] S5. Load the sample into the liquid chromatograph and run the analysis method S6. Calculation of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol content in the vaccine (1) Establishment of standard curve The peak area values of the calibration standard solution samples of BSA, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol were used to establish calibration standard curves in which the concentrations of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol were proportional to the peak area.

[0215] BSA protein calibration standard solution sample test results and standard curve, the standard curve is as follows Figure 9 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 protein peak area standard curve: y = 60497x - 224266, R 2 =0.9999.

[0216] The test results and standard curve of the sample of Quillaja saponin QS-21 calibration standard solution are as follows: Figure 10 shown QS-21 calibration standard solution Peak area (A+B isomer) (mAU*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.

[0217] The test results and standard curve of dioleoylphosphatidylcholine (DOPC) and cholesterol calibration standard solution samples are shown in the figure below. Figure 11 and Figure 12 shown DOPC calibration standard solution Peak area (mAU*min) tailing factor Standard curve solution-1 (500 μg / ml) 840670 1.766 Standard curve solution-2 (750 μg / ml) 1255779 1.553 Standard curve solution-3 (1000 μg / ml) 1710671 1.173 Standard curve solution-4 (1250 μg / ml) 2141153 0.962 Standard curve solution-5 (1500 μg / ml) 2577165 0.994 DOPC peak area standard curve: y = 1743.3x - 38258, R 2 =0.9999. Cholesterol calibration standard solution Peak area (mAU*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, R 2 =0.9998.

[0218] (2) Content calculation Substitute the peak area values of gE, Quillaja saponin QS-21, dioleoylphosphatidylcholine and cholesterol of the vaccine sample into the calibration standard curve formula of the corresponding components, and then multiply them by the dilution factor of the vaccine (two times) to calculate the content of gE, QS-21, dioleoylphosphatidylcholine and cholesterol in the vaccine. Figure 13 , the retention peak and the front of each substance are completely separated, which meets the requirements; Figure 14 , the detection value of each substance is close to the theoretical value and meets the requirements. 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 detecting the content of the main components of protein and composite adjuvant in a recombinant protein vaccine, characterized in that: The following steps are involved: S1. Prepare protein calibration standard solution sample; S2. Prepare QS-21 calibration standard solution sample; S3, preparing dioleoylphosphatidylcholine and cholesterol calibration standard solution samples; S4, preparing test samples; S5. Load the sample into the liquid chromatograph and run the analysis method; S6. Calculate the content of the main components of protein and adjuvant in the vaccine.

2. The method for detecting the content of the main components of protein and composite adjuvant in recombinant protein vaccine according to claim 1, characterized in that: The recombinant protein vaccine is composed of a recombinant protein and a composite adjuvant; the recombinant protein is the extracellular region of herpes virus glycoprotein E or its fusion protein prepared by a eukaryotic expression system; the herpes virus includes varicella-zoster virus, herpes simplex virus type I, herpes simplex virus type II, Epstein-Barr virus, human cytomegalovirus, Kaposi's sarcoma-associated herpes virus, pseudorabies virus, and Marek's disease virus; an optional molecular structure of the extracellular region gE of the glycoprotein E of the varicella-zoster virus from N-terminus to C-terminus is AA31-AA544, and the amino acid sequence is shown in SEQ ID NO.1; an optional protein of the fusion protein of the extracellular region of the glycoprotein E of the varicella-zoster virus is PADRE-gE-P2, and an optional molecular structure of the extracellular region gE of the varicella-zoster virus from N-terminus to C-terminus is: PADRE-GSGSG-gE-GGS-P2, and the amino acid sequence is shown in SEQ ID NO. As shown in IDNO.2; the composite adjuvant is XA-401, whose main components are dioleoylphosphatidylcholine, cholesterol and Quillaja saponin QS-21.

3. The method for detecting the content of the main components of protein and composite adjuvant in recombinant protein 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.

4. The method for detecting the content of the main components of protein and composite adjuvant in a recombinant protein vaccine according to claim 1, characterized in that: The concentration of the Quillaja 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.

5. The method for detecting the content of the main components of protein and composite adjuvant in recombinant protein vaccine according to claim 1, characterized in that: The chromatographic column of the analysis method in S5 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 S5 include but are not limited to: column temperature, injection volume, flow rate, and detector parameters; the column temperature is 40°C to 60°C; the injection volume is 1μl to 100μl; the flow rate is 0.1 ml / min to 2.5 ml / min; when the detector is a UV detector, the detection wavelength is 200 to 400nm.

6. The method for detecting the content of the main components of protein and composite adjuvant in a recombinant protein vaccine according to claim 1, characterized in that: The elution procedure of the analytical method in S5 includes: an elution method for eluting the retention peaks of Quillaja saponin QS-21 and protein; 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 protein is gradient elution, wherein the acetonitrile concentration gradient 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%.

7. The method for detecting the content of the main components of protein and composite adjuvant in recombinant protein vaccine according to claim 6, characterized in that: 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 detecting the content of the main components of protein and composite adjuvant in a recombinant protein vaccine according to claim 1, characterized in that: The calculation of the content of the main components of protein and adjuvant in the vaccine in S6 includes two calculation methods: Calculation method 1: First, establish calibration standard curves in which the concentrations of protein, Quillaja saponin QS-21, dioleoylphosphatidylcholine, and cholesterol are proportional to their peak areas. Then, substitute the peak area values of protein, 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 "protein" or "Quillaja saponin QS-21" or "dioleoylphosphatidylcholine" or "cholesterol".

9. Application of the method for detecting the content of the main components of protein and composite adjuvant in a recombinant protein vaccine according to any one of claims 1 to 8 in simultaneously detecting components in a recombinant herpes virus glycoprotein E extracellular domain or glycoprotein E extracellular domain fusion protein vaccine, characterized in that: The herpes viruses include varicella-zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, human cytomegalovirus, Kaposi's sarcoma-associated herpes virus, pseudorabies virus, and Marek's disease virus; the composite adjuvant in the recombinant protein vaccine is a composite adjuvant containing neutral liposomes and the immune enhancer QS-21.

10. Application of the method for detecting the content of the main components of protein and composite adjuvant in a recombinant protein vaccine according to claim 9 in the simultaneous detection of components in a recombinant varicella-zoster virus vaccine, characterized in that: Used to detect the content of protein or fusion protein and / or Quillaja saponin QS-21 and / or dioleoylphosphatidylcholine and / or cholesterol in recombinant varicella-zoster vaccine.

Images