Method for qualitatively and quantitatively detecting protein in recombinant varicella-herpes zoster vaccine and application

Abstract

The invention discloses a method for qualitatively and quantitatively detecting protein in a recombinant varicella-herpes zoster vaccine and application. The method comprises the following steps: preparing a protein calibration standard solution sample; preparing a detection sample; loading the sample into a liquid chromatograph, and operating an analysis method; calculating the relative deviation of the protein retention time in the vaccine; and calculating the protein content in the vaccine. The technical problem that in the prior art, two different methods are needed for twice detection in qualitative and quantitative detection of the protein in the vaccine is solved, and the technical problem that in an existing protein content detection technology, the protein in the vaccine can only be quantitatively detected and cannot be qualitatively detected is also solved. According to the high performance liquid chromatography disclosed by the invention, the protein in the vaccine can be qualitatively and quantitatively detected only by one-time sample loading detection, so that the vaccine verification efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for qualitatively and quantitatively detecting proteins in a recombinant varicella-zoster vaccine and its application. Background Art

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

[0003] GlaxoSmithKline's recombinant protein zoster vaccine Approved by the FDA in 2017, it is used to prevent herpes zoster in adults over 50 years old. 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 immunopotentiator 3D-MPL, the immunopotentiator Quillaja saponin QS-21, and liposomes; the main components of the liposomes are dioleoylphosphatidylcholine (DOPC) and cholesterol).

[0004] Compared with the live attenuated vaccines of Merck and Biopharma, GSK's adjuvanted subunit vaccine The protection rate is higher, but the clinical side effects are more serious. Therefore, the immunogenicity is better than However, the clinical side effects were lower than The recombinant varicella-zoster vaccine is a promising candidate vaccine in clinical trials (immunogenicity data are available in CN 116747298B and CN 117003896 B): the candidate vaccine contains gE fusion protein (PADRE-gE-P2) antigen and XA-401 composite adjuvant [XA-401 composite adjuvant consists of the immunopotentiator Quillaja saponin QS-21 and liposomes; the main components of 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.

[0005] 3D-MPL is a Toll-like receptor (TLR) 4 agonist. After binding to TLR4, it activates myeloid differentiation primary response 88 (MyD88) and downstream signaling pathways, leading to the activation of nuclear factor-κB (NFκB) and activator protein (AP)-1, as well as the expression of proinflammatory cytokine genes. The production process of 3D-MPL is very complex, and the production process and quality control are quite difficult. The production process of 3D-MPL powder includes: fermentation and cultivation of Salmonella Minnesota R595 strain, LPS extraction, LPS acid hydrolysis detoxification, LPS alkaline hydrolysis detoxification, chromatographic purification, protonation, salification, and lyophilization. The LPS acid hydrolysis and alkaline hydrolysis detoxification steps are particularly difficult. 3D-MPL quality control is also difficult: it is necessary to control the distribution ratio of 3D-MPL tetraacyl analogs, pentaacyl analogs, hexaacyl analogs, and heptaacyl analogs in the 3D-MPL powder and AS01B adjuvant system.

[0006] Quillaja saponin QS-21 is an immunopotentiator extracted from the bark of the Chilean soap tree, Quillaja saponaria. In aqueous solution, the quillaja saponin QS-21A isomer partially converts to the QS-21B isomer, but QS-21A remains the predominant molecular form. Both QS-21A and QS-21B are known to exhibit similar adjuvant activity.

[0007] Currently, the recombinant herpes zoster vaccine is available on the market. In the quality control, qualitative and quantitative detection Two different detection methods are required for proteins in vaccines: 1. Lowry method for detecting protein content in vaccines (quantitative detection of proteins); 2. ELISA method for qualitative detection (identification) of proteins in vaccines. When the existing technology (lowry method) quantitatively detects proteins in vaccines, it will be interfered with by other components in the vaccine, resulting in large detection errors; the ELISA method can only qualitatively detect gE on the PADRE-gE-P2 fusion protein, and cannot completely qualitatively detect the PADRE-gE-P2 fusion protein. Therefore, it is necessary to establish a qualitative detection method that can distinguish between the PADRE-gE-P2 fusion protein and gE. In short, quantitative detection The method for protein in vaccine has large error and qualitative detection The ELISA method for proteins in the vaccine is not sufficient to characterize the PADRE-gE-P2 fusion protein: it cannot distinguish between the PADRE-gE-P2 fusion protein and gE. Summary of the Invention

[0008] The purpose of the present invention is to provide a method and application for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine, so as to solve the technical problems of the existing methods for detecting proteins in vaccines, which are cumbersome, time-consuming, material-consuming, labor-consuming and inefficient.

[0009] To achieve the above objectives, the present invention provides the following technical solutions:

[0010] The present invention provides a method for qualitatively and quantitatively detecting proteins in a recombinant varicella-zoster vaccine, which uses a liquid chromatography method. The protein includes the amino acid sequence of the extracellular segment of varicella-zoster virus (VZV) glycoprotein E (gE), the universal DR Th epitope peptide PADRE (PADRE), and the tetanus toxin Th epitope P2 (P2). The gE, PADRE, and P2 are connected by a connecting peptide, and the connecting peptide is GGS and / or GGGS and / or GGGGS and / or GSGSG connecting peptide.

[0011] Furthermore, the protein is a PADRE-gE-P2 fusion protein, an optional molecular structure of which is (from N-terminus to C-terminus): PADRE-GSGSG (connecting peptide)-gE (AA31-AA544)-GGS (connecting peptide)-P2, and the amino acid sequence is shown in SEQ ID NO.1.

[0012] Furthermore, the varicella-zoster vaccine also includes a composite adjuvant, which is XA-401 composite 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.

[0013] Furthermore, the protein content of the recombinant varicella-zoster vaccine is 10 μg / ml to 400 μg / ml, the dioleoylphosphatidylcholine content is 400 μg / ml to 4000 μg / ml, the cholesterol content is 100 μg / ml to 1000 μg / ml, and the QS-21 content is 20 μg / ml to 200 μg / ml.

[0014] Furthermore, the recombinant varicella-zoster vaccine contains a buffer component for maintaining the pH value of the vaccine stable and a component for regulating the osmotic pressure of the vaccine.

[0015] 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.

[0016] 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.

[0017] Furthermore, the recombinant varicella-zoster vaccine may also contain components for maintaining protein stability in the vaccine.

[0018] Furthermore, the component for maintaining 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.

[0019] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra high performance liquid chromatography.

[0020] Furthermore, the liquid chromatography method is a reverse phase liquid chromatography method, wherein the chromatographic column contains an alkyl or phenyl reverse phase chromatography filler, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water, and the mobile phase may also contain methanol.

[0021] 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).

[0022] Furthermore, the liquid chromatography method is a liquid chromatography internal standard method or a liquid chromatography external standard method.

[0023] Furthermore, the liquid chromatography external standard method comprises the following steps:

[0024] S1. Prepare protein calibration standard solution sample;

[0025] S2, preparing test samples;

[0026] S3, loading the sample into the liquid chromatograph and running the analytical method;

[0027] S4. Calculate the relative deviation of protein retention time in the vaccine;

[0028] S5. Calculate the protein content in the vaccine.

[0029] Furthermore, in the preparation of protein calibration standard solution samples in S1, calibration standard solution samples of one or two proteins can be prepared, and a PADRE-gE-P2 fusion protein calibration standard solution sample (for qualitative and quantitative purposes) must be prepared. In addition, a calibration standard solution sample of another protein (for quantitative purposes) may also be prepared. The other 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), and varicella-zoster virus (VZV) glycoprotein E (gE) extracellular segment (gE).

[0030] Furthermore, the protein calibration standard solution sample in S1 has a protein concentration of 1 μg / ml to 200 μg / ml.

[0031] Furthermore, the protein calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0032] Furthermore, the vaccine used to prepare the test sample in S2 has a protein content of 10 μg / ml to 400 μg / ml, a dioleoylphosphatidylcholine (DOPC) 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.

[0033] Furthermore, the concentration of the vaccine protein used to prepare the test sample in S2 is 1 μg / ml to 200 μg / ml.

[0034] Furthermore, the test sample prepared in S2 may contain DMSO at a concentration of 0.5% to 5%.

[0035] Furthermore, the detector of the liquid chromatograph in S3 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0036] Furthermore, the analysis method in S3 includes a chromatographic column, liquid chromatography parameters, and an elution procedure.

[0037] Furthermore, the chromatographic column used in the analysis method in S3 is a reverse phase chromatographic column.

[0038] Furthermore, the liquid chromatography parameters of the analysis method in S3 include column temperature, injection volume, flow rate, detector parameters, etc.

[0039] Further, the column temperature is 20°C to 70°C, such as 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 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, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C.

[0040] 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.

[0041] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the injection volume is 5 μl to 30 μl, such as 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, or 30 μl.

[0042] 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.

[0043] 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.

[0044] Further, the detector parameters are characterized in that 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.

[0045] 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.

[0046] 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.

[0047] Furthermore, the elution procedure of the analytical method in S3 at least includes an elution method for eluting the protein retention peak.

[0048] Furthermore, the elution method for eluting the protein retention peak can also elute the Quillaja saponin QS-21 retention peak.

[0049] Further, the elution method for eluting the protein retention peak is gradient elution, and the acetonitrile concentration gradient in the mobile phase is 5% to 100%, such as 5% to 100%, 5% to 95%, 5% to 90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 10% to 100%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 15% to 100%, 15% to 95%, 15% to 90%, 15% to 85%, 15% to 8 0%, 15%-75%, 15%-70%, 15%-65%, 15%-60%, 15%-55%, 15%-50%, 20%-100%, 20%-95%, 20%-90%, 20%-85%, 20%-80%, 20%-75%, 20%-70%, 20%-65%, 20%-60%, 20%-55%, 20%-50%, 25%-100%, 25%-95%, 25%-90%, 25%-85%, 25%-80%, 25%-75%, 25%-70%, 25%-65%, 25%-60%, 25%-55%, 25%-50%, 30%-100%, 30%-95%, 30% ~90%、30%~85%、30%~80%、30%~75%、30%~70%、30%~65%、30%~60%、30%~55%、30%~50%、35%~100%、35%~95%、35%~90%、35%~85%、35%~80%、35%~75%、35%~70 %, 35%-65%, 35%-60%, 35%-55%, 35%-50%, 40%-100%, 40%-95%, 40%-90%, 40%-85%, 40%-80%, 40%-75%, 40%-70%, 40%-65%, 40%-60%, 40%-55%, 40%-50%, 45 %~100%, 45%~95%, 45%~90%, 45%~85%, 45%~80%, 45%~75%, 45%~70%, 45%~65%, 45%~60%, 45%~55%, 45%~50%, 50%~55%, 50%~60%, 50%~65%, 50%~70%, 50%~7 5%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 50%-100%, 55%-60%, 55%-65%, 55%-70%, 55%-75%, 55%-80%, 55%-85%, 55%-90%, 55%-95%, 55%-100%, 60%-65%,60% to 70%, 60% to 75%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, or 65% to 75%, 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%.

[0050] Furthermore, the elution procedure of the analytical method in S3 may also include an elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol.

[0051] Furthermore, the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol is isocratic elution or gradient elution.

[0052] 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%.

[0053] 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%.

[0054] Furthermore, if the elution program includes an elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, an isocratic or gradient elution method can be added between the elution method for eluting the protein retention peak and the elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol.

[0055] Furthermore, in the elution procedure of the S3 analysis method, a pre-equilibration method and / or a pre-elution method may be added before the elution method for eluting the protein retention peak.

[0056] Furthermore, a pre-equilibrium method is added before the elution method of eluting the protein retention peak, and the acetonitrile concentration in the mobile phase is not higher than the minimum value of the acetonitrile concentration gradient of the eluting protein retention peak.

[0057] Furthermore, a pre-equilibrium method is added before the elution method of eluting 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%, and the equilibration time is 1 to 960 minutes, such as 1 minute, 2 minutes, 4 minutes, 8 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, The mobile phase is stirred for 10 minutes at 40°C for 30 minutes, 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%.

[0058] Further, a pre-elution method is added before the elution method of eluting the protein retention peak: 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% to 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%, and the gradient elution time is 1-960 min, such as 1 min, The mobile phase may be stirred for 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%.

[0059] Furthermore, in the elution procedure of the analysis method in S3, a chromatographic column regeneration method and / or a post-equilibration method may be added after the elution method for eluting the protein retention peak.

[0060] Furthermore, if the elution program includes an elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol, the column regeneration method and / or post-equilibrium method should be after the elution method for eluting the retention peaks of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0061] 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.

[0062] Furthermore, a chromatographic column regeneration method is added after the elution method for eluting the protein retention peak: the acetonitrile concentration in the mobile phase is 98% to 100%, such as 98%, 98.5%, 99%, 99.5% or 100%, 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%.

[0063] 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 protein retention peak.

[0064] Furthermore, the elution method for eluting the protein retention peak is followed by a post-equilibrium method: 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%, and 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 The mobile phase is stirred for 10 minutes at 40°C for 30 minutes, and the concentration of trifluoroacetic acid in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0065] Furthermore, the relative deviation of the protein retention time in the vaccine is calculated in S4, and the calculation formula is: [|retention time of vaccine protein retention peak - retention time average value| / retention time average value]×100%, where the retention time average value is the average of the retention time of the vaccine protein retention peak and the retention time of the protein calibration standard solution sample retention peak.

[0066] Furthermore, the criterion for determining whether the protein in the vaccine is a PADRE-gE-P2 fusion protein is that the relative deviation of the protein retention time in the vaccine is no more than 2%.

[0067] Furthermore, the method for calculating the protein content in the vaccine in S5 includes:

[0068] Calculation method 1: First, establish a calibration standard curve in which the protein concentration is proportional to the peak area; then, substitute the peak area value of the protein in the vaccine sample into the calibration standard curve formula to calculate the concentration of the protein in the test sample; then, multiply the concentration of the protein in the test sample by the dilution factor to calculate the concentration of the protein in the vaccine;

[0069] Calculation method 2: Directly calculate the protein content in the vaccine. Protein concentration in the vaccine = protein peak area of ​​the test sample * protein calibration standard solution sample concentration * dilution factor / protein calibration standard solution sample peak area.

[0070] The present invention also provides a method for qualitatively and quantitatively detecting proteins in a recombinant varicella-zoster vaccine and its application in detecting components in the recombinant varicella-zoster vaccine, including but not limited to the detection of PADRE-gE-P2 fusion protein.

[0071] Furthermore, it was used to quantitatively detect proteins in the PADRE-gE-P2 fusion protein vaccine.

[0072] Furthermore, it was used to qualitatively detect proteins in the PADRE-gE-P2 fusion protein vaccine.

[0073] Furthermore, it was used to simultaneously qualitatively and quantitatively detect proteins in the PADRE-gE-P2 fusion protein vaccine.

[0074] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0075] The present invention provides a method for qualitatively and quantitatively detecting proteins in a recombinant varicella-zoster vaccine. This method not only addresses the technical problem of existing techniques requiring two separate methods for qualitative and quantitative detection of vaccine proteins, but also addresses the technical problem of existing protein content detection techniques being able to only quantitatively detect proteins in vaccines, not qualitatively. The high-performance liquid chromatography method disclosed in the present invention enables both qualitative and quantitative detection of vaccine proteins with only a single sample loading, significantly improving vaccine testing efficiency.

[0076] The present invention provides a method for qualitatively and quantitatively detecting proteins in a recombinant varicella-zoster vaccine, which can quantitatively detect the protein content in the recombinant varicella-zoster vaccine; can identify whether the protein in the vaccine is a PADRE-gE-P2 fusion protein; and can simultaneously qualitatively and quantitatively detect proteins in the PADRE-gE-P2 fusion protein vaccine (XA-401 composite adjuvant). BRIEF DESCRIPTION OF THE DRAWINGS

[0077] 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.

[0078] Figure 1 This is a liquid chromatogram of the main components of the vaccine under different mobile phase pH adjusters in Example 1 of the present invention;

[0079] The left chromatogram is acetic acid; the right chromatogram is trifluoroacetic acid.

[0080] Figure 2 1 is a liquid chromatogram of the PADRE-gE-P2 fusion protein and Quillaja saponin QS-21 in the vaccine under different elution conditions of the PADRE-gE-P2 fusion protein according to Example 2 of the present invention;

[0081] From left to right are the chromatograms of 5% to 95% acetonitrile gradient elution for 15 min, 50% to 75% acetonitrile gradient elution for 10 min, 50% to 75% acetonitrile gradient elution for 20 min, and 50% to 75% acetonitrile gradient elution for 30 min.

[0082] Figure 3 This is a comparison chart of the retention peak separation of the main components of the vaccine under different elution methods of the PADRE-gE-P2 fusion protein in Example 2 of the present invention.

[0083] Figure 4 This is a liquid chromatogram of the PADRE-gE-P2 fusion protein and Quillaja saponin QS-21 in the vaccine under different column temperature conditions in Example 3 of the present invention, wherein from left to right are the chromatograms at column temperatures of 30°C, 40°C, 50°C, and 60°C.

[0084] Figure 5 This is a comparison chart of the separation of the PADRE-gE-P2 fusion protein retention peaks under different column temperature conditions in Example 3 of the present invention.

[0085] Figure 6This is a comparison chart of the tailing factors of the PADRE-gE-P2 fusion protein retention peak under different column temperature conditions in Example 3 of the present invention.

[0086] Figure 7 This is a comparison chart of the separation of the retained peaks of the PADRE-gE-P2 fusion protein under different injection volume conditions in Example 4 of the present invention.

[0087] Figure 8 This is a comparison chart of the tailing factors of the PADRE-gE-P2 fusion protein retention peak under different injection volume conditions in Example 4 of the present invention.

[0088] Figure 9 This is a liquid chromatogram of the PADRE-gE-P2 fusion protein and Quillaja saponin QS-21 in the vaccine under different flow rate conditions in Example 5 of the present invention, wherein from left to right are the chromatograms at flow rates of 1.0 ml / min, 1.5 ml / min, and 2.0 ml / min.

[0089] Figure 10 This is a comparison chart of the separation of the PADRE-gE-P2 fusion protein retention peaks under different flow rate conditions in Example 5 of the present invention.

[0090] Figure 11 This is a comparison chart of the tailing factors of the PADRE-gE-P2 fusion protein retention peak under different flow rate conditions in Example 5 of the present invention.

[0091] Figure 12 This is a standard curve of the concentration and peak area of ​​the PADRE-gE-P2 fusion protein of Example 6 of the present invention. The mobile phase for eluting DOPC and cholesterol is an acetonitrile and water system.

[0092] Figure 13 This is a liquid chromatogram of detecting the protein content in the vaccine sample according to Example 6 of the present invention. The mobile phase for eluting DOPC and cholesterol is an acetonitrile and water system.

[0093] Figure 14 This is a comparison chart of the liquid chromatography method of Example 6 of the present invention (PADRE-gE-P2 fusion protein is the protein standard; the mobile phase for eluting DOPC and cholesterol is an acetonitrile and water system) and the Lowry method for detecting protein content in vaccine samples.

[0094] Figure 15 This is the standard curve of PADRE-gE-P2 fusion protein concentration and peak area in Example 8 of the present invention (the mobile phase for eluting DOPC and cholesterol is a methanol and water system).

[0095] Figure 16 This is a liquid chromatogram of Example 8 of the present invention for detecting the protein content in the vaccine sample (the mobile phase for eluting DOPC and cholesterol is a methanol and water system).

[0096] Figure 17 This is a comparison chart of the protein content in the vaccine sample detected by the liquid chromatography method of Example 8 of the present invention (PADRE-gE-P2 fusion protein is the protein standard; the mobile phase for eluting DOPC and cholesterol is a methanol and water system) and the Lowry method.

[0097] Figure 18 This is the standard curve of bovine serum albumin (BSA) protein concentration and peak area in Example 10 of the present invention (the mobile phase for eluting DOPC and cholesterol is a methanol and water system).

[0098] Figure 19 This is a comparison chart of the protein content in the vaccine sample detected by the liquid chromatography method (BSA is a protein standard; the mobile phase for eluting DOPC and cholesterol is a methanol and water system) and the Lowry method in Example 10 of the present invention. DETAILED DESCRIPTION

[0099] 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.

[0100] 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.

[0101] 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.

[0102] A liquid chromatography method for the simultaneous qualitative and quantitative detection of proteins in a recombinant varicella-zoster vaccine containing PADRE-gE-P2 fusion protein and XA-401 composite adjuvant.

[0103] An optional molecular structure of the PADRE-gE-P2 fusion protein is (from N-terminus to C-terminus): PADRE-GSGSG (connecting peptide)-gE (AA31-AA544)-GGS (connecting peptide)-P2, its nucleic acid sequence is shown in CN 117003896 B, and its amino acid sequence is shown in SEQ ID NO.1.

[0104] A PADRE-gE-P2 fusion protein is prepared and the protein concentration of the fusion protein solution is detected; liposomes are prepared and the concentrations of dioleoylphosphatidylcholine and cholesterol in the liposomes are detected; a Quillaja saponin QS-21 solution is prepared and the concentration of Quillaja saponin QS-21 is detected; and the PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine is prepared using the PADRE-gE-P2 fusion protein, liposomes and Quillaja saponin QS-21 solution.

[0105] A Waters reversed-phase chromatography column (Symmetry300TMC18 5μm 4.6*150mm) and the PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine were used to study the type of acidic pH regulator in the mobile phase, the protein elution method, column temperature, injection volume, and flow rate of the liquid chromatography method to screen out the analytical method that met the requirements. The screening criteria were: the PADRE-gE-P2 fusion protein had a retention peak; the resolution of the PADRE-gE-P2 fusion protein retention peak had to be greater than 1.5; and the tailing factor had to be between 0.8 and 1.8.

[0106] The optimal liquid chromatography parameters and elution methods when detecting target substances (such as PADRE-gE-P2 fusion protein in vaccines) using reversed-phase chromatography columns containing different chromatographic fillers may be different, which is known to those skilled in the art.

[0107] Liquid chromatography was used to quantitatively detect the PADRE-gE-P2 fusion protein in the PADRE-gE-P2 fusion protein vaccine and compared with the results of existing techniques. The comparison results showed that both the liquid chromatography method and the existing techniques can be used to quantitatively detect the protein in the vaccine, but the liquid chromatography method is more accurate.

[0108] In addition, this liquid chromatography analysis method can also qualitatively detect proteins in the PADRE-gE-P2 fusion protein vaccine.

[0109] The most convenient thing is that this liquid chromatography analysis method can perform qualitative and quantitative detection at the same time, greatly improving the detection efficiency.

[0110] Example 1: Study on the Type of Acidic pH Regulator in Mobile Phase

[0111] 1) Preparation of PADRE-gE-P2 fusion protein and protein concentration detection

[0112] 1. Fusion protein codon optimization and whole gene synthesis

[0113] For details, see patent CN 117003896 B.

[0114] 2. Construction of fusion protein expression plasmid

[0115] For details, see patent CN 117003896 B.

[0116] 3. Construction of Stable Cell Lines

[0117] For details, see patent CN 117003896 B.

[0118] 4. Expression of target product

[0119] For details, see patent CN 117003896 B.

[0120] 5. Fusion Protein Purification

[0121] For details, see patent CN 117003896 B.

[0122] VI. Fusion protein (vaccine stock solution) concentration detection [(Lowry method) first method]

[0123] (1) Reagent preparation

[0124] 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.

[0125] (2) Preparation of protein reference working solution

[0126] 200 μg / ml Protein Reference Solution (e.g., BSA): Dissolve one vial of Protein Reference 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. Upon use, dilute the Protein Reference Solution by two-fold and mix thoroughly to create the Reference Working Solution (100 μg / ml).

[0127] (3) Protein sample concentration detection

[0128] Test sample detection: Accurately pipette an appropriate amount of the sample solution to be tested, add water to dilute until the protein content is within the calibration 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).

[0129] Protein control assay: Precisely pipette 0.2ml, 0.4ml, 0.6ml, 0.8ml, and 1.0ml of the 100μg / ml protein control working solution into test tubes in duplicate. For any solution less than 1.0ml, add water to 1.0ml. Repeat the procedure starting with "Add 1.0ml of alkaline copper solution." Precisely pipette another 1.0ml of water and repeat the procedure starting with "Add 1.0ml of alkaline copper solution" to serve as a blank control.

[0130] (4) Calculation of protein sample concentration

[0131] 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. 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.

[0132] 2) Liposome preparation and detection of dioleoylphosphatidylcholine and cholesterol concentrations

[0133] 1. Liposome Preparation (Theoretical Concentrations of Dioleoylphosphatidylcholine and Cholesterol are 4 mg / ml and 1 mg / ml, respectively)

[0134] 400 mg of dioleoylphosphatidylcholine (DOPC; Japan Fine Chemicals Co., Ltd.) and 100 mg of cholesterol (Japan Fine Chemicals Co., Ltd.) were weighed separately; the dioleoylphosphatidylcholine (DOPC) and cholesterol were completely dissolved in 10 ml of anhydrous ethanol and mixed evenly to obtain 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; a high-pressure microfluidizer was used to granulate the liposomes to a particle size of approximately 100 nm; the ethanol was then removed by dialysis; and finally, the liposomes were filtered and sterilized using a 0.22 μm sterilizing filter to obtain the finished liposome product.

[0135] 2. Dioleoylphosphatidylcholine (DOPC) and cholesterol concentration detection

[0136] (1) Mobile phase preparation

[0137] 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.

[0138] (2) Preparation of mixed dioleoylphosphatidylcholine (DOPC) and cholesterol standard solution sample

[0139] Preparation of 90% ethanol solution: Accurately measure 45 ml of anhydrous ethanol, mix with 5 ml of ultrapure water and shake well.

[0140] Preparation of 89.3% ethanol solution: Accurately measure 44.65 ml of anhydrous ethanol, mix with 5.35 ml of ultrapure water and shake well.

[0141] Preparation of mixed dioleoylphosphatidylcholine and cholesterol reference substance 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 scale with anhydrous ethanol, shake well, and obtain.

[0142] Mix dioleoylphosphatidylcholine and cholesterol standard solution sample preparation: Pipette 1.667ml of reference stock solution into a 25ml volumetric flask, dilute to 25ml with 89.3% ethanol solution, shake well, and obtain a standard solution with a cholesterol concentration of 100μg / ml and a dioleoylphosphatidylcholine (DOPC) concentration of 400μg / ml⑤; Pipette 7.5ml, 5ml, 2.5ml and 1.25ml of standard solution⑤ respectively into a 10ml volumetric flask, dilute to 10ml with 90% ethanol solution, shake well, and obtain standard Quasi-solution ④ [cholesterol concentration of 75 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration of 300 μg / ml], standard solution ③ [cholesterol concentration of 50 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration of 200 μg / ml], standard solution ② [cholesterol concentration of 25 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration of 100 μg / ml)] and standard solution ① [cholesterol concentration of 12.5 μg / ml, dioleoylphosphatidylcholine (DOPC) concentration of 50 μg / ml].

[0143] (3) Preparation of test sample solution

[0144] 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.

[0145] (4) Instrument parameters and elution procedures

[0146]

[0147] (5) Calculation of dioleoylphosphatidylcholine and cholesterol concentrations

[0148] 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 of the concentration and peak area of ​​dioleoylphosphatidylcholine and cholesterol, respectively. 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.

[0149] 3) Preparation and concentration determination of Quillaja saponin QS-21 solution

[0150] 1. Preparation of Quillaja saponin QS-21 solution (theoretical concentration 4 mg / ml)

[0151] 40 mg of Quillaja saponin QS-21 (Desert King) was weighed and completely dissolved in 4 ml of phosphate solution (10 mM, pH 6.0). The volume was then made up to 10 ml with phosphate solution (10 mM, pH 6.0) and mixed well to obtain a Quillaja saponin QS-21 solution.

[0152] 2. Concentration detection of Quillaja saponin QS-21 solution

[0153] (1) Mobile phase preparation

[0154] 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.

[0155] 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 to obtain the mobile phase.

[0156] (2) Preparation of standard solution sample of Quillaja saponin QS-21

[0157] 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.

[0158] Standard solution: Pipette 150 μl of the reference substance stock solution into a 1.5 ml centrifuge tube, add 600 μl of 1.25% DMSO solution, and mix to obtain a solution with a concentration of 1 mg / ml; Pipette 0.5 ml of the above solution into a 5 ml volumetric flask, dilute to the scale with 1% DMSO solution, and mix to obtain a solution with a concentration of 100 μg / ml; then pipette 875 μl, 750 μl, 625 μl, 500 μl, 375 μl, and 250 μl of the above solution into different injection vials, and then add 125 μl, 250 μl, 375 μl, 500 μl, 625 μl, and 750 μl of 1% DMSO solution, respectively, and mix to obtain standard solutions with concentrations of 87.5 μg / ml, 75 μg / ml, 62.5 μg / ml, 50 μg / ml, 37.5 μg / ml, and 25 μg / ml, respectively.

[0159] (3) Preparation of Quillaja saponin QS-21 sample solution

[0160] 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.

[0161] (4) Instrument parameters and elution procedures

[0162]

[0163]

[0164] (5) Calculation of concentration of Quillaja saponin QS-21

[0165] 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.

[0166] 4) Vaccine sample preparation

[0167] 1. Preparation of adjuvant

[0168] 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.

[0169] 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]

[0170] 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: the mass of the added PADRE-gE-P2 fusion protein (μg) / the concentration of the PADRE-gE-P2 fusion protein (μg / ml).

[0171] 5) Study the effect of acidic pH regulators (trifluoroacetic acid and acetic acid) in the mobile phase on the retention peak of PADRE-gE-P2 fusion protein

[0172] 1. Solution preparation

[0173] (1) Preparation of mobile phase (0.05% trifluoroacetic acid)

[0174] 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 degas by ultrasonication for 5 minutes.

[0175] Mobile phase B [acetonitrile: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and ultrasonically degas for 5 minutes to obtain the mobile phase.

[0176] (2) Preparation of mobile phase (0.05% acetic acid)

[0177] 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.

[0178] 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 to obtain the mobile phase.

[0179] (3) Preparation of sample dilution solution

[0180] 1% dimethyl sulfoxide (DMSO) solution: Measure 99 ml of ultrapure water, add 1 ml of dimethyl sulfoxide solution, and mix well.

[0181] 1.25% dimethyl sulfoxide (DMSO) solution: Measure 98.75 ml of ultrapure water, add 1.25 ml of dimethyl sulfoxide solution, and mix well.

[0182] 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix well.

[0183] 3.5% dimethyl sulfoxide (DMSO) solution: Measure 96.5 ml of ultrapure water, add 3.5 ml of dimethyl sulfoxide solution, and mix well.

[0184] 2. Sample Preparation

[0185] Test sample: Pipette 0.5 ml of vaccine into a sample vial, add 0.5 ml of 2% DMSO solution, cover the vial, and mix well.

[0186] 3. Chromatographic column, instrument parameters and elution procedure

[0187]

[0188]

[0189] 4. Results Analysis

[0190] The liquid chromatography method used for qualitative and quantitative detection of proteins in vaccines has the following characteristics: the protein has a retention peak; the resolution of the protein retention peak must be greater than 1.5; and the tailing factor must be between 0.8 and 1.8.

[0191] (1) The acidic pH regulator in the mobile phase is acetic acid

[0192]

[0193] Note: “No peak” means “there is no retention peak of the substance on the chromatogram”.

[0194] (2) The acidic pH regulator in the mobile phase is trifluoroacetic acid

[0195]

[0196] like Figure 1 As shown, when acetic acid was used as the mobile phase pH adjuster, there was no retention peak for the PADRE-gE-P2 fusion protein.

[0197] Example 2: Study on the method of eluting PADRE-gE-P2 fusion protein

[0198] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0199] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0200] 3) The preparation of Quillaja saponin QS-21 solution and the concentration detection were the same as those in Example 1.

[0201] 4) The vaccine sample preparation was the same as in Example 1.

[0202] 5) Study the method of eluting PADRE-gE-P2 fusion protein

[0203] 1. Solution preparation

[0204] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0205] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0206] 2. The preparation of test samples is the same as that in Example 1.

[0207] 3. Chromatographic column, instrument parameters and elution procedure

[0208] (1) Chromatographic column and instrument parameters

[0209]

[0210] (2) Elution procedure 1: Gradient elution of acetonitrile concentration 5% to 95% (5% mobile phase B to 95% mobile phase B) for 15 minutes to elute the protein

[0211]

[0212] (3) Elution procedure 2: Gradient elution with acetonitrile concentration of 50% to 75% (50% mobile phase B to 75% mobile phase B) for 10 min to elute the protein

[0213]

[0214] (4) Elution procedure 3: Gradient elution of acetonitrile concentration 50% to 75% (50% mobile phase B to 75% mobile phase B) for 20 min to elute the protein

[0215]

[0216] (5) Elution procedure 4: Gradient elution with acetonitrile concentration of 50% to 75% (50% mobile phase B to 75% mobile phase B) for 30 min to elute the protein.

[0217]

[0218] 4. Results Analysis

[0219] The liquid chromatography method used for qualitative and quantitative detection of proteins in vaccines has the following characteristics: the protein has a retention peak; the resolution of the protein retention peak must be greater than 1.5; and the tailing factor must be between 0.8 and 1.8.

[0220] (1) Elution procedure 1: Gradient elution with acetonitrile concentration of 5% to 95% for 15 minutes to elute the protein

[0221]

[0222] (2) Elution procedure 2: Gradient elution with acetonitrile concentration of 50% to 75% for 10 minutes to elute the protein

[0223]

[0224] (3) Elution procedure 3: Gradient elution with acetonitrile concentration of 50% to 75% for 20 minutes to elute the protein

[0225]

[0226] (4) Elution procedure 4: Gradient elution with acetonitrile concentration of 50% to 75% for 30 minutes to elute the protein

[0227]

[0228] like Figure 2 As shown, when the method for eluting the PADRE-gE-P2 fusion protein is 5% to 95% acetonitrile gradient elution for 15 minutes, the retention peak of the PADRE-gE-P2 fusion protein and the front peak (QS-21A isomer retention peak) cannot be completely separated, which does not meet the requirements.

[0229] like Figure 3 As shown, when the method for eluting the PADRE-gE-P2 fusion protein was 5% to 95% acetonitrile gradient elution for 15 min, the separation degree of the PADRE-gE-P2 fusion protein retention peak was less than 1.5, which did not meet the requirements.

[0230] Example 3: Study on column temperature

[0231] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0232] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0233] 3) The preparation of Quillaja saponin QS-21 solution and the concentration detection were the same as those in Example 1.

[0234] 4) The vaccine sample preparation was the same as in Example 1.

[0235] 5) Study column temperature

[0236] 1. Solution preparation

[0237] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0238] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0239] 2. The preparation of test samples is the same as that in Example 1.

[0240] 3. Chromatographic column, instrument parameters and elution procedure

[0241]

[0242] 4. Results Analysis

[0243] The liquid chromatography method used for qualitative and quantitative detection of proteins in vaccines has the following characteristics: the protein has a retention peak; the resolution of the protein retention peak must be greater than 1.5; and the tailing factor must be between 0.8 and 1.8.

[0244] (1) Column temperature 30°C

[0245]

[0246] (2) Column temperature 40°C

[0247]

[0248] (3) Column temperature 50°C

[0249]

[0250] (4) Column temperature 60°C

[0251]

[0252] like Figure 4 As shown in the figure, when the column temperature is 60°C, the retention peak of the PADRE-gE-P2 fusion protein and the front peak (QS-21A isomer retention peak) cannot be completely separated, which does not meet the requirements.

[0253] like Figure 5 As shown in the figure, when the column temperature is 60°C, the separation degree of the retention peak of the PADRE-gE-P2 fusion protein is less than 1.5, which does not meet the requirements.

[0254] like Figure 6 As shown in the figure, when the column temperature was 60°C, the software did not calculate the tailing factor of the PADRE-gE-P2 fusion protein retention peak, which did not meet the requirements.

[0255] Example 4: Study of injection volume

[0256] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0257] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0258] 3) The preparation of Quillaja saponin QS-21 solution and the concentration detection were the same as those in Example 1.

[0259] 4) The vaccine sample preparation was the same as in Example 1.

[0260] 5) Study the injection volume

[0261] 1. Solution preparation

[0262] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0263] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0264] 2. The preparation of test samples is the same as that in Example 1.

[0265] 3. Chromatographic column, instrument parameters and elution procedure

[0266]

[0267]

[0268] 4. Results Analysis

[0269] The liquid chromatography method used for qualitative and quantitative detection of proteins in vaccines has the following characteristics: the protein has a retention peak; the resolution of the protein retention peak must be greater than 1.5; and the tailing factor must be between 0.8 and 1.8.

[0270] (1) Injection volume 50 μl

[0271]

[0272] (2) Injection volume 75 μl

[0273]

[0274] (3) Injection volume 100 μl

[0275]

[0276] Example 5: Study flow rate

[0277] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0278] 2) The preparation of liposomes and the detection of dioleoylphosphatidylcholine and cholesterol concentrations were the same as in Example 1.

[0279] 3) The preparation of Quillaja saponin QS-21 solution and the concentration detection were the same as those in Example 1.

[0280] 4) The vaccine sample preparation was the same as in Example 1.

[0281] 5) Study flow rate

[0282] 1. Solution preparation

[0283] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0284] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0285] 2. The preparation of test samples is the same as that in Example 1.

[0286] 3. Chromatographic column, instrument parameters and elution procedure

[0287]

[0288]

[0289] 4. Results Analysis

[0290] The liquid chromatography method used for qualitative and quantitative detection of proteins in vaccines has the following characteristics: the protein has a retention peak; the resolution of the protein retention peak must be greater than 1.5; and the tailing factor must be between 0.8 and 1.8.

[0291] (1) Flow rate: 1.0 ml / min

[0292]

[0293] (2) Flow rate: 1.5 ml / min

[0294]

[0295] (3) Flow rate: 2.0 ml / min

[0296]

[0297] like Figure 9 As shown, when the flow rate is 1.0 ml / min, the retention peak of the PADRE-gE-P2 fusion protein and the front peak (QS-21A isomer retention peak) cannot be completely separated, which does not meet the requirements.

[0298] Example 6: Detection of PADRE-gE-P2 fusion protein content in vaccine samples (mobile phase for eluting DOPC and cholesterol is acetonitrile and water system)

[0299] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0300] 2) The vaccine sample preparation was the same as in Example 1.

[0301] 3) Detect the content of PADRE-gE-P2 fusion protein in vaccine samples.

[0302] 1. Solution preparation

[0303] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0304] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0305] 2. Vaccine Sample Testing

[0306] S1. Preparation of protein calibration standard solution samples

[0307] 658 μl of PADRE-gE-P2 fusion protein reference solution (concentration of 1.52 mg / ml) was placed in a 5 ml volumetric flask and dissolved and diluted to the scale with phosphate solution (10 mM; pH 6.0). Shake well to obtain a solution with a concentration of 200 μg / ml. Then, 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution were transferred to different centrifuge tubes, and 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0) were added, respectively, and mixed 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 respectively, cover the vials, 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.

[0308] S2. Preparation of test samples

[0309] Pipette 0.5 ml of the vaccine sample solution to be tested into a sampling vial, add 0.5 ml of 2% DMSO solution, cover the vial cap, and mix well.

[0310] S3. Load the sample into the liquid chromatograph and run the analysis method

[0311] 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.

[0312]

[0313]

[0314] S5. Calculate the protein content in the vaccine

[0315] (1) Establishment of standard curve

[0316] The peak area values ​​of the PADRE-gE-P2 fusion protein calibration standard solution samples were used to establish a calibration standard curve in which the protein concentration was proportional to the peak area, such as Figure 12 shown.

[0317] PADRE-gE-P2 fusion protein calibration standard solution sample test results and standard curve

[0318] Protein calibration standard solution Retention time (min) Peak area (μAU*min) tailing factor Calibration standard solution-1 (50 μg / ml) 11.757 3029134 1.101 Calibration standard solution-2 (62.5 μg / ml) 11.721 3792102 1.111 Calibration standard solution-3 (75 μg / ml) 11.697 4508465 1.115 Calibration standard solution-4 (87.5 μg / ml) 11.677 5294522 1.117 Calibration standard solution-5 (100 μg / ml) 11.667 6093204 1.102

[0319] PADRE-gE-P2 fusion protein peak area standard curve: y = 60930x-43848, R 2 =0.9999.

[0320] (2) Content calculation

[0321] like Figure 13 As shown, the retained peak of PADRE-gE-P2 fusion protein was completely separated from the front peak, which met the requirements.

[0322] The protein content in the vaccine was calculated by substituting the peak area value of the protein in the vaccine sample into the calibration standard curve formula and multiplying it by the dilution factor of the vaccine (twice).

[0323]

[0324] 4) Compare the differences in the detection of PADRE-gE-P2 fusion protein content in vaccine samples by liquid chromatography (the mobile phase for eluting DOPC and cholesterol is acetonitrile and water system) and Lowry method, such as Figure 14 shown

[0325] 1. Lowry method for detecting protein content in vaccine samples (existing technology)

[0326] 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.

[0327] (A) Reagent preparation

[0328] 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.

[0329] (B) Preparation of protein reference working solution

[0330] 200 μg / ml Protein Reference Solution (Bovine Serum Albumin Solution): Dissolve one vial of Protein Reference 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).

[0331] (C) XA-401 adjuvant control formulation

[0332] 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; 4.76 ml of liposomes are added with 0.24 ml of Quillaja saponin QS-21 solution, and finally the solution is made up to 10 ml with phosphate solution (10 mM; pH 6.0), and stirred evenly to obtain the product.

[0333] (D) Protein concentration detection

[0334] Preparation and testing of test samples: Accurately pipette an appropriate amount of vaccine sample and dilute with water until the protein content is within the standard curve range; accurately pipette 1.0 ml of the above solution into a test tube in duplicate, add 1.0 ml of alkaline copper solution, mix well, and let it stand at room temperature for 10 minutes. Add 4.0 ml of folin phenol test solution, mix immediately, let it stand at room temperature for 30 minutes, and after color development, measure the absorbance at a wavelength of 650 nm by UV-visible spectrophotometry (after color development, if turbidity is found, centrifuge at 3000 rpm / min for 15 minutes, and then take the supernatant for determination).

[0335] 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".

[0336] 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 of the vaccine sample); accurately pipette 1.0 ml of the above solution into a test tube, perform two replicates, and proceed in the same manner starting from "add 1.0 ml of alkaline copper solution" to serve as the adjuvant control.

[0337] (E) Calculation of protein concentration in vaccine

[0338] 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. Vaccine sample protein content (μg / ml) = 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 vaccine sample dilution factor.

[0339] 2. Comparison of the differences in the detection of PADRE-gE-P2 fusion protein content in vaccine samples by liquid chromatography (acetonitrile as the mobile phase for eluting DOPC and cholesterol) and Lowry method

[0340]

[0341] Example 7: Qualitative Detection of Protein in Vaccine Samples (Mobile Phase for Elution of DOPC and Cholesterol: Acetonitrile and Water System)

[0342] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0343] 2) The vaccine sample preparation was the same as in Example 1.

[0344] 3) Qualitative detection of PADRE-gE-P2 fusion protein in vaccine samples

[0345] 1. Solution preparation

[0346] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 1.

[0347] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0348] 2. Vaccine Sample Testing

[0349] S1. Preparation of protein calibration standard solution samples

[0350] Place 658 μl of the PADRE-gE-P2 fusion protein reference solution (1.52 mg / ml) in a 5 ml volumetric flask and dilute to the mark with 10 mM phosphate solution (pH 6.0). Shake well to obtain a 200 μg / ml solution. Pipette 1 ml of this 200 μg / ml solution into a centrifuge tube, add 0.33 ml of 10 mM phosphate solution (pH 6.0), and mix well to obtain a 150 μg / ml solution. Pipette 0.5 ml of this solution into a sample vial, add 0.5 ml of 2% DMSO solution, cap the vial, and mix well to obtain a 75 μg / ml protein solution.

[0351] S2. Preparation of test samples

[0352] The same as "S2. Preparation of test samples" in Example 6.

[0353] S3. Load the sample into the liquid chromatograph and run the analysis method

[0354] The same as "S3, loading the sample into the liquid chromatograph and running the analysis method" in Example 6.

[0355] S4. Calculate the relative deviation of protein retention time in vaccines

[0356] Calculate the relative deviation of the retention time of the vaccine protein retention peak and the qualitative standard retention peak. The calculation formula is relative deviation = [|retention time of vaccine protein retention peak - average retention time| / average retention time] × 100%. Note: The average retention time is the average retention time of the vaccine protein retention peak and the retention time of the qualitative standard (PADRE-gE-P2 fusion protein calibration standard solution sample). Judgment standard: if the relative deviation is not higher than 2%, it can be determined that the protein of the vaccine sample is PADRE-gE-P2 fusion protein.

[0357]

[0358] Example 8: Detection of PADRE-gE-P2 fusion protein content in vaccine samples (mobile phase for eluting DOPC and cholesterol is methanol and water system)

[0359] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0360] 2) The vaccine sample preparation was the same as in Example 1.

[0361] 3) Detection of PADRE-gE-P2 fusion protein content in vaccine samples

[0362] 1. Solution preparation

[0363] (1) Preparation of mobile phase (trifluoroacetic acid)

[0364] 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 degas by ultrasonication for 5 minutes.

[0365] Mobile phase B [acetonitrile: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and ultrasonically degas for 5 minutes to obtain the mobile phase.

[0366] 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.

[0367] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0368] 2. Vaccine Sample Testing

[0369] S1. Preparation of protein calibration standard solution samples

[0370] The same as “S1. Preparation of protein calibration standard solution sample” in Implementation Case 6.

[0371] S2. Preparation of test samples

[0372] Same as “S2. Preparation of test samples” in Implementation Case 6.

[0373] S3. Load the sample into the liquid chromatograph and run the analysis method

[0374] 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.

[0375]

[0376]

[0377] S5. Calculate the protein content in the vaccine

[0378] (1) Establishment of standard curve

[0379] The peak area values ​​of the PADRE-gE-P2 fusion protein calibration standard solution samples were used to establish a calibration standard curve in which the protein concentration was proportional to the peak area, such as Figure 15 shown.

[0380] PADRE-gE-P2 fusion protein calibration standard solution sample test results and standard curve

[0381]

[0382]

[0383] Peak area standard curve of PADRE-gE-P2 fusion protein: y=60464x-36883, R2=0.9998.

[0384] (2) Content calculation

[0385] like Figure 16As shown, the retained peak of PADRE-gE-P2 fusion protein was completely separated from the front peak, which met the requirements.

[0386] The protein content in the vaccine was calculated by substituting the peak area value of the protein in the vaccine sample into the calibration standard curve formula and multiplying it by the dilution factor of the vaccine (twice).

[0387]

[0388] 4) Compare the differences in the detection of PADRE-gE-P2 fusion protein content in vaccine samples by liquid chromatography (PADRE-gE-P2 fusion protein as protein standard; the mobile phase for eluting DOPC and cholesterol is methanol and water system) and Lowry method, such as Figure 17 shown

[0389] 1. Lowry method for detecting protein content in vaccine samples (existing technology)

[0390] The same as the item “4)” in Example 6 “1. Lowry method for detecting protein content in vaccine samples (existing technology)”.

[0391] 2. Comparison of the differences in the detection of PADRE-gE-P2 fusion protein content in vaccine samples by liquid chromatography (methanol as the mobile phase for eluting DOPC and cholesterol) and Lowry method

[0392]

[0393] Example 9: Qualitative Detection of Protein in Vaccine Samples (Mobile Phase for Elution of DOPC and Cholesterol: Methanol and Water System)

[0394] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0395] 2) The vaccine sample preparation was the same as in Example 1.

[0396] 3) Qualitative detection of PADRE-gE-P2 fusion protein in vaccine samples

[0397] 1. Solution preparation

[0398] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 8.

[0399] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0400] 2. Vaccine Sample Testing

[0401] S1. Preparation of protein calibration standard solution samples

[0402] The same as "S1, preparation of protein calibration standard solution sample" in Example 7.

[0403] S2. Preparation of test samples

[0404] The same as "S2. Preparation of test samples" in Example 6.

[0405] S3. Load the sample into the liquid chromatograph and run the analysis method

[0406] The same as "S3, loading the sample into the liquid chromatograph and running the analysis method" in Example 8.

[0407] S4. Calculate the relative deviation of protein retention time in vaccines

[0408]

[0409] Example 10. Simultaneous qualitative and quantitative detection of proteins in vaccine samples (BSA as a protein standard; the mobile phase for eluting DOPC and cholesterol is a methanol and water system)

[0410] 1) The preparation of PADRE-gE-P2 fusion protein and the detection of protein concentration were the same as those in Example 1.

[0411] 2) The vaccine sample preparation was the same as in Example 1.

[0412] 3) Simultaneous qualitative and quantitative detection of PADRE-gE-P2 fusion protein in vaccine samples

[0413] 1. Solution preparation

[0414] (1) The mobile phase preparation (trifluoroacetic acid) was the same as that in Example 8.

[0415] (2) The preparation of vaccine sample dilution solution is the same as that in Example 1.

[0416] 2. Vaccine Sample Testing

[0417] S1. Preparation of protein calibration standard solution samples

[0418] 1. Preparation of PADRE-gE-P2 fusion protein calibration standard solution

[0419] The same as "S1, preparation of protein calibration standard solution sample" in Example 7.

[0420] 2. Preparation of BSA Calibration Standard Solution

[0421] 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). 457 μl of BSA reference stock solution (concentration of 2.19 mg / ml) was placed in a 5 ml volumetric flask and diluted to the mark with phosphate solution (10 mM; pH 6.0). Shake well to obtain a solution with a concentration of 200 μg / ml. Then, 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution were transferred to different centrifuge tubes. Then, 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0) were added, respectively, and mixed 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.

[0422] S2. Preparation of test samples

[0423] Same as "S2, preparation of test samples" in Example 6

[0424] S3. Load the sample into the liquid chromatograph and run the analysis method

[0425] The same as “S3, loading the sample into the liquid chromatograph and running the analysis method” in Implementation Case 8.

[0426] S4. Calculate the relative deviation of protein retention time in vaccines

[0427]

[0428] S5. Calculate the protein content in the vaccine

[0429] (1) Establishment of standard curve

[0430] Use the peak area values ​​of the BSA calibration standard solution samples to establish a calibration standard curve in which protein concentration is proportional to peak area, such as Figure 18 shown.

[0431] BSA calibration standard solution sample test results and standard curve

[0432]

[0433]

[0434] BSA protein peak area standard curve: y = 55701x-54266, R 2 =0.9993.

[0435] (2) Content calculation

[0436] The protein content in the vaccine was calculated by substituting the peak area value of the protein in the vaccine sample into the calibration standard curve formula and multiplying it by the dilution factor of the vaccine (twice).

[0437]

[0438] 4) Comparison of the differences in the detection of PADRE-gE-P2 fusion protein content in vaccine samples by liquid chromatography (BSA as protein standard; methanol and water as mobile phase for elution of DOPC and cholesterol) and Lowry method

[0439] 1. Lowry method for detecting protein content in vaccine samples (existing technology)

[0440] The same as the item “4)” in Example 6 “1. Lowry method for detecting protein content in vaccine samples (existing technology)”.

[0441] Second, the differences in the detection of PADRE-gE-P2 fusion protein content in vaccine samples were compared between liquid chromatography (methanol system for elution of DOPC and cholesterol) and Lowry method. Figure 19 shown.

[0442]

[0443] Finally, it should be noted that:

[0444] 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 qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine, characterized in that: The following steps are involved: S1. Prepare protein calibration standard solution sample; S2, preparing test samples; S3, loading the sample into the liquid chromatograph and running the analytical method; S4. Calculate the relative deviation of protein retention time in the vaccine; S5. Calculate the protein content in the vaccine.

2. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 1, characterized in that: The recombinant varicella-zoster vaccine consists of a fusion protein and a composite adjuvant. The fusion protein includes the extracellular segment gE of varicella-zoster virus glycoprotein E, the universal DR Th epitope peptide PADRE, and the tetanus toxin Th epitope P2 amino acid sequence. The gE, PADRE, and P2 are connected by a connecting peptide, and the connecting peptide is GGS and / or GGGS and / or GGGGS and / or GSGSG connecting peptide. The composite adjuvant is a neutral liposome and an immunopotentiator, Quillaja saponin QS-21. The neutral liposome is composed of dioleoylphosphatidylcholine and cholesterol.

3. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 2, characterized in that: The fusion protein is a PADRE-gE-P2 fusion protein, 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.

1.

4. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 1, characterized in that: The protein concentration of the protein calibration standard solution sample in S1 is 1 μg / ml to 200 μg / ml.

5. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 1, characterized in that: The chromatographic column of the liquid chromatograph in S3 contains an alkyl or phenyl reverse-phase chromatographic filler, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water; the liquid chromatograph parameters of the analytical method include but are not limited to: column temperature, injection volume, flow rate, and detector parameters; the column temperature is 20°C to 70°C; the injection volume is 5μl to 100μl; the flow rate is 0.1 ml / min to 2.5 ml / min; when the detector is an ultraviolet detector, the detection wavelength is 200 to 400nm.

6. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 1, characterized in that: The elution procedure of the analytical method in S3 includes an elution method for eluting the protein retention peak, the elution method for eluting the protein retention peak is gradient elution, the acetonitrile concentration gradient in the mobile phase is 5% to 100%, 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 qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 6, characterized in that: The elution procedure of the analytical method further includes an elution method for eluting the retention peaks of dioleoylphosphatidylcholine and cholesterol.

8. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 1, characterized in that: The relative deviation of protein retention time is calculated in S4 as follows: The relative deviation of the protein retention time in the vaccine = [|retention time of the vaccine protein retention peak - average retention time| / average retention time] × 100%, where the average retention time is the average retention time of the vaccine protein retention peak and the retention time of the protein calibration standard solution sample retention peak; the criterion for determining whether the protein in the vaccine sample is the PADRE-gE-P2 fusion protein is: the relative deviation is not higher than 2%.

9. The method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine according to claim 1, characterized in that: The method for calculating the protein content in the vaccine in S5 includes: Calculation method 1: First, establish a calibration standard curve in which the protein concentration is proportional to the peak area; then, substitute the peak area value of the protein in the vaccine sample into the calibration standard curve formula to calculate the concentration of the protein in the test sample; then, multiply the concentration of the protein in the test sample by the dilution factor to calculate the concentration of the protein in the vaccine; Calculation method 2: Protein concentration in vaccine = protein peak area of ​​test sample * protein calibration standard solution sample concentration * dilution factor / protein calibration standard solution sample peak area.

10. Application of a method for qualitative and quantitative detection of proteins in recombinant varicella-zoster vaccine in the detection of components in recombinant varicella-zoster vaccine, characterized in that: Including but not limited to the detection of PADRE-gE-P2 fusion protein.

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