A liquid chromatography method for simultaneously qualitatively and quantitatively detecting proteins in a PADRE-gE-P2 fusion protein stock solution and application

Abstract

This invention discloses a high-performance liquid chromatography (HPLC) method and its application for the simultaneous qualitative and quantitative detection of proteins in PADRE-gE-P2 fusion protein stock solution. The method includes the following steps: preparing a protein calibration standard solution sample; preparing a test sample; loading the sample into the HPLC instrument and running the analytical method; calculating the relative deviation of protein retention time; and calculating the protein content. This method not only solves the technical problem of numerous manual steps leading to significant errors in existing technologies, but also addresses the inability of existing technologies to distinguish between PADRE-gE-P2 and gE. The HPLC method disclosed in this invention has a higher degree of automation than existing technologies, can distinguish between PADRE-gE-P2 and gE, and can simultaneously perform qualitative and quantitative detection of PADRE-gE-P2 fusion protein stock solution, greatly improving detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a liquid chromatography method and its application for simultaneous qualitative and quantitative detection of proteins in PADRE-gE-P2 fusion protein stock solution. Background Technology

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

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

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

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

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

[0007] To control vaccine quality, it is essential to qualitatively and quantitatively detect the proteins in the protein antigen solutions (stock solutions) used to prepare the vaccines. Therefore, it is necessary to establish a method for qualitative and quantitative detection of proteins in the PADRE-gE-P2 fusion protein solution (stock solution).

[0008] Currently, the recombinant zoster vaccine already on the market In quality control, qualitative and quantitative detection Two different detection methods are needed for gE protein solutions (gE stock solution) in vaccines: 1. The Lowry method for quantitative protein detection (lowry method); 2. The ELISA method for qualitative detection (identification) of proteins in the stock solution. Current technology (the Lowry method) requires numerous manual steps in quantitative protein detection, resulting in low automation, significant errors, and poor stability of results. The ELISA method can only qualitatively detect gE on the PADRE-gE-P2 fusion protein, not the PADRE-gE-P2 fusion protein itself. Therefore, a qualitative detection method that can distinguish between the PADRE-gE-P2 fusion protein and gE must be established. In summary, current methods for quantitatively detecting protein in gE stock solution have low automation, numerous manual steps, large detection errors, and poor stability of results. Furthermore, the qualitative detection method for gE stock solution cannot adequately identify the PADRE-gE-P2 fusion protein. Summary of the Invention

[0009] The purpose of this invention is to provide a liquid chromatography method and application for simultaneous qualitative and quantitative detection of protein in PADRE-gE-P2 fusion protein stock solution, in order to solve the problems of unstable results and large errors in quantitative detection methods and the inability of qualitative detection methods to fully identify PADRE-gE-P2 fusion protein in the prior art.

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

[0011] This invention provides a liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in a PADRE-gE-P2 fusion protein stock solution. The PADRE-gE-P2 fusion protein includes the extracellular region of varicella-zoster virus (VZV) glycoprotein E (gE), the amino acid sequence of the universal DR Th epitope peptide PADRE (PADRE), and the tetanus toxin Th epitope P2 (P2). The gE, PADRE, and P2 are linked by a linker peptide, which is a GGS and / or GGGS and / or GGGGS and / or GGSSG linker peptide.

[0012] Furthermore, one alternative molecular structure of the PADRE-gE-P2 fusion protein is (from N-terminus to C-terminus): PADRE—GSGSG (linker peptide)—gE (AA31-AA544)—GGS (linker peptide)—P2, with the amino acid sequence shown in SEQ ID NO.1.

[0013] Furthermore, the PADRE-gE-P2 fusion protein solution (stock solution) has a protein content of 0.25 mg / ml to 16 mg / ml.

[0014] Furthermore, the PADRE-gE-P2 fusion protein solution (stock solution) may contain buffering components to maintain pH stability.

[0015] Furthermore, the buffer component for maintaining pH stability is selected from any one or more of the following substances, but is not limited to: disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, histidine, histidine hydrochloride, glycine, hydrochloric acid, sodium hydroxide, sodium carbonate, potassium carbonate, sodium citrate, citric acid, succinic acid, sodium succinate, acetic acid, sodium acetate, tris(hydroxymethyl)aminomethane, and 4-hydroxyethylpiperazine ethanesulfonic acid.

[0016] Furthermore, the PADRE-gE-P2 fusion protein solution (stock solution) may also contain components that maintain protein stability.

[0017] Furthermore, the component that maintains protein stability may be selected from any one or more of the following substances, but is not limited to: polysorbate 80, polysorbate 20, and poloxamer 188.

[0018] Furthermore, the liquid chromatography method is high performance liquid chromatography or ultra-high performance liquid chromatography.

[0019] Furthermore, the liquid chromatography method is reversed-phase liquid chromatography, wherein the chromatographic column contains alkyl or phenyl reversed-phase chromatographic packing material, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water.

[0020] Furthermore, the alkyl reversed-phase chromatography packing material can be selected from any of the following chromatography packing materials, but is not limited to: butylsilane bonded silica gel (C4), hexaalkylsilane bonded silica gel (C6), octaalkylsilane bonded silica gel (C8), hexadecylsilane bonded silica gel (C16), and octadecylsilane bonded silica gel (C18).

[0021] Furthermore, the liquid chromatography method is either the internal standard method or the external standard method.

[0022] Furthermore, the liquid chromatography external standard method includes the following steps:

[0023] S1. Prepare protein calibration standard solution samples;

[0024] S2. Prepare the test sample;

[0025] S3. Load the sample into the liquid chromatograph and run the analytical method;

[0026] S4. Calculate the relative deviation of protein retention time;

[0027] S5. Calculate the protein content.

[0028] Furthermore, in step S1, the preparation of protein calibration standard solution samples can involve preparing calibration standard solution samples of one or two proteins. A PADRE-gE-P2 fusion protein calibration standard solution sample must be prepared (for qualitative analysis; possibly for quantitative analysis). Additionally, another protein calibration standard solution sample may be prepared (possibly for quantitative analysis). The other protein used can be selected from any of the following, but is not limited to: bovine serum albumin (BSA), bovine gamma-globulin (BGG), and the extracellular region of varicella-zoster virus (VZV) glycoprotein E (gE).

[0029] Furthermore, the preparation method for the PADRE-gE-P2 fusion protein calibration standard solution sample (for qualitative analysis; possibly for quantitative analysis) can be selected from any of the following methods, but is not limited to: 1. Accurately weigh the target weight of PADRE-gE-P2 fusion protein powder, dissolve it in water or buffer solution, and finally bring the volume up to the target volume with water or buffer solution and mix well; 2. Detect the protein concentration of the PADRE-gE-P2 fusion protein solution using methods such as Lowry method, BCA method, biuret method, Kjeldahl method, Coomassie brilliant blue method, and liquid chromatography, and then calibrate its protein concentration.

[0030] Furthermore, the preparation of another protein calibration standard solution sample (potentially for quantification) can be selected from any of the following methods, but is not limited to: 1. Accurately weigh the target weight of the other protein powder, dissolve it in water or buffer solution, and finally dilute to the target volume with water or buffer solution and mix well; 2. Use methods such as Lowry method, BCA method, biuret method, Kjeldahl method, Coomassie brilliant blue method, liquid chromatography, etc. to detect the protein concentration of the other protein solution, and then calibrate its protein concentration.

[0031] Furthermore, the protein calibration standard solution sample in S1 has a protein concentration of 0.4 μg / ml to 1000 μg / ml.

[0032] Furthermore, the protein calibration standard solution sample may contain DMSO at a concentration of 0.5% to 5%.

[0033] Furthermore, the PADRE-gE-P2 fusion protein solution (stock solution) used in S2 to prepare the test sample has a protein content of 0.25 mg / ml to 16 mg / ml.

[0034] Furthermore, the protein concentration of the sample detected in S2 is 0.4 μg / ml to 4 mg / ml.

[0035] Furthermore, the sample prepared in step S2 may contain DMSO at a concentration of 0.5% to 5%.

[0036] Furthermore, the detector of the liquid chromatograph in S3 is an ultraviolet detector, an electrospray ionization detector (CAD), or an evaporative light detector.

[0037] Furthermore, the analytical method in S3 includes a chromatographic column, liquid chromatograph parameters, and elution program.

[0038] Furthermore, the chromatographic column used in the analytical method in S3 is a reversed-phase chromatographic column.

[0039] Furthermore, the liquid chromatography parameters of the analytical method in S3 include column temperature, injection volume, flow rate, detector parameters, etc.

[0040] Furthermore, the column temperature is 20℃ to 70℃, such as 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, or 70℃.

[0041] 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.

[0042] 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.

[0043] Furthermore, when the instrument is a high-performance liquid chromatograph, the flow rate is 1.0 ml / min to 2.5 ml / min, such as 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, 1.6 ml / min, 1.7 ml / min, 1.8 ml / min, 1.9 ml / min, 2.0 ml / min, 2.1 ml / min, 2.2 ml / min, 2.3 ml / min, 2.4 ml / min, or 2.5 ml / min.

[0044] Furthermore, when the instrument is an ultra-high performance liquid chromatograph, the flow rate is 0.1 ml / min to 1.0 ml / min, such as 0.1 ml / min, 0.2 ml / min, 0.3 ml / min, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, or 1.0 ml / min.

[0045] Furthermore, the detector parameters are characterized as follows: when the detector is an ultraviolet detector, the detection wavelength is 200nm to 400nm, such as 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, or 400nm.

[0046] Furthermore, when the detector is a CAD detector, the atomization temperature is 35℃~70℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃.

[0047] Furthermore, when the detector is an evaporative photodetector, the atomization temperature is 35℃~70℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃.

[0048] Furthermore, the elution procedure of the analytical method in S3 includes at least an elution method for eluting protein retention peaks.

[0049] Furthermore, the elution method for the protein retention peak is gradient elution, with an acetonitrile concentration gradient in the mobile phase of 5%–100%, such as 5%–100%, 5%–95%, 5%–90%, 5%–85%, 5%–80%, 5%–75%, 5%–70%, 5%–65%, 5%–60%, 5%–55%, 5%–50%, 10%–100%, 10%–95%, 10%–90%, 10%–85%, 10%–80%, 10%–75%, 10%–70%, 10%–65%, 10%–60%, 10%–55%, 10%–50%, 15%–100%, 15%–95%, 15%–90%, 15%–85%, 15%–15%. ~80%, 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 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%–7 0%, 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%, 4 5%–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%–70% 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%,The concentrations of trifluoroacetic acid in the mobile phase are 60%–70%, 60%–75%, 60%–80%, 60%–85%, 60%–90%, 60%–95%, 60%–100%, or 65%–75%, with gradient elution times ranging from 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. The concentration of trifluoroacetic acid in the mobile phase is 0.01%–0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0050] Furthermore, the elution method for the protein retention peak is isocratic elution, with an acetonitrile concentration gradient in the mobile phase of 50%–100%, such as 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 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%, 94%, 95%, 96%, 97%. 98%, 99%, or 100%, isocratic elution time of 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, with trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0051] Furthermore, in the elution procedure of the S3 analysis method, a pre-equilibration method and / or a pre-elution method can be added before the elution method for eluting the protein retention peak.

[0052] Furthermore, a pre-equilibration method is added before the elution method for the protein retention peak, wherein the acetonitrile concentration in the mobile phase is not higher than the acetonitrile concentration of the protein retention peak.

[0053] Furthermore, a pre-equilibration method is added before eluting the protein retention peak: the acetonitrile concentration in the mobile phase is 5%–60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, and the equilibration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc. The timeframes are 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, with the trifluoroacetic acid concentration in the mobile phase being 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0054] Furthermore, a pre-elution method is added before the elution of the protein retention peak, wherein the acetonitrile concentration in the mobile phase is not higher than the acetonitrile concentration of the protein retention peak.

[0055] Furthermore, a pre-elution method is added before the elution of the protein retention peak: the acetonitrile gradient concentration in the mobile phase is 5%–60%, such as 5%–10%, 5%–15%, 5%–20%, 5%–25%, 5%–30%, 5%–35%, 5%–40%, 5%–45%, 5%–50%, 5%–55%, 5%–60%, 10%–15%, 10%–20%, 10%–25%, 10%–30%, 10%–35%, 10%–40%, 10%–45%, 10%–50%, 10%–55%. 10%–60%, 15%–20%, 15%–25%, 15%–30%, 15%–35%, 15%–40%, 15%–45%, 15%–50%, 15%–55%, 15%–60%, 20%–25%, 20%–30%, 20%–35%, 20%–40%, 20%–45%, 20%–50%, 20%–55%, 20%–60%, 25%–30%, 25%–35%, 25%–40%, 25%–45%, 25%–50%, 25%–55%, 25%– 60%, 30%–35%, 30%–40%, 30%–45%, 30%–50%, 30%–55%, 30%–60%, 35%–40%, 35%–45%, 35%–50%, 35%–55%, 35%–60%, 40%–45%, 40%–50%, 40%–55%, 40%–60%, 45%–50%, 45%–55%, 45%–60%, 50%–55%, 50%–60%, or 55%–60%, with gradient elution times of 1–960 min, such as 1 min. The timeframes are 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, with a trifluoroacetic acid concentration in the mobile phase of 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0056] Furthermore, in the elution procedure of the analytical method in S3, a column regeneration method and / or a post-equilibration method can be added after the elution method for eluting the protein retention peak.

[0057] Furthermore, when both the column regeneration method and the post-equilibration method exist simultaneously, the column regeneration method should precede the post-equilibration method.

[0058] Furthermore, after eluting the protein retention peak, a column regeneration method is added: the acetonitrile concentration in the mobile phase is 98%–100%, such as 98%, 98.5%, 99%, 99.5%, or 100%; the column regeneration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min; the mobile phase may also contain trifluoroacetic acid at a concentration of 0.01%–0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0059] Furthermore, after eluting the protein retention peak, an equilibration method is added: the acetonitrile concentration in the mobile phase is 5%–60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, and the equilibration time is 1–960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc. The timeframes are 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, with the trifluoroacetic acid concentration in the mobile phase being 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0060] Furthermore, in S4, the relative deviation of protein retention time in the vaccine is calculated using the following formula: [|protein retention peak retention time - average retention time| / average retention time] × 100%. Note: the average retention time is the average of the retention time of the protein retention peak and the retention time of the protein calibration standard solution sample.

[0061] Furthermore, the criterion for determining whether a protein is a PADRE-gE-P2 fusion protein is: the relative deviation of protein retention time in the vaccine is not higher than 1%.

[0062] Furthermore, the method for calculating the protein content in the vaccine in S5 includes:

[0063] Calculation Method 1: First, establish a calibration standard curve in which the protein concentration is proportional to the peak area; then, substitute the protein peak area value of the test sample into the calibration standard curve formula to calculate the protein concentration in the test sample; multiply the protein concentration of the test sample by the dilution factor to calculate the protein content (concentration) in the PADRE-gE-P2 fusion protein solution (stock solution).

[0064] Calculation Method 2: Directly calculate the protein content in the vaccine. The protein content (concentration) in the PADRE-gE-P2 fusion protein solution (stock solution) = peak area of ​​the protein in the test sample * concentration of the protein calibration standard solution * dilution factor / peak area of ​​the protein calibration standard solution.

[0065] The present invention also provides the application of a liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in protein stock solutions in the detection of protein stock solution components, including but not limited to the qualitative and / or quantitative detection of PADRE-gE-P2 fusion protein.

[0066] Furthermore, it is used to detect the protein content (quantitative detection) in PADRE-gE-P2 fusion protein solution (stock solution).

[0067] Furthermore, it is used for qualitative detection of proteins in PADRE-gE-P2 fusion protein vaccines.

[0068] Furthermore, it can be used for the simultaneous qualitative and quantitative detection of protein content in PADRE-gE-P2 fusion protein solution (stock solution).

[0069] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0070] (1) Quantitatively detect the protein content in the PADRE-gE-P2 fusion protein stock solution.

[0071] (2) Identify whether the protein in the PADRE-gE-P2 fusion protein stock solution is the PADRE-gE-P2 fusion protein or qualitatively detect the protein in the PADRE-gE-P2 fusion protein stock solution.

[0072] (3) Simultaneously, qualitative and quantitative detection of proteins in the PADRE-gE-P2 fusion protein stock solution was performed.

[0073] (4) The liquid chromatography method and application provided by this invention for simultaneous qualitative and quantitative detection of proteins in PADRE-gE-P2 fusion protein stock solution are similar to those of Shingrix. Compared to the quantitative detection method used for gE stock solution, this method has a higher degree of automation, fewer manual steps, smaller error in protein content detection, and better stability. Furthermore, while quantitatively detecting PADRE-gE-P2 fusion protein stock solution, it can also qualitatively detect the protein stock solution. Additionally, when used for qualitative protein detection, this method can distinguish between PADRE-gE-P2 fusion protein and gE. The ELSI method used for qualitative detection of gE stock solution could not distinguish between PADRE-gE-P2 fusion protein and gE. Attached Figure Description

[0074] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0075] Figure 1 This is a liquid chromatogram of the BSA calibration standard solution sample from Example 1 of the present invention.

[0076] Figure 2 This is the standard curve of BSA concentration versus peak area in Example 1 of this invention.

[0077] Figure 3 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) by 4 times in Example 1 of this invention.

[0078] Figure 4 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) of Example 1 of the present invention by 8 times.

[0079] Figure 5 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) of Example 1 of the present invention by 16 times.

[0080] Figure 6 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) 32 times according to Example 1 of the present invention.

[0081] Figure 7 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) of Example 1 of the present invention by 64 times.

[0082] Figure 8 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) of Example 1 of the present invention by 128 times.

[0083] Figure 9 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) of Example 1 of the present invention by 256 times.

[0084] Figure 10 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) of Example 1 of the present invention by 512 times.

[0085] Figure 11 This is a comparison chart of the RSD values ​​of the PADRE-gE-P2 fusion protein solution (stock solution) detected by liquid chromatography and Lowry method in Example 1 of this invention.

[0086] Figure 12 This is a liquid chromatogram of the calibration standard solution sample of the PADRE-gE-P2 fusion protein in Example 2 of this invention.

[0087] Figure 13 This is a liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) 32 times according to Example 2 of the present invention.

[0088] Figure 14 This is a comparison chart of the retention times of the PADRE-gE-P2 fusion protein and the gE retention peak in Example 2 of this invention. Detailed Implementation

[0089] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0090] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0091] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0092] A liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in PADRE-gE-P2 fusion protein stock solution.

[0093] The PADRE-gE-P2 fusion protein comprises the extracellular region of varicella-zoster virus (VZV) glycoprotein E (gE), the universal DR Th epitope peptide PADRE (PADRE), and the amino acid sequence of tetanus toxin Th epitope P2 (P2). The gE, PADRE, and P2 are linked by a linker peptide, which is a GGS and / or GGGS and / or GGGGS and / or GGSSG linker peptide. The gene sequences encoding gE, PADRE, and P2 can express the gE fusion protein containing PADRE and P2 in genetically engineered cells.

[0094] An optional molecular structure of the PADRE-gE-P2 fusion protein (from N-terminus to C-terminus) is: PADRE-GSGSG (linker peptide)-gE(AA31-AA544)-GGS (linker peptide)-P2, the nucleic acid sequence of which is shown in CN 117003896 B and the amino acid sequence of which is shown in SEQ ID NO.1.

[0095] After codon optimization and full-gene synthesis of the nucleic acid sequence of the PADRE-gE-P2 fusion protein, a fusion protein expression plasmid was constructed, followed by the construction of a stable cell line, expression of the target product (PADRE-gE-P2 fusion protein), and finally purification to obtain the PADRE-gE-P2 fusion protein. The protein concentration of the fusion protein solution was then detected.

[0096] The PADRE-gE-P2 fusion protein (XA-401 adjuvant) vaccine showed superior immunogenicity in mice compared to GSK's recombinant herpes zoster vaccine Shingrix, as detailed in CN 116747298 B and CN 117003896 B.

[0097] The protein in the PADRE-gE-P2 fusion protein solution (stock solution) was quantitatively detected using liquid chromatography (LC), and the results were compared with those of existing techniques. The comparison results show that both the LC method and existing techniques can be used for the quantitative detection of protein in the PADRE-gE-P2 fusion protein solution (stock solution). However, the LC method has a higher degree of automation, reducing errors caused by manual operation and resulting in more stable detection results. Furthermore, the optimal LC parameters and elution methods may differ when using reversed-phase columns with different packing materials to detect target substances (such as the PADRE-gE-P2 fusion protein), which is known to those skilled in the art.

[0098] In addition, this liquid chromatography method can also qualitatively detect proteins in the PADRE-gE-P2 fusion protein solution (stock solution) and can distinguish between the PADRE-gE-P2 fusion protein and gE.

[0099] Most conveniently, this liquid chromatography method can simultaneously perform qualitative and quantitative detection of PADRE-gE-P2 fusion protein solution (stock solution), greatly improving detection efficiency.

[0100] Example 1: Quantitative detection of protein in PADRE-gE-P2 fusion protein solution (stock solution)

[0101] 1) Preparation of PADRE-gE-P2 fusion protein solution (stock solution)

[0102] I. Codon Optimization and Whole Genome Synthesis of Fusion Proteins

[0103] See patent CN 117003896 B for details.

[0104] II. Construction of Fusion Protein Expression Plasmids

[0105] See patent CN 117003896 B for details.

[0106] III. Construction of Stable Cell Lines

[0107] See patent CN 117003896 B for details.

[0108] IV. Expression of the Target Product

[0109] See patent CN 117003896 B for details.

[0110] V. Fusion Protein Purification

[0111] Cell culture supernatant was adjusted to pH 7.5. Affinity chromatography protein A was equilibrated to UV absorption baseline using 40 mM PB buffer (pH 7.5, containing 150 mM sodium chloride). After pH stabilization, the cell supernatant was passed through the column and equilibrated to UV absorption baseline using the same buffer. The target analyte was then eluted with acetate-sodium acetate buffer (pH 3.0–4.0). The purified product was inactivated at low pH (pH 3.0–4.0, 18–25°C for 60 min). After inactivation, 1 M ammonium sulfate was added, and the pH was adjusted to 7.5. The hydrophobic chromatography column (Capto Phenyl ImpRes) was equilibrated to UV absorption baseline using 50 mM PB buffer (pH 7.5, + 1 M ammonium sulfate) and equilibrated to pH stabilization. The inactivation solution was then passed through the column and equilibrated with the same buffer. Finally, the target analyte was linearly eluted with 50 mM PB buffer (pH 7.5). The hydrophobic purified product was purified by molecular sieve chromatography using a Sephacryl S-300 High Resolution molecular sieve, and the solution was changed to obtain the purified protein. The purified protein was then subjected to nanofiltration through a 15 nm filter and sterile filtration through a 0.22 μm filter membrane to obtain the PADRE-gE-P2 fusion protein solution (stock solution).

[0112] 2) Detect the protein content in the PADRE-gE-P2 fusion protein solution (stock solution).

[0113] I. Solution Preparation

[0114] (1) Preparation of mobile phase (trifluoroacetic acid)

[0115] Mobile phase A [water:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of trifluoroacetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0116] Mobile phase B [acetonitrile:trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and sonicate to degas for 5 min to obtain the mobile phase.

[0117] (2) 2% DMSO solution

[0118] 2% Dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, mix well, and the solution is ready.

[0119] II. Detection of PADRE-gE-P2 fusion protein solution (stock solution) samples

[0120] S1. Preparation of protein calibration standard solution samples

[0121] Preparation of BSA reference stock solution: Weigh 21.9 mg of BSA (China National Institutes for Food and Drug Control) into a 10 ml volumetric flask, dissolve it in 6 ml of phosphate solution (10 mM; pH 6.0); then dilute to the mark with phosphate solution (10 mM; pH 6.0) and mix well; finally, dispense into 500 μl tubes to obtain the BSA reference stock solution (concentration 2.19 mg / ml). 457 μl of BSA reference stock solution (concentration 2.19 mg / ml) was placed in a 5 ml volumetric flask and diluted to the mark with phosphate solution (10 mM; pH 6.0). The solution was shaken well to obtain a concentration of 200 μg / ml. Then, 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200 μg / ml solution were transferred to different centrifuge tubes, and 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0) were added respectively. The solutions were mixed well to obtain concentrations of 175 μg / ml, 150 μg / ml, 125 μg / ml, and 100 μg / ml, respectively. Take 0.5 ml of each of the five concentration solutions and place them in different vials. Then add 0.5 ml of 2% DMSO solution to each vial and mix well to obtain calibration standard solutions with protein concentrations of 100 μg / ml, 87.5 μg / ml, 75 μg / ml, 62.5 μg / ml and 50 μg / ml, respectively.

[0122] S2. Preparation of test samples

[0123] Dilute 1 ml of the sample solution to be tested in a two-fold serial dilution, to the maximum dilution factor of 2. 8 256 times (2 times). From 2... 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 Dilute the sample solution by 0.5 ml into eight vials, then add 0.5 ml of 2% DMSO solution to each vial. Cap the vials and mix well. This yields the dilution factors of 2... 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 The test samples.

[0124] S3. Load the sample into the liquid chromatograph and run the analytical method.

[0125] The vials containing the protein calibration standard solution and the vials containing the test sample were loaded into the injection tray of the high-performance liquid chromatograph (HPLC), and the analytical method was run using the HPLC. The HPLC chromatogram of the BSA calibration standard solution sample is shown below. Figure 1 As shown.

[0126]

[0127]

[0128] S5. Calculate protein content

[0129] (1) Establishment of the standard curve

[0130] A calibration standard curve proportional to protein concentration and peak area was established using the peak area values ​​of the BSA calibration standard solution samples. Figure 2 As shown.

[0131] Calibration standard solution-1 (50 μg / ml) 7.315 2727058 1.780 Calibration standard solution - 2 (62.5 μg / ml) 7.309 3529660 1.774 Calibration standard solution - 3 (75 μg / ml) 7.311 4280222 1.745 Calibration standard solution-4 (87.5 μg / ml) 7.299 4981774 1.750 Calibration standard solution - 5 (100 μg / ml) 7.300 5688019 1.763

[0132] BSA concentration versus peak area standard curve: y = 58943x - 183075, R 2 =0.9992.

[0133] (2) Content Calculation

[0134] The target sample is selected based on the protein peak area within the range of the standard curve. The protein peak area of ​​the target sample is then substituted into the calibration standard curve formula and multiplied by the dilution factor of the target sample to calculate the protein content in the PADRE-gE-P2 fusion protein solution (stock solution). The chromatogram is shown in Figure 3-10.

[0135]

[0136] 3) Compare the differences in protein content between liquid chromatography and the Lowry method in the detection of PADRE-gE-P2 fusion protein solution (stock solution).

[0137] I. Lowry method for determining protein content in PADRE-gE-P2 fusion protein solution (stock solution) (existing technology)

[0138] (1) Reagent preparation

[0139] Folin-Ciocalteu solution: Add 5.0 ml of phenol reagent to 75 ml of water and mix well; Solution A: Weigh 10.0 g of sodium hydroxide and 50.0 g of sodium carbonate into a plastic reagent bottle, add 400 ml of water to dissolve, and mix well; Potassium tartrate solution (0.5 g → 50 ml): Weigh 0.5 g of potassium tartrate into a reagent bottle, add 50 ml of water to dissolve, and mix well; Copper sulfate solution (0.25 g → 30 ml): Weigh 0.39 g of copper sulfate pentahydrate into a reagent bottle, add 30 ml of water to dissolve, and mix well; Solution B: Prepare according to the ratio of potassium tartrate solution (0.5 g → 50 ml): copper sulfate solution (0.25 g → 30 ml) = 5:3 (volume ratio), and mix well; Alkaline copper solution: Prepare according to the ratio of Solution A: Solution B: water = 40:8:2 (volume ratio), and mix well.

[0140] (2) Preparation of working solution for protein reference standard

[0141] 200 μg / ml protein reference solution (e.g., bovine serum albumin solution): Take one vial of protein reference standard, reconstitute it with purified water to prepare a 200 μg / ml protein reference solution, aliquot it into 5 ml plastic tubes, and store at -20℃ or below. When using, dilute the protein reference solution twice and mix well to obtain the working solution (100 μg / ml).

[0142] (3) Protein sample concentration detection

[0143] Test sample detection: Accurately transfer an appropriate amount of the sample solution to be tested, dilute with water until the protein content is within the standard curve range; accurately transfer 1.0 ml of the above solution into two test tubes, add 1.0 ml of alkaline copper solution, mix well, let stand at room temperature for 10 min, add 4.0 ml of Folin-Ciocalteu solution, mix immediately, let stand at room temperature for 30 min for color development, and then measure the absorbance at a wavelength of 650 nm using ultraviolet-visible spectrophotometry (if turbidity is found after color development, centrifuge at 3000 rpm / min for 15 min, and then take the supernatant for measurement).

[0144] Protein standard detection: Accurately transfer 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of 100 μg / ml protein standard working solution into test tubes, making duplicates. For solutions less than 1.0 ml, add water to bring the volume to 1.0 ml. Proceed with the same procedure starting from "Add 1.0 ml of alkaline copper solution". Separately, accurately transfer 1.0 ml of water and proceed with the same procedure starting from "Add 1.0 ml of alkaline copper solution" as a blank control.

[0145] (4) Protein sample concentration calculation

[0146] A linear regression was performed with the protein content of a series of protein reference standards as the X-axis and the corresponding mean absorbance as the Y-axis to obtain the linear regression equation. The mean absorbance of the test sample solution was then substituted into the linear regression equation to calculate the protein content of the test sample. The protein content of the sample (μg / ml) = A*n; where: A is the protein content of the test sample obtained by substituting the measured absorbance into the linear regression equation, in μg / ml; and n is the dilution factor of the test sample.

[0147] II. Comparison of protein content differences in PADRE-gE-P2 fusion protein solution (stock solution) between liquid chromatography and the Lowry method (existing technology)

[0148]

[0149] 4) Compare the stability of protein content in PADRE-gE-P2 fusion protein solution (stock solution) using liquid chromatography and the Lowry method (existing technology), such as... Figure 11 As shown

[0150] The stability of the PADRE-gE-P2 fusion protein solution (stock solution) was compared by repeatedly testing it using liquid chromatography and the Lowry method (existing technology).

[0151]

[0152]

[0153] The analysis revealed potential factors contributing to the higher stability of liquid chromatography compared to the Lowry method (existing technology). The results showed significant differences in the operational processes of the two methods: liquid chromatography exhibited a higher degree of automation.

[0154]

[0155]

[0156] Example 2: Qualitative detection of protein in PADRE-gE-P2 fusion protein solution (stock solution)

[0157] 1) The preparation of PADRE-gE-P2 fusion protein solution (stock solution) is the same as in “1)” in Example 1.

[0158] 2) Qualitative detection of protein in PADRE-gE-P2 fusion protein solution (stock solution)

[0159] I. Solution Preparation

[0160] Same as "I. Solution Preparation" in item "2)" of Example 1.

[0161] II. Sample Testing

[0162] S1. Preparation of protein calibration standard solution samples

[0163] 658 μl of PADRE-gE-P2 fusion protein control solution (Lowry method protein concentration of 1.52 mg / ml) was placed in a 5 ml volumetric flask and diluted to the mark with phosphate solution (10 mM; pH 6.0). The solution was shaken well to obtain a concentration of 200 μg / ml. 1 ml of the 200 μg / ml solution was then transferred to a centrifuge tube, and 0.33 ml of phosphate solution (10 mM; pH 6.0) was added. The mixture was then mixed to obtain a concentration of 150 μg / ml. 0.5 ml of the 150 μg / ml solution was transferred to a vial, and 0.5 ml of 2% DMSO solution was added. The mixture was then mixed to obtain a calibration standard solution with a protein concentration of 75 μg / ml.

[0164] S2. Preparation of test samples

[0165] Same as "S2" in Example 1.

[0166] S3. Load the sample into the liquid chromatograph and run the analytical method. The chromatogram is shown in Figure 12.

[0167]

[0168]

[0169] S4. Calculate the relative deviation of protein retention time.

[0170] From the samples at different dilution ratios, the sample with the peak area closest to that of the protein calibration standard solution was selected as the target sample. Then, the relative deviation of the retention time of the protein retention peak in the target sample was calculated using the formula: Relative Deviation = [|Retention Time of Target Sample Protein Retention Peak – Average Retention Time| / Average Retention Time] × 100%. Note: The average retention time is the average of the retention times of the protein retention peak in the target sample and the protein retention peak in the calibration standard solution. Judgment Criterion: If the relative deviation is not higher than 1%, the protein in the original sample can be determined to be the PADRE-gE-P2 fusion protein. The chromatogram of the original sample diluted 32 times is shown in Figure 13.

[0171]

[0172]

[0173] 3) Compare the differences in retention time between the PADRE-gE-P2 fusion protein and the gE retention peak.

[0174] I. Preparation and Detection of gE Solution

[0175] 198 μl of gE solution (concentration detected by liquid chromatography: 5.063 mg / ml) was placed in a 5 ml volumetric flask and diluted to the mark with phosphate solution (10 mM; pH 6.0). The solution was shaken well to obtain a concentration of 200 μg / ml. 1 ml of this 200 μg / ml solution was then transferred to a centrifuge tube, and 0.33 ml of phosphate solution (10 mM; pH 6.0) was added. The mixture was then stirred to obtain a concentration of 150 μg / ml. 0.5 ml of this solution was transferred to a sample vial, and 0.5 ml of 2% DMSO solution was added. The vial was capped and stirred well to obtain a protein concentration of 75 μg / ml. The detection method was the same as in "S3".

[0176] II. Compare the retention times of the PADRE-gE-P2 fusion protein and the gE retention peak, such as... Figure 14 As shown, the retention times of the PADRE-gE-P2 fusion protein and the gE retention peak differ significantly.

[0177]

[0178] Example 3: Simultaneous qualitative and quantitative detection of proteins in the original solution sample

[0179] 1) The preparation of PADRE-gE-P2 fusion protein solution (stock solution) is the same as in “1)” in Example 1.

[0180] 2) Simultaneous qualitative and quantitative detection of proteins in the original solution sample

[0181] I. Solution Preparation

[0182] Same as "I. Solution Preparation" in item "2)" of Example 1.

[0183] II. Testing of raw solution samples

[0184] S1. Preparation of protein calibration standard solution samples

[0185] (1) Preparation of calibration standard solution for PADRE-gE-P2 fusion protein

[0186] Same as "S1" in Example 2.

[0187] (2) Preparation of BSA calibration standard solution

[0188] Same as "S1" in Example 1.

[0189] S2. Preparation of test samples

[0190] Same as "S2" in Example 1.

[0191] S3. Load the sample into the liquid chromatograph and run the analytical method.

[0192] Same as "S3" in Example 2.

[0193] S4. Calculate the relative deviation of protein retention time.

[0194] Same as "S4" in Example 2.

[0195] S5. Calculate protein content

[0196] A calibration standard curve was established using the peak area values ​​of BSA calibration standard solution samples, showing that protein concentration is proportional to peak area.

[0197] Calibration standard solution-1 (50 μg / ml) 7.691 2693734 1.752 Calibration standard solution - 2 (62.5 μg / ml) 7.673 3551053 1.740 Calibration standard solution - 3 (75 μg / ml) 7.676 4323296 1.716 Calibration standard solution-4 (87.5 μg / ml) 7.668 5033768 1.740 Calibration standard solution - 5 (100 μg / ml) 7.672 5800613 1.751

[0198] BSA concentration versus peak area standard curve: y = 61521x - 337391, R 2 =0.9989.

[0199] II. Content Calculation

[0200] Select the sample whose protein peak area is within the range of the standard curve as the target sample, then substitute the protein peak area value of the target sample into the calibration standard curve formula, and multiply it by the dilution factor of the target sample to calculate the protein content in the PADRE-gE-P2 fusion protein solution (stock solution).

[0201]

[0202]

[0203] Finally, it should be noted that:

[0204] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid chromatography method for simultaneously qualitatively and quantitatively detecting proteins in a stock solution of PADRE gEP2 fusion protein, characterized in that, Includes the following steps: S1. Prepare protein calibration standard solution samples; S2. Prepare the test sample; S3. Load the sample into the liquid chromatograph and run the analytical method; S4. Calculate the relative deviation of protein retention time; S5. Calculate protein content; The PADREgEP2 fusion protein comprises the extracellular segment gE of varicella-zoster virus glycoprotein E, the universal DR Th epitope peptide PADRE, and the amino acid sequence of tetanus toxin Th epitope P2; gE, PADRE, and P2 are linked by a linker peptide, which is GGS and / or GGGS and / or GGGGS and / or GGSSG linker peptide. The chromatographic column of the analytical method in S3 contains alkyl or phenyl reversed-phase chromatographic packing material, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water; the liquid chromatograph parameters of the analytical method in S3 include, but are not limited to: column temperature, injection volume, flow rate, and detector parameters; the column temperature is 20℃~70℃; the injection volume is 5μl~100μl; the flow rate is 0.1 ml / min~2.5 ml / min; when the detector is an ultraviolet detector, the detection wavelength is 200~400nm; The elution procedure of the analytical method in S3 includes an elution method for eluting protein retention peaks. The elution method for eluting protein retention peaks is gradient elution or isocratic elution, wherein the concentration gradient of acetonitrile in the mobile phase is 5% to 100%, the gradient elution time is 2 min to 1920 min, and the concentration of trifluoroacetic acid in the mobile phase is 0.01% to 0.10%.

2. The liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in the PADREgEP2 fusion protein stock solution according to claim 1, characterized in that, An optional molecular structure of the PADREgEP2 fusion protein from the N-terminus to the C-terminus is PADREGSGSGgEGGSP2, the amino acid sequence of which is shown in SEQ ID NO.

1.

3. The liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in the PADREgEP2 fusion protein stock solution according to claim 1, characterized in that, The protein concentration of the protein calibration standard solution sample in S1 is 0.4 μg / ml to 1 mg / ml.

4. The liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in the PADREgEP2 fusion protein stock solution according to claim 1, characterized in that, In step S4, the relative deviation of protein retention time is calculated as follows: Relative deviation of protein retention time = [|Vaccine protein retention peak retention time - Average retention time| / Average retention time] × 100%, where the average retention time is the average of the retention time of the original protein retention peak and the retention time of the protein calibration standard solution sample retention peak; wherein the protein in the vaccine sample is the PADREgEP2 fusion protein, and the relative deviation is not higher than 1%.

5. The liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in the PADREgEP2 fusion protein stock solution 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 test sample into the calibration standard curve formula to calculate the protein concentration in the test sample; finally, multiply the protein concentration in the test sample by the dilution factor to calculate the protein concentration in the stock solution. Calculation Method 2: Protein concentration in stock solution = protein peak area of ​​test sample * protein calibration standard solution concentration * dilution factor / protein calibration standard solution peak area.

6. The liquid chromatography method for simultaneous qualitative and quantitative detection of proteins in the PADREgEP2 fusion protein stock solution according to claim 1, characterized in that, Applications include the qualitative and / or quantitative detection of PADREgEP2 fusion protein in the component detection of recombinant varicella-zoster vaccine.

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