Liquid chromatography method for quantitatively detecting protein and application

Abstract

The invention discloses a liquid chromatography method for quantitatively detecting protein 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; and calculating the protein content. The technical problem that in the prior art, a large number of manual operation steps exist in a method for quantitatively detecting protein, and consequently large errors are caused is solved. Compared with the prior art, the high performance liquid chromatography disclosed by the invention has the advantages of higher automation degree, smaller error and better stability when being used for quantitatively detecting the protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically relates to a liquid chromatography method for quantitatively detecting proteins and applications thereof. Background Art

[0002] Varicella-zoster virus (VZV), also known as human herpesvirus type 3, is a human α-herpesvirus. The genome size of VZV is approximately 125 kb and encodes about 69 proteins, including 8 glycoproteins, namely gB, gC, gE, gH, gI, gK, gL, and gM; among them, glycoprotein E (gE) is the most abundant and immunogenic glycoprotein on the viral envelope and host cell membrane, and it can induce cellular immunity and humoral immunity.

[0003] The recombinant protein zoster vaccine Shingrix from GlaxoSmithKline ® (Shingrix ® ) was approved by the FDA for marketing in 2017 for the prevention of herpes zoster in adults aged 50 and above. The vaccine consists of two parts: one is the truncated VZV glycoprotein E (gE) expressed by CHO cells, and the other is the AS01B adjuvant system [the AS01B adjuvant system consists of the immunopotentiator 3D-MPL, the immunopotentiator saponin QS-21, and liposomes; the main components of liposomes are dioleoyl phosphatidylcholine (DOPC) and cholesterol].

[0004] Compared with the live attenuated vaccines from Merck & Co., Inc. and Changsheng Bio-tech Co., Ltd., the recombinant protein vaccine Shingrix from GSK ® has a higher protection rate. Additionally, a recombinant varicella-zoster vaccine that is superior to Shingrix ® in immunogenicity but has lower expected clinical side effects than Shingrix ® is a promising candidate vaccine in clinical trials (immunogenicity data can be found in CN 116747298 B and CN117003896 B): The candidate vaccine contains the gE fusion protein (PADRE-gE-P2) antigen and the XA-401 complex adjuvant [the XA-401 complex adjuvant consists of the immunopotentiator saponin QS-21 and liposomes; the main components of liposomes are dioleoyl phosphatidylcholine (DOPC) and cholesterol]; compared with Shingrix ® , this candidate vaccine removes the immunopotentiator 3D-MPL (3-O-deacylated-4'-monophosphoryl lipid A) that exacerbates clinical side effects.

[0005] To control the quality of vaccines, it is necessary to quantitatively detect the protein in the protein antigen solution (stock solution) used to formulate the vaccines. Therefore, a method for quantitatively detecting the protein in the protein solution (stock solution) needs to be established.

[0006] Currently, for the quality control of the already marketed recombinant zoster vaccine Shingrix ® the detection method used for quantitatively detecting the gE protein solution (gE stock solution) in the Shingrix ® vaccine is the Lowry method. During the process of quantitatively detecting proteins by the existing technology (Lowry method), a large amount of manual operation is required, the degree of automation is low, there are relatively large errors, and the stability of the detection results is poor. Summary of the Invention

[0007] The object of the present invention is to provide a liquid chromatography method and application for quantitatively detecting proteins to solve the problems in the existing technology.

[0008] To achieve the above object, the present invention provides the following technical solutions: A liquid chromatography method for quantitatively detecting a protein provided by the present invention, wherein the protein is gE or a gE fusion protein; the gE is the extracellular region of varicella-zoster virus glycoprotein E; the gE fusion protein is a fusion protein formed by the fusion expression of gE and an immune enhancing peptide / protein sequence; the immune enhancing peptide / protein sequence includes, but is not limited to, the universal DR Th epitope peptide PADRE and / or the tetanus toxin Th epitope peptide P2 and / or the Fc segment (Fc) of human immunoglobulin γ (IgG) and / or a human interleukin sequence and / or a human interferon sequence; the human interleukin includes interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), interleukin-36 (IL-36), interleukin-37 (IL-37), interleukin-38 (IL-38); the human interferon includes interferon α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ); the gE, PADRE and / or P2 and / or Fc and / or human interleukin and / or human interferon sequence in the gE fusion protein are connected by a linker peptide, and the linker peptide is a GGS and / or GGGS and / or GGGGS and / or GSGSG linker peptide.

[0009] Furthermore, for the gE or gE fusion protein, the gE may also be the extracellular domain of glycoprotein E of herpes simplex virus (HSV) type I, herpes simplex virus (HSV) type II, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus (KSHV), pseudorabies virus (PRV), or Marek's disease virus (MDV).

[0010] Furthermore, the gE is the extracellular domain of varicella-zoster virus glycoprotein E. An optional molecular structure of the gE from the N-terminus to the C-terminus is: AA31 - AA544, and the amino acid sequence is as shown in SEQ ID NO.1; an optional fusion protein of the gE fusion protein is PADRE-gE(AA31 - AA544)-P2, and its molecular structure from the N-terminus to the C-terminus is: PADRE-GSGSG-gE-GGS-P2, and the amino acid sequence is as shown in SEQ ID NO.2.

[0011] Furthermore, a liquid chromatography method for quantitatively detecting a protein, where the protein is the protein in a protein solution / stock solution.

[0012] Furthermore, for the protein solution / stock solution, the protein content is 0.25 mg / ml to 16 mg / ml.

[0013] Furthermore, the protein solution / stock solution may contain buffer components for maintaining a stable pH value.

[0014] Furthermore, the buffer components for maintaining a stable pH value are selected from any one or more of the following substances, but are 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, tris(hydroxymethyl)aminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0015] Furthermore, the protein solution / stock solution may also contain components for maintaining protein stability.

[0016] Furthermore, the components for maintaining protein stability can be selected from any one or more of the following substances, but are not limited to the following substances: polysorbate 80, polysorbate 20, poloxamer 188.

[0017] Furthermore, the liquid chromatography method is high-performance or ultra-high-performance liquid chromatography.

[0018] Furthermore, the liquid chromatography method is reverse-phase liquid chromatography, and its chromatographic column contains an alkyl or phenyl reverse-phase chromatographic packing material, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water.

[0019] Furthermore, the alkyl reversed-phase chromatographic packing material can be selected from any of the following chromatographic packing materials, but is not limited to the following: butylsilyl-bonded silica gel (C4), hexylsilyl-bonded silica gel (C6), octylsilyl-bonded silica gel (C8), cetylsilyl-bonded silica gel (C16), octadecylsilyl-bonded silica gel (C18).

[0020] Furthermore, the liquid chromatography method is the internal standard method or the external standard method of liquid chromatography.

[0021] Furthermore, the external standard method of liquid chromatography includes the following steps: S1. Prepare a protein calibration standard solution sample; S2. Prepare a test sample; S3. Load the sample into the liquid chromatograph and run the analysis method; S4. Calculate the protein content.

[0022] Furthermore, for the preparation of the protein calibration standard solution sample in S1, the protein used for preparing the protein calibration standard solution sample can be selected from any of the following, but is not limited to the following: gE fusion protein (such as: PADRE-gE-P2 fusion protein), bovine serum albumin (BSA), bovine Gamma-globulin (BGG), extracellular region (gE) of varicella-zoster virus (VZV) glycoprotein E (gE).

[0023] Furthermore, for the preparation of the protein calibration standard solution sample, the preparation method can be selected from any of the following, but is not limited to the following: 1. Accurately weigh the target weight of protein powder, then dissolve it in water or buffer solution, and finally make up the volume to the target volume with water or buffer solution and mix well to obtain; 2. Use methods such as the lowry method, BCA method, biuret method, Kjeldahl method, coomassie brilliant blue method, liquid chromatography method, etc. to detect the protein concentration of the protein solution, and then calibrate its protein concentration to obtain.

[0024] Furthermore, for the protein calibration standard solution sample in S1, the protein concentration is 0.4 μg / ml to 1000 μg / ml.

[0025] Furthermore, the protein calibration standard solution sample may contain DMSO, and the concentration is 0.5% to 5%.

[0026] Furthermore, for the protein solution / stock solution used for preparing the test sample in S2, the protein content is 0.25 mg / ml to 16 mg / ml.

[0027] Furthermore, for the test sample in S2, the protein concentration is 0.4 μg / ml to 4 mg / ml.

[0028] Further, the sample detected in S2 may contain DMSO, with a concentration of 0.5% to 5%.

[0029] Further, the detector of the liquid chromatograph in S3 is an ultraviolet detector, an electrospray detector (CAD), or an evaporative light scattering detector.

[0030] Further, the analysis method in S3 includes a chromatographic column, liquid chromatograph parameters, and an elution program.

[0031] Further, the chromatographic column of the analysis method in S3 is a reversed-phase chromatographic column.

[0032] Further, the liquid chromatograph parameters of the analysis method in S3 include column temperature, injection volume, flow rate, detector parameters, etc.

[0033] 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.

[0034] Further, 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.

[0035] Further, when the instrument is an ultra-high performance liquid chromatograph, the injection volume is 1 μl to 30 μl, such as 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, or 30 μl.

[0036] 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.

[0037] Further, 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.

[0038] Further, for the detector parameters, when the detector is an ultraviolet detector, the detection wavelength is 200 nm to 400 nm, such as 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm or 400 nm.

[0039] Further, when the detector is a CAD detector, the nebulization 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.

[0040] Further, when the detector is an evaporative light scattering detector, the nebulization 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.

[0041] Further, the elution program of the analysis method in S3 includes at least the elution method for eluting the protein retention peak.

[0042] Further, the elution method for the protein retention peak is gradient elution, and the acetonitrile concentration gradient in the mobile phase is 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% - 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% - 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% - 75%, 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 duration 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, the concentration of the pH regulator in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid, acetic acid.

[0043] Furthermore, the elution method for the elution protein retention peak is isocratic elution, the acetonitrile concentration gradient in the mobile phase is 50% to 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%, the isocratic elution duration 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, the concentration of the pH regulator in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid, acetic acid.

[0044] Furthermore, in the elution program of the S3 analysis method, a pre-equilibration method and / or a pre-elution method can be added before the elution method for the elution protein retention peak.

[0045] Further, a pre-equilibration method is added before the elution method for the elution peak of the retained protein, and the acetonitrile concentration in the mobile phase is not higher than that of the elution peak of the retained protein.

[0046] Further, a pre-equilibration method is added before the elution method for the elution peak of the retained protein: the acetonitrile concentration in the mobile phase is 5% to 60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, the equilibration time is 1 to 960 min, such as 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 120 min, 240 min, 480 min, or 960 min, and the concentration of the pH regulator in the mobile phase is 0.01% to 0.10%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid and acetic acid.

[0047] Further, a pre-elution method is added before the elution method for the elution peak of the retained protein, and the acetonitrile concentration in the mobile phase is not higher than that of the elution peak of the retained protein.

[0048] Further, a pre-elution method is added before the elution method for the elution peak of the retained protein: the gradient concentration of acetonitrile 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%, the gradient elution duration 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, and the concentration of the pH regulator 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%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid and acetic acid.

[0049] Further, in the elution program of the analysis method in S3, a column regeneration method and / or a post-equilibration method can be added after the elution method for the elution peak of the retained protein.

[0050] Further, when the column regeneration method and the post-equilibration method exist simultaneously, the column regeneration method should be before the post-equilibration method.

[0051] Further, a chromatographic column regeneration method is added after the elution method for the protein retention peak: the concentration of acetonitrile in the mobile phase is 98% - 100%, such as 98%, 98.5%, 99%, 99.5% or 100%, the chromatographic 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, and the mobile phase may also contain a pH regulator with 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%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid and acetic acid.

[0052] Further, a post - equilibration method is added after the elution method for the protein retention peak: the concentration of acetonitrile in the mobile phase is 5% - 60%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, 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, 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 contains a pH regulator with 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%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid and acetic acid.

[0053] Further, in S4, the protein content is calculated by calculation method 1 and calculation method 2.

[0054] Further, calculation method 1 is: first establish a calibration standard curve in which the concentration of the protein is proportional to the peak area, and then calculate the protein content (concentration).

[0055] Further, calculation method 1: first establish a calibration standard curve in which the concentration of the protein is proportional to the peak area; then substitute the protein peak area value of the sample into the calibration standard curve formula to calculate the concentration of the protein in the test sample; multiply the concentration of the protein in the test sample by the dilution factor to calculate the protein content (concentration) in the protein solution / stock solution.

[0056] Further, the second calculation method is: directly calculate the protein content (concentration).

[0057] Further, the second calculation method: The calculation formula is that the protein content (concentration) in the protein solution / stock solution = the peak area of the protein in the test sample * the sample concentration of the protein calibration standard solution * the dilution factor ∕ the peak area of the protein calibration standard solution sample.

[0058] The present invention also provides an application of a liquid chromatography method for quantitatively detecting proteins in the detection of the components of a protein solution / stock solution, including but not limited to quantitatively detecting the protein in a gE or gE fusion protein solution / stock solution.

[0059] Based on the above technical solutions, the embodiments of the present invention can at least produce the following technical effects: A liquid chromatography method for quantitatively detecting proteins provided by the present invention can detect the concentration of the protein in a gE solution / stock solution.

[0060] A liquid chromatography method for quantitatively detecting proteins provided by the present invention can detect the concentration of the protein in a PADRE-gE-P2 fusion protein solution / stock solution.

[0061] A liquid chromatography method for quantitatively detecting proteins provided by the present invention, compared with the existing method (Lowry method) for quantitatively detecting gE stock solution, has a higher degree of automation, fewer manual operation steps, smaller errors in detecting protein content, and better stability. Description of the Drawings

[0062] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0063] Figure 1 It is the liquid chromatography diagram of the BSA calibration standard solution sample in Embodiment 1 of the present invention.

[0064] Figure 2 It is the standard curve of the BSA concentration and the peak area in Embodiment 1 of the present invention.

[0065] Figure 3 It is the liquid chromatography diagram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) 4 times in Embodiment 1 of the present invention.

[0066] Figure 4 It is the liquid chromatography diagram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) 8 times in Embodiment 1 of the present invention.

[0067] Figure 5 It is the liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) in Example 1 of the present invention by 16 times.

[0068] Figure 6 It is the liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) in Example 1 of the present invention by 32 times.

[0069] Figure 7 It is the liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) in Example 1 of the present invention by 64 times.

[0070] Figure 8 It is the liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) in Example 1 of the present invention by 128 times.

[0071] Figure 9 It is the liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) in Example 1 of the present invention by 256 times.

[0072] Figure 10 It is the liquid chromatogram of the test sample after diluting the PADRE-gE-P2 fusion protein solution (stock solution) in Example 1 of the present invention by 512 times.

[0073] Figure 11 It is the comparison chart of the RSD values for detecting the protein content in the PADRE-gE-P2 fusion protein stock solution by liquid chromatography (using BSA as the protein standard) and the Lowry method in Example 1 of the present invention.

[0074] Figure 12 It is the liquid chromatogram of the test sample after diluting the gE solution (stock solution) in Example 2 of the present invention by 64 times.

[0075] Figure 13 It is the comparison chart of the RSD values for detecting the protein content in the gE stock solution by liquid chromatography (using BSA as the protein standard) and the Lowry method in Example 2 of the present invention. Detailed implementation manners

[0076] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0077] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which will be apparent to those skilled in the art. Other embodiments obtained from the specification of the present invention will be apparent to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0078] The liquid chromatography analysis method was used to quantitatively detect the proteins in the gE and gE fusion protein solutions / stocks, and the results were compared with those of the prior art. The comparison results showed that both the liquid chromatography analysis method and the prior art could be used to quantitatively detect the proteins in the gE and gE fusion protein solutions / stocks. However, the liquid chromatography analysis method had a higher degree of automation, could reduce the errors caused by manual operation, and had more stable detection results. In addition, the optimal liquid chromatography instrument parameters and elution methods may be different when using reverse-phase chromatography columns with different chromatographic packings to detect the target substances (such as gE and gE fusion proteins), which is known to those skilled in the art.

[0079] SEQ ID NO.1: SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGG SEQ ID NO.2: AKFVAAWTLKAAAGSGSGSVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGGGSQYIKANSKFIGITE Example 1. Detection of protein in the PADRE-gE-P2 fusion protein solution (stock solution) quantitatively 1) Preparation of PADRE-gE-P2 fusion protein and detection of protein concentration I. Codon optimization and gene synthesis of the fusion protein See Patent CN 117003896 B for details.

[0080] II. Construction of the expression plasmid of the fusion protein See Patent CN 117003896 B for details.

[0081] III. Construction of the stable cell line See Patent CN 117003896 B for details.

[0082] IV. Expression of the target product See Patent CN 117003896 B for details.

[0083] V. Purification of the fusion protein The cell culture supernatant was adjusted to pH 7.5, and the affinity chromatography protein A was equilibrated with 40 mM PB buffer containing 150 mM sodium chloride at pH 7.5 to the baseline level of ultraviolet absorption. After the pH was stabilized, the cell supernatant was passed through the column. After equilibration to the baseline level of ultraviolet absorption with the same equilibration buffer, the target substance was eluted with acetic acid-sodium acetate buffer at pH 3.0 - 4.0. The purified product was inactivated at low pH (pH 3.0 - 4.0, placed at 18 - 25 °C for 60 min). After inactivation, 1 M ammonium sulfate was added to the product and the pH was adjusted to 7.5. The hydrophobic chromatography column Capto Phenyl ImpRes was equilibrated with 50 mM PB buffer + 1 M ammonium sulfate buffer at pH 7.5 to the baseline level of ultraviolet absorption. After the pH was stabilized, the inactivated solution was passed through the column, and the chromatography column was equilibrated with the same buffer. Finally, the target substance was linearly eluted with 50 mM PB buffer at pH 7.5. The hydrophobic purified product was purified by molecular sieve chromatography Sephacryl S-300 High Resolution molecular sieve chromatography and the buffer was exchanged to obtain the purified protein. After the purified protein was nanofiltered through a 15 nm filter and sterilized by filtration through a 0.22 μm filter membrane, a PADRE-gE-P2 fusion protein solution (stock solution) was obtained.

[0084] 2) Detect the protein content in the PADRE-gE-P2 fusion protein solution (stock solution) I. Solution preparation (1) Preparation of mobile phase (trifluoroacetic acid) Mobile phase A [water: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of ultrapure water, add 0.5 ml of trifluoroacetic acid solution, shake well, and degas by ultrasonic for 5 min to obtain.

[0085] Mobile phase B [acetonitrile: trifluoroacetic acid (100:0.05)]: Measure 1000 ml of acetonitrile, add 0.5 ml of trifluoroacetic acid solution, shake well, and degas by ultrasonic for 5 min to obtain.

[0086] (2) 2% DMSO solution 2% dimethyl sulfoxide (DMSO) solution: Measure 98 ml of ultrapure water, add 2 ml of dimethyl sulfoxide solution, and mix evenly to obtain.

[0087] II. Detection of PADRE-gE-P2 fusion protein solution (stock solution) sample S1. Preparation of protein calibration standard solution sample Preparation of BSA reference stock solution: Weigh 21.9 mg of BSA (National Institutes for Food and Drug Control) and place it in a 10-ml volumetric flask. Dissolve it in 6 ml of phosphate solution (10 mM; pH 6.0); then make up the volume to the mark with phosphate solution (10 mM; pH 6.0) and mix well. Finally, aliquot it into tubes at a specification of 500 μl / tube, which is the BSA reference stock solution (concentration: 2.19 mg / ml). Take 457 μl of the BSA reference stock solution (concentration: 2.19 mg / ml) and place it in a 5-ml volumetric flask. Dilute it to the mark with phosphate solution (10 mM; pH 6.0) and shake well to obtain a solution with a concentration of 200 μg / ml. Then transfer 1 ml, 1 ml, 0.5 ml, and 0.5 ml of the above 200-μg / ml solution into different centrifuge tubes, and add 0.14 ml, 0.33 ml, 0.30 ml, and 0.50 ml of phosphate solution (10 mM; pH 6.0) respectively, and mix well to obtain solutions with concentrations of 175 μg / ml, 150 μg / ml, 125 μg / ml, and 100 μg / ml respectively. Transfer 0.5 ml of each of the above 5-concentration solutions into different sample vials, and then add 0.5 ml of 2% DMSO solution respectively, 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.

[0088] S2. Preparation of test samples Perform two-fold serial dilution on 1 ml of the test sample solution until the maximum dilution factor is 2 8 times (256 times). Take 0.5 ml of the sample solution diluted 2 1 times, 2 2 times, 2 3 times, 2 4 times, 2 5 times, 2 6 times, 2 7 times, 2 8 times and place them in 8 sample vials. Then add 0.5 ml of 2% DMSO solution to each vial, cap the vials, and mix well to obtain test samples with dilution factors of 2 2 times, 2 3 times, 2 4 times, 2 5 times, 2 6 times, 2 7 times, 2 8 times, 2 9 times respectively.

[0089] S3. Load the samples into the liquid chromatograph and run the analytical method Load the vials containing the protein calibration standard solution and the vials of the test sample onto the injection tray of a high-performance liquid chromatograph, and use the liquid chromatograph to run the analytical method. The liquid chromatogram of the BSA calibration standard solution sample is as shown in Figure 1 shown. S4. Calculate the protein content (1) Establishment of the standard curve Use the peak area values of the BSA calibration standard solution sample to establish a calibration standard curve in which the protein concentration is proportional to the peak area. The chromatogram is as shown in Figure 1 shown, and the standard curve diagram is as shown in Figure 2 shown. Protein calibration standard solution Retention time (min) Peak area (μAU*min) Tailing factor 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 Standard curve of BSA concentration vs. peak area: y = 58943x - 183075, R 2 = 0.9992.

[0090] (2) Content calculation Select the test samples with protein peak areas within the range of the standard curve as the target test samples, then substitute the protein peak area values of the target test samples into the calibration standard curve formula, and multiply by the dilution factor of the target test samples to calculate the protein content in the PADRE-gE-P2 fusion protein solution (stock solution). The chromatogram is as shown in Figure 3-10. Test sample Retention time (min) Peak area (μAU*min) Tailing factor Protein content in stock solution (mg / ml) Test sample 1 (stock solution diluted 4 times) 9.970 30831704 1.306 Rejected Test sample 2 (stock solution diluted 8 times) 9.998 15985163 1.225 Rejected Test sample 3 (stock solution diluted 16 times) 10.025 8156248 1.184 Rejected Test sample 4 (stock solution diluted 32 times) 10.034 4094907 1.164 2.323 Test sample 5 (stock solution diluted 64 times) 10.038 2019655 1.165 Rejected Test sample 6 (stock solution diluted 128 times) 10.035 1020283 1.179 Rejected Test sample 7 (stock solution diluted 256 times) 10.043 510518 1.136 Rejected Test sample 8 (stock solution diluted 512 times) 10.046 327332 1.236 Rejected 3) Compare the differences in the protein content detected by liquid chromatography and the Lowry method in the PADRE-gE-P2 fusion protein solution (stock solution) I. Detection of the protein content in the PADRE-gE-P2 fusion protein solution (stock solution) by the Lowry method (prior art) (1) Reagent preparation Folin-Ciocalteu reagent: Take 5.0 ml of phenol reagent and add 75 ml of water, mix well to obtain; Solution A: Weigh 10.0 g of sodium hydroxide and 50.0 g of sodium carbonate, place them in a plastic reagent bottle, add 400 ml of water to dissolve, and mix well to obtain; Potassium tartrate solution (0.5 g → 50 ml): Weigh 0.5 g of potassium tartrate, place it in a reagent bottle, add 50 ml of water to dissolve, and mix well to obtain; Copper sulfate solution (0.25 g → 30 ml): Weigh 0.39 g of copper sulfate pentahydrate, place it in a reagent bottle, add 30 ml of water to dissolve, and mix well to obtain; Solution B: Prepare according to potassium tartrate solution (0.5 g → 50 ml): copper sulfate solution (0.25 g → 30 ml) = 5:3 (volume ratio), and mix well to obtain; Alkaline copper solution: Prepare according to Solution A: Solution B: water = 40:8:2 (volume ratio), and mix well to obtain.

[0091] (2) Preparation of the working solution of the protein reference substance 200 μg / ml Protein Reference Solution (BSA Solution): Take 1 vial of protein reference substance, reconstitute it with purified water to prepare a 200 μg / ml protein reference solution, dispense it into 5-ml plastic tubes, and store it at -20 °C or below. When in use, dilute the protein reference solution two-fold, mix well, and it is the working reference solution (100 μg / ml).

[0092] (3) Detection of Protein Sample Concentration Test sample detection: Accurately pipette an appropriate amount of the test sample solution, dilute it 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, let stand at room temperature for 10 min, add 4.0 ml of Folin-Ciocalteu reagent, immediately mix well, and let stand at room temperature for 30 min for color development. Then, according to the ultraviolet-visible spectrophotometry, measure the absorbance at a wavelength of 650 nm (after color development, if turbidity is found, centrifuge at 3000 rpm for 15 min and take the supernatant for measurement).

[0093] Protein reference substance detection: Accurately pipette 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of the 100 μg / ml protein reference working solution into test tubes, in duplicate. For the solution less than 1.0 ml, make up the volume to 1.0 ml with water. Starting from "add 1.0 ml of alkaline copper solution", operate in the same way. Separately, accurately pipette 1.0 ml of water and operate in the same way starting from "add 1.0 ml of alkaline copper solution" as the blank control.

[0094] (4) Calculation of Protein Sample Concentration Use the content of the series of protein reference substances as the X-axis and the corresponding average absorbance as the Y-axis to make a linear regression to obtain the linear regression equation; substitute the average absorbance of the test sample solution into the linear regression equation to calculate the protein content of the test sample. The protein content of the test sample (μg / ml) = A * n; where: A is the protein content of the test sample obtained by substituting the measured absorbance of the test sample into the linear regression equation, in the unit of μg / ml; n is the dilution factor of the test sample.

[0095] II. Compare the differences in the protein content detected by liquid chromatography and the Lowry method (prior art) in the PADRE-gE-P2 fusion protein solution (stock solution), as Figure 11 shown 4) Compare the stability of the protein content detected by liquid chromatography and the Lowry method (prior art) in the PADRE-gE-P2 fusion protein solution (stock solution) Use liquid chromatography and the Lowry method (prior art) to detect the PADRE-gE-P2 fusion protein solution (stock solution) multiple times, and compare the stability of the two methods. Analyze the possible factors for the higher stability of the analytical liquid chromatography method compared to the Lowry method (prior art); the analysis results show that there are significant differences in the operation processes of the two methods: the operation process of the liquid chromatography method has a higher degree of automation. Example 2. Quantitative detection of protein in the gE solution (stock solution) 1) Preparation of the gE solution (stock solution) I. Protein codon optimization and gene synthesis Optimize the codons of the gE gene: avoid common restriction enzyme cleavage sites; according to the codon preference in CHO cells, replace synonymous codons with low frequency with those with high frequency to control rare codons; control the GC content in the sequence at 40% - 60% to improve the transcription efficiency of mRNA, and at the same time avoid the high GC content affecting the secondary structure of mRNA and further affecting the translation efficiency. Add the gene sequence encoding the signal peptide in front of the optimized gene sequence; introduce the Hind III restriction enzyme cleavage site at the upstream of the sequence, add the stop codon and the BamH I restriction enzyme cleavage site at the downstream of the sequence, and perform gene synthesis of the nucleotide sequence.

[0096] II. Construction of the protein expression plasmid The cloning vector containing the gene synthesis sequence is transformed into DH5α competent bacteria, and then amplified in large quantities. After extracting the plasmid, the cloning vector is double digested with the restriction enzymes Hind III and BamH I, and at the same time, the expression vector pXNM3.0 is double digested with the restriction enzymes Hind III and BamH I. The gene part of the fusion protein is recovered by gel extraction of the double-digested cloning vector, and the backbone part is recovered by gel extraction of the expression vector. The two are ligated with T4 ligase and then transformed into DH5α competent bacteria. The plate containing ampicillin resistance is coated for screening. The positive colonies are picked for amplification and plasmid extraction, and then identified by double digestion with Hind III and BamH I, and the correct recombinant expression vector is verified by sequencing.

[0097] III. Construction of the stable cell line The correctly identified recombinant expression vector is amplified in large quantities after increasing the bacteria, and the recombinant expression vector is single digested with Pvμ I, and the digested and linearized vector is recovered by gel extraction, and then filtered and sterilized for use. After the CHO-K1 cells are resuscitated, they are passaged continuously for more than two times, and the cell viability is greater than 95%. In the clean bench, add 0.6 ml of the cell suspension (about 1×10 7200 μl of the linearized recombinant expression vector (about 50 μg), and the electroporation conditions were set as a voltage of 300 V and a capacitance of 900 μF. After electroporation, the cells were transferred to a cell shake flask containing 30 ml of CD CHO medium and cultured at 37 °C, 5% carbon dioxide, and a rotation speed of 125 rpm for 24 hours. The electroporated cell suspension was centrifuged at 100 g for 10 minutes, the supernatant was discarded, the cells were resuspended in CD CHO medium containing 25 μM MSX and 200 μg / ml bleomycin, and then inoculated into a 24-well plate. After 3 weeks of culture, the expression level was detected by ELISA. The three cell wells with high expression levels were mixed and inoculated into a 96-well plate by the limiting dilution method for monoclonal screening, and photos were taken on days 0, 1, 2, 3, 7, and 15 with a single-cell imaging device. After 15 days, the expression level was detected by ELISA, and the three cell strains with high expression levels were cryopreserved. After stability studies, the cell line used for vaccine preparation was determined, and then a two-tier cell bank was established.

[0098] IV. Expression of the target product A cryopreserved working seed was revived with OPM-CHO CDP9 medium, amplified step by step in a shake flask, and finally transferred into a 5-L bioreactor for culture. The inoculation density was 0.8×10 6 cells / ml, and the culture parameters were set as a temperature of 37 °C, a pH of 7.0, a rotation speed of 150 rpm, and a dissolved oxygen concentration of 40%. Samples were taken daily to detect the cell viability, density, lactate content, and glucose content. On the 3rd day of culture, when the viable cell density reached 3×106 cells / ml, the feeding media CDF18 and CDF26 were added, 250 ml and 25 ml respectively. Then the same volume of the feeding medium was added every 1 day. The glucose content in the culture broth was maintained above 2 g / L, and when it was lower than this concentration, the glucose concentration was supplemented to 4 g / L. After culturing for about 15 days, when the cell viability dropped to 70%, the culture was terminated. The cells and cell debris were removed by deep filtration, and the supernatant of the cell culture was collected.

[0099] V. Protein purification Deep filter the cell culture supernatant with a filter having a cut-off range of 0.2 to 2 μm. Then adjust the pH of the filtered cell culture supernatant to 7.5; equilibrate the anion exchange column Capto Q with 20 mM PB buffer at pH 7.5 to the baseline level of ultraviolet absorption and stable pH, then pass the cell supernatant through the column, after equilibrating to the baseline level of ultraviolet absorption with the same buffer, perform linear elution with 20 mM PB elution buffer containing 1 M sodium chloride at pH 7.5, and collect the target product. Inactivate the anion purification product at low pH (pH 3.0 - 4.0, place at 18 - 25 °C for 60 min). Add ammonium sulfate (final concentration 1 M) to the inactivated product and adjust the pH to 7.5; equilibrate the hydrophobic chromatography column Capto PhenylImpRes with 50 mM PB buffer + 1 M ammonium sulfate buffer at pH 7.5 to the baseline level of ultraviolet absorption and stable pH; then pass the inactivated solution after pH adjustment through the column and equilibrate the chromatography column with the same buffer; finally, linearly elute the target product with 50 mM PB buffer at pH 7.5. Purify the hydrophobic purification product by Sephacryl S-300 High Resolution molecular sieve chromatography and change the solution to obtain the purified protein. After nanofiltration of the purified protein through a 15 nm filter and sterilizing filtration through a 0.22 μm filter membrane, obtain the gE solution (stock solution).

[0100] 2) Detect the protein content in the gE solution (stock solution) I. Solution preparation is the same as in Example 1.

[0101] II. Detection of the gE solution (stock solution) sample S1. Preparation of the protein calibration standard solution sample is the same as in Example 1.

[0102] S2. Preparation of the detection sample is the same as in Example 1.

[0103] S3. Load the sample onto the liquid chromatograph and run the analysis method the same as in Example 1.

[0104] S4. Calculate the protein content (1) Establish the standard curve the same as in Example 1.

[0105] (2) Content calculation Select the detection sample with the protein peak area within the standard curve range as the target detection sample, then substitute the protein peak area value of the target detection sample into the calibration standard curve formula, and multiply by the dilution factor of the target detection sample to calculate the protein content in the gE solution (stock solution). The chromatogram is as Figure 12 (shown for the gE stock solution diluted 64 times). Test sample Retention time (min) Peak area (μAU*min) Tailing factor Protein content in stock solution (mg / ml) Test sample 1 (stock solution diluted 4 times) 9.851 61095496 1.604 Rejected Test sample 2 (stock solution diluted 8 times) 9.916 34289601 1.361 Rejected Test sample 3 (stock solution diluted 16 times) 9.951 17839490 1.267 Rejected Test sample 4 (stock solution diluted 32 times) 9.968 8900786 1.194 Rejected Test sample 5 (stock solution diluted 64 times) 9.987 4479746 1.178 5.063 Test sample 6 (stock solution diluted 128 times) 9.984 2217324 1.176 Rejected Test sample 7 (stock solution diluted 256 times) 9.998 1091682 1.166 Rejected Test sample 8 (stock solution diluted 512 times) 10.009 509004 1.166 Rejected 3) Compare the differences in protein content detection of gE solution (stock solution) by liquid chromatography and Lowry method I. The detection of protein content in gE solution (stock solution) by Lowry method (prior art) is the same as that in Example 1.

[0106] II. Compare the differences in protein content detection of gE solution (stock solution) by liquid chromatography and Lowry method (prior art) 4) Compare the stability of protein content detection of gE solution (stock solution) by liquid chromatography and Lowry method (prior art), as Figure 13 shown Use liquid chromatography and Lowry method (prior art) to detect gE solution (stock solution) multiple times respectively, and compare the stabilities of the two methods. The reason for the higher stability of liquid chromatography than Lowry method (prior art) may be that the degree of automation in the operation process of liquid chromatography is higher.

[0107] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. A liquid chromatography method for quantitatively detecting proteins, characterized in that It includes the following steps: S1. Prepare a protein calibration standard solution sample; S2. Prepare a test sample; S3. Load the sample into a liquid chromatograph and run the analytical method; S4. Calculate the protein content.

2. The liquid chromatography method for quantitatively detecting proteins according to claim 1, wherein The protein is gE or a gE fusion protein; the gE is the extracellular domain of varicella-zoster virus glycoprotein E; the gE fusion protein is a fusion protein formed by the fusion expression of gE and an immune enhancing peptide / protein sequence; the immune enhancing peptide / protein sequence includes the universal DR Th epitope peptide PADRE and / or the tetanus toxin Th epitope peptide P2 and / or the Fc segment of human immunoglobulin γ and / or a human interleukin sequence and / or a human interferon sequence; the human interleukin includes interleukin-1 to interleukin-38; the human interferon includes interferon α, interferon β, and interferon γ; between the gE and the immune enhancing peptide / protein sequence of the gE fusion protein is connected by a linker peptide, and the linker peptide is a GGS and / or GGGS and / or GGGGS and / or GSGSG linker peptide.

3. The liquid chromatography method for quantitatively detecting proteins according to claim 2, wherein An optional molecular structure of the gE from the N-terminus to the C-terminus is: AA31-AA544, and the amino acid sequence is as shown in SEQ ID NO.1; an optional protein of the gE fusion protein is PADRE-gE-P2, and its molecular structure from the N-terminus to the C-terminus is: PADRE-GSGSG-gE-GGS-P2, and the amino acid sequence is as shown in SEQ ID NO.

2.

4. The liquid chromatography method for quantitatively detecting proteins according to claim 1, wherein The protein concentration of the protein calibration standard solution sample in S1 is 0.4 μg / ml to 1 mg / ml.

5. The liquid chromatography method for quantitatively detecting proteins according to claim 1, wherein The chromatographic column of the analytical method in S3 contains an alkyl or phenyl reversed-phase chromatographic packing material, and the mobile phase contains acetonitrile, trifluoroacetic acid, and water; the parameters of the liquid chromatograph in 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°C to 70°C; the injection volume is 1 μl to 100 μl; 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 400 nm.

6. The liquid chromatography method for quantitatively detecting proteins according to claim 1, wherein, The elution program of the analytical method in S3 includes an elution method for eluting the protein retention peak, and the elution method for eluting the protein retention peak is gradient elution or isocratic elution, the acetonitrile concentration gradient in the mobile phase is 5% to 100%, the gradient elution time is 2 min to 1920 min, and the concentration of the pH regulator in the mobile phase is 0.01% to 0.10%; the pH regulator is an acidic substance, including but not limited to trifluoroacetic acid and acetic acid.

7. The liquid chromatography method for quantitatively detecting proteins according to claim 1, wherein In S4 for calculating the protein content, the calculation method includes: Calculation method 1: First establish a calibration standard curve in which the concentration of the protein 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 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 stock solution. Calculation method 2: The calculation formula is the concentration of the protein in the stock solution = the peak area of the protein in the test sample * the concentration of the protein calibration standard solution sample * the dilution factor ∕ the peak area of the protein calibration standard solution sample.

8. Use of a liquid chromatography method for quantitatively detecting proteins in the detection of components in a protein solution / stock solution, characterized in that, Including but not limited to quantitatively detecting proteins in the stock solution of gE and / or gE fusion proteins.

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