CN113984947A

CN113984947A - Specific peptide segment and method for determining content of antigen protein in 9-valent HPV vaccine

Info

Publication number: CN113984947A
Application number: CN202010732651.5A
Authority: CN
Inventors: 王佑春; 龙珍; 黄维金; 李长坤; 李晓玉; 聂建辉; 李月琪; 黄涛宏
Original assignee: Shimadzu Enterprise Management China Co ltd; National Institutes for Food and Drug Control
Current assignee: Shimadzu Enterprise Management China Co ltd; National Institutes for Food and Drug Control
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2022-01-28
Anticipated expiration: 2040-07-27
Also published as: CN113984947B

Abstract

The invention discloses specific peptide sections for measuring the content of antigen protein in a 9-valent HPV vaccine, which comprise 9 peptide sections for measuring HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58. The invention also discloses a method for determining the content of the antigen protein in the 9-valent HPV vaccine, which comprises the following steps: and (3) carrying out trypsin enzymolysis on the sample to be detected to obtain an enzymolysis solution, carrying out high performance liquid chromatography tandem mass spectrometry on the enzymolysis solution to obtain a chromatogram, calculating the response peak areas of the 9 specific peptide fragments, substituting the response peak areas into a standard working curve, and calculating to obtain the content of each antigen protein of the sample to be detected. The invention screens out 9 quantitative peptide fragments suitable for antigen protein by enzymolysis condition optimization, mass spectrum condition optimization, stability, sensitivity, linearity, repeatability and recovery rate test. The method can be used for content determination of 9 antigen proteins in 9-valent HPV vaccines of different manufacturers and different batches, and has reference significance for improving vaccine research and development, production and quality evaluation.

Description

Specific peptide segment and method for determining content of antigen protein in 9-valent HPV vaccine

Technical Field

The invention relates to a specific peptide segment and a method for determining the content of antigen protein in a 9-valent HPV vaccine, in particular to a peptide segment and a method for determining the content of antigen proteins HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58 in a 9-valent HPV vaccine, and belongs to the technical field of vaccine quality evaluation.

Background

Human papillomavirus is the leading cause of cervical cancer in women and the fourth leading cause of cancer death in women worldwide. The emergence of human papillomavirus vaccines (i.e., HPV vaccines) has played a great role in preventing cervical lesions caused by human papillomavirus infection. Clinical data show that the 2-valent HPV vaccine (HPV-16/18) can prevent 70% of cervical early-stage canceration infection, and the 9-valent HPV vaccine (HPV-6/11/16/18/31/33/45/52/58) can prevent more than 95% of cervical early-stage canceration infection. The content of antigenic proteins in the vaccine is directly related to the effectiveness and safety of the vaccine: if the content of the antigen protein is too low, the immunogenicity of the vaccine is poor, and the aim of stimulating an immune system to generate antibodies cannot be achieved, so that the effectiveness is poor; if the antigen protein is too high, certain toxic and side effects can be caused, and the safety of the vaccine is influenced. Therefore, the content determination of the antigen protein in the HPV vaccine is one of the key parameters for the quality control of the HPV vaccine.

Because different types of HPV antigen proteins have different disease prevention, the content and the proportion of different types of antigen proteins in the HPV vaccine need to be strictly controlled, so that the safety and the effectiveness of the HPV vaccine are ensured. The method for measuring HPV antigen protein by using ELISA method is reported in literature, but the method is greatly influenced by sample matrix composition, test condition and antibody quality, thereby resulting in poor repeatability of the method. In addition, the method is difficult to interpret the causes of the differences affecting the quality of the vaccine, and thus cannot provide guidance and improvement advice for vaccine production. The Lowry method can also be used for content determination of antigen protein, but the method can only detect the total protein content and cannot realize content determination of different antigen proteins in a complex matrix. The laboratory of the inventor of the invention opens a method for measuring the content of 2-valent HPV vaccine antigen protein based on synthetic peptide fragments in early research, the method can realize the content measurement of HPV-16/18 antigen protein to a certain extent, but the method needs to purchase the synthetic peptide fragments, the cost is higher, and the test accuracy is influenced by the enzymolysis efficiency and the stability of the synthetic peptide fragments.

Disclosure of Invention

Aiming at the prior art, the invention provides a specific peptide fragment for measuring the content of the antigen protein in the 9-valent HPV vaccine and a specific measuring method. The invention screens specific peptide sections suitable for quantitative determination of antigen protein, establishes a high-throughput, high-selectivity and high-sensitivity quantitative method for the effective component HPV-6/11/16/18/31/33/45/52/58 of the 9-valent HPV vaccine by adopting a high performance liquid chromatography tandem mass spectrometry method, and is used for quantitative analysis of HPV-6/11/16/18/31/33/45/52/58 in the 9-valent HPV vaccine. The method can be used for screening quantitative reference products of the 9-valent HPV vaccine, evaluating the stability of vaccine finished products of different batches from the same manufacturer, comparing the vaccine finished products from different manufacturers and detecting the free antigen protein in the supernatant of the vaccine.

The invention is realized by the following technical scheme:

specific peptide sections for measuring the content of antigen proteins in a 9-valent HPV vaccine comprise 9 peptide sections for measuring HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58 (9 antigen proteins contained in the HPV vaccine), and are as follows:

determination of peptide fragment of HPV 6: at least one selected from SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR;

determination of peptide fragment of HPV 11: at least one selected from GQPLGVGVSGHPLLNK, LFFYLR, AGTVGEPVPDDLLVK, FSSELDQFPLGR;

determination of peptide fragment of HPV 16: at least one selected from LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK;

determination of peptide fragment of HPV 18: at least one selected from QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR;

determination of peptide fragment of HPV 31: at least one selected from LLTVGHPYYSIPK, GSPCSNNAITPGDCPPLELK, SNVPLDICNSICK, SGTVGESVPTDLYIK, FLLQAGYR;

determination of peptide fragment of HPV 33: at least one selected from SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK;

determination of peptide fragment of HPV 45: at least one selected from GQPLGIGLSGHPFYNK, GTLCKPAQLQPGDCPPLELK, FWTVDLK, FSSDLDQYPLGR;

determination of peptide fragment of HPV 52: at least one selected from LLTVGHPYFSIK, FGFPDTSFYNPETQR, SDVPIDICSSVCK;

determination of peptide fragment of HPV 58: at least one selected from LLAVGNPYFSIK, SDVPIDICNSTCK, LGEAVPDDLYIK, FLLQSGLK.

Further, the specific peptide fragment for determining the content of the antigen protein in the 9-valent HPV vaccine comprises the following peptide fragments:

determination of peptide fragment of HPV 6: SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR, respectively;

determination of peptide fragment of HPV 11: GQPLGVGVSGHPLLNK, LFFYLR, AGTVGEPVPDDLLVK, FSSELDQFPLGR, respectively;

determination of peptide fragment of HPV 16: LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK, respectively;

determination of peptide fragment of HPV 18: QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR, respectively;

determination of peptide fragment of HPV 31: LLTVGHPYYSIPK, GSPCSNNAITPGDCPPLELK, SNVPLDICNSICK, SGTVGESVPTDLYIK, FLLQAGYR, respectively;

determination of peptide fragment of HPV 33: SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK, respectively;

determination of peptide fragment of HPV 45: GQPLGIGLSGHPFYNK, GTLCKPAQLQPGDCPPLELK, FWTVDLK, FSSDLDQYPLGR, respectively;

determination of peptide fragment of HPV 52: LLTVGHPYFSIK, FGFPDTSFYNPETQR, SDVPIDICSSVCK, respectively;

determination of peptide fragment of HPV 58: LLAVGNPYFSIK, SDVPIDICNSTCK, LGEAVPDDLYIK, FLLQSGLK are provided.

The specific peptide fragment is used as a quantitative peptide fragment for determining the content of the antigen protein in the 9-valent HPV vaccine.

A method for determining the content of antigen protein in a 9-valent HPV vaccine comprises the following steps: and (2) carrying out trypsin enzymolysis on the sample to be detected to obtain an enzymolysis solution, carrying out high performance liquid chromatography tandem mass spectrometry (LC-MS/MS analysis) on the enzymolysis solution to obtain a chromatogram, calculating the response peak areas of the 9 specific peptide fragments, substituting the response peak areas into a standard working curve, and calculating to obtain the content of HPV-6/11/16/18/31/33/45/52/58 in the sample to be detected. It will be understood by those skilled in the art that for any specific peptide fragment, if more than one peptide fragment is included, the calculated response peak area is the sum of the response peak areas of the individual peptide fragments.

The standard working curve is obtained by the following method: taking a standard reference substance of 9 antigen proteins, wherein the 9 antigen proteins are respectively as follows: HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58 (9 antigen proteins contained in HPV vaccine); mixing uniformly, preparing standard solution with series concentration, carrying out trypsin enzymolysis to obtain enzymolysis solution, carrying out high performance liquid chromatography tandem mass spectrometry on the enzymolysis solution to obtain a chromatogram, calculating the response peak areas of the 9 specific peptide fragments, and making a standard working curve by using the concentration-response peak areas (taking the sum of the peak areas of the peptide fragments as a vertical coordinate and the corresponding protein concentration as a horizontal coordinate and making a standard curve).

Further, the specific steps of performing trypsin enzymolysis on the sample to be detected are as follows:

taking a sample to be detected, adding a protein denaturant or a solution thereof, and incubating for 15min at 60 ℃;

(ii) adding a disulfide bond cleavage reagent or a solution thereof, and reacting at 60 ℃ for 60 min;

(iii) cooling to room temperature, adding iodoacetamide or a solution thereof, keeping out of the sun at room temperature, and reacting for 30 min;

(iv) adding trypsin or a solution thereof and an nSMOL solution, and reacting for 10-14 h at 37 ℃;

(v) adding inorganic acid or its solution, and reacting at 37 deg.C for 30 min;

and (vi) centrifuging, taking the supernatant, concentrating to be dry, dissolving with a proper amount of water, and analyzing by LC-MS/MS.

Further, the protein denaturant is selected from urea, sodium dodecyl sulfate, sodium octane sulfonate, RapiGest^SFAnd (3) solution.

Further, the concentration of the protein denaturant solution is 10 mmol/L-10 mol/L.

Further, the disulfide bond cleaving agent is selected from Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP).

Further, the concentration of the disulfide bond cleavage reagent solution is 10 mmol/L-2 mol/L.

Further, the concentration of the iodoacetamide solution is 10 mmol/L-2 mol/L, preferably 1 mol/L.

Further, the concentration of the trypsin solution was 0.1 mg/ml.

Further, the inorganic acid is selected from formic acid, acetic acid and trifluoroacetic acid.

Further, in the inorganic acid solution, the volume ratio of the inorganic acid to water is 1: 0.1-1000.

Further, the specific conditions of centrifugation are as follows: centrifuge at 14000r/min for 15 min.

Preferably, the specific steps of performing trypsin enzymolysis on the sample to be detected are as follows:

taking 150 mu L of sample to be detected, placing the sample in a centrifuge tube, and adding an equal volume of RapiGest^SFPlacing the solution in a column incubator, and incubating for 15min at 60 ℃;

(ii) adding 10 mu L of dithiothreitol solution, and reacting for 60min at 60 ℃; the concentration of the dithiothreitol solution is 0.5 mol/L;

(iii) cooling to room temperature, adding 10 mu L of iodoacetamide solution, keeping out of the sun at room temperature, and reacting for 30 min; the concentration of the iodoacetamide solution is 1 mol/L;

(iv) adding 5 mu L of trypsin solution and nSMOL solution, and reacting for 10-14 h at 37 ℃; the concentration of the trypsin solution is 0.1 mg/ml;

(v) adding 5. mu.L of formic acid solution, and reacting at 37 ℃ for 30 min; the volume ratio of formic acid to water in the formic acid solution is 1: 200;

(vi) 14000r/min for 15min, taking the supernatant, concentrating to be dry, dissolving with 200 mu L of water, and then using for LC-MS/MS analysis.

Further, the specific operation mode for preparing the standard solution with the series of concentrations is as follows: taking 150 μ L, 120 μ L, 100 μ L, 80 μ L, 50 μ L and 25 μ L of the mother liquor of the standard substance, and adding 0 μ L, 30 μ L, 50 μ L, 70 μ L, 100 μ L and 125 μ L of ammonium bicarbonate solution; the concentration of the ammonium bicarbonate solution is 10 mmol/L-500 mmol/L, preferably 50-100 mmol/L.

Further, in the LC-MS/MS analysis, the chromatographic conditions were as follows:

stationary phase: stationary phase 1 or/and stationary phase 2;

column temperature: 30 ℃;

mobile phase: a-an aqueous ion exchanger solution; b-an aqueous organic solvent solution containing an ion exchanger;

the ion exchanger is a mass spectrum compatible acid or salt, or a mixture of the acid and the salt;

the salt is selected from ammonium formate or ammonium acetate; the acid is selected from formic acid, acetic acid, trifluoroacetic acid;

the organic solvent is selected from methanol, acetonitrile and ethanol;

in the organic solvent water solution, the volume ratio of the organic solvent to water is 60-100: 0 to 40 parts by weight;

the concentration of the ion exchanger in the mobile phase A is 0-20 mmol/L, and the concentration of the ion exchanger in the mobile phase B is 0-20 mmol/L.

Gradient: 0-8min, 5% B-40% B; 8-8.1min, 40% B-100% B; 8.1-10min, 100% B; 10-10.1min, 100% B-5% B; 10.1-15min, 5% B.

Flow rate: 0.2-0.5 mL/min;

sample introduction volume: 10 mu L of the solution;

the stationary phase 1 is a biocompatible C18 chromatographic column; the stationary phase 2 is a biocompatible C8 chromatographic column.

Further, in the LC-MS/MS analysis, the mass spectrum conditions are as follows:

an ion source: an ES + mode; mass spectrometry: a triple quadrupole mass spectrometer; flow rate of the atomizer: 3L/min;

flow rate of heater: 10L/min; interface temperature: 200 ℃; DL temperature: 235 ℃; heating module temperature: 400 ℃;

flow rate of drying gas: 10L/min; interface voltage: 3 kV.

The spray voltage was 1.5kv and the spray needle length was 0.5mm, deviating by 3 mm.

The mass spectrometric detector detection mode is multi-ion selective monitoring (MRM) and the mass spectrometric detection parameters are shown in table 1.

Quantitative peptide fragment of table 19 antigen proteins and mass spectrum parameters thereof

Is a quantitative ion

The specific peptide segment for determining the content of the antigen protein in the 9-valent HPV vaccine is obtained by experimental screening, and has strong specificity, high sensitivity, good linearity and good repeatability (every 3-4 specific peptide segments correspond to one antigen protein). The invention screens out 9 quantitative peptide fragments suitable for antigen protein through enzymolysis condition optimization, mass spectrum condition optimization, stability, sensitivity, linearity, repeatability and recovery rate tests; and then, the optimized method is used for investigating the stability and batch repeatability of the vaccine finished product, and the vaccine finished product which is suitable to be used as a 9-valent vaccine antigen quantitative standard product is screened out. The method can be used for content determination of 9 antigen proteins in 9-valent HPV vaccines of different manufacturers and different batches (for example, the method is used for stability evaluation of vaccine finished products of different batches to provide references for enterprises, for contrast of vaccine finished products of different manufacturers to provide references for vaccine inspection institutions, and for detection of free antigen proteins in vaccine supernatant to provide references for application in research and development, production and quality evaluation links), and has reference significance for improvement of research and development, production and quality evaluation of vaccines. Compared with an ELISA method, the method has high selectivity and high flux, and can be used for evaluating vaccine products of different batches and different manufacturers. Compared with the Lowry method, the method can realize the simultaneous quantitative determination of the 9 types of HPV antigen proteins. Compared with the method based on the synthetic peptide segment, the method has the following advantages:

1) the HPV antigen protein which can be quantified simultaneously is more, only two antigen proteins can be quantified by the peptide fragment synthesis method, and the method can realize the content determination of 9 antigen proteins.

2) The method does not need to synthesize peptide fragments, and avoids the problem of cost increase of enterprises caused by the synthesis of peptide fragments.

3) The method avoids the problem of antigen protein content measurement deviation caused by posttranslational modification of the peptide fragment in the storage process.

4) The problem of content measurement deviation caused by incomplete proteolysis of antigen is avoided.

The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.

Drawings

FIG. 1: 9 kinds of antigen protein quantitative peptide fragment spectrogram.

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example one

1. Laboratory apparatus and equipment:

the device comprises a high-pressure binary pump, a degasser, an automatic sample injector, a column incubator and a triple quadrupole mass spectrometer.

2. Experimental reagent:

HPV-6/11/16/18/31/33/45/52/58 antigen protein control.

3. Detection conditions are as follows:

a chromatographic column: a biocompatible C18 chromatography column;

mobile phase: a-formic acid: water (1:1000, v/v); b-formic acid: acetonitrile (1:1000, v/v);

gradient: 0-8min 5% B-40% B, 8-8.1min 40% -100% B, 8.1-10min 100% B,10-10.1min 100% -5% B, 10.1-15min 5% B; column temperature: 35 ℃; flow rate: 0.2-0.5 mL/min; sample introduction volume: 10 μ L.

Mass spectrum conditions:

an ion source: an ES + mode; mass spectrometry: a triple quadrupole mass spectrometer; flow rate of the atomizer: 3L/min; flow rate of heater: 10L/min; interface temperature: 200 ℃; DL temperature: 235 ℃; heating module temperature: 400 ℃; flow rate of drying gas: 10L/min; interface voltage: 3 kV; the spraying voltage is 1.5kv, the length of the spraying needle is 0.5mm, and the deviation is 3 mm; the mass spectrometric detector detection mode is multi-ion selective monitoring (MRM) and the mass spectrometric detection parameters are shown in table 1.

4. And (3) investigating the types of enzymolysis enzymes: preparing reference substance solutions of 9 HPV antigen proteins respectively, wherein two parts of each solution with the same volume are prepared, one part is subjected to enzymolysis by trypsin, and the other part is subjected to enzymolysis by chymotrypsin.

The trypsin enzymolysis step is as follows:

taking 9 antigen protein reference substances, and diluting the reference substances into a solution with the concentration of 100 mu g/mL by using 50mmol/L ammonium bicarbonate solution; taking 150 mu L of reference substance solution, placing the reference substance solution in a centrifuge tube, and adding an equal volume of RapiGest^SFPlacing the solution in a column incubator, and incubating for 15min at 60 ℃;

(iv) adding 5. mu.L of trypsin solution, and reacting at 37 ℃ for 12 h; the concentration of the trypsin solution is 0.1 mg/ml;

The chymotrypsin enzymolysis step is the same as the above, except that: (iv) adding 5 mu L of chymotrypsin solution, and reacting for 12h at 25 ℃; the concentration of the chymotrypsin solution is 0.1 mg/ml.

Comparing the peptide fragments obtained by enzymolysis of trypsin and chymotrypsin, the result shows that trypsin can obtain more peptide fragments with better response. Carrying out enzymolysis on HPV (human papilloma Virus) antigen proteins by using trypsin, wherein each protein can obtain at least five peptide segments with better mass spectrum response, the antigen proteins with the same concentration are subjected to enzymolysis, only 1-2 peptide segments with the same mass spectrum response strength can be obtained from chymotrypsin enzymolysis liquid, and the specificity of the obtained peptide segments is not high: if the chymotrypsin enzymatic hydrolysate does not contain the specific peptide fragment of HPV58, the peptide fragment detected in the chymotrypsin enzymatic hydrolysate of HPV58 can be detected in other HPV antigen proteins. Therefore, trypsin is selected as the enzymolysis reagent in the invention.

5. Reagent investigation is improved in enzymolysis efficiency: and (3) investigating whether the influence of an enzymolysis reagent enhancer nSMOL solution on the enzymolysis efficiency is added in the sample. The addition of the nSMOL solution into the trypsin solution can obviously improve the enzymolysis efficiency. As shown in table 2, the mass spectrum response intensity of the peptide fragments is significantly improved after the addition of the nSMOL solution.

Table 2 mass spectrum response intensity of peptide fragments before and after addition of nSMOL solution

6. Sensitivity: analyzing the sample obtained by the enzymolysis of the trypsin in the step 4 by using a mass spectrum, selecting 4-5 peptide fragments with the highest sensitivity for each HPV antigen protein as a quasi-quantitative peptide fragment 1 of the antigen protein, wherein the related information is as follows:

HPV6 corresponding to a particular peptide stretch: SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR, QTQLCMVCAPPLGEHWGK, NLSFWEVNLK, YDYLQMAADFYGDR are provided.

HPV11 corresponding to a particular peptide stretch: GQPLGVGVSGHPLLNK, LFFYLR, AGTVGEPVPDDLLVK, FSSELDQFPLGR, LLAVGHPYYSIK, FALPDSSLFDPPTQR, LVWACTGLEVGR, QTQLCMVGCAPPLGEAWGK are provided.

HPV16 corresponding to a particular peptide stretch: LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK, FLLQAGLK, YTFWEVKLK, LVWACCGVEVGR, MVSEPYGDSLFFYLR are provided.

HPV18 corresponding to a particular peptide stretch: QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR, EQLFAR, SRPLSQGDCPPLELK, FLVQAGLR are provided.

HPV31 corresponding to a particular peptide stretch: LLTVGHPYYSIPK, GSPCSNNAITPGDCPPLELK, SNVPLDICNSICK, SGTVGESVPTDLYIK, FLLQAGYR, FGFPDTSFYNPETQR, GQPLGVGISGHPLLNK, QTQLCLLGCKPPIGEHWGK, AQGHNNGICWGNQLFVTVVDTTR, HGEEFDLQFIFQLCK, FSADLDQFPLGR, SDVDIDICSSTCK, YPDYLK are provided.

HPV33 corresponding to a particular peptide stretch: SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK, LLTVGNPYFR, LVWACVGLEIGR, YDDYLK, FSADLDQFPLGR are provided.

HPV45 corresponding to a particular peptide stretch: GQPLGIGLSGHPFYNK, GTLCKPAQLQPGDCPPLELK, FWTVDLK, FSSDLDQYPLGR, LLTVGNPYFR, EQLFAR, GTLCKPAQLQPRDCPPLELK, EQLFAR, CEVPLDICQSICK, LLTVGNPYFR are provided.

HPV52 corresponding to a particular peptide stretch: LLTVGHPYFSIK, FGFPDTSFYNPETQR, SDVPIDICSSVCK, TSIYYYAGSSR, FGFDDTSFYNPETQR, AGEEFDLQR, FSADLDQFPLGR, FLLQAGLQARPK are provided.

HPV58 corresponding to a particular peptide stretch: LLAVGNPYFSIK, SDVPIDICNSTCK, LGEAVPDDLYIK, FLLQSGLK, TSIYYYAGSSR, FGFPDTSFYNPDTQR, LVWACVGLEIGR, GSGNTAVIQSSAR, MASEPYGDSLFFFLR are provided.

7. Specificity: the quasi-quantitative peptide fragment 1 of the antigen protein obtained in the 6 is used for searching a protein database and comparing with peptide fragments obtained by other types of antigen proteins of HPV. Peptide fragments that could also be obtained in other HPV antigenic proteins were knocked out as follows: HPV 6: QTQLCMVCAPPLGEHWGK, respectively; HPV 11: LVWACTGLEVGR, QTQLCMVGCAPPLGEAWGK, respectively; HPV 16: FLLQAGLK, YTFWEVKLK; HPV 18: EQLFAR; HPV 31: FGFPDTSFYNPETQR, GQPLGVGISGHPLLNK, AQGHNNGICWGNQLFVTVVDTTR, HGEEFDLQFIFQLCK, FSADLDQFPLGR, YPDYLK, respectively; HPV 33: LLTVGNPYFR, FSADLDQFPLGR, respectively; HPV 45: EQLFAR, cevpldicsk, LLTVGNPYFR; HPV 52: TSIYYYAGSSR, AGEEFDLQR, FSADLDQFPLGR, respectively; HPV 58: TSIYYYAGSSR, FGFPDTSFYNPDTQR, LVWACVGLEIGR are provided.

The peptide segment which can only be obtained in the antigen protein is used as a specific peptide segment and is used as a quasi-quantitative peptide segment 2 of the antigen protein, and the related peptide segments are as follows:

HPV6：SDVPIDICGTTCK，AGEVGEPVPDTLIIK，FSSELDQYPLGR，NLSFWEVNLK,YDYLQMAADFYGDR；

HPV11：GQPLGVGVSGHPLLNK，LFFYLR，AGTVGEPVPDDLLVK，FSSELDQFPLGR，LLAVGHPYYSIK,FALPDSSLFDPPTQR；

HPV16：LLAVGHPYFPIK，SEVPLDICTSICK，AGAVGENVPDDLYIK,LVWACCGVEVGR，MVSEPYGDSLFFYLR；

HPV18：QTQLCILGCAPAIGEHWAK，AGTMGDTVPQSLYIK，FSLDLDQYPLGR，SRPLSQGDCPPLELK,FLVQAGLR；

HPV31：LLTVGHPYYSIPK，GSPCSNNAITPGDCPPLELK，SNVPLDICNSICK，SGTVGESVPTDLYIK，FLLQAGYR,QTQLCLLGCKPPIGEHWGK,SDVDIDICSSTCK；

HPV33：SDVPIDICGSTCK，YTFWEVDLK，FLLQAGLK,LVWACVGLEIGR,YDDYLK；

HPV45：GQPLGIGLSGHPFYNK，GTLCKPAQLQPGDCPPLELK，FWTVDLK，FSSDLDQYPLGR,EQLFAR,GTLCKPAQLQPRDCPPLELK；

HPV52：LLTVGHPYFSIK，FGFPDTSFYNPETQR，SDVPIDICSSVCK,FGFDDTSFYNPETQR,FLLQAGLQARPK；

HPV58：LLAVGNPYFSIK，SDVPIDICNSTCK，LGEAVPDDLYIK，FLLQSGLK,GSGNTAVIQSSAR,MASEPYGDSLFFFLR。

8. linearity: the quasi-quantitative peptide fragment 2 of the antigen protein obtained in the above 7 was used for linear examination, and the preparation steps for examining the sample were as follows:

(1) mixing the mother liquor of the standard solution: mixing 9 HPV antigen protein reference substances, and diluting the mixture into a solution with the concentration of 100 mu g/mL by using 50mmol/L ammonium bicarbonate solution as a mother solution of a standard substance for later use;

(2) preparation of a working curve:

taking 150 mu L, 120 mu L, 100 mu L, 80 mu L, 50 mu L and 25 mu L of standard substance mother liquor, and adding 0 mu L, 30 mu L, 50 mu L, 70 mu L, 100 mu L and 125 mu L of ammonium bicarbonate solution (with the concentration of 50mmol/L) respectively; placing in a centrifuge tube, adding an equal volume of RapiGest^SFPlacing the solution in a column incubator, and incubating for 15min at 60 ℃;

(iv) adding 5 mu L of trypsin solution and nSMOL solution, and reacting for 12h at 37 ℃; the concentration of the trypsin solution is 0.1 mg/ml;

(vi) 14000r/min for 15min, taking the supernatant, concentrating to be dry, dissolving with 200 mu L of water, and analyzing by LC-MS/MS to obtain a spectrogram, which is shown in figure 1;

(3) drawing a standard working curve: obtaining a standard working curve according to the relation between the solution concentration and the corresponding chromatographic peak area, and removing peptide fragments with poor linearity; the peptide fragments rejected were as follows: HPV 6: NLSFWEVNLK, respectively; HPV 11: LLAVGHPYYSIK, respectively; HPV 16: LVWACCGVEVGR, respectively; HPV 18: SRPLSQGDCPPLELK, respectively; HPV 31: SDVDIDICSSTCK, respectively; HPV 33: LVWACVGLEIGR, respectively; HPV 45: GTLCKPAQLQPRDCPPLELK, respectively; HPV 52: FLLQAGLQARPK, respectively; HPV 58: MASEPYGDSLFFFLR are provided.

And (3) screening out a quasi-quantitative peptide segment 3 serving as a quasi-quantitative antigen protein with better linearity, wherein the related peptide segments are as follows:

HPV6 corresponding to a particular peptide stretch: SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR, YDYLQMAADFYGDR, respectively;

HPV11 corresponding to a particular peptide stretch: GQPLGVGVSGHPLLNK, LFFYLR, AGTVGEPVPDDLLVK, FSSELDQFPLGR, FALPDSSLFDPPTQR, respectively;

HPV16 corresponding to a particular peptide stretch: LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK, MVSEPYGDSLFFYLR, respectively;

HPV18 corresponding to a particular peptide stretch: QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR, FLVQAGLR, respectively;

HPV31 corresponding to a particular peptide stretch: LLTVGHPYYSIPK, GSPCSNNAITPGDCPPLELK, SNVPLDICNSICK, SGTVGESVPTDLYIK, FLLQAGYR, QTQLCLLGCKPPIGEHWGK, respectively;

HPV33 corresponding to a particular peptide stretch: SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK, YDDYLK, respectively;

HPV45 corresponding to a particular peptide stretch: GQPLGIGLSGHPFYNK, GTLCKPAQLQPGDCPPLELK, FWTVDLK, FSSDLDQYPLGR, EQLFAR, respectively;

HPV52 corresponding to a particular peptide stretch: LLTVGHPYFSIK, FGFPDTSFYNPETQR, SDVPIDICSSVCK, FGFDDTSFYNPETQR, respectively;

HPV58 corresponding to a particular peptide stretch: LLAVGNPYFSIK, SDVPIDICNSTCK, LGEAVPDDLYIK, FLLQSGLK, GSGNTAVIQSSAR are provided.

9. Repeatability: taking 9 HPV antigen protein solutions of 120 mu g/ml, 60 mu g/ml and 30 mu g/ml, sampling three parts of each solution in parallel, carrying out enzymolysis by using the 4 parts, and removing peptide fragments with poor repeatability from the quasi-quantitative peptide fragment 3 of the antigen protein obtained by the 7 parts, wherein the removed peptide fragments are as follows: HPV 6: YDYLQMAADFYGDR, respectively; HPV 11: FALPDSSLFDPPTQR, respectively; HPV 16: MVSEPYGDSLFFYLR, respectively; HPV 18: FLVQAGLR; HPV 31: QTQLCLLGCKPPIGEHWGK, respectively; HPV 33: YDDYLK; HPV 45: EQLFAR; HPV 52: FGFDDTSFYNPETQR, respectively; HPV 58: GSGNTAVIQSSAR are provided.

And (3) screening out the peptide fragment with better repeatability (the peak area RSD% is less than 20%) as the quantitative peptide fragment of the antigen protein, wherein the peptide fragment is shown as SEQ ID NO. 1-32, and is shown as table 1, table 3 and table 4.

TABLE 3 quantification parameters for each antigenic protein

TABLE 4 results of methods repeatability and enzymatic repeatability

10. Optimizing mass spectrum conditions: the optimal spray voltage of each peptide fragment was 1.5kV by selecting 1kV, 1.5kV, 2.0kV, 2.5kV, 3.0kV, 3.5kV and 4.0kV as the spray voltage and examining the response of the antigen protein quantitative peptide fragments obtained in the above 8. The influence of the lengths of the spray needles of 0.5mm,1.0mm,1.5mm and 2.0mm on the response intensity of the peptide fragments and the influence of the positions of the spray needles of 1mm, 2mm,3mm and 4mm on the response intensity of the peptide fragments are examined. The result shows that when the length of the spray needle is 0.5mm and the deviation position is 3mm, the response intensity of the peptide fragments is the highest, and the response intensity of all the peptide fragments is improved by 8-10 times compared with that of the conventional mass spectrum condition. The conclusion is obviously different from the common parameters (the length of the spray needle is 1-2 mm, and the deviation position is 1mm) of the mass spectrum, and the conclusion is obtained through a large number of experiments. The comparative data are as follows:

TABLE 5 comparison of the typical parameters of mass spectrometry (spray voltage 4.0kv, spray needle length 1mm, deviation from 1mm) and parameters used according to the invention (spray voltage 1.5kv, spray needle length 0.5mm, deviation from 3mm) for the response intensity of the individual peptide fragments

11. Adding a standard and recovering: the results of the detection of the recovery rate of the antigen protein after mixing the antigen protein enzymatic hydrolysate with the vaccine enzymatic hydrolysate at a ratio of 1:2, 1:1 and 2:1 are shown in table 6.

Table 6 recovery test of quantitative peptide fragments in vaccine matrix

a mixing ratio of vaccine sample and antigen protein sample

Example two

1. Laboratory apparatus and equipment:

2. Experimental reagent:

9 antigen protein controls, manufacturer 1HPV9 vaccine (batch 3), manufacturer 2HPV9 vaccine (batch 2).

3. Detection conditions are as follows:

a chromatographic column: stationary phase 1 (biocompatible C18 chromatography column);

mobile phase: a-acetic acid: water (1:1000, v/v); b-acetic acid: acetonitrile (1:1000, v/v);

Mass spectrum conditions:

4. Drawing a standard curve:

(1) mixing the mother liquor of the standard solution: mixing 9 HPV antigen protein reference substances, diluting with 100mmol/L ammonium bicarbonate solution to obtain solution as standard mother solution (the concentration of each antigen protein in the standard mother solution is 100 μ g/mL), and keeping;

(2) preparation of a working curve:

taking 150 mu L, 120 mu L, 100 mu L, 80 mu L, 50 mu L and 25 mu L of standard substance mother liquor, and adding 0 mu L, 30 mu L, 50 mu L, 70 mu L, 100 mu L and 125 mu L of ammonium bicarbonate solution (the concentration is 100mmol/L) respectively; placing in a centrifuge tube, adding an equal volume of RapiGest^SFPlacing the solution in a column incubator, incubating at 60 deg.CCulturing for 15 min;

(ii) adding 10. mu.L of TCEP solution, and reacting at 60 ℃ for 60 min; the concentration of the TCEP solution is 0.5 mol/L;

(iv) adding 5. mu.L of trypsin solution and nSMOL solution, and reacting at 37 ℃ for 12 h; the concentration of the trypsin solution is 0.1 mg/ml;

(v) adding 5. mu.L of trifluoroacetic acid solution, and reacting at 37 ℃ for 30 min; the volume ratio of trifluoroacetic acid to water in the trifluoroacetic acid solution is 1: 500;

(vi) 14000r/min for 15min, taking supernatant, concentrating to be dry, dissolving with 200 mu L of water, and analyzing by LC-MS/MS to obtain a spectrogram;

(3) drawing a standard working curve: and obtaining a standard working curve according to the relation between the concentration of the solution and the peak area of the corresponding chromatogram.

5. Sample detection: and performing enzymolysis on a sample in a mode of a reference substance, analyzing and detecting by using a liquid chromatography tandem mass spectrum, and obtaining the content of the component to be detected according to the response peak area and the standard working curve of the component to be detected. The sample test is completed within 5 days, the relative standard deviation of the content of each antigen protein obtained by the test is calculated, as shown in table 7, the antigen proteins of four manufacturers have better test repeatability (RSD is less than 20%), which indicates that the method has better repeatability and the tested sample is more stable. As a reference, not only sample stabilization but also production process stabilization is required, i.e. batch reproducibility of the sample is required to be good. Of the four manufacturer samples, manufacturer 1 had better batch reproducibility, as shown in table 8, with the relative standard deviation of the test content not exceeding 10%. In summary, the product of manufacturer 1 was selected as the control.

TABLE 74 manufacturer's samples Multi-day content measurement repeatability

TABLE 8 batch repeatability of manufacturer 1 and manufacturer 2

EXAMPLE III

1. Laboratory apparatus and equipment:

2. Experimental reagent:

HPV antigen protein reference, manufacturer 1-4HPV vaccine.

3. Detection conditions are as follows:

a chromatographic column: stationary phase 2 (biocompatible C8 chromatography column);

Mass spectrum conditions:

4. The control curve and the enzymatic assay of the samples were the same as in example two. The sample test results are shown in table 9. All 4 manufacturer samples showed a tendency that HPV16 was the highest antigen protein. The trend of the content of the antigen protein of the manufacturer 1, the manufacturer 3 and the manufacturer 4 is the same, the HPV16 is the antigen protein with the highest content, and the HPV11 and the HPV18 are arranged in the second place. The trend of manufacturer 2 is much different from the rest of the manufacturers.

TABLE 9 test results of the content of 9 antigens in 1-4HPV vaccines from manufacturers

Example 4

1. Laboratory apparatus and equipment:

2. Experimental reagent:

HPV9 antigen protein standard, TCEP, Iodoacetamide (IAA), ammonium bicarbonate, RapidGestTM, trypsin, and nSMOL solution.

3. Detection conditions are as follows:

Mass spectrum conditions:

4. The method for preparing the comparison curve is the same as that of the second embodiment. Compared with the two examples, the following steps are added to the sample used in the example before enzymolysis: 4 factory HPV vaccine samples were taken and centrifuged at 14000r/min for 10 min. Taking 100 mu L of supernatant for enzymolysis. The sample enzymolysis step is the same as the second embodiment. No obvious antigen protein is detected in the supernatant of the plants 1-4, which shows that the aluminum adjuvant adsorption processes of the four plants can completely adsorb the antigen protein and have less free antigen protein.

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Sequence listing

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Claims

1. The specific peptide fragment for determining the content of the antigen protein in the 9-valent HPV vaccine is characterized in that: comprises the determination of 9 peptide segments of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58, which are as follows:

2. The specific peptide fragment for determining the antigen protein content in a 9-valent HPV vaccine according to claim 1, wherein: the specific peptide fragment for determining the content of the antigen protein in the 9-valent HPV vaccine comprises the following peptide fragments:

3. Use of the specific peptide fragment of claim 1 or 2 as a quantitative peptide fragment for determining the content of antigen protein in a 9-valent HPV vaccine.

The method for measuring the content of the antigen protein in the 4.9-valent HPV vaccine is characterized by comprising the following steps: carrying out trypsin enzymolysis on a sample to be detected to obtain an enzymolysis solution, carrying out high performance liquid chromatography tandem mass spectrometry on the enzymolysis solution to obtain a chromatogram, calculating the response peak area of the 9 specific peptide fragments according to claim 1 or 2, substituting the response peak area into a standard working curve, and calculating to obtain the content of HPV-6/11/16/18/31/33/45/52/58 in the sample to be detected;

the standard working curve is obtained by the following method: taking a standard reference substance of 9 antigen proteins, wherein the 9 antigen proteins are respectively as follows: HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58; mixing to prepare standard solutions with a series of concentrations, carrying out trypsin enzymolysis to obtain an enzymolysis solution, carrying out high performance liquid chromatography tandem mass spectrometry on the enzymolysis solution to obtain a chromatogram, calculating the response peak area of the 9 specific peptide fragments according to claim 1 or 2, and making a standard working curve by using the concentration-response peak area.

5. The method of determining the content of antigen protein in a 9-valent HPV vaccine of claim 4, wherein: the specific steps of carrying out trypsin enzymolysis on a sample to be detected are as follows:

(v) adding inorganic acid or its solution, and reacting at 37 deg.C for 30 min;

6. The method of determining the content of antigen protein in a 9-valent HPV vaccine of claim 5, wherein: the protein denaturant is selected from urea, sodium dodecyl sulfate, sodium octane sulfonate and RapiGest^SFA solution;

or/and: the concentration of the protein denaturant solution is 10 mmol/L-10 mol/L;

or/and: the disulfide bond cleaving agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine;

or/and: the concentration of the disulfide bond breaking reagent solution is 10 mmol/L-2 mol/L;

or/and: the concentration of the iodoacetamide solution is 10 mmol/L-2 mol/L;

or/and: the concentration of the trypsin solution is 0.1 mg/ml;

or/and: the inorganic acid is selected from formic acid, acetic acid and trifluoroacetic acid;

or/and: in the inorganic acid solution, the volume ratio of the inorganic acid to water is 1: 0.1-1000;

or/and: the specific conditions of the centrifugation are as follows: centrifuge at 14000r/min for 15 min.

7. The method of determining the content of an antigen protein in a 9-valent HPV vaccine of claim 5 or 6, wherein: the specific steps of carrying out trypsin enzymolysis on a sample to be detected are as follows:

8. The method of determining the content of antigen protein in a 9-valent HPV vaccine of claim 4, wherein: in the LC-MS/MS analysis, the chromatographic conditions were as follows:

stationary phase: stationary phase 1 or/and stationary phase 2;

column temperature: 30 ℃;

the organic solvent is selected from methanol, acetonitrile and ethanol;

Flow rate: 0.2-0.5 mL/min;

sample introduction volume: 10 mu L of the solution;

9. The method of determining the content of antigen protein in a 9-valent HPV vaccine of claim 4, wherein: in the LC-MS/MS analysis, the mass spectrum conditions are as follows:

flow rate of drying gas: 10L/min; interface voltage: 3 kV;

10. The method of determining the content of an antigen protein in a 9-valent HPV vaccine according to claim 4 or 9, wherein: the mass spectrometric detector detection mode is multi-ion selective monitoring (MRM), and the mass spectrometric detection parameters are shown in Table 1;

quantitative peptide fragment of table 19 antigen protein and its mass spectrum parameter (as quantitative ion)