CN113984947B

CN113984947B - Specific peptide fragment and method for determining antigen protein content in 9-valent HPV vaccine

Info

Publication number: CN113984947B
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: 2024-05-17
Anticipated expiration: 2040-07-27
Also published as: CN113984947A

Abstract

The invention discloses a specific peptide segment for determining the content of antigen proteins in 9-valent HPV vaccine, which comprises 9 peptide segments for determining 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 measuring the content of antigen protein in the 9-valent HPV vaccine, which comprises the following steps: carrying out trypsin enzymolysis on a sample to be detected to obtain enzymolysis liquid, carrying out high performance liquid chromatography tandem mass spectrometry analysis on the enzymolysis liquid to obtain a chromatogram, calculating the response peak areas of 9 specific peptide fragments, substituting the response peak areas into a standard working curve, and calculating the content of each antigen protein of the sample to be detected. According to the invention, through enzymolysis condition optimization, mass spectrum condition optimization, stability, sensitivity, linearity, repeatability and recovery rate tests, 9 proper quantitative peptide fragments of antigen proteins are screened out. The invention can be used for measuring the content 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 fragment and method for determining antigen protein content in 9-valent HPV vaccine

Technical Field

The invention relates to a specific peptide fragment and a method for determining the content of antigen proteins in a 9-valent HPV vaccine, in particular to a peptide fragment 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 papillomaviruses are the leading cause of cervical cancer in women and the fourth leading cause of cancer death in women worldwide. The advent of human papillomavirus vaccines (i.e., HPV vaccines) has played a great role in preventing cervical lesions caused by human papillomavirus infection. Clinical data shows that the 2-valent HPV vaccine (HPV-16/18) can prevent 70% of cervical early-stage cancerous 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 cancerous infection. The content of antigenic proteins in a 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 the immune system to produce 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 antigen proteins in HPV vaccines is one of the key parameters for HPV vaccine quality control.

Because different types of HPV antigen proteins have differences in disease prevention, the content and the proportion of the 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 ELISA method is reported to be used for measuring HPV antigen proteins, but the method is greatly influenced by the matrix composition of a sample, the test condition and the quality of an antibody, so that the repeatability of the method is poor. In addition, this approach is difficult to interpret the reasons that affect vaccine quality differences, and thus fails to provide guidance and improvement advice for vaccine production. The Lowry method can also be used for measuring the content of antigen proteins, but the method can only detect the total protein content and can not realize the detection of the content of different antigen proteins in a complex matrix. The inventor of the invention opens a method for measuring the antigen protein content of 2-valent HPV vaccine based on synthetic peptide in the previous research in laboratory, the method can realize the antigen protein content measurement of HPV-16/18 to a certain extent, but the method needs to purchase synthetic peptide, has higher cost, and the test accuracy is influenced by enzymolysis efficiency and stability of the synthetic peptide.

Disclosure of Invention

In view of the above prior art, the present invention provides specific peptide fragments for determining the content of antigenic proteins in a 9-valent HPV vaccine, as well as specific methods of determination. The invention screens out a specific peptide segment suitable for quantitative determination of antigen protein, and establishes a quantitative method of the effective component HPV-6/11/16/18/31/33/45/52/58 of the 9-valent HPV vaccine with high flux, high selectivity and high sensitivity by adopting a high performance liquid chromatography tandem mass spectrometry method, which 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 9-valent HPV vaccine quantitative reference substances, evaluating the stability of vaccine finished products of different batches of the same manufacturer, comparing vaccine finished products of different manufacturers, and detecting free antigen proteins in vaccine supernatant.

The invention is realized by the following technical scheme:

Specific peptide fragments for determining the content of antigen proteins in a 9-valent HPV vaccine, comprising 9 peptide fragments for determining 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) are specified as follows:

Determination of peptide fragments of HPV 6: at least one selected from SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR;

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

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

Determination of peptide fragments of HPV 18: at least one selected from QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR;

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

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

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

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

determination of peptide fragments of HPV 6: SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR;

Determination of peptide fragments of HPV 11: GQPLGVGVSGHPLLNK, LFFYLR, AGTVGEPVPDDLLVK, FSSELDQFPLGR;

Determination of peptide fragments of HPV 16: LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK;

Determination of peptide fragments of HPV 18: QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR;

Determination of peptide fragments of HPV 31: LLTVGHPYYSIPK, GSPCSNNAITPGDCPPLELK, SNVPLDICNSICK, SGTVGESVPTDLYIK, FLLQAGYR;

determination of peptide fragments of HPV 33: SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK;

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

determination of peptide fragments of HPV 52: LLTVGHPYFSIK, FGFPDTSFYNPETQR, SDVPIDICSSVCK;

determination of peptide fragments of HPV 58: LLAVGNPYFSIK, SDVPIDICNSTCK, LGEAVPDDLYIK, FLLQSGLK.

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

A method for measuring the content of antigen proteins in a 9-valent HPV vaccine comprises the following steps: carrying out trypsin enzymolysis on a sample to be detected to obtain enzymolysis liquid, carrying out high performance liquid chromatography tandem mass spectrometry (LC-MS/MS analysis) on the enzymolysis liquid 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 the content of HPV-6/11/16/18/31/33/45/52/58 in the sample to be detected. It will be appreciated by those skilled in the art that for any one 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 standard reference substances of 9 antigen proteins, wherein the 9 antigen proteins are respectively: 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); uniformly mixing, preparing a standard substance solution with a series of concentrations, carrying out trypsin enzymolysis to obtain an enzymolysis solution, carrying out high performance liquid chromatography tandem mass spectrometry analysis on the enzymolysis solution to obtain a chromatogram, calculating the response peak areas of the 9 specific peptide fragments, and preparing a standard working curve by using the concentration-response peak areas (adding the peak areas of the peptide fragments to obtain an ordinate, and preparing a standard curve by using the corresponding protein concentration as an abscissa).

Further, the specific steps of carrying out trypsin enzymolysis on the sample to be tested are as follows:

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

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

(iii) cooling to room temperature, adding iodoacetamide or a solution thereof, and reacting for 30min at room temperature in the absence of light;

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

(v) adding inorganic acid or solution thereof, and reacting for 30min at 37 ℃;

(vi) centrifuging, collecting supernatant, concentrating to dryness, dissolving with appropriate amount of water, and analyzing with LC-MS/MS.

Further, the protein denaturant is selected from urea, sodium dodecyl sulfonate, sodium octane sulfonate, rapiGest ^SF solution.

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

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

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

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

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

Further, the mineral acid is selected from formic acid, acetic acid, 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: centrifuging at 14000r/min for 15min.

Preferably, the specific steps of carrying out trypsin enzymolysis on the sample to be tested are as follows:

Taking 150 mu L of a sample to be detected, placing the sample into a centrifuge tube, adding RapiGest ^SF solution with equal volume, placing the sample into a column incubator, and incubating for 15min at 60 ℃;

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

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

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

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

(vi) centrifugation at 14000r/min for 15min, collecting supernatant, concentrating to dryness, dissolving with 200 μl water, and analyzing with LC-MS/MS.

Further, the specific operation mode of preparing the standard substance solution with the series of concentration is as follows: respectively 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 respectively 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 of the ammonium bicarbonate solution is 10mmol/L to 500mmol/L, preferably 50 mmol/L to 100mmol/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 solution of an organic solvent containing an ion exchanger;

the ion exchanger is an acid or a salt compatible with mass spectrum 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 and trifluoroacetic acid;

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

in the organic solvent aqueous solution, the volume ratio of the organic solvent to the water is 60-100: 0 to 40 percent;

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, 5-40% 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 injection volume: 10. Mu.L;

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, mass spectrometry conditions were as follows:

ion source: es+ mode; mass spectrometer: triple quadrupole mass spectrometer; atomizer flow rate: 3L/min;

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

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

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

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

Table 19 antigen protein quantitative peptide fragment and mass spectrum parameter thereof

* To quantify ions

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

1) More HPV antigen proteins can be quantified simultaneously, and the synthetic peptide method can only quantify two antigen proteins, so that the content of 9 antigen proteins can be measured by the method.

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

3) The problem of deviation in the measurement of the content of the antigen protein caused by post-translational modification of the peptide fragment in the storage process is avoided.

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

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

Drawings

Fig. 1:9 antigen protein quantitative peptide fragment spectra.

Detailed Description

The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.

The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.

Example 1

1. Experimental instrument and equipment:

High pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.

2. Experimental reagent:

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

3. Detection conditions:

Chromatographic column: a biocompatible C18 chromatographic 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: 0.2-0.5mL/min; sample injection volume: 10 mu L.

Mass spectrometry conditions:

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

4. And (5) investigating enzymolysis enzyme types: respectively preparing 9 HPV antigen proteins as reference solutions, wherein each solution has the same volume of two parts, 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 9 antigen protein reference substances into a solution with the concentration of 100 mug/mL by using 50mmol/L ammonium bicarbonate solution; taking 150 mu L of reference substance solution, placing the reference substance solution into a centrifuge tube, adding RapiGest ^SF solution with equal volume, placing the solution into a column incubator, and incubating for 15min at 60 ℃;

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

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

Comparing the peptide fragments obtained by enzymolysis of trypsin and chymotrypsin, the result shows that more peptide fragments with better response can be obtained by trypsin. The HPV antigen proteins are subjected to enzymolysis by trypsin, at least five peptide fragments with better mass spectrum response can be obtained from each protein, antigen proteins with the same concentration are subjected to enzymolysis, only 1-2 peptide fragments with the same mass spectrum response intensity can be obtained from chymotrypsin enzymolysis liquid, and the obtained peptide fragments have low specificity: if the specific peptide segment of HPV58 is not contained in the chymotrypsin enzymolysis liquid, the peptide segment detected in the HPV58 chymotrypsin enzymolysis liquid can be detected in other HPV antigen proteins. The present invention therefore selects trypsin as the enzymatic reagent.

5. And (3) investigating an enzymolysis efficiency improving reagent: whether the enzymolysis efficiency is influenced by adding the enzymolysis reagent improver nSMOL solution into the sample is examined. The nSMOL solution is added into the trypsin solution, so that the enzymolysis efficiency can be remarkably improved. As shown in table 2, the mass spectral response intensity of the peptide fragments was significantly improved after the nSMOL solutions were added.

TABLE 2 Mass Spectrometry response intensity of peptide fragments before and after adding nSMOL solutions

6. Sensitivity: analyzing the sample obtained by trypsin enzymolysis in the step 4 by mass spectrometry, wherein 4-5 peptide fragments with highest sensitivity are selected for each HPV antigen protein to be used as quasi-quantitative peptide fragment 1 of the antigen protein, and the related information is as follows:

specific peptide fragment corresponding to HPV 6: SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR, QTQLCMVCAPPLGEHWGK, NLSFWEVNLK, YDYLQMAADFYGDR.

Specific peptide fragment corresponding to HPV11 ：GQPLGVGVSGHPLLNK,LFFYLR,AGTVGEPVPDDLLVK,FSSELDQFPLGR,LLAVGHPYYSIK,FALPDSSLFDPPTQR,LVWACTGLEVGR,QTQLCMVGCAPPLGEAWGK.

Specific peptide fragment corresponding to HPV 16: LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK, FLLQAGLK, YTFWEVKLK, LVWACCGVEVGR, MVSEPYGDSLFFYLR.

Specific peptide segment corresponding to HPV 18: QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR, EQLFAR, SRPLSQGDCPPLELK, FLVQAGLR.

Specific peptide segment corresponding to HPV31 ：LLTVGHPYYSIPK,GSPCSNNAITPGDCPPLELK,SNVPLDICNSICK,SGTVGESVPTDLYIK,FLLQAGYR,FGFPDTSFYNPETQR,GQPLGVGISGHPLLNK,QTQLCLLGCKPPIGEHWGK,AQGHNNGICWGNQLFVTVVDTTR,HGEEFDLQFIFQLCK,FSADLDQFPLGR,SDVDIDICSSTCK,YPDYLK.

Specific peptide fragment corresponding to HPV 33: SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK, LLTVGNPYFR, LVWACVGLEIGR, YDDYLK, FSADLDQFPLGR.

Specific peptide segment corresponding to HPV45 ：GQPLGIGLSGHPFYNK,GTLCKPAQLQPGDCPPLELK,FWTVDLK,FSSDLDQYPLGR,LLTVGNPYFR,EQLFAR,GTLCKPAQLQPRDCPPLELK,EQLFAR,CEVPLDICQSICK,LLTVGNPYFR.

Specific peptide segment corresponding to HPV52 ：LLTVGHPYFSIK,FGFPDTSFYNPETQR,SDVPIDICSSVCK,TSIYYYAGSSR,FGFDDTSFYNPETQR,AGEEFDLQR,FSADLDQFPLGR,FLLQAGLQARPK.

Specific peptide fragment corresponding to HPV58 ：LLAVGNPYFSIK,SDVPIDICNSTCK,LGEAVPDDLYIK,FLLQSGLK,TSIYYYAGSSR,FGFPDTSFYNPDTQR,LVWACVGLEIGR,GSGNTAVIQSSAR,MASEPYGDSLFFFLR.

7. Specificity: the quasi-quantitative peptide 1 of the antigen protein obtained in the step 6 is used for searching a protein database and comparing peptide obtained by other antigen proteins of HPV types. The peptide fragments which can be obtained in other HPV antigen proteins are removed, and the removed peptide fragments are as follows ：HPV6：QTQLCMVCAPPLGEHWGK;HPV11：LVWACTGLEVGR,QTQLCMVGCAPPLGEAWGK;HPV16：FLLQAGLK,YTFWEVKLK;HPV18：EQLFAR;HPV31：FGFPDTSFYNPETQR,GQPLGVGISGHPLLNK,AQGHNNGICWGNQLFVTVVDTTR,HGEEFDLQFIFQLCK,FSADLDQFPLGR,YPDYLK;HPV33：LLTVGNPYFR,FSADLDQFPLGR;HPV45：EQLFAR,CEVPLDICQSICK,LLTVGNPYFR;HPV52：TSIYYYAGSSR,AGEEFDLQR,FSADLDQFPLGR;HPV58：TSIYYYAGSSR,FGFPDTSFYNPDTQR,LVWACVGLEIGR.

The peptide fragment which can be obtained only in the antigen protein is used as a specific peptide fragment and is used as the antigen protein quasi-quantitative peptide fragment 2, and the related peptide fragments 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 investigation, and the preparation steps of the investigation sample were as follows:

(1) Mixing standard substance solution mother liquor: mixing 9 HPV antigen protein reference substances, diluting with 50mmol/L ammonium bicarbonate solution to obtain 100 μg/mL solution as standard mother solution;

(2) Preparation of the working curve:

150. Mu.L, 120. Mu.L, 100. Mu.L, 80. Mu.L, 50. Mu.L and 25. Mu.L of standard stock solution were respectively added with 0. Mu.L, 30. Mu.L, 50. Mu.L, 70. Mu.L, 100. Mu.L and 125. Mu.L of ammonium bicarbonate solution (concentration: 50 mmol/L); placing into a centrifuge tube, adding RapiGest ^SF solution with equal volume, placing into a column incubator, and incubating at 60deg.C for 15min;

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

(vi) centrifuging at 14000r/min for 15min, collecting supernatant, concentrating to dry, dissolving with 200 μl water, and analyzing with LC-MS/MS to obtain spectrogram shown in FIG. 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 a peptide segment with poor linearity; the deleted peptide fragments were as follows ：HPV6：NLSFWEVNLK;HPV11：LLAVGHPYYSIK;HPV16：LVWACCGVEVGR;HPV18：SRPLSQGDCPPLELK;HPV31：SDVDIDICSSTCK;HPV33：LVWACVGLEIGR;HPV45：GTLCKPAQLQPRDCPPLELK;HPV52：FLLQAGLQARPK;HPV58：MASEPYGDSLFFFLR.

Screening out quasi-quantitative peptide 3 serving as quasi-quantitative antigen protein with good linearity, wherein the related peptide is as follows:

Specific peptide fragment corresponding to HPV 6: SDVPIDICGTTCK, AGEVGEPVPDTLIIK, FSSELDQYPLGR, YDYLQMAADFYGDR;

Specific peptide fragment corresponding to HPV 11: GQPLGVGVSGHPLLNK, LFFYLR, AGTVGEPVPDDLLVK, FSSELDQFPLGR, FALPDSSLFDPPTQR;

specific peptide fragment corresponding to HPV 16: LLAVGHPYFPIK, SEVPLDICTSICK, AGAVGENVPDDLYIK, MVSEPYGDSLFFYLR;

Specific peptide segment corresponding to HPV 18: QTQLCILGCAPAIGEHWAK, AGTMGDTVPQSLYIK, FSLDLDQYPLGR, FLVQAGLR;

Specific peptide fragment corresponding to HPV 31: LLTVGHPYYSIPK, GSPCSNNAITPGDCPPLELK, SNVPLDICNSICK, SGTVGESVPTDLYIK, FLLQAGYR, QTQLCLLGCKPPIGEHWGK;

Specific peptide fragment corresponding to HPV 33: SDVPIDICGSTCK, YTFWEVDLK, FLLQAGLK, YDDYLK;

Specific peptide segment corresponding to HPV 45: GQPLGIGLSGHPFYNK, GTLCKPAQLQPGDCPPLELK, FWTVDLK, FSSDLDQYPLGR, EQLFAR;

Specific peptide fragment corresponding to HPV 52: LLTVGHPYFSIK, FGFPDTSFYNPETQR, SDVPIDICSSVCK, FGFDDTSFYNPETQR;

specific peptide fragment corresponding to HPV 58: LLAVGNPYFSIK, SDVPIDICNSTCK, LGEAVPDDLYIK, FLLQSGLK, GSGNTAVIQSSAR.

9. Repeatability: taking 9 HPV antigen protein solutions of 120 mug/ml, 60 mug/ml and 30 mug/ml, sampling each solution in parallel for three times, carrying out enzymolysis by using the above 4, removing peptide fragments with poor repeatability from quasi-quantitative peptide fragment 3 of the antigen protein obtained by the above 7, wherein the removed peptide fragments are as follows ：HPV6：YDYLQMAADFYGDR;HPV11：FALPDSSLFDPPTQR;HPV16：MVSEPYGDSLFFYLR;HPV18：FLVQAGLR;HPV31：QTQLCLLGCKPPIGEHWGK;HPV33：YDDYLK;HPV45：EQLFAR;HPV52：FGFDDTSFYNPETQR;HPV58：GSGNTAVIQSSAR.

Peptide fragments with better repeatability (peak area RSD% < 20%) are screened out and used as quantitative peptide fragments of antigen proteins, as shown in SEQ ID NO. 1-32, and as shown in tables 1,3 and 4.

TABLE 3 quantitative parameters of antigen proteins

TABLE 4 method repeatability and enzymatic hydrolysis repeatability test results

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

TABLE 5 comparison of the response intensity of individual peptide fragments for the parameters commonly used in mass spectrometry (spray voltage 4.0kv, spray needle length 1mm, offset 1 mm) and the parameters used in the present invention (spray voltage 1.5kv, spray needle length 0.5mm, offset 3 mm)

/>

11. And (3) marking and recycling: the antigen protein recovery rate was measured after mixing the antigen protein enzymatic hydrolysate with the vaccine enzymatic hydrolysate in a ratio of 1:2,1:1 and 2:1, and the results are shown in Table 6.

TABLE 6 recovery test of quantitative peptide fragments in vaccine matrices

Mixing ratio of vaccine sample and antigen protein sample

Example two

1. Experimental instrument and equipment:

2. Experimental reagent:

9 antigen protein controls, vendor 1HPV9 vaccine (3 batches), vendor 2HPV9 vaccine (2 batches).

3. Detection conditions:

Chromatographic column: stationary phase 1 (biocompatible C18 chromatographic column);

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

Mass spectrometry conditions:

4. Drawing a standard curve:

(1) Mixing standard substance solution mother liquor: mixing 9 HPV antigen protein reference substances, diluting with 100mmol/L ammonium bicarbonate solution to obtain a solution, and taking the solution as standard mother liquor (the concentration of each antigen protein in the standard mother liquor is 100 mug/mL) for standby;

(2) Preparation of the working curve:

150. Mu.L, 120. Mu.L, 100. Mu.L, 80. Mu.L, 50. Mu.L and 25. Mu.L of standard stock solution were respectively added with 0. Mu.L, 30. Mu.L, 50. Mu.L, 70. Mu.L, 100. Mu.L and 125. Mu.L of ammonium bicarbonate solution (concentration 100 mmol/L); placing into a centrifuge tube, adding RapiGest ^SF solution with equal volume, placing into a column incubator, and incubating at 60deg.C for 15min;

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

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

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

(vi) centrifuging at 14000r/min for 15min, collecting supernatant, concentrating to dry, dissolving with 200 μl water, and analyzing with LC-MS/MS to obtain 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 corresponding chromatographic peak area.

5. Sample detection: and (3) taking a sample for enzymolysis in a reference substance mode, then analyzing and detecting by using a liquid chromatography-tandem mass spectrometry, 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. Sample testing was completed within 5 days, and the relative standard deviation was calculated for each antigen protein content obtained from the test, as shown in table 7, with good test reproducibility (RSD < 20%) for all four manufacturers, indicating that the method has good reproducibility, and also that the tested samples were relatively stable. As a reference, not only is the sample required to be stable, but also the production process is required to be stable, namely, the batch repeatability of the sample is required to be good. Of the four manufacturer's samples, manufacturer 1 has good batch reproducibility, as shown in Table 8, with a relative standard deviation of no more than 10%. In summary, the product from manufacturer 1 was selected as a control.

Table 7 4 samples from factories for repeated measurement of content over multiple days

Table 8 factory 1 and factory 2 batch reproducibility

Example III

1. Experimental instrument and equipment:

2. Experimental reagent:

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

3. Detection conditions:

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

Mass spectrometry conditions:

4. The control curve and the sample enzymolysis test method are the same as in the second embodiment. The results of the sample testing are shown in table 9. The 4 manufacturer samples all showed a tendency that HPV16 is the highest antigen protein. The content trends of antigen proteins of manufacturer 1, manufacturer 3 and manufacturer 4 are the same, and HPV16 is the antigen protein with the highest content, and then HPV11 and HPV18 are used. The trend of manufacturer 2 is quite different from the rest of the manufacturers.

TABLE 9 results of testing the content of 9 antigens in manufacturer 1-4HPV vaccine

/>

Example 4

1. Experimental instrument and equipment:

2. Experimental reagent:

HPV9 antigen protein standards, TCEP, iodoacetamide (IAA), ammonium bicarbonate, RAPIGESTTM, trypsin, nSMOL solution.

3. Detection conditions:

Mass spectrometry conditions:

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

The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.

Sequence listing

<110> National food and drug verification institute

Shimadzu corporation of management (China)

<120> Specific peptide fragment and method for determining content of antigen protein in 9-valent HPV vaccine

<141> 2020-07-27

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 1

Ser Asp Val Pro Ile Asp Ile Cys Gly Thr Thr Cys Lys

1 5 10

<210> 2

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 2

Ala Gly Glu Val Gly Glu Pro Val Pro Asp Thr Leu Ile Ile Lys

1 5 10 15

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 3

Phe Ser Ser Glu Leu Asp Gln Tyr Pro Leu Gly Arg

1 5 10

<210> 4

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 4

Gly Gln Pro Leu Gly Val Gly Val Ser Gly His Pro Leu Leu Asn Lys

1 5 10 15

<210> 5

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 5

Leu Phe Phe Tyr Leu Arg

1 5

<210> 6

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 6

Ala Gly Thr Val Gly Glu Pro Val Pro Asp Asp Leu Leu Val Lys

1 5 10 15

<210> 7

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 7

Phe Ser Ser Glu Leu Asp Gln Phe Pro Leu Gly Arg

1 5 10

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 8

Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys

1 5 10

<210> 9

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 9

Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys

1 5 10

<210> 10

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 10

Ala Gly Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys

1 5 10 15

<210> 11

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 11

Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His

1 5 10 15

Trp Ala Lys

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 12

Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys

1 5 10 15

<210> 13

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 13

Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg

1 5 10

<210> 14

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 14

Leu Leu Thr Val Gly His Pro Tyr Tyr Ser Ile Pro Lys

1 5 10

<210> 15

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 15

Gly Ser Pro Cys Ser Asn Asn Ala Ile Thr Pro Gly Asp Cys Pro Pro

1 5 10 15

Leu Glu Leu Lys

20

<210> 16

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 16

Ser Asn Val Pro Leu Asp Ile Cys Asn Ser Ile Cys Lys

1 5 10

<210> 17

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 17

Ser Gly Thr Val Gly Glu Ser Val Pro Thr Asp Leu Tyr Ile Lys

1 5 10 15

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 18

Phe Leu Leu Gln Ala Gly Tyr Arg

1 5

<210> 19

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 19

Ser Asp Val Pro Ile Asp Ile Cys Gly Ser Thr Cys Lys

1 5 10

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 20

Tyr Thr Phe Trp Glu Val Asp Leu Lys

1 5

<210> 21

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 21

Phe Leu Leu Gln Ala Gly Leu Lys

1 5

<210> 22

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 22

Gly Gln Pro Leu Gly Ile Gly Leu Ser Gly His Pro Phe Tyr Asn Lys

1 5 10 15

<210> 23

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 23

Gly Thr Leu Cys Lys Pro Ala Gln Leu Gln Pro Gly Asp Cys Pro Pro

1 5 10 15

Leu Glu Leu Lys

20

<210> 24

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 24

Phe Trp Thr Val Asp Leu Lys

1 5

<210> 25

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 25

Phe Ser Ser Asp Leu Asp Gln Tyr Pro Leu Gly Arg

1 5 10

<210> 26

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 26

Leu Leu Thr Val Gly His Pro Tyr Phe Ser Ile Lys

1 5 10

<210> 27

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 27

Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu Thr Gln Arg

1 5 10 15

<210> 28

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 28

Ser Asp Val Pro Ile Asp Ile Cys Ser Ser Val Cys Lys

1 5 10

<210> 29

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 29

Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys

1 5 10

<210> 30

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 30

Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys Lys

1 5 10

<210> 31

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 31

Leu Gly Glu Ala Val Pro Asp Asp Leu Tyr Ile Lys

1 5 10

<210> 32

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 32

Phe Leu Leu Gln Ser Gly Leu Lys

1 5

Claims

1. A specific peptide fragment for determining the content of antigenic proteins in a 9-valent HPV vaccine, characterized in that: comprising the determination of 9 peptide fragments of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58, in particular as follows:

2. The specific peptide fragment for determining the content of antigenic proteins in a 9-valent HPV vaccine of claim 1, wherein the peptide fragment is selected from the group consisting of: the specific peptide segment for determining the content of the antigen protein in the 9-valent HPV vaccine comprises the following peptide segments:

3. Use of a specific peptide fragment according to claim 1 or 2 as a quantitative peptide fragment for determining the antigen protein content 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 of: carrying out trypsin enzymolysis on a sample to be detected to obtain an enzymolysis liquid, carrying out high performance liquid chromatography tandem mass spectrometry analysis on the enzymolysis liquid to obtain a chromatogram, calculating the response peak areas of the 9 peptide fragments according to claim 1 or 2, substituting the response peak areas into a standard working curve, and calculating 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 standard reference substances of 9 antigen proteins, wherein the 9 antigen proteins are respectively: HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58; mixing to prepare standard substance solutions with serial concentrations, carrying out trypsin enzymolysis to obtain enzymolysis liquid, carrying out high performance liquid chromatography tandem mass spectrometry analysis on the enzymolysis liquid to obtain a chromatogram, calculating the response peak areas of the 9 peptide fragments according to claim 1 or2, and preparing a standard working curve according to the concentration-response peak areas.

5. The method for determining the content of antigen proteins in a 9-valent HPV vaccine according to claim 4, characterized by the following steps: the specific steps of carrying out trypsin enzymolysis on the sample to be tested are as follows:

6. The method for determining the content of antigen proteins in a 9-valent HPV vaccine according to claim 5, characterized by the following steps: the protein denaturant is selected from urea, sodium dodecyl sulfonate, sodium octane sulfonate and RapiGest ^SF solution;

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

Or/and: the disulfide bond cleavage reagent 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 0.1mg/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 the water is 1:0.1-1000;

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

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

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

8. The method for determining the content of antigen proteins in a 9-valent HPV vaccine according to claim 4, characterized by the following steps: 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;

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, 5-40% 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 injection volume: 10. Mu.L;

9. The method for determining the content of antigen proteins in a 9-valent HPV vaccine according to claim 4, characterized by the following steps: in the LC-MS/MS analysis, the mass spectrometry conditions were as follows:

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

10. The method for determining the content of antigen proteins in a 9-valent HPV vaccine according to claim 4, characterized by the following steps: the detection mode of the mass spectrum detector is multi-ion selection monitoring, and the mass spectrum detection parameters are shown in table 1, wherein the mass spectrum detection parameters are quantitative ions;

Table 19 peptide fragments and mass spectrum parameters thereof