Method for detecting purity of bordetella pertussis adhesive

Abstract

The invention provides a method for detecting the purity of bordetella pertussis stickins, which comprises the step of detecting the purity of a to-be-detected sample containing bordetella pertussis stickins by using a molecular exclusion high performance liquid chromatography, according to the molecular exclusion high performance liquid chromatography, rigid spherical silica gel particles with a diol functional group are adopted as a filling agent of a chromatographic column, and a Tris-HCl buffer solution is adopted as a mobile phase for elution. According to the method disclosed by the invention, the purity of the bordetella pertussis adhesive in the to-be-detected sample is detected by adopting the molecular exclusion high-performance liquid chromatography, so that the PRN antigen polymer can be detected while the purity of the PRN antigen monomer is detected, and the interference of the PRN antigen polymer on purity detection is reduced; the detection accuracy and precision of the purity of the bordetella pertussis mucin are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection. Specifically, the present invention relates to a method for detecting the purity of pertactin of Bordetella pertussis. Background Art

[0002] Pertussis is a highly contagious severe acute respiratory infectious disease caused by Bordetella pertussis. It is highly infectious and the general population is susceptible, especially infants and young children. It is one of the major infectious diseases seriously threatening human health.

[0003] Bordetella pertussis can produce many virulence factors and can undergo phenotypic changes due to changes in environmental conditions, and the expression levels of virulence factors are also different. These factors include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), heat-stable endotoxin (ET), heat-labile toxin (HLT), tracheal cytotoxin (TCT), adenylate cyclase toxin (ACT) and other bioactive substances. When the bacteria invade the respiratory tract of susceptible individuals through airborne droplets, the filamentous hemagglutinin of the bacteria adheres to the surface of ciliated epithelial cells of the pharynx to the bronchiolar mucosa. Subsequently, the bacteria multiply locally and produce various toxins, such as pertussis toxin and adenylate cyclase toxin, which cause ciliary paralysis and cell degeneration of epithelial cells, reduce their protein synthesis, cause necrosis and shedding of epithelial cells, and trigger systemic reactions.

[0004] In the production process of pertussis vaccine, the traditional detoxification method is to use formaldehyde or glutaraldehyde and lysine detoxification method. After the acellular pertussis vaccine detoxified with formaldehyde is treated at 37°C, most of them show obvious toxicity reversal. Glutaraldehyde is a cross-linking agent with an intervening arm and can freely bind to two binding sites of the toxin or two toxin molecules to form a stable conjugate. Lysine may have two functions. One is to prevent excessive cross-linking between toxin molecules to form clots; the other is to block residual reactive aldehyde groups to terminate the detoxification effect and prevent toxicity reversal or over-detoxification from affecting immunogenicity (Liu Dezheng, Xiao Zhanrong, Li Wanchen, Wang Xingyu, Zhao Baoming, etc., Study on the detoxification of pertussis antigen with glutaraldehyde [J], Chinese Journal of Biologicals, 1990, 3(4): 181-184). Since the detoxified sample will be mixed with formaldehyde or glutaraldehyde cross-linking agent, it will cause a corresponding increase in molecular weight and affect the detection of the purity of the detoxified sample.

[0005] Bordetella pertussis adhesin (PRN) is an outer membrane protein (OMP) exposed on the surface of virulent strains of Bordetella pertussis. It is a precursor polypeptide with a molecular weight of 93KD encoded by the prn gene of Bordetella pertussis. After hydrolysis of this polypeptide, a mature protein of approximately 69KD at the N-terminus and a C-terminal P30 protein of 30KD are obtained. Moreover, the Bordetella pertussis adhesin (PRN) protein is also prone to form multimers in solution, doubling its molecular weight. The changes in the protein structure of PRN before and after detoxification and the appearance of PRN multimers require a highly accurate and sensitive detection method to accurately quantify the purity of PRN before and after detoxification or multimeric Bordetella pertussis PRN.

[0006] Currently, neither the Chinese Pharmacopoeia nor the pharmacopoeias of other countries in the world record the purity determination methods for pertussis toxin, filamentous hemagglutinin, Bordetella pertussis adhesin, etc. The lack of purity determination methods has caused difficulties in the quality evaluation of pertussis toxin products and diphtheria-tetanus-pertussis vaccines. The currently existing method for detecting the purity of Bordetella pertussis PRN antigen is polyacrylamide gel electrophoresis, which has a long detection time, low sensitivity, and cannot accurately detect the presence of a small amount of PRN multimers. For the precise detection of Bordetella pertussis PRN antigen, Patent CN110672732A uses a method of high performance liquid chromatography tandem mass spectrometry to determine the content of PRN antigen by detecting the PRN characteristic peptide fragments decomposed. However, PRN multimers can also be decomposed into PRN characteristic peptide fragments. Using this method will count PRN multimers into the content of PRN antigen monomers. When performing purity detection, PRN multimers as impurities should not be counted. Using this method will lead to inaccurate purity detection of PRN multimers, seriously affecting the precision and accuracy of purity detection, and is not suitable for purity detection.

[0007] In summary, an effective method for detecting the purity of Bordetella pertussis PRN (Bordetella pertussis adhesin) antigen remains an urgent problem to be solved. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for detecting the purity of Bordetella pertussis adhesin. This method uses size exclusion high performance liquid chromatography. Compared with the commonly used polyacrylamide gel electrophoresis method in the prior art, it can detect PRN antigen multimers while detecting the purity of PRN antigen monomers, reduce the interference of PRN antigen multimers on purity detection, and improve the accuracy and precision of the purity detection of Bordetella pertussis adhesin.

[0009] Definition:

[0010] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the definitions of terms in this field, those skilled in the art can specifically refer to Current Protocols in Molecular Biology (Ausubel).

[0011] Although the present invention shows numerical ranges and parameter approximations in a wide range, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "2 to 40" should be considered to include any and all sub-ranges between the minimum value of 2 and the maximum value of 40 (including the endpoints), that is, all sub-ranges starting from the minimum value of 2 or greater, such as 2 to 6.1, and sub-ranges ending at the maximum value of 40 or less, such as 5.5 to 40. In addition, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0012] The term "or" used herein can be interchangeably used with the term "and / or", unless the context clearly indicates otherwise.

[0013] The above object of the present invention is achieved by the following technical solutions:

[0014] A method for detecting the purity of Bordetella pertussis adhesin, which comprises using size exclusion high performance liquid chromatography to detect the purity of a sample to be tested containing Bordetella pertussis adhesin, wherein the size exclusion high performance liquid chromatography uses rigid spherical silica gel particles with a diol functional group as the packing material of the chromatographic column and elutes with Tris-HCl buffer as the mobile phase.

[0015] In some embodiments of the present invention, the size exclusion high performance liquid chromatography uses a Wxl type chromatographic column, preferably a TSKgel G2000SWxl chromatographic column.

[0016] In some embodiments of the present invention, the size exclusion high performance liquid chromatography performs isocratic elution with Tris-HCl buffer as the mobile phase.

[0017] In some embodiments of the present invention, the mobile phase further contains preferably Tween 80.

[0018] In the method of the present invention, the mass concentration of Tween 80 contained in the mobile phase can be, for example, or any range composed of any two of the above point values or any point value therein.

[0019] In some embodiments of the present invention, the concentration of the Tris-HCl buffer is 20 to 100 mmol / L, preferably 40 to 60 mmol / L.

[0020] In the method of the present invention, the concentration of the Tris-HCl buffer can be, for example, 20 mmol / L, 30 mmol / L, 40 mmol / L, 41 mmol / L, 42 mmol / L, 43 mmol / L, 44 mmol / L, 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, 56 mmol / L, 57 mmol / L, 58 mmol / L, 59 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, or any range composed of any two of the above point values or any point value therein.

[0021] In some embodiments of the present invention, the pH value of the mobile phase is 7.0 to 9.0, preferably 7.5 to 8.5.

[0022] In the method of the present invention, the pH value of the mobile phase can be, for example, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range composed of any two of the above point values or any point value therein.

[0023] In some embodiments of the present invention, the mobile phase does not contain inorganic salts, such as sodium chloride.

[0024] In some embodiments of the present invention, the flow rate of the mobile phase is 0.35 mL / min to 0.65 mL / min, preferably 0.5 mL / min.

[0025] In some embodiments of the present invention, the column temperature of the chromatographic column is 25°C to 35°C, preferably 30°C.

[0026] In some embodiments of the present invention, the elution time is less than 40 min, preferably 30 to 40 min.

[0027] In some embodiments of the present invention, the detection wavelength of the size exclusion high performance liquid chromatography is 288 nm.

[0028] In some embodiments of the present invention, the concentration of pertussis adhesin in the sample to be tested is not less than 300 μg / mL.

[0029] In some embodiments of the present invention, the sample to be tested is a sample before detoxification containing PRN, and a solution with a PRN concentration of 0.3 - 2 mg / ml is prepared using the mobile phase as the solvent. Before detection, it can be filtered through a microporous membrane (such as 0.22 μm) and placed in the injection vial of the liquid chromatograph.

[0030] In some embodiments of the present invention, the injection volume of the sample to be tested is 40 μL - 120 μL, preferably 50 μL.

[0031] In the method of the present invention, in the molecular exclusion high performance liquid chromatography, calculated by the pertussis adhesin peak, the theoretical plate number is not less than 3000.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The present invention provides a method for detecting the purity of pertussis adhesin. Different from the polyacrylamide gel electrophoresis method in the prior art, the present invention uses the molecular exclusion high performance liquid chromatography method, and the purity can be calculated by the area normalization method. It can detect the PRN antigen multimer while detecting the purity of the PRN antigen monomer, reduce the interference of the PRN antigen multimer on the purity detection, and improve the accuracy and precision of the pertussis adhesin purity detection;

[0034] 2) In the method of the present invention, the change in the retention time of the PRN protein before and after detoxification can detect the purity of the PRN protein before and after detoxification at the same time;

[0035] 3) The method of the present invention is simple, feasible, easy to operate, low in cost, suitable for large-scale detection, and no organic reagents are used in the detection method of the present invention, reducing the harm to the operators and significantly improving the detection method of pertussis adhesin.

[0036] 4) The shortest detection period of the method of the present invention can be 30 minutes. The detection time is short. Compared with the detection period of the commonly used purity detection method (polyacrylamide gel electrophoresis method) in the field, which is at least 4 hours, the detection period is greatly shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0038] Figure 1 Showing the liquid chromatogram obtained using the TSKgel G2000SWxl chromatographic column in Example 1;

[0039] Figure 2Show the liquid chromatogram obtained using the TSKgel G3000PWxl chromatographic column in Example 1;

[0040] Figure 3 Show the liquid chromatogram obtained in Example 2 using 50 mM Tris-HCl with pH 8.5 and added Tween 80 as the mobile phase;

[0041] Figure 4 Show the liquid chromatogram obtained in Example 2 using 50 mM Tris-HCl with pH 8.5 and added Tween 80 as the mobile phase;

[0042] Figure 5 Show the liquid chromatogram obtained in Example 2 using 50 mM Tris-HCl with pH 8.5 and added Tween 80 as the mobile phase;

[0043] Figure 6 Show the liquid chromatogram obtained in Example 2 using 50 mM Tris-HCl with pH 8.5 and added Tween 80 as the mobile phase;

[0044] Figure 7 Show the liquid chromatogram obtained in Example 3 using 50 mM PB with pH 8.5 and added 130 mM NaCl as the mobile phase;

[0045] Figure 8 Show the liquid chromatogram obtained in Example 3 using 50 mM PB with pH 8.5 and added 50 mM NaCl as the mobile phase;

[0046] Figure 9 Show the liquid chromatogram obtained in Example 3 using 50 mM PB with pH 8.5 and added 50 mM NaCl and Tween 80 as the mobile phase;

[0047] Figure 10 Show the liquid chromatogram obtained in Example 4 using 50 mM Tris-HCl with pH 8.5 as the mobile phase;

[0048] Figure 11 Show the liquid chromatogram obtained in Example 4 using 50 mM Tris-HCl with pH 8.5 and added 50 mM NaCl as the mobile phase;

[0049] Figure 12 Show the liquid chromatogram obtained in Example 5 using 50 mM Tris-HCl with pH 7.5 and added Liquid chromatogram obtained with 50 mM Tris-HCl in Tween 80 as the mobile phase;

[0050] Figure 13 In Example 5, pH 8.0 was used and the addition Liquid chromatogram obtained with 50 mM Tris-HCl in Tween 80 as the mobile phase;

[0051] Figure 14 In Example 5, pH 8.5 was used and the addition Liquid chromatogram obtained with 50 mM Tris-HCl in Tween 80 as the mobile phase;

[0052] Figure 15 The liquid chromatogram obtained from the post-detoxification detection test in Example 6 is shown;

[0053] Figure 16 shows the liquid chromatogram obtained from the polymer detection experiment in Example 6;

[0054] Figure 17 The liquid chromatography comparison diagrams before and after detoxification and the polymer in Example 6 are shown;

[0055] Figure 18 The line graph for Example 7 is shown. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0057] The information of some reagents, drugs and instruments used in the examples of the present invention are listed as follows:

[0058] Reagents and drugs: Tris(hydroxymethyl)aminomethane was purchased from Vidayouer, batch number 21A2856285, analytical grade; HCl was purchased from Shanghai Test, batch number 20221202, analytical grade; sodium chloride was purchased from Aladdin, batch number I2114144, analytical grade; Tween 80 was purchased from Shanghai Test, batch number 20221202, analytical grade; sodium dihydrogen phosphate was purchased from Shanghai Test, batch number 20210908, analytical grade; disodium hydrogen phosphate was purchased from Shanghai Test, batch number 20211116, analytical grade.

[0059] Instruments: Thermo Fisher Ultimate 3000 high performance liquid chromatograph (equipped with DAD detector, temperature-controlled column oven, temperature-controlled autosampler, USA); TSKgel G2000SWxl chromatographic column purchased from TOSOH, model 7.8 mm*30 cm, 5 μm; TSKgel G3000PWxl chromatographic column purchased from TOSOH, model 7.8 mm*30 cm, 5 μm.

[0060] Preparation method of the sample to be tested: Take 1 mL of the sample containing PRN, where the solvent is the mobile phase, filter it using a 0.22 μm microporous filter membrane, and transfer it to an injection vial, which is the sample to be tested. Among them, the above-mentioned sample containing PRN is the sample purified before detoxification. The concentration of PRN in the sample to be tested is shown in the descriptions of the respective examples.

[0061] Preparation method of 50 mM Tris-HCl: Weigh 6.05 g of Tris powder, dissolve it in 1 L of pure water, stir evenly, and adjust the pH to 8.5 using HCl.

[0062] Preparation method of 50 mM PB buffer: Weigh 21.8502 g of Na2HPO4·12H2O powder and 10.76 g of NaH2PO4·H2O powder, dissolve them fully, measure the pH to be 8.5, and make up the volume to 1 L.

[0063] In the following examples, calculated based on the pertussis adhesin peak, the theoretical plate number of molecular exclusion high performance liquid chromatography is not less than 3000.

[0064] Example 1 Chromatographic column screening

[0065] In order to investigate the influence of different chromatographic columns on the detection of PRN, a TSKgel G2000SWxl chromatographic column and a TSKgel G3000PWxl chromatographic column were selected for a comparative experiment. According to the chromatographic conditions listed in Table 1 below, molecular exclusion high performance liquid chromatography detection was carried out on the sample to be tested. Among them, the concentration of PRN in the sample to be tested used was 762 μg / mL.

[0066] Table 1 Chromatographic conditions

[0067] Mobile phase 50 mM Tris-HCl + 130 mM NaCl, pH 7.5 Flow rate 0.5 ml / min Column temperature 30℃ Wavelength 280 nm Sample tray temperature 8℃ Injection volume 100 μl Running time Isocratic elution for 30 min

[0068] From Figure 1 it can be seen that when using the TSKgel G2000SWxl chromatographic column for detection, the elution time of PRN was 13.890 min. Using the area normalization method, the peak area of PRN was 7391831, and the proportion was 99.80%; from Figure 2 it can be seen that when using the TSKgel G3000PWxl chromatographic column for detection, the elution time of PRN was 14.275 min. Using the area normalization method, the peak area of PRN was 2974143, and the proportion was 89.63%. It can be seen that the determination result when using the TSKgel G3000PWxl chromatographic column for purity detection was significantly lower than that of the TSKgel G2000SWxl chromatographic column, indicating that different chromatographic columns have a greater influence on the detection of PRN. Therefore, the TSKgel G2000SWxl chromatographic column was selected for the PRN purity inspection.

[0069] Example 2 Screening of the Concentration of Tween 80 in the Mobile Phase

[0070] To investigate the effect of the concentration of Tween 80 in the mobile phase on the detection of PRN, size exclusion high performance liquid chromatography (HPLC) was performed on the test samples according to the chromatographic conditions listed in Table 2 below and the mobile phase design shown in Table 3 below, and comparison and screening were carried out. Among them, the concentration of PRN in the test samples and the injection volume of the samples are shown in Table 3 below.

[0071] Table 2 Chromatographic Conditions

[0072] Chromatographic column TSKgel G2000SWxl chromatographic column Flow rate 0.5 ml / min Column temperature 30℃ Wavelength 280 nm Sample tray temperature 8℃ Running time Isocratic elution for 30 min or 40 min

[0073] Table 3 Screening of the Concentration of Tween 80

[0074]

[0075] The test results of the above screening experiment are shown in Table 4 below:

[0076] Table 4 Summary of the Test Results of the Mobile Phase Screening Experiment

[0077]

[0078]

[0079] The obtained liquid chromatogram is as Figures 3 to 6 shown, and it can be seen from Figures 3 to 6 and Table 4 above that:

[0080] As the concentration of Tween 80 increases, the peak height will also increase accordingly. However, when the concentration of Tween 80 increases to , the peak height no longer continues to increase, and when it increases to , the baseline will drift upward. Therefore, it is preferred to add of Tween 80 to the mobile phase.

[0081] Example 3 Screening of the Buffer System

[0082] To investigate the effect of different buffer systems on the detection of PRN, size exclusion HPLC was performed on the test samples according to the chromatographic conditions listed in Table 2 above and the mobile phase design shown in Table 5 below, and comparison and screening were carried out. Among them, the concentration of PRN in the test samples and the injection volume of the samples are shown in Table 5 below.

[0083] Table 5 Screening of the Buffer System

[0084]

[0085] Note: PB in the table represents phosphate buffer.

[0086] The test results of the above screening experiment are shown in Table 6 below:

[0087] Table 6 Summary of Test Results of Mobile Phase Screening Experiment

[0088]

[0089]

[0090] The obtained liquid chromatogram is as Figures 7 to 9 shown, and it can be seen from Figures 7 to 9 and Table 6 above that:

[0091] When using the PB system as the mobile phase, there is no obvious improvement compared with using the Tris-HCl system as the mobile phase. Instead, it becomes worse. The chromatographic peak areas of the PB buffer system are 4,934,864, 4,929,638, and 4,973,168 respectively, which are significantly lower than the peak area of 7,391,831 of the initial mobile phase Tris-HCl system, indicating that regardless of the content of inorganic salt sodium chloride and whether Tween 80 is added, the relative content of the PRN sample detected is relatively low and the accuracy is poor. Therefore, compared with the PB system mobile phase, it is better to choose the Tris-HCl system mobile phase.

[0092] Example 4 Screening of Inorganic Salt System

[0093] In order to investigate the influence of the inorganic salt system of the mobile phase on the detection of PRN, according to the chromatographic conditions listed in Table 2 above and the mobile phase design shown in Table 7 below, size exclusion high performance liquid chromatography detection was carried out on the sample to be tested, and comparison and screening were carried out. Among them, the concentration of PRN in the sample to be tested and the injection volume of the sample are shown in Table 7 below.

[0094] Table 7 Screening of Inorganic Salt System

[0095]

[0096] The test results of the above screening experiment are shown in Table 8 below:

[0097] Table 8 Summary of Test Results of Mobile Phase Screening Experiment

[0098]

[0099]

[0100] The obtained liquid chromatogram is as Figure 10 and Figure 11 shown, and it can be seen from Figure 10 and Figure 11 and Table 8 above that:

[0101] When using the Tris-HCl system as the mobile phase, there is no significant difference in the number and area of peaks between adding inorganic salts and not adding inorganic salts in the mobile phase. It can be seen that whether to add inorganic salts has little effect on the mobile phase. In order to simplify the experimental operation and reduce the components of the mobile phase, the mobile phase without adding inorganic salts is selected.

[0102] Example 5 pH Value Screening

[0103] In order to investigate the influence of the pH value of the mobile phase on the PRN detection, according to the chromatographic conditions listed in Table 2 above and the mobile phase design shown in Table 9 below, size exclusion high performance liquid chromatography detection is carried out on the sample to be tested, and comparison and screening are carried out. Among them, the concentration of PRN in the sample to be tested and the injection volume of the sample are shown in Table 9 below.

[0104] Table 9 pH Value Screening

[0105]

[0106] The detection results of the above screening experiment are shown in Table 10 below:

[0107] Table 10 Summary of Detection Results of Mobile Phase Screening Experiment

[0108]

[0109]

[0110] The obtained liquid chromatogram is as Figures 12 to 14 shown, and it can be seen from Figures 12 to 14 and Table 8 above that:

[0111] When using the Tris-HCl system with different pH values as the mobile phase, the change in the peak area of PRN obtained from the three experiments is not significant, with no obvious difference, and the proportion is between 93-94%. Continuing to increase or decrease the pH value will affect the service life of the chromatographic column. Therefore, considering comprehensively, the pH range of the mobile phase is selected to be between 7.5-8.5.

[0112] Example 6 Determination of Bordetella pertussis Adhesin and Bordetella pertussis Adhesin Polymer

[0113] According to the screening experiments of Examples 1-5 above, the best chromatographic conditions are screened out, as shown in Table 11 below:

[0114] Table 11 Best Chromatographic Conditions

[0115]

[0116] Detection after Detoxification:

[0117] The detoxified sample was obtained by adding a formaldehyde crosslinking agent to the purified sample before detoxification, mixing evenly, and incubating at 37°C and 70 rpm for 7 days. In the detoxified sample, the concentration of PRN was 1003.75 μg / mL. The above-mentioned detoxified sample was aspirated and detected according to the chromatographic conditions in Table 11, and the obtained chromatogram was as Figure 15 shown.

[0118] Result analysis: Since the product will carry some formaldehyde crosslinking agent after detoxification, the molecular weight of the PRN protein after detoxification increases, resulting in the retention time of the PRN protein after detoxification moving forward. As Figure 15 shown, the PRN proteins before and after detoxification can be effectively separated and detected by the method of the present invention.

[0119] Detection of polymers:

[0120] The sample to be tested (the concentration of PRN was 1 mg / ml) was aspirated and detected according to the chromatographic conditions in Table 11, and the obtained chromatogram was as Figure 16 shown, and the liquid chromatography comparison diagrams of PRN before detoxification, after detoxification and polymers were as Figure 17 shown.

[0121] Result analysis: Compared with the PRN protein monomer, the molecular weight of the PRN protein polymer will increase several times. The retention time of the PRN protein polymer is 11.502. Compared with the retention time of the PRN protein monomer, the peak emergence time moves forward by about 2 minutes, while the retention time of the PRN protein after detoxification is 13.091, and the retention time of the PRN protein before detoxification is 13.980. It can be seen that the method of the present application can simultaneously detect the purity of the PRN protein monomer and the PRN protein polymer, and can also effectively separate PRN before detoxification, after detoxification and the PRN protein polymer.

[0122] In summary, there are PRN proteins before detoxification, after detoxification and PRN protein polymers in the purification process of pertussis PRN protein. These are different forms of manifestation of the PRN protein. The method of the present application can detect the PRN protein before and after detoxification, as well as the PRN protein before detoxification and its polymer, and all have good separation degrees. For different existing forms of the PRN protein, the method of Patent CN110672732A cannot detect its purity and cannot achieve effective separation.

[0123] Methodology verification of Example 7

[0124] According to the chromatographic conditions in Table 11 above, the detection method was verified for specificity, repeatability, linearity and accuracy. Among them, the concentration of PRN in the sample to be tested was 1 mg / ml.

[0125] Specificity

[0126] Respectively aspirate the mobile phase solution and the sample to be tested, with a concentration of 1 mg / ml, and inject 50 μl of each into the chromatograph according to the above method to examine the specificity. It is required that the solvent and buffer have no interference with the elution peak of the main peak.

[0127] Result analysis: The elution peak position of Bordetella pertussis adhesin (PRN) before detoxification is at 13.87 min, the elution peak position of PRN after detoxification is at 13.091 min, and the elution peak position of the PRN polymer is at 11.502 min. No suspicious peaks were found at this position for the solvent and buffer, and the specificity is good.

[0128] Repeatability

[0129] Prepare 6 samples to be tested (batch number: 18-1PRNP202310062, concentration 1 mg / ml) respectively, aspirate 50 μl of each and inject it into the chromatograph to examine the repeatability. It is required that the RSD of the 6 samples to be tested ≤ 2.0%. The results are shown in Table 12 below:

[0130] Table 12 Repeatability results

[0131]

[0132] Result analysis: The RSD of the 6 test solution samples is 0.24%, which meets the requirements and the repeatability is good.

[0133] Linearity

[0134] Prepare three test solution samples with concentrations of 50%, 80%, 100%, 120%, 150%, 180%, and 200% respectively. Aspirate 50 μl of each of the above test solution samples with different concentrations and inject them into the chromatograph to examine the linearity. It is required that R 2 > 0.99. The results are shown in Table 13 below:

[0135] Table 13 Linearity results

[0136]

[0137] Based on the results in Table 13 above, the linear equation is obtained, as Figure 18 shown. It can be seen from Figure 18 that for the purified sample with a concentration range of 0% - 200%, the linear equation is y = 4950.6x - 54378, and R 2 = 0.9996, which meets the requirements and the linear relationship is good.

[0138] Accuracy

[0139] The purity determination range of the Bordetella pertussis adhesin sample to be tested is 50% - 200%. The recovery rate within the linear range is between 95 - 105% indicates that this range is valid. The results are shown in Table 14 below:

[0140] Table 14 Recovery Rate Results

[0141]

[0142]

[0143] Result Analysis: The recovery rates of the above PRN concentrations between 500 - 2000 μg / ml are between 97.01 - 103.29%, meeting the requirement of the recovery rate in the range of 95 - 105%. The detection method of the present invention has good accuracy.

[0144] The above are only several exemplary embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, making some changes or modifications using the technical content disclosed above are equivalent to equivalent embodiments and all fall within the scope of the technical solution of the present invention.

Claims

1. A method for detecting the purity of Bordetella pertussis adhesin, which comprises using size exclusion high performance liquid chromatography to detect the purity of a sample to be tested containing Bordetella pertussis adhesin, wherein the size exclusion high performance liquid chromatography uses rigid spherical silica gel particles with a diol functional group as the filler of the chromatographic column and elutes with Tris-HCl buffer as the mobile phase.

2. The method according to claim 1, wherein The size exclusion high performance liquid chromatography uses a Wxl type chromatographic column, preferably a TSKgel G2000SWxl chromatographic column.

3. The method according to claim 1 or 2, wherein The size exclusion high performance liquid chromatography performs isocratic elution with Tris-HCl buffer as the mobile phase.

4. The method according to any one of claims 1 to 3, wherein The mobile phase further contains Tween 80 with a mass concentration of preferably .

5. The method according to any one of claims 1 to 4, wherein The concentration of the Tris-HCl buffer is 20 - 100 mmol / L, preferably 40 - 60 mmol / L; Preferably, the pH value of the mobile phase is 7.0 - 9.0, preferably 7.5 - 8.

5.

6. The method according to any one of claims 1 to 5, wherein The mobile phase does not contain inorganic salts, such as sodium chloride.

7. The method according to any one of claims 1 to 6, wherein, The flow rate of the mobile phase is 0.35 mL / min - 0.65 mL / min, preferably 0.5 mL / min; Preferably, the column temperature of the chromatographic column is 25°C - 35°C, preferably 30°C.

8. The method according to any one of claims 1 to 7, wherein The elution time is less than 40 min, preferably 30 - 40 min; Preferably, the detection wavelength of the size exclusion high performance liquid chromatography is 288 nm.

9. The method according to any one of claims 1 to 8, wherein The concentration of Bordetella pertussis adhesin in the sample to be tested is not less than 300 μg / mL.

10. The method according to any one of claims 1 to 9, wherein, The injection volume of the sample to be tested is 40 μL - 120 μL, preferably 50 μL.

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