Method for determining purity of pertussis Bordetella adhesin protein by SEC-HPLC (single electron chromatography-high performance liquid chromatography) method
The purity of Bordetella pertussis adhesin protein was determined by SEC-HPLC, which solved the problem of insufficient precision of SDS-PAGE and achieved high-precision purity detection, making it suitable for vaccine production quality control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN INST OF BIOLOGICAL PROD CO LTD
- Filing Date
- 2025-12-12
- Publication Date
- 2026-05-01
AI Technical Summary
The existing SDS-PAGE method is not precise enough in detecting the purity of Bordetella pertussis adhesin protein, making it difficult to meet the accuracy requirements for purity detection in vaccine production.
The SEC-HPLC method was used for detection. The mobile phase was phosphate buffer II containing sodium chloride with a pH of 6.5-7.5. Isocratic elution was performed at a flow rate of 0.3-1 mL/min. The size exclusion column was a TSKgel G2000 SWXL. The detector was a differential refractive index detector at a detection wavelength of 280 nm.
It enables accurate detection of the purity of adhesin protein in Bordetella pertussis. The operation is simple, highly sensitive, and has good repeatability. The separation degree and tailing factor meet the requirements, ensuring the accuracy and repeatability of the test results.
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Figure CN121955221A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of Bordetella pertussis detection technology, specifically relating to a method for determining the purity of Bordetella pertussis adhesin protein by SEC-HPLC. Background Technology
[0002] Pertussis, or whooping cough, is an acute respiratory infectious disease caused by Bordetella pertussis (BP). It is one of the leading infectious diseases causing death in children. Vaccination is an effective means of preventing and controlling the incidence of pertussis. The effective antigenic components of Bordetella pertussis mainly include pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertussis adhesin (PRN). Among them, PRN can mediate adhesion to host cells and is an indispensable component of fractional pertussis vaccines. The key process in the development and production of fractional pertussis vaccines is purification, and antigen purity testing is an essential part of quality control in vaccine research and production.
[0003] Currently, the method for detecting the purity of PRN is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For example, the paper "Validation of SDS-PAGE Detection Method for Purity of Purified Antigens of Pertussis Toxin, Filament Hemagglutinin and Pertussis Adhesin" (DOI: 10.3321 / j.issn:0001-6209.2008.03.010) discloses the use of SDS-PAGE to detect the purity of purified antigens of pertussis toxin, filament hemagglutinin and pertussis adhesin, and validates the method. The results show that the protein concentration of the purified PRN antigen exhibits good correlation with the optical density value of its corresponding band within the low concentration range of 10.00–50.00 μg / mL and the high concentration range of 50.00–600.00 μg / mL. The accuracy and reproducibility of impurity levels are both within the range of 80%–120%, and the intermediate precision validation RSD is ≤10%. However, the precision of this method is still not high enough, which is not conducive to the accurate analysis of PRN protein purity. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a method for determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC (size exclusion high-performance liquid chromatography). Compared with the SDS-PAGE method, this method features simple operation, high sensitivity, good repeatability, and good intermediate precision.
[0005] Specifically, in order to achieve the above objectives, the present invention adopts the following technical solution: A method for determining the purity of Bordetella pertussis adhesin protein by SEC-HPLC includes the following steps: diluting Bordetella pertussis adhesin protein into a sample solution, and then performing SEC-HPLC to determine the purity of Bordetella pertussis adhesin protein; the mobile phase used in SEC-HPLC is phosphate buffer II containing sodium chloride with a pH of 6.5-7.5, the elution method is isocratic elution, and the flow rate of the mobile phase is 0.3-1 mL / min; the column temperature of the size exclusion column is 20-30℃; the size exclusion column is a TSKgel G2000 SWXL.
[0006] In a preferred embodiment, the flow rate of the mobile phase is 0.3~0.65 mL / min.
[0007] In a further preferred embodiment, the flow rate of the mobile phase is 0.3 mL / min.
[0008] In a preferred embodiment, the column temperature of the size exclusion chromatographic column is 30°C. In a preferred embodiment, the diluent for diluting the Bordetella pertussis adhesin protein is buffer I with a pH of 4 to 5.5; the buffer I contains 0.005 to 0.015 mol / L sodium acetate and 0.15 to 0.25 mol / L NaCl.
[0009] In a preferred embodiment, the concentration of Bordetella pertussis adhesin protein in the sample solution is 100-300 μg / mL. In a further preferred embodiment, the concentration of Bordetella pertussis adhesin protein in the sample solution is 300 μg / mL.
[0010] In a preferred embodiment, the injection volume of the sample solution is 50~200μL.
[0011] In a further preferred embodiment, the injection volume of the sample solution is 50 μL.
[0012] In a preferred embodiment, the phosphate buffer II contains 0.04~0.06 mol / L of phosphate and 0.25~0.35 mol / L of NaCl.
[0013] In the preferred embodiment, the detector used in SEC-HPLC is a differential refractive index detector.
[0014] In a further preferred embodiment, the detection wavelength is 280 nm.
[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) In this invention, the purity of Bordetella pertussis adhesin protein is detected by SEC-HPLC method. Compared with SDS-PAGE method, it has the characteristics of simple operation, high sensitivity, good repeatability and high intermediate precision. Therefore, it can accurately detect the purity of Bordetella pertussis adhesin protein in intermediate products (such as crude extract and purified solution) at each stage of vaccine production, and provide quality assurance for vaccine production and research.
[0016] (2) In this invention, by adjusting the flow rate of the mobile phase, the amount of sample solution injected and the temperature of the detection column, the resolution and tailing factor are controlled, which effectively avoids the peaks of impurities and the peaks of the target protein from overlapping into one peak, thus ensuring the accuracy of the detection results. The peak area and the RSD of the purity of the obtained HPLC chromatogram are both <2.0%. Attached Figure Description
[0017] Figure 1 The chromatograms are those of PRN sample solutions detected under different flow rates, injection volumes, and column temperatures in Examples 1-9 of the present invention.
[0018] Figure 2 This is a chromatogram of the detection of PRN sample solution and blank control solution in Example 10 of the present invention.
[0019] Figure 3 This is a chromatogram of six repeated tests on a PRN sample in Example 11 of the present invention.
[0020] Figure 4 This is a chromatogram of six repeated tests on a PRN sample in Example 12 of the present invention.
[0021] Figure 5 This is a chromatogram of a sample solution of PRN at different concentrations in Example 13 of the present invention.
[0022] Figure 6 This is a chromatogram of a PRN sample solution detected using mobile phases with different pH values in Example 14 of the present invention.
[0023] Figure 7This is a chromatogram of a PRN sample solution detected using mobile phases with different phosphate concentrations in Example 15 of the present invention.
[0024] Figure 8 This is a chromatogram of a PRN sample solution detected using a mobile phase containing different concentrations of NaCl in Example 16 of the present invention.
[0025] Figure 9 The chromatogram of the PRN sample solution was obtained by diluting PRN with different pH values (buffer I) in Example 17 of the present invention.
[0026] Figure 10 The chromatogram of the PRN sample solution after diluting PRN with different sodium acetate concentrations (buffer I) in Example 18 of the present invention is shown.
[0027] Figure 11 The chromatogram of the PRN sample solution detected after diluting PRN with a diluent (buffer I) containing different concentrations of NaCl in Example 19 of the present invention is shown.
[0028] Figure 12 This is a chromatogram of the PRN sample solution detected in Comparative Example 1 of the present invention. Detailed Implementation
[0029] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.
[0030] The Bordetella pertussis adhesin protein in the following examples was provided by Wuhan Institute of Biological Products Co., Ltd. In the specific embodiments and accompanying drawings of this application, "PRN" represents the Bordetella pertussis adhesin protein.
[0031] A specific embodiment of the present invention provides a method for determining the purity of Bordetella pertussis adhesin protein by SEC-HPLC, comprising the following steps: S1. Preparation: Dilute the concentration of Bordetella pertussis adhesin protein to 1.23-300 μg / mL using Buffer I (containing 0.005-0.015 mol / L sodium acetate and 0.15-0.25 mol / L NaCl) with pH 4-5.5 to obtain the Bordetella pertussis adhesin protein sample solution. The preparation method of Buffer I is as follows: Weigh 0.41-1.23 g of sodium acetate and 8.77-14.61 g of NaCl, dissolve in 800 mL of purified water, adjust the pH to 4-5.5 with hydrochloric acid solution, bring the volume to 1 L, filter through a 0.22 μm membrane, and set aside for later use.
[0032] Prepare a 0.04–0.06 mol / L phosphate buffer II (containing 0.25–0.35 mol / L NaCl) with a pH of 6.5–7.5 as the mobile phase. The preparation method for phosphate buffer II is as follows: Weigh 14.61–20.45 g NaCl, 1.072–1.479 g NaH₂PO₄·H₂O, and 4.409–6.766 g Na₂HPO₄, dissolve them in 800 mL of purified water, adjust the pH to 6.5–7.5 with sodium hydroxide solution, and bring the volume to 1 L. Filter through a 0.22 μm membrane before use.
[0033] S2. Detection: Set appropriate chromatographic conditions. The size exclusion column was a TSKgel G2000 SWXL gel column, 7.8 mm ID × 30 cm, with a packing particle size of 5 μm and a silica matrix. The flow rate was 0.3–1 mL / min, the injection volume was 50–200 μL, and the column temperature was 10–30℃. The detector was a WanYi RID3300 differential refractive index detector with a detection wavelength of 280 nm. Isocratic elution was performed with the mobile phase for 60 min. The *Bordetella pertussis* adhesin protein sample solution and blank control solution were injected into the SEC-HPLC instrument for chromatographic analysis. The sample peak was collected using the detector to obtain the purity of the *Bordetella pertussis* adhesin protein. The blank control solution was buffer I prepared in step S1.
[0034] Examples 1-9 Examples 1-9 describe the purity of Bordetella pertussis adhesin protein tested according to the above steps, investigating the effects of different flow rates, injection volumes, and column temperatures on the results. In step S1, buffer I had a pH of 5.0 and contained 0.01 mol / L sodium acetate and 0.2 mol / L NaCl; the concentration of the Bordetella pertussis adhesin protein sample solution was 300 μg / mL; and phosphate buffer II had a pH of 7.0, a concentration of 0.05 mol / L, and contained 0.3 mol / L NaCl. The flow rate, injection volume, and column temperature conditions in step S2 are shown in Table 1. The detection results are as follows: Figure 1As shown.
[0035] Calculate the separation degree and tailing factor using the following formulas: Resolution = 2(TR2-TR1) / (W1+W2), where TR2 is the retention time of the latter peak among two adjacent chromatographic peaks; TR1 is the retention time of the former peak among two adjacent chromatographic peaks; and W1 and W2 are the peak base widths of the two adjacent peaks mentioned above.
[0036] Tail trail factor = W 0.05h / 2d1, W 0.05h d1 is the peak width at 5% of the peak height, and d1 is the distance between the peak apex and the peak front.
[0037] according to Figure 1 The resolution and tailing factor of Examples 1 to 9 were calculated based on the detection results. The calculation results are shown in Table 1.
[0038] Table 1. Flow rate, injection volume, column temperature, and detection results for Examples 1-9.
[0039] Since the Chinese Pharmacopoeia lacks a quality management standard for the HPLC detection method of Bordetella pertussis adhesin protein, while the 2020 edition of the Chinese Pharmacopoeia stipulates that the resolution for HPLC detection of protein purity must be greater than 1.5 and the tailing factor should be 0.95–1.4, this invention adopts this standard as the quality management standard for the HPLC detection method of Bordetella pertussis adhesin protein. Table 1 shows that a flow rate of 0.3–1 mL / min, an injection volume of 50–200 μL, and a column temperature of 20–30℃ all meet the requirements for resolution and tailing factor. Furthermore, at a flow rate of 0.3–0.65 mL / min, the resolution is greater than 1.8; even further, at a flow rate of 0.3 mL / min, the resolution is greater than 2. Further still, at a column temperature of 30℃ and a flow rate of 0.3 mL / min, the resolution is greater than 2. The optimal chromatographic conditions were those with the highest resolution and the tailing factor closest to 1.0, i.e., the detection conditions in Example 5 were the optimal chromatographic conditions.
[0040] Example 10 This embodiment uses the detection conditions of Example 5 to verify the specificity of the SEC-HPLC method for determining the purity of Bordetella pertussis adhesin protein. Bordetella pertussis adhesin protein sample solution and blank control solution were injected into the SEC-HPLC instrument for chromatographic analysis. The presence of an absorption peak in the blank control solution within the integration time of the target protein peak was observed. The results are as follows: Figure 2 As shown. The blank control solution is buffer I prepared in step S1.
[0041] from Figure 2 As can be seen above, the retention time of the main peak of *Bordetella pertussis* adhesin protein was approximately 18.559 min, the peak start time was approximately 17.8 min, and the peak end time was approximately 20.1 min, with an integration range of 0–25 min. The blank control solution (buffer I) showed no peak within the set integration range. Both the blank control solution (buffer I) and the *Bordetella pertussis* adhesin protein solution showed typical salt peaks between 27.5 and 29.6 min. This is due to the inconsistency in composition between the blank control solution (buffer I) and the mobile phase solution, indicating that the method of determining the purity of *Bordetella pertussis* adhesin protein using SEC-HPLC has good specificity.
[0042] Example 11 This embodiment uses the detection conditions of Example 5 to study the reproducibility of the SEC-HPLC method for determining the purity of Bordetella pertussis adhesin protein. The same batch of Bordetella pertussis adhesin protein sample solution was repeatedly tested 6 times by Analyst 1 on the first day. The test results are shown in […]. Figure 3 .according to Figure 3 The RSD value is calculated from the test results, RSD = S / X and S are the standard deviations. X represents the average value, and the results are shown in Table 2.
[0043] Table 2. Repeatability verification results of the PRN detection method in Example 11
[0044] As shown in Table 2, when the detection method of the present invention is used to detect the adhesin protein of Bordetella pertussis, the peak area RSD value of the adhesin protein is 1.40%, the purity RSD value is 0.53%, and the retention time RSD value is 1.55%. The RSD values of peak area and purity are both <2%, indicating that the method provided by the present invention for detecting the purity of Bordetella pertussis adhesin protein has good repeatability.
[0045] Example 12 This embodiment uses the detection conditions of Example 5 to determine the purity of Bordetella pertussis adhesin protein by SEC-HPLC, and verifies the intermediate precision of the method in conjunction with the results of Example 11. The same batch of Bordetella pertussis adhesin protein sample solution as in Example 11 was repeatedly tested 6 times by Analyst 2 on the second day. The results are shown in […]. Figure 4 .according to Figure 4 The RSD value is calculated from the test results, RSD = S / X and S are the standard deviations. X represents the average value, and the detection results are shown in Table 3.
[0046] Table 3. Validation results of the intermediate precision of the PRN detection method
[0047] As shown in Table 3, when different operators repeatedly tested the adhesin protein of Bordetella pertussis using the method of this invention, the peak area RSD values of the adhesin protein were 1.40% and 1.72%, respectively; the purity RSD values were 0.53% and 0.41%, respectively; and the retention time RSD values were 1.55% and 1.14%, respectively. The RSD values of peak area and purity were both <2%, indicating that the method for detecting the purity of adhesin protein of Bordetella pertussis provided by this invention has good intermediate precision.
[0048] Example 13 This example investigated the limits of detection and quantitation (LODs) for determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC. The detection method differed from Example 5 in that, in step S1, the PRN concentration was diluted to 100 μg / mL, 33 μg / mL, 11 μg / mL, 3.7 μg / mL, and 1.23 μg / mL. The detection results are as follows: Figure 5 As shown. According to Figure 5 The linear relationship between the signal-to-noise ratio and the concentration of the PRN sample solution and the peak area was calculated based on the detection results. The sample concentration with a signal-to-noise ratio ≥3:1 was the limit of detection, and the sample concentration with a signal-to-noise ratio ≥10:1 was the limit of quantitation. The results are shown in Table 4.
[0049] Table 4. Detection results of PRN sample solutions of different concentrations in Example 13
[0050] As shown in Table 4, when the PRN sample solution concentration is 11 μg / mL, the signal-to-noise ratio is greater than 10:1. Therefore, a concentration of 11 μg / mL and an injection volume of 50 μL (0.55 μg) are set as the limit of quantitation. When the PRN sample solution concentration is 3.7 μg / mL, the signal-to-noise ratio is greater than 3:1. Therefore, a concentration of 3.7 μg / mL and an injection volume of 50 μL (0.185 μg) are set as the limit of detection. From the limits of detection and quantitation, it can be seen that the method provided by this invention for detecting the purity of Bordetella pertussis adhesin protein has good sensitivity. Furthermore, when the PRN sample solution concentration is 100 μg / mL and the injection volume is 50 μL, the resolution and tailing factor both meet the requirements. Therefore, diluting PRN to a concentration of 100-300 μg / mL yields better detection results.
[0051] Example 14 This example investigated the effect of mobile phase pH changes on the detection results when determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC. The detection method differs from Example 5 in that the pH values of phosphate buffer II (mobile phase) in step S1 were 6.5, 7.0, and 7.5, respectively. The detection results are shown below. Figure 6 .according to Figure 6 The purity, retention time, peak area RSD, resolution, and tailing factor were calculated from the test results, and the results are shown in Table 5.
[0052] Table 5 Results of PRN sample solution detection using mobile phases with different pH values
[0053] The results showed that when using phosphate buffer II (mobile phase) at different pH values to detect Bordetella pertussis adhesin protein, the purity RSD was 0.44%, the retention time RSD was 1.93%, and the peak area RSD was 1.98%. The RSDs for purity, retention time, and peak area were all <2.0%, and the resolution was greater than 1.5. The tailing factor was within the range of 0.95–1.4, indicating that small fluctuations in the pH of phosphate buffer II (mobile phase) (6.5–7.5) had little effect on the purity detection of Bordetella pertussis adhesin protein. Therefore, phosphate buffer II (mobile phase) with a pH range of 6.5–7.5 can be used for the SEC-HPLC method to determine the purity of Bordetella pertussis adhesin protein.
[0054] Example 15 This example investigated the effect of phosphate concentration in the mobile phase on the detection results when determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC. The detection method differs from Example 5 in that the phosphate concentration in phosphate buffer II (mobile phase) in step S1 was 0.04 mol / L, 0.05 mol / L, and 0.06 mol / L, respectively. The detection results are shown below. Figure 7 .according to Figure 7 The RSD of purity, retention time and peak area were calculated from the test results, and the results are shown in Table 6.
[0055] Table 6. Detection results of PRN sample solutions using mobile phases with different phosphate concentrations.
[0056] The results showed that when using phosphate buffer II (mobile phase) with different phosphate concentrations to detect Bordetella pertussis adhesin protein, the RSD for purity was 0.34%, the RSD for retention time was 1.15%, and the RSD for peak area was 1.31%. The RSDs for purity, retention time, and peak area were all <2.0%, and the resolution was greater than 1.5. The tailing factor was in the range of 0.95–1.4, indicating that small fluctuations in the phosphate concentration in phosphate buffer II (mobile phase) (0.04–0.06 mol / L) had little effect on the purity detection of Bordetella pertussis adhesin protein. Therefore, phosphate buffer II (mobile phase) with a phosphate concentration range of 0.04–0.06 mol / L can be used for the SEC-HPLC method to determine the purity of Bordetella pertussis adhesin protein.
[0057] Example 16 This example investigated the effect of varying NaCl content in the mobile phase on the determination of Bordetella pertussis adhesin protein purity using SEC-HPLC. The detection method differed from Example 5 in that the NaCl concentration in phosphate buffer II (mobile phase) in step S1 was 0.25 mol / L, 0.3 mol / L, and 0.35 mol / L, respectively. The detection results are shown below. Figure 8 .according to Figure 8 The RSD of purity, retention time and peak area were calculated from the test results, and the results are shown in Table 7.
[0058] Table 7. Detection results of PRN sample solutions using mobile phases containing different concentrations of NaCl.
[0059] The results showed that when using phosphate buffer II (mobile phase) containing different concentrations of NaCl to detect the adhesin protein of Bordetella pertussis, the RSD of purity was 0.50%, the RSD of retention time was 1.90%, and the RSD of peak area was 1.90%. The RSDs for purity, retention time, and peak area were all <2.0%, the resolution was greater than 1.5, and the tailing factor was in the range of 0.95–1.4. This indicates that small fluctuations in the NaCl concentration in phosphate buffer II (mobile phase) (0.25–0.35 mol / L) have little effect on the purity detection of Bordetella pertussis adhesin protein. Therefore, phosphate buffer II (mobile phase) with NaCl concentrations of 0.25–0.35 mol / L can be used for the SEC-HPLC method to determine the purity of Bordetella pertussis adhesin protein.
[0060] Example 17 This example investigated the effect of the pH value of buffer I on the detection results when determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC. The detection method differs from Example 5 in that the pH values of buffer I (diluent) in step S1 were 4.0, 4.5, 5.0, and 5.5, respectively. The detection results are as follows: Figure 9 As shown. According to Figure 9 The RSD of purity, retention time and peak area were calculated from the test results, and the results are shown in Table 8.
[0061] Table 8. Detection results of PRN after dilution with diluents of different pH values.
[0062] The results showed that when Bordetella pertussis adhesin protein was diluted with buffer I (diluent) at different pH values, the RSD for purity was 0.35%, the RSD for retention time was 1.97%, and the RSD for peak area was 1.88%. The RSDs for purity, retention time, and peak area were all <2.0%, and the resolution was greater than 1.5. The tailing factor was within the range of 0.95–1.4, indicating that small fluctuations in the pH of buffer I (diluent) (4.0–5.5) had little effect on the purity determination of Bordetella pertussis adhesin protein. Therefore, buffer I (diluent) with a pH range of 4.0–5.5 can be used for the SEC-HPLC method to determine the purity of Bordetella pertussis adhesin protein.
[0063] Example 18 This example investigated the effect of sodium acetate concentration in buffer I on the detection results when determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC. The detection method differs from Example 5 in that the sodium acetate concentration in buffer I (diluent) in step S1 was 0.005 mol / L, 0.01 mol / L, and 0.015 mol / L, respectively. The detection results are shown below. Figure 10 .according to Figure 10 The RSD of purity, retention time and peak area were calculated from the test results, and the results are shown in Table 9.
[0064] Table 9. Detection results of PRN after dilution with sodium acetate solutions of different concentrations.
[0065] The results showed that when Bordetella pertussis adhesin protein was diluted with buffer I (diluent) containing different concentrations of sodium acetate for detection, the RSD for purity was 0.71%, the RSD for retention time was 1.71%, and the RSD for peak area was 1.99%. The RSDs for purity, retention time, and peak area were all <2.0%, the resolution was greater than 1.5, and the tailing factor was in the range of 0.95–1.4. This indicates that small fluctuations in the sodium acetate concentration in buffer I (diluent) (0.005–0.015 mol / L) have little impact on the purity detection of PRN. Therefore, a sodium acetate concentration in buffer I (diluent) ranging from 0.005 to 0.015 mol / L can be used for the SEC-HPLC method to determine the purity of Bordetella pertussis adhesin protein.
[0066] Example 19 This example investigated the effect of NaCl concentration in buffer I on the detection results when determining the purity of Bordetella pertussis adhesin protein using SEC-HPLC. The detection method was basically the same as in Example 5, except that the NaCl concentration in buffer I (diluent) in step S1 was 0.15 mol / L, 0.2 mol / L, and 0.25 mol / L, respectively. The detection results are as follows: Figure 11 As shown. According to Figure 11 The RSD of purity, retention time and peak area were calculated from the test results, and the results are shown in Table 10.
[0067] Table 10 Detection results of PRN after dilution with diluents containing different concentrations of NaCl
[0068] The results showed that when Bordetella pertussis adhesin protein was diluted with buffer I (diluent) containing different concentrations of NaCl for detection, the RSD for purity was 0.29%, the RSD for retention time was 1.71%, and the RSD for peak area was 1.91%. The RSDs for purity, retention time, and peak area were all <2.0%, the resolution was greater than 1.5, and the tailing factor was in the range of 0.95–1.4. This indicates that small fluctuations in the NaCl concentration in buffer I (diluent) (NaCl concentration range of 0.15–0.25 mol / L) have little impact on the purity detection of PRN. Therefore, a NaCl concentration range of 0.15–0.25 mol / L in buffer I (diluent) can be used for the determination of Bordetella pertussis adhesin protein purity by SEC-HPLC.
[0069] Comparative Example 1 In this comparative example, the purity of Bordetella pertussis adhesin protein was determined by SEC-HPLC. The determination method was basically the same as that in Example 5, except that the chromatographic column in Example 5 was replaced with a Zenix SEC300.
[0070] Test results as follows Figure 12 As shown, the Zenix SEC300 column could not separate the impurity peaks from the target protein, therefore the Zenix SEC300 cannot be selected as the column for determining the purity of Bordetella pertussis adhesin protein by SEC-HPLC.
[0071] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of this application and the content of the specification should be included within the scope of protection of this application.
Claims
1. A method for determining the purity of Bordetella pertussis adhesin protein by SEC-HPLC, characterized in that, Includes the following steps: After diluting the Bordetella pertussis adhesin protein into a sample solution, the purity of the Bordetella pertussis adhesin protein was determined by SEC-HPLC. The mobile phase used in SEC-HPLC was phosphate buffer II containing sodium chloride with a pH of 6.5-7.5, and the elution method was isocratic elution. The flow rate of the mobile phase was 0.3-1 mL / min. The column temperature of the size exclusion column was 20℃-30℃. The size exclusion column was a TSKgel G2000 SWXL.
2. The method according to claim 1, characterized in that, The diluent for the Bordetella pertussis adhesin protein is buffer I with a pH of 4-5.5; buffer I contains 0.005-0.015 mol / L sodium acetate and 0.15-0.25 mol / L NaCl.
3. The method according to claim 1, characterized in that, The concentration of the Bordetella pertussis adhesin protein in the sample solution is 100~300 μg / mL.
4. The method according to claim 3, characterized in that, The concentration of the Bordetella pertussis adhesin protein in the sample solution was 300 μg / mL.
5. The method according to claim 1, characterized in that, The injection volume of the sample solution is 50~200μL.
6. The method according to claim 5, characterized in that, The injection volume of the sample solution was 50 μL.
7. The method according to claim 1, characterized in that, The phosphate buffer II contains 0.04-0.06 mol / L of phosphate and 0.25-0.35 mol / L of NaCl.
8. The method according to claim 1, characterized in that, The detector used in SEC-HPLC is a differential refractive index detector.
9. The method according to claim 8, characterized in that, The detection wavelength is 280nm.
10. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.3~0.65 mL / min.