Method for detecting purity of hepatitis A virus based on IEX-HPLC

Abstract

The invention discloses a method for detecting the purity of hepatitis A virus based on IEX-HPLC, and the method comprises the following steps: filtering a hepatitis A virus purified liquid sample through a filter membrane; a strong anion exchange chromatographic column is adopted, a mixed solution of a mobile phase A and a mobile phase B is used as a mobile phase, the pH range of the mobile phase is 7.5-8.5, NaCl is adopted for gradient elution, the flow velocity is 0.5-1.5 mL / min, the column temperature is 25-40 DEG C, and the detection wavelength is 260-280 mm; and identifying the target peak by adopting an SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) method and a Western-Blot method, identifying the target peak of the hepatitis A virus, and calculating the purity of the hepatitis A virus. According to the method, sample treatment, target chromatographic peak qualification and detection condition optimization are combined, an efficient hepatitis A virus antigen purity analysis method is established, and the key technical problem of hepatitis A virus purity analysis in actual production is solved.

Description

Technical Field

[0001] The present invention relates to the field of virus detection, and in particular to a method for detecting the purity of hepatitis A virus based on IEX-HPLC. Background Art

[0002] Hepatitis A virus (HAV) is an important foodborne enterovirus that can cause severe hepatitis in humans. The virus belongs to the genus Hepatovirus of the family Picornaviridae. The virus particles are non-enveloped and have an icosahedral symmetric structure with a diameter of 27-32nm. It contains a positive-strand RNA genome with a length of about 7.5kb. HAV is mainly transmitted through fecal-oral transmission. Direct contact between people or consumption of contaminated food and water are considered to be the main ways of HAV transmission. Worldwide, approximately 1 million people are infected with HAV each year, but the source of infection in 50% of these cases is uncertain. Hepatitis A poses a serious threat to people's lives and health.

[0003] Enteroviruses such as hepatitis A virus have relatively stable physical and chemical properties and can survive for a long time in the environment and food, but they cannot replicate. The virus content in contaminated environments and foods is relatively low (10-100 infectious particles), and it is spread non-randomly. Therefore, it is often difficult to identify the source of the virus in hepatitis A outbreaks. However, once ingested by the human body, even low doses of the virus can cause human disease and seriously endanger human health. Therefore, it is crucial to develop a highly sensitive method for detecting hepatitis A virus. The classic cell culture method has a long detection cycle and low sensitivity, and is not suitable for the detection of hepatitis A virus in the environment and food.

[0004] High performance liquid chromatography (HPLC) is an analytical method with simple operation, high efficiency and high sensitivity, and fast analysis speed. The detection of virus particles directly reflects the true state of the sample. And the detection process is visualized, that is, the chromatographic absorption peak of the virus particles is intuitively presented on the chromatogram, and the equipped ultraviolet detector can intuitively reflect the virus content of different inactivated antigens through the size of the integral area of ​​the chromatographic peak. The sample does not need special treatment, the detection time is short, and the results can be obtained within half an hour. The operation is simple, it belongs to the instrumental analysis method, the human operation error is small, the relative standard deviation of repeated injections is generally less than 2%, and the repeatability is very good. It is very suitable for intermediate quality control and finished product testing. Therefore, the use of HPLC detection technology for hepatitis A virus purity analysis is conducive to the quality control of hepatitis A virus antigen production, and thus plays a role in improving the quality of hepatitis A virus vaccine products. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for detecting the purity of hepatitis A virus which has the advantages of simple operation, high sensitivity and good repeatability.

[0006] In order to solve the above technical problems, the technical solution of the present invention is as follows: a method for detecting the purity of hepatitis A virus based on IEX-HPLC, the method is operated as follows:

[0007] (1) Preparation of test solution

[0008] Use the purified hepatitis A virus sample and filter it through a filter membrane (generally 0.22um);

[0009] (2) Chromatographic conditions

[0010] The chromatographic column is a strong anion exchange column

[0011] Mobile phase A: Tris-HCl solution;

[0012] Mobile phase B: Tris-HCl+NaCl solution;

[0013] The mobile phase is a mixture of mobile phases A and B, the mobile phase pH range is 7.5-8.5, NaCl gradient elution is used, the flow rate is 0.5-1.5 mL / min, the column temperature is 25-40°C, and the detection wavelength is 260 nm.

[0014] (3) Qualitative identification of chromatographic peaks

[0015] The SDS-PAGE method and / or Western-Blot method are used to identify multiple target peaks, recognize the hepatitis A virus target peak, and calculate the hepatitis A virus purity.

[0016] As a further description of the above scheme: the specifications of the strong anion chromatographic column are: the column length is between 50-250 mm, the inner diameter of the chromatographic column is between 1-10 mm, and the particle size is between 1-10 um.

[0017] As a further preference for the above scheme: the strong anion chromatography column is a quaternary ammonium chromatography column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 um.

[0018] As a further description of the above scheme: the concentration of the mobile phase A Tris-HCl solution is 0.01-0.05 mol / L, the concentration of Tris-HCl in the mobile phase B Tris-HCl+NaCl solution is 0.01-0.05 mol / L, and the concentration of the NaCl solution is 0.5-1.5 mol / L.

[0019] As a further preferred embodiment of the above scheme: the mobile phase A Tris-HCl solution is 0.02 mol / L, the mobile phase B Tris-HCl+NaCl solution has a Tris-HCl concentration of 0.02 mol / L, and the concentration of the NaCl solution is 1 mol / L.

[0020] As a further preference for the above scheme: in the step (2), the anion chromatographic column is a quaternary ammonium chromatographic column, and the mobile phase is a mixed solution of phase A Tris-HCl solution and phase B Tris-HCl+NaCl solution, phase A is 0.02mol / L Tris-HCl solution, and phase B is 0.02mol / L Tris-HCl+1mol / L NaCl.

[0021] As a further description of the above scheme: the hepatitis A virus sample is a purified hepatitis A virus solution, which comes from the research and development or production of purified hepatitis A virus intermediates.

[0022] The characteristics of the present invention are as follows: According to the structural characteristics of hepatitis A virus, the present invention designs a high-performance liquid chromatography detection method for separation by anion exchange column. The hepatitis A virus is negatively charged and attached to the anion exchange column in a mobile phase of pH 7.5-8.5. After elution with mobile phases of different gradients, it can be well separated from impurities. The target peak is qualitatively identified by SDS-PAGE and Western-Blot methods. The retention time of the hepatitis A virus antigen on the chromatogram is 12 minutes. The present invention also optimizes the chromatographic conditions, so that the detection accuracy is significantly improved. The present invention achieves superior technical effects with innovative technical improvements, and at the same time has high sensitivity and is easy to operate.

[0023] Compared with the prior art, the present invention has the following beneficial effects: the present invention combines sample processing, target chromatographic peak qualitative analysis and detection condition optimization to establish an efficient purity analysis method for hepatitis A virus antigen. According to the purity analysis method for hepatitis A virus provided by the present invention, since the IEX-HPLC method is used to detect the virus, the virus and impurities can be effectively separated to achieve the purpose of detecting the virus. It is more suitable for product detection and quality control in the actual production process. Through the purity analysis of hepatitis A virus, the production of vaccines can be effectively guided, which has great guiding significance for improving the quality of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the spectrum of the purified hepatitis A virus solution of batch 042-UF2-20230614-10-23;

[0025] Figure 2 It is the SDS-PAGE silver staining picture;

[0026] Figure 3is the Western-Blot image;

[0027] Figure 4 This is the spectrum of batch 042-20231009 of hepatitis A virus purified liquid. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0029] The Tris-HCl described in the following examples refers to a buffer solution of tris(hydroxymethyl)aminomethane and hydrochloric acid, Tris-HCl+NaCl refers to a mixed solution of Tris-HCl and sodium chloride, and IEX-HPLC refers to ion exchange high performance liquid chromatography.

[0030] The hepatitis A virus described in the present invention is a hepatitis A virus intermediate or purified liquid that is developed or produced and purified and extracted.

[0031] Example 1 Qualitative identification of hepatitis A chromatographic peaks

[0032] (1) Instruments and chromatographic conditions

[0033] High performance liquid chromatography: ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0034] Chromatographic column model: Thermo Fisher Scientific ProPac SAX-10HT (4*50mm).

[0035] Prepare 0.02mol / L Tris-HCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02mol / LTris-HCl and 1mol / L NaCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase B. The flow rate is 1mL / min, and the column temperature is 30℃.

[0036] Detection wavelength: 260nm.

[0037] Chromatographic conditions: see Table 1.

[0038] Table 1 Mobile phase gradient

[0039]

[0040] (2) Experimental procedures

[0041] Preparation of test solution

[0042] The homemade hepatitis A virus purified solution (batch number: 042-UF2-20230614-10-23) was concentrated 10 times using a 50KDa 15mL ultrafiltration tube and filtered through a 0.22um filter membrane.

[0043] Take 100uL of the above sample, inject it into the liquid chromatograph, and record the chromatogram. From the chromatogram, it can be seen that the hepatitis A virus and impurities can be separated and an effective separation degree can be achieved. The peak time and peak area percentage of the hepatitis A virus and impurities are as follows: Figure 1 And as shown in Table 2.

[0044] The sample was injected repeatedly 20 times according to the detection method of Example 1, and the chromatographic peaks at RT = 9.0 min, RT = 10.08 min, and RT = 12.02 min were collected. The collected chromatographic peak solutions were concentrated using a 30 KDa 15 mL ultrafiltration tube under the conditions of 4000 rpm, 2-8 ° C, 10 min, and 1 mL of concentrated solution was obtained.

[0045] The above samples were subjected to SDS-PAGE silver staining and Western-Blot.

[0046] SDS-PAGE silver staining

[0047] 1. Sample Preparation

[0048] Take 40uL of hepatitis A virus purified solution (batch number: 042-UF2-20230614-10-23), ten-fold concentrated sample (042-UF2-20230614-10-23C10), RT=9.0min, RT=10.08min, RT=12.02min, RT=9.0min concentrated, RT=10.08min concentrated, and RT=12.02min concentrated samples, add 10uL of 5*leading buffer, mix well, and heat at 98°C in a metal bath for 10min to obtain SDS-PAGE loading samples.

[0049] 2.SDS-PAGE

[0050] Load the above samples into the prepared electrophoresis gel holes respectively, voltage: 120V, and end the electrophoresis when bromofen blue runs to the bottom of the electrophoresis gel.

[0051] 3. Silver staining

[0052] 3.1 Fixation: Take out the electrophoresis gel and add fixative (the fixative is: 50% methanol, 10% acetic acid, add 50uL 37% formaldehyde per 100mL) for at least 1h.

[0053] 3.2 Washing: Add 50% ethanol and wash three times, 20 minutes each time.

[0054] 3.3 Pre-dyeing: rinse with 0.2g / L Na2S2O3.5H2O for 1 min.

[0055] 3.4 Water washing: Wash three times with distilled water, 20 seconds each time.

[0056] 3.5 Staining: Add silver staining solution (silver staining solution is: 2g / L AgNO3, add 75uL 37% formaldehyde for every 100mL solution) and stain for 30min.

[0057] 3.6 Water washing: Wash three times with distilled water, 20 seconds each time.

[0058] 3.7 Color development: Add color developing solution for color development. (The color developing solution is: add Na2S2O3.5H2O to 60g / L Na2CO3 solution to a final concentration of 4mg / L, and add 50uL 37% formaldehyde per 100mL solution)

[0059] 3.8 After color development, wash with water first, then add stop solution (stop solution is: 50% methanol, 10% glacial acetic acid), the result is shown in Figure 2 .

[0060] Western-Blot

[0061] 1. Sample Preparation

[0062] Take 40uL of hepatitis A virus purified solution (batch number: 042-UF2-20230614-10-23), ten-fold concentrated sample (042-UF2-20230614-10-23C10), RT=9.0min, RT=10.08min, RT=12.02min, RT=9.0min concentrated, RT=10.08min concentrated, and RT=12.02min concentrated samples, add 10uL of 5*leading buffer, mix well, and heat at 98°C in a metal bath for 10min to obtain SDS-PAGE loading samples.

[0063] 2.SDS-PAGE

[0064] Load the above samples into the prepared electrophoresis gel holes respectively, voltage: 120V, and end the electrophoresis when bromofen blue runs to the bottom of the electrophoresis gel.

[0065] 3. Transfer

[0066] The electrophoresis gel was removed and the protein was transferred to a PVDF membrane.

[0067] 4. Closure

[0068] 5% skimmed milk powder was used as the blocking solution. The PVDF membrane was placed in the blocking solution and blocked at room temperature for 2 h. The blocking solution was then discarded and the membrane was washed 3 times with TBST, each time for 10 min.

[0069] 5. Primary Antibody Incubation

[0070] The antibody (mouse anti-HAV) was dissolved in 5% skim milk powder at a dilution ratio of 1:1000, incubated at room temperature for 2 h, the primary antibody was recovered, and the membrane was washed 3 times with TBST, each time for 10 min.

[0071] 6. Secondary Antibody Incubation

[0072] The antibody (goat anti-mouse) was dissolved in 5% skim milk powder at a dilution ratio of 1:1000, incubated at room temperature for 2 h, and the membrane was washed three times with TBST, each time for 10 min.

[0073] 7. Development

[0074] Use ECL color developing solution for color development and use developer to take pictures. Figure 3 .

[0075] From the results of SDS-PAGE silver staining and Western-Blot, it was determined that the chromatographic peak of hepatitis A virus in IEX-HPLC was the chromatographic peak at RT=12.02min; the purity of hepatitis A was 54.93% obtained by peak area normalization method.

[0076] Figure 4 This is the spectrum of another batch of hepatitis A virus purified liquid, batch 042-20231009, with a purity of 99.37%.

[0077] Example 2

[0078] (1) Instruments and chromatographic conditions

[0079] High performance liquid chromatography: ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0080] Chromatographic column model: Thermo Fisher Scientific ProPac SAX-10HT (4*50mm).

[0081] Prepare 0.02mol / L Tris-HCl solution, adjust the pH to 7.5 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02mol / LTris-HCl and 1mol / L NaCl solution, adjust the pH to 7.5 with concentrated hydrochloric acid as mobile phase B. The flow rate is 1mL / min, and the column temperature is 30℃.

[0082] Detection wavelength: 260nm.

[0083] Chromatographic conditions: see Table 2.

[0084] Table 2 Mobile phase gradient

[0085]

[0086] (2) Experimental procedures

[0087] Preparation of test solution

[0088] A homemade purified hepatitis A virus sample was used and filtered through a 0.22 um filter membrane.

[0089] Take 100uL of the above sample, inject it into the liquid chromatograph, and record the chromatogram.

[0090] Example 3

[0091] (1) Instruments and chromatographic conditions

[0092] High performance liquid chromatography: ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0093] Chromatographic column model: Thermo Fisher Scientific ProPac SAX-10HT (4*50mm).

[0094] Prepare 0.02mol / L Tris-HCl solution, adjust the pH to 8.5 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02mol / LTris-HCl and 1mol / L NaCl solution, adjust the pH to 8.5 with concentrated hydrochloric acid as mobile phase B. The flow rate is 1.0mL / min, and the column temperature is 30℃.

[0095] Detection wavelength: 260nm.

[0096] Chromatographic conditions: see Table 3.

[0097] Table 3 Mobile phase gradient

[0098]

[0099] (2) Experimental procedures

[0100] Preparation of test solution

[0101] A homemade purified hepatitis A virus sample was used and filtered through a 0.22 um filter membrane.

[0102] Take 100uL of the above sample, inject it into the liquid chromatograph, and record the chromatogram.

[0103] Example 4

[0104] (1) Instruments and chromatographic conditions

[0105] High performance liquid chromatography: ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0106] Chromatographic column model: Thermo Fisher Scientific ProPac SAX-10HT (4*50mm).

[0107] Prepare 0.02mol / L Tris-HCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02mol / LTris-HCl and 1mol / L NaCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase B. The flow rate is 1.0mL / min, and the column temperature is 25℃.

[0108] Detection wavelength: 260nm;

[0109] Chromatographic conditions: see Table 4.

[0110] Table 4 Mobile phase gradient

[0111]

[0112] (2) Experimental procedures

[0113] Preparation of test solution

[0114] A homemade purified hepatitis A virus sample was used and filtered through a 0.22 um filter membrane.

[0115] Take 100uL of the above sample, inject it into the liquid chromatograph, and record the chromatogram.

[0116] Example 5

[0117] (1) Instruments and chromatographic conditions

[0118] High performance liquid chromatography: ACCHROM S6000 high performance liquid chromatography system and chemical workstation.

[0119] Chromatographic column model: Thermo Fisher Scientific ProPac SAX-10HT (4*50mm).

[0120] Prepare 0.02mol / L Tris-HCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase A. Prepare 0.02mol / LTris-HCl and 1mol / L NaCl solution, adjust the pH to 8.0 with concentrated hydrochloric acid as mobile phase B. The flow rate is 1.0mL / min, and the column temperature is 35℃.

[0121] Detection wavelength: 260nm.

[0122] Chromatographic conditions: see Table 5.

[0123] Table 5 Mobile phase gradient

[0124]

[0125] (2) Experimental procedures

[0126] Preparation of test solution

[0127] A homemade purified hepatitis A virus sample was used and filtered through a 0.22 um filter membrane.

[0128] Take 100uL of the above sample, inject it into the liquid chromatograph, and record the chromatogram.

[0129] According to the chromatographic conditions provided in Example 1-5, the hepatitis A virus was tested by injection to obtain a chromatogram.

[0130] The differences of hepatitis A virus under different chromatographic conditions are shown in Table 6.

[0131] Table 6 Differences in hepatitis A virus under different chromatographic conditions

[0132]

[0133]

[0134] It can be seen from the above table that in the above 5 examples, the hepatitis A virus was not interfered by other substances under different chromatographic conditions, which proves that the method has good specificity.

[0135] Example 6 Hepatitis A virus solution stability test

[0136] Samples of the purified hepatitis A virus solution were injected at 0 h, 3 h, 5 h, 12 h, and 24 h, the chromatograms were recorded, and the relative standard deviation of the hepatitis A virus peak area was calculated. The results are shown in Table 7.

[0137] Table 7 Hepatitis A virus solution stability test results

[0138] Time (h) Retention time (min) Peak area 0h 12.12 32773 3h 12.09 32985 5h 12.09 32790 12h 12.07 33887 24h 12.07 32231 average value 12.09 32933.2 RSD% 0.17％ 1.83％

[0139] As can be seen from the above table, when the hepatitis A virus purified solution was placed at 2-8°C for 24 hours, the peak area RSD% of the hepatitis A virus purified solution was less than 2%, proving that the solution had good stability.

[0140] Example 7 Detection accuracy and precision

[0141] The IEX-HPLC method for detecting the purity of hepatitis A virus provided in Example 1 was used to repeat the injection of the same batch of hepatitis A virus purified liquid for 6 times to verify the good precision of the method. The results are shown in Table 8.

[0142] Table 8 Repeatability test results

[0143]

[0144]

[0145] As can be seen from the above table, the RSD% of the hepatitis A virus peak area detected by 6 injections is less than 2%, proving that the method has good precision.

[0146] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting the purity of hepatitis A virus based on IEX-HPLC, characterized in that: The method operates as follows: (1) Preparation of test solution The purified sample of hepatitis A virus was filtered through a membrane; (2) Chromatographic conditions The chromatographic column is a strong anion exchange column Mobile phase A: Tris-HCl solution; Mobile phase B: Tris-HCl+NaCl solution; The mobile phase was a mixture of mobile phase A and B, the mobile phase pH range was 7.5-8.5, NaCl gradient elution was used, the flow rate was 0.5-1.5 mL / min, the column temperature was 25-40 ° C, and the detection wavelength was 260 nm; (3) Qualitative identification of chromatographic peaks The SDS-PAGE method and / or Western-Blot method are used to identify multiple target peaks, recognize the hepatitis A virus target peak, and calculate the hepatitis A virus purity.

2. The method for detecting the purity of hepatitis A virus based on IEX-HPLC according to claim 1, characterized in that: The specifications of the strong anion chromatographic column are as follows: the column length is between 50-250 mm, the inner diameter of the chromatographic column is between 1-10 mm, and the particle size is between 1-10 um.

3. The method for detecting the purity of hepatitis A virus based on IEX-HPLC according to claim 2, characterized in that: The strong anion chromatographic column is a quaternary ammonium chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 um.

4. The method for detecting the purity of hepatitis A virus based on IEX-HPLC according to claim 1, characterized in that: The concentration of the mobile phase A Tris-HCl solution is 0.01-0.05 mol / L, the concentration of Tris-HCl in the mobile phase B Tris-HCl+NaCl solution is 0.01-0.05 mol / L, and the concentration of the NaCl solution is 0.5-1.5 mol / L.

5. The method for detecting the purity of hepatitis A virus based on IEX-HPLC according to claim 4, characterized in that: The mobile phase A Tris-HCl solution has a concentration of 0.02 mol / L, the mobile phase B Tris-HCl+NaCl solution has a concentration of 0.02 mol / L, and the concentration of the NaCl solution is 1 mol / L.

6. The method for detecting the purity of hepatitis A virus based on IEX-HPLC according to claim 1, characterized in that: In the step (2), the anion chromatography column is a quaternary ammonium chromatography column, and the mobile phase is a mixed solution of phase A Tris-HCl solution and phase B Tris-HCl+NaCl solution, wherein phase A is 0.02 mol / L Tris-HCl solution, and phase B is 0.02 mol / L Tris-HCl and 1 mol / L NaCl.

7. The method for detecting the purity of hepatitis A virus based on IEX-HPLC according to claim 1, characterized in that: The hepatitis A virus sample is a purified hepatitis A virus liquid, which is derived from the research and development or production of purified hepatitis A virus intermediates.

Images