A cell-derived influenza virus split vaccine and a method for detecting recombinant trypsin content in the production process thereof

Abstract

The application relates to the field of vaccine detection, and specifically discloses a cell influenza virus split vaccine and a detection method for the content of recombinant trypsin in the production process of the cell influenza virus split vaccine. The method comprises the following steps: taking the influenza virus split vaccine or an intermediate product in the production process of the influenza virus split vaccine for pretreatment to obtain a test sample solution; taking the recombinant trypsin as a control sample to prepare a control sample solution; performing HPLC analysis on the control sample solution and the test sample solution; and calculating the content of the recombinant trypsin in the test sample solution by using an external standard method. The detection method has strong specificity and small interference; the detection limit is 0.5 ug / ml, the method has high sensitivity; and the linear relationship is good within the concentration range of 10-45 ug / ml, which is helpful for rapidly detecting the content of the recombinant trypsin in the influenza virus split vaccine and a culture system in the production process of the vaccine, so as to further improve the production efficiency and biological safety of the influenza virus split vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccine detection, more particularly, it relates to a cell-based influenza virus split vaccine and a method for detecting the content of recombinant trypsin in the production process thereof. BACKGROUND

[0002] The glycoprotein hemagglutinin (HA) on the surface of influenza virus plays an important role in the process of influenza virus infection. The HA is initially synthesized in the form of a precursor, which is cleaved by a host protease to form HA1 and HA2 when the virus infects a host cell, thereby acquiring membrane fusion ability and mediating the infection of the host cell by the influenza virus.

[0003] During the culture of influenza virus using MDCK cells as the substrate, the MDCK cells themselves contain less protease, so exogenous trypsin is added to mediate the infection of the host cell by the influenza virus and increase the proliferation effect of the influenza virus.

[0004] Recombinant trypsin can be used as an exogenous protease to mediate the infection of the host cell by the influenza virus. When the virus is proliferated in the host cell culture system, an appropriate amount of recombinant trypsin is added at regular intervals. However, the recombinant trypsin may also excessively hydrolyze the proteins on the surface of the cell membrane, affecting the activity of the host cell. Therefore, the amount of recombinant trypsin in the culture system needs to be continuously monitored during the virus proliferation process, for the optimization of the upstream virus culture process. Meanwhile, as a biologically active substance, it is also necessary to monitor the removal of the recombinant trypsin in the downstream purification process, to reduce the quality risk of the final product. However, at present, there is only a method for detecting the purity of recombinant trypsin as a specific biological raw material / animal and auxiliary material. This method is only suitable for the purity detection of recombinant trypsin as a raw material for the production of biological products, requires a high sample concentration, and has a high detection limit. Therefore, it is not suitable for the detection of the content (trace residues) of recombinant trypsin in the cell-based influenza virus split vaccine and the production process thereof. Therefore, at present, there is a lack of effective detection methods for the residual amount of recombinant trypsin in the influenza virus split vaccine and the production process thereof. SUMMARY

[0005] In order to effectively monitor the content of recombinant trypsin in the culture system during the production process of the influenza virus split vaccine and detect the residual amount of recombinant trypsin in the influenza virus split vaccine, the present application provides a method for detecting the content of recombinant trypsin in a cell-based influenza virus split vaccine and the production process thereof.

[0006] The present application adopts the following technical solution:

[0007] A method for detecting the content of recombinant trypsin in an influenza virus split vaccine and the production process thereof, comprising:

[0008] Taking the influenza virus split vaccine or intermediate product in its production process as a pretreatment, a test sample solution is obtained;

[0009] Taking the recombinant trypsin as a control sample, a control sample solution is prepared;

[0010] The control sample solution and the test sample solution are analyzed by HPLC, and the content of the recombinant trypsin in the test sample solution is calculated by an external standard method;

[0011] The chromatographic conditions of the HPLC analysis are as follows:

[0012] The chromatographic column is a C18 silica gel column;

[0013] The mobile phase A is a 0.05-0.15 vol% phosphoric acid-water solution;

[0014] The mobile phase B is a 0.05-0.15 vol% phosphoric acid-acetonitrile solution;

[0015] The detection wavelength is 275-285 nm;

[0016] The column temperature is 37-42°C;

[0017] The flow rate is 0.3-1.0 mL / min;

[0018] The injection volume is 8-20 μL.

[0019] Further, the gradient elution program of the HPLC is as follows:

[0020] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70～80 20～30 18～22 56～66 34～44 23～27 8～12 88～92 28～32 8～12 88～92 29～33 70～80 20～30 39～43 70～80 20～30

[0021] Preferably, the gradient elution program of the HPLC is as follows:

[0022] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 20 61 39 25 10 90 30 10 90 31 75 25 41 75 25

[0023] Further, the packing agent of the chromatographic column is octadecylsilane group bonded silica gel, the column length is 250-460 mm, the column inner diameter is 4-5 mm, and the packing particle size is 3-4 μm.

[0024] Further, the method for preparing the test sample solution comprises:

[0025] Taking the influenza virus split vaccine or intermediate product in its production process, the pH is adjusted to 4-5, and the solution is filtered by a 0.2-0.3 μm filter membrane.

[0026] Further, the method for preparing the control sample solution comprises:

[0027] Take the recombinant trypsin control and dilute it with cell culture medium with a pH of 4-6 to prepare a solution of recombinant trypsin with a concentration of 20-50 μg / mL. Filter the solution through a 0.2-0.3 μm filter membrane.

[0028] Furthermore, the process of calculating the recombinant trypsin content in the test solution using the external standard method requires the preparation of a standard curve. Methods for preparing a standard curve include:

[0029] The reference solution was diluted to prepare a series of standard solutions of different concentrations. HPLC analysis was performed under the same chromatographic conditions. Linear regression was performed with the peak area of ​​the obtained chromatographic peak as the ordinate and the concentration of recombinant trypsin in the standard solution as the abscissa to obtain the standard curve.

[0030] Furthermore, the detection method also includes system suitability verification of HPLC analysis using a reference solution.

[0031] Preferably, in the system suitability verification of HPLC analysis, when the peak area RSD of recombinant trypsin in the obtained chromatogram is less than 10% after 5 to 8 repeated injections of the reference solution according to the chromatographic conditions, the system suitability is determined to meet the detection requirements.

[0032] Furthermore, the influenza virus split vaccine was prepared by culturing MDCK cells as a substrate.

[0033] In summary, this application has the following beneficial effects:

[0034] The detection method provided in this application involves pretreatment of influenza virus split vaccine or intermediate products in its production process to obtain a test solution. Recombinant trypsin is used as a reference standard, and a C18 silica gel column is used for separation. By optimizing chromatographic parameters and elution procedures, the test solution and reference solution are analyzed by HPLC. Finally, the content of recombinant trypsin is calculated using the external standard method. This detection method exhibits high specificity and low interference; the detection limit is 0.5 μg / ml, and the method has high sensitivity. Furthermore, it demonstrates good linearity within the concentration range of 10–45 μg / ml, facilitating rapid detection of recombinant trypsin content in influenza virus split vaccines and the culture system during vaccine production, thereby further improving the production efficiency and biosafety of influenza virus split vaccines. Attached Figure Description

[0035] Figure 1 This is a liquid chromatogram of the reference standard used in the system suitability test provided in the embodiments of this application;

[0036] Figure 2 This is a liquid chromatogram of the blank solution used in the system suitability test provided in this application embodiment;

[0037] Figure 3 This is a liquid chromatogram of the test solution 1 (i.e., the intermediate product virus harvest liquid in the production process of influenza virus split vaccine) provided in the embodiments of this application;

[0038] Figure 4 This is a liquid chromatogram of the test solution 2 (i.e., influenza virus split vaccine) provided in the embodiments of this application. Detailed Implementation

[0039] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] In the production of influenza virus vaccines, MDCK cells are often used as the culture medium. Since MDCK cells lack certain proteases, exogenous recombinant trypsin needs to be added during the culture process to mediate influenza virus infection of host cells. However, the amount of recombinant trypsin in the culture system must be controlled within an appropriate range; otherwise, it will affect the activity of host cells. Therefore, the amount of recombinant trypsin in the culture system needs to be continuously monitored during virus proliferation. Furthermore, recombinant trypsin needs to be removed during the final production of the influenza virus split vaccine. If the residual amount of recombinant trypsin in the final influenza virus split vaccine is high, it will negatively affect the stability, safety, and bioavailability of the vaccine. Therefore, it is also necessary to accurately determine the residual amount of recombinant trypsin in the influenza virus split vaccine.

[0041] In view of this, this embodiment provides an influenza virus split vaccine and a method for detecting the content of recombinant trypsin during the production process. The main quality control target of this method is the cellular influenza virus split vaccine, and more importantly, the influenza virus split vaccine prepared by culturing MDCK cells as a substrate.

[0042] The detection method includes the following steps:

[0043] Step S1: Take the influenza virus split vaccine or its intermediate products in the production process and pre-treat them to obtain the test solution.

[0044] Because influenza virus split vaccines require MDCK cells as a substrate for virus culture during their production, the composition of influenza virus split vaccines and their intermediate products is complex, containing a large amount of cell debris and viral proteins. To improve the binding rate of recombinant proteases to the liquid chromatography column, sample pretreatment is necessary before HPLC analysis to remove cell debris and some biomolecules that may interfere with detection.

[0045] Furthermore, the pretreatment method for preparing the test solution includes: taking an influenza virus split vaccine or an intermediate product from its production process, adjusting the pH to 4-5, and filtering it through a 0.2-0.3 μm filter membrane. Adjusting the pH to 4-5 facilitates the binding of recombinant trypsin to the chromatographic column without causing degradation of the recombinant trypsin; selecting a filter membrane with a pore size of 0.2-0.3 μm helps remove insoluble substances and extends the lifespan of the liquid chromatograph and the chromatographic column.

[0046] Preferably, the pretreatment method is as follows: take the influenza virus split vaccine or the intermediate product in the production process, adjust the pH to 4-5 (preferably 4.4-4.8) with phosphoric acid, and filter it with a 0.2-0.3 μm filter membrane (preferably, a 0.22 μm microporous filter membrane is used for filtration).

[0047] Step S2: Prepare a control solution using recombinant trypsin as a control.

[0048] Further, the method for preparing the reference solution includes: taking recombinant trypsin reference standard, diluting it with cell culture medium with a pH of 4-6 to prepare a solution with a concentration of 20-50 μg / mL of recombinant trypsin, and filtering it through a 0.2-0.3 μm filter membrane. Preferably, it is diluted with cell culture medium with a pH of 4.4-4.8 to prepare a solution containing 30-40 μg per 1 ml, and filtered through a 0.22 μm filter membrane to obtain the solution.

[0049] Step S3: Perform HPLC analysis on the reference solution and the test solution, and calculate the content of recombinant trypsin in the test solution using the external standard method.

[0050] The chromatographic conditions for HPLC analysis are as follows:

[0051] Chromatographic column: C18 silica gel column; preferably, the column packing material is octadecylsilane-bonded silica gel, the column length is 250-460 mm, the column inner diameter is 4-5 mm, and the packing particle size is 3-4 μm; more preferably, the column specifications are: 3.6 μm, 4.6 mm × 250 mm. More specifically, a Phenomenx C18 column was selected.

[0052] Detection wavelength: 275-285nm, preferably 277-283nm, and more preferably 280nm.

[0053] Column temperature: 37-42℃, preferably 39-41℃, and more preferably 40℃.

[0054] Flow rate: 0.3-1.0 mL / min, preferably 0.5-1.0 mL / min, more preferably 0.8-1.0 mL / min, for example, 0.9 mL / min or 1.0 mL / min.

[0055] Injection volume: 8–20 μL, preferably 10–20 μL, for example, 15 μL or 20 μL.

[0056] Mobile phase A: 0.05-0.15 vol% phosphoric acid-water solution; preferably, the phosphoric acid content is 0.07-0.13 vol%, more preferably 0.08-0.12 vol%, for example, it can be 0.09 vol%, 0.10 vol%, or 0.11 vol%.

[0057] Mobile phase B: 0.05-0.15 vol% phosphoric acid-acetonitrile solution; preferably, the phosphoric acid content is 0.07-0.13 vol%, more preferably 0.08-0.12 vol%, for example, 0.09 vol%, 0.10 vol%, or 0.11 vol%.

[0058] In HPLC analysis, to achieve good separation of components in the sample, the following gradient elution procedure is used:

[0059] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70～80 20～30 18～22 56～66 34～44 23～27 8～12 88～92 28～32 8～12 88～92 29～33 70～80 20～30 39～43 70～80 20～30

[0060] More preferably, elution is performed according to the following gradient elution program: 0 min → 20 min → 25 min → 30 min → 31 min → 41 min, during which time periods, mobile phase A: 75% → 61% → 10% → 10% → 75% → 75%; mobile phase B: 25% → 39% → 90% → 90% → 25% → 25%.

[0061] Furthermore, the process of calculating the recombinant trypsin content in the test solution using the external standard method requires the preparation of a standard curve. The method for preparing the standard curve includes: diluting the reference solution into a series of standard solutions of different concentrations, performing HPLC analysis under the same chromatographic conditions, and using the peak area of ​​the obtained chromatographic peak as the ordinate and the concentration of recombinant trypsin in the standard solution as the abscissa to perform linear regression and obtain the standard curve.

[0062] Furthermore, the detection method also includes system suitability verification of HPLC analysis using a reference solution.

[0063] Preferably, in the system suitability verification of HPLC analysis, when the reference solution is repeatedly injected and analyzed 5 to 8 times (preferably 6 to 7 times) according to the chromatographic conditions, and the peak area RSD of recombinant trypsin in the obtained chromatogram is less than 10%, the system suitability is determined to meet the detection requirements.

[0064] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0065] Example

[0066] This embodiment provides a method for detecting the recombinant trypsin content in the virus harvest fluid during the production of influenza virus split vaccines, including the following steps:

[0067] 1. Instruments, reagents and test samples

[0068] Instrument: Shimadzu High Performance Liquid Chromatograph (LC-2010A)

[0069] Reagents: Phosphoric acid (Fisher Chemical, chromatographic grade, batch number: 158625); Acetonitrile (Fisher Chemical, chromatographic grade, batch number: 175161); Recombinant trypsin; MDCK cell influenza lysate vaccine virus harvest fluid;

[0070] 2. Solution preparation:

[0071] Mobile phase A: Accurately measure 1 ml of phosphoric acid, place it in a 1000 ml volumetric flask, dilute to volume with ultrapure water, shake well, and degas by sonication to obtain the mobile phase A solution.

[0072] Mobile phase B: Accurately measure 1 ml of phosphoric acid, place it in a 1000 ml volumetric flask, dilute to volume with acetonitrile, shake well, and degas by sonication to obtain the mobile phase B.

[0073] Cell culture medium (pH 4.6): Add an appropriate amount of phosphate to the cell culture medium to make its pH value 4.6.

[0074] 3. Sample pretreatment

[0075] (1) Take an appropriate amount of the virus harvested liquid from the influenza virus split vaccine produced using MDCK cells as a matrix, adjust the pH to 4.6 with phosphoric acid, and filter it through a 0.22 μm filter membrane to obtain test solution 1.

[0076] (2) Take an appropriate amount of the final product of the influenza virus split vaccine, adjust the pH to 4.6 with phosphoric acid, filter it with a 0.22μm filter membrane to obtain test solution 2.

[0077] 4. Pretreatment of reference standards

[0078] Take an appropriate amount of recombinant trypsin solution, dilute it with cell culture medium at pH 4.6 to prepare a solution with a final concentration of 30 μg / ml, filter it through a 0.22 μm filter membrane to obtain the control solution.

[0079] 5. Chromatographic conditions:

[0080] Chromatographic column: Phenomenx C18 column, 3.6 μm, 4.6 mm × 250 mm.

[0081] Detection wavelength: 280nm;

[0082] Column temperature 40℃;

[0083] Flow rate 1 ml / min;

[0084] Injection volume: 20 μl;

[0085] Mobile phase: 0.1% phosphoric acid aqueous solution was used as mobile phase A, and 0.1% acetonitrile phosphoric acid solution was used as mobile phase B; elution was performed according to a gradient elution method: 0 min → 20 min → 25 min → 30 min → 31 min → 41 min. During the above time period, mobile phase A: 75% → 61% → 10% → 10% → 75% → 75%; mobile phase B: 25% → 39% → 90% → 90% → 25% → 25%.

[0086] 6. Test results of the test sample

[0087] (1) System suitability and specificity test

[0088] The pretreated reference solution was injected six times under chromatographic conditions. Chromatograms were recorded, and the peak area RSD of the recombinant trypsin peak in the six chromatograms was calculated. The RSD was less than 10%, indicating that the impurities did not interfere with the determination. System suitability and specificity chromatograms are attached. Figures 1-2 The comparison shows that the retention time of β-recombinant trypsin is 13.937, while that of α-recombinant trypsin is 13.247.

[0089] (2) Linear range

[0090] Standard curves were prepared with concentrations of 10, 15, 20, 30, 36, and 45 μg / ml. Analysis was performed under chromatographic conditions. A linear regression was performed with the peak area of ​​each sample as the ordinate and the concentration of the added standard solution as the abscissa. The regression equation was Y = 2285.9X - 1060.9 (r = 0.999). 2 Recombinant trypsin showed good linearity in the concentration range of 10–45 μg / ml.

[0091] 7. Methodological Examination:

[0092] (1) Recovery rate test:

[0093] Accurately measure six portions of the pretreated test sample and add the same concentration (30 μg / ml) of reference solution to each. Simultaneously, take the reference solution, test sample solution 1, and these six test samples for determination. Calculate the recovery rate according to the law: Recovery rate % = (Measured amount - Sample amount) / Added amount × 100%. The acceptable recovery rate should be between 80% and 120%. The calculation results are shown in Table 1.

[0094] Table 1 Results of the recovery rate test

[0095]

[0096]

[0097] As shown in Table 1, the average recovery rate of the six spikings was 96.5%, which is in the range of 80-120%. This indicates that the method has low loss of recombinant trypsin during the detection process and the detection results are highly accurate.

[0098] (2) Precision test:

[0099] Six pretreated test samples were precisely measured and analyzed by HPLC. The RSD of recombinant trypsin residue in the test samples was calculated to be 4.5%, indicating good method repeatability.

[0100] Another technician performed the same procedure the following day and calculated the RSD of recombinant trypsin in the test sample to be 3.8%. This indicates that the intermediate precision of the method is good. The results of the precision test are shown in Table 2.

[0101] Table 2. Precision Test Results

[0102]

[0103] (3) Limit of quantitation and limit of detection

[0104] The reference solution was gradually diluted and injected under the above chromatographic conditions. The limit of quantification of recombinant trypsin was 10 μg / ml (S / N≥10), and the limit of detection was 0.5 μg / ml (S / N≥3). The detection sensitivity is high, and it can effectively detect trace amounts of recombinant trypsin residues in samples.

[0105] (4) Durability test

[0106] The effects of minute changes in system parameters on the determination of recombinant trypsin residues were investigated within the pH range of the reference solution (4.6±0.2) and column temperature (40±2)℃. The results are as follows:

[0107] Table 3. Precision Test Results

[0108]

[0109]

[0110] As shown in Table 3, the RSD of the test results is less than 10% within the range of pH (4.6±0.2) and column temperature (40±2)℃, which indicates that the detection method has good robustness.

[0111] 8. Test results of the test sample

[0112] Take test solution 1 and test solution 2 respectively, and perform HPLC analysis according to the above chromatographic conditions. The chromatogram of test solution 1 is shown below. Figure 3 As shown, the peak area is 34044. Substituting this into the standard curve, the recombinant trypsin content in the intermediate product virus harvest liquid was calculated to be 15.4 μg / ml. No target peak was found around the retention time of 13.5 min in the chromatogram of test solution 2, indicating that the residual amount of recombinant trypsin in the influenza virus split vaccine was below the detection limit of 0.5 μg / ml.

[0113] In summary, the HPLC detection method provided in this application is effective for detecting influenza virus split vaccines and intermediate products in their production process. The system exhibits good adaptability, and the recombinant trypsin shows good linearity within the concentration range of 10–45 μg / ml. It also demonstrates good recovery and precision, providing reliable and highly reproducible results. Furthermore, the detection limit is 0.5 μg / ml, indicating high sensitivity. The method is robust; minor changes in parameters such as pH and column temperature have minimal impact on the determination of recombinant trypsin residues. Therefore, this detection method facilitates rapid detection of recombinant trypsin content in influenza virus split vaccines and the culture system during vaccine production, thereby further improving the production efficiency and biosafety of influenza virus split vaccines.

[0114] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for detecting the content of recombinant trypsin in an influenza virus split vaccine and its production process, characterized in that, The quality control target of the detection method is the influenza virus split vaccine and its intermediate products prepared by culturing MDCK cells as a matrix. The method is used to detect the residual amount of recombinant trypsin in the quality control target. The limit of quantification of the detection method for recombinant trypsin is 10 µg / ml and the limit of detection is 0.5 μg / ml. It includes: Take the influenza virus split vaccine or its intermediate product in the production process for pretreatment, wherein the intermediate product is the virus harvest liquid, adjust the pH to 4-5, filter it with a 0.2-0.3 μm filter membrane to obtain the test solution; A control solution was prepared using recombinant trypsin as a reference standard. The reference solution and the test solution were analyzed by HPLC, and the content of recombinant trypsin in the test solution was calculated by the external standard method. The chromatographic conditions for the HPLC analysis are as follows: Chromatographic column: C18 silica gel column; Mobile phase A: 0.05~0.15 vol% phosphoric acid-water solution; Mobile phase B: 0.05~0.15 vol% phosphoric acid-acetonitrile solution; Detection wavelength: 275-285 nm; Column temperature: 37-42 ℃; Flow rate: 0.3~1.0 mL / min; Injection volume: 8~20 μL; The gradient elution program of the HPLC is as follows: 。 2. The influenza virus split vaccine according to claim 1 and the method for detecting the content of recombinant trypsin during its production process, characterized in that, The chromatographic column is packed with octadecylsilane-bonded silica gel, with a column length of 250-460 mm, an inner diameter of 4-5 mm, and a particle size of 3-4 μm.

3. The influenza virus split vaccine according to claim 1 and the method for detecting the content of recombinant trypsin during its production process, characterized in that, The method for preparing the reference solution includes: Take the recombinant trypsin control and dilute it with cell culture medium with a pH of 4-6 to prepare a solution of recombinant trypsin with a concentration of 20-50 μg / mL. Filter the solution through a 0.2-0.3 μm filter membrane.

4. The influenza virus split vaccine according to claim 1 and the method for detecting the content of recombinant trypsin during its production process, characterized in that, The process of calculating the recombinant trypsin content in the test solution using the external standard method requires the preparation of a standard curve. The method for preparing the standard curve includes: The reference solution was diluted to prepare a series of standard solutions of different concentrations. The same chromatographic conditions were used for HPLC analysis. The peak area of ​​the obtained chromatographic peak was used as the ordinate and the concentration of recombinant trypsin in the standard solution was used as the abscissa to perform linear regression and obtain the standard curve.

5. The influenza virus split vaccine according to claim 1 and the method for detecting the content of recombinant trypsin during its production process, characterized in that, It also includes using the reference solution to verify the system suitability of the HPLC analysis.

6. The influenza virus split vaccine according to claim 4 and the method for detecting the content of recombinant trypsin during its production process, characterized in that, In the system suitability verification of the HPLC analysis, if the peak area RSD of recombinant trypsin in the obtained chromatogram is less than 10% after 5 to 8 repeated injections of the reference solution according to the chromatographic conditions, the system suitability is determined to meet the detection requirements.

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