Method for detecting cpG content in double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris)

Abstract

The present application relates to the field of CpG content detection method, particularly to a method for detecting CpG content in a double-adjuvant recombinant novel coronavirus vaccine (Pichia pastoris), comprising the following steps: S1: taking 1ml of the novel coronavirus vaccine (Pichia pastoris) test sample and loading it into a 10ml EP tube, adding 1ml of 3% sodium hydroxide solution, ensuring that the seal is intact, heating to clarity in a boiling water bath for 15 minutes, shaking well, precisely taking 100ul of the mixed solution and loading it into a 10ml EP tube, adding 4.9ml of water, shaking well, and taking it as the test sample solution, a total dilution of 100 times, the present application has the following beneficial effects: low cost, saving test solution, only using sodium hydroxide; after treatment, there is no electrostatic adsorption and coordination reaction in the test sample, and the CpG content can be accurately determined.

Description

Technical Field

[0001] This invention relates to the field of CpG content detection methods, and in particular to a method for detecting CpG content in a dual-adjuvant recombinant novel coronavirus vaccine (Pichia pastoris). Background Technology

[0002] CpGISS1018 is a 22-mer unmethylated CpG-B oligonucleotide that stimulates the activation of B cells and NK cells. CpG-B is located in the transferrin receptor 1 (TFR1) endosome and induces IFNα production via a TLR9-dependent mechanism. Immunotherapy with soluble CpG-B and antigen induces the activation of NF-κB to produce pro-inflammatory and Th1-type cytokines via a TLR9-dependent MyD88 activation mechanism in dendritic cells. CpG is currently a novel adjuvant that has been clinically proven to be safe and effective. CpGISS1018 is modified with a phosphate thioester (PS) backbone, where a sulfur atom replaces a non-bridging oxygen atom in the phosphate ester backbone. The PS modification enhances resistance to nuclease degradation and makes the phosphate backbone more hydrophobic and stable.

[0003] Aluminum hydroxide adjuvant exists as aluminum hydroxyl groups, forming fibrous particles with an isoelectric point typically around 10.0-11.0. In neutral pH solutions, it exists as positively charged micron-sized particles, effectively adsorbing negatively charged CpG and negatively charged antigens. Amino acids possess lone pairs of electrons; adding metal ions to the solution can trigger a coordination reaction. Therefore, when determining CpG content, the adsorption reaction between aluminum hydroxide and CpG leads to a lower detection limit and thus a lower measured value when directly measuring CpG content. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting CpG content in a dual-adjuvant recombinant novel coronavirus vaccine (Pichia pastoris), thereby solving the above problems.

[0005] To achieve the above objectives, the following technical solutions are provided:

[0006] A method for detecting CpG content in a dual-adjuvant recombinant novel coronavirus vaccine (Pichia pastoris) includes the following steps:

[0007] S1: Take 1 ml of the novel coronavirus vaccine (Pichia pastoris) test sample and put it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution. After ensuring that the seal is intact, heat it in a boiling water bath for 15 minutes until it is clear. Shake well. Accurately measure 100 μl of the mixed solution and put it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This is the test sample solution. Dilute it 100 times in total.

[0008] S2: Set the wavelength of the UV-Vis spectrophotometer to 260nm, use purified water as a blank control, and measure the absorbance of the standard curve solution, the test solution, and the diluted antigen.

[0009] S3: Plot a standard curve using the absorbance values ​​corresponding to the CpG standard solution content (μg / ml), and find the linear regression equation (Conc=aX+b). Substitute the measured absorbance value of the test sample into the linear regression equation.

[0010] Preferably, the CpG content detection further includes specificity testing to ensure that the analytical method can accurately determine the analyte even in the presence of other components (such as impurities, degradation products, excipients, etc.). The specific steps are as follows:

[0011] S1: Accurately measure 1 ml of the test sample and put it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution. After ensuring a good seal, heat the solution in a boiling water bath for 15 minutes until clear. Shake well. Accurately measure 100 μl of the mixed solution and put it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This solution is the test sample solution. Dilute it 100 times in total.

[0012] S2: Measure 40 μl of the 2 mg / ml CpG reference working solution and place it in a 5 ml EP tube. Add purified water to make up to 4 ml, which means the CpG content is 20 μg / ml.

[0013] S3: Prepare 1 ml of antigen-aluminum hydroxide-sorbitol solution (blank solution), adjust it to have the same content of antigen, aluminum hydroxide and sorbitol as in the test sample, measure 50 μl of the mixed solution and 4.95 ml of purified water, mix well, and dilute 100 times in total.

[0014] S4: The CpG content of the solution in step S3 is determined by steps S2-S3 in claim 1.

[0015] Preferably, the CpG content detection further includes a linearity test to determine the ability of the linear test result to be directly proportional to the concentration of the analyte in the sample within a designed range. The specific steps are as follows:

[0016] S1: Measure 20 μl, 30 μl, 40 μl, 50 μl, and 60 μl of the 2 mg / ml CpG reference working solution and place them in 5 ml EP tubes respectively. Add purified water to make up to 4 ml, which means that the CpG content is 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, and 30 μg / ml respectively.

[0017] S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1.

[0018] Preferably, the CpG content detection further includes repeatability and intermediate precision testing, which measures the degree of similarity between results obtained from multiple sampling and determination of the same homogeneous sample under specified determination conditions. The precision of results obtained by the same analyst is called repeatability, and the precision of results obtained under different conditions in the same laboratory is called intermediate precision. The specific steps are as follows:

[0019] S1: Take a batch of test samples. Place 1 ml of the test sample into a 10 ml EP tube, add 1 ml of 3% sodium hydroxide solution, ensure a tight seal, and heat in a boiling water bath for 15 minutes until clear. Shake well. Accurately measure 100 μl of the mixed solution into a 10 ml EP tube, add 4.9 ml of water, and shake well. This is the test solution, diluted 100 times. Prepare 6 parallel samples for each test sample.

[0020] S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1, and the relative standard deviation (RSD) of the CpG content of the 6 test solutions is determined.

[0021] S3: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1, and the RSD value of the CpG content of each test solution is determined by experimenter A and experimenter B respectively.

[0022] Preferably, the CpG content detection further includes an accuracy test, which measures the degree to which the result determined by the established method closely approximates the true value or reference value, and expresses this as a recovery rate (%). The specific steps are as follows:

[0023] S1: Prepare test seedlings with CpG concentrations of 1 mg / ml, 2 mg / ml, and 3 mg / ml. Take 1 ml of the test sample and put it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution. After ensuring a good seal, heat in a boiling water bath for 15 minutes until clear. Shake well. Accurately measure 100 μl of the mixed solution and put it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This is the test sample solution. Dilute it 100 times. Prepare 3 parallel samples for each test sample.

[0024] S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1.

[0025] Preferably, the CpG content detection also includes a robustness test, which tests the degree to which the measurement results are unaffected by minor changes in the measurement conditions, providing a basis for the established method to be used in routine testing. The boiling time and sodium hydroxide solution were varied, and the specific steps are as follows:

[0026] S1: The difference between this step and step S1 in claim 1 is that the boiling time is changed to 14 min and 16 min, and the concentration of sodium hydroxide solution is changed to 2.8% and 3.2%.

[0027] S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1.

[0028] Preferably, the vaccine contains the following components: 1.5 mg / dose of aluminum hydroxide, 1.0 mg / dose of CpG, 10% / dose of sorbitol, and 50 μg / dose of antigen.

[0029] The beneficial effects of this invention are as follows:

[0030] (1) Low cost, saves test solution, only sodium hydroxide is used;

[0031] (2) There is no electrostatic adsorption and coordination reaction in the treated sample, so the CpG content can be accurately determined. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] A method for detecting CpG content in a dual-adjuvant recombinant novel coronavirus vaccine (Pichia pastoris) includes the following steps:

[0034] S1: Take 1 ml of the novel coronavirus vaccine (Pichia pastoris) test sample and put it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution. After ensuring that the seal is intact, heat it in a boiling water bath for 15 minutes until it is clear. Shake well. Accurately measure 100 μl of the mixed solution and put it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This is the test sample solution. Dilute it 100 times in total.

[0035] S2: Set the wavelength of the UV-Vis spectrophotometer to 260nm, use purified water as a blank control, and measure the absorbance of the standard curve solution, the test solution, and the diluted antigen.

[0036] S3: Plot a standard curve using the absorbance values ​​corresponding to the CpG standard solution content (μg / ml), and find the linear regression equation (Conc=aX+b). Substitute the measured absorbance value of the test sample into the linear regression equation.

[0037] In some embodiments, CpG content detection also includes specificity testing to ensure that the analytical method can accurately determine the analyte even in the presence of other components (such as impurities, degradation products, excipients, etc.). The specific steps are as follows:

[0038] S1: Accurately measure 1 ml of the test sample and put it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution. After ensuring a good seal, heat the solution in a boiling water bath for 15 minutes until clear. Shake well. Accurately measure 100 μl of the mixed solution and put it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This solution is the test sample solution. Dilute it 100 times in total.

[0039] S2: Measure 40 μl of the 2 mg / ml CpG reference working solution and place it in a 5 ml EP tube. Add purified water to make up to 4 ml, which means the CpG content is 20 μg / ml.

[0040] S3: Prepare 1 ml of antigen-aluminum hydroxide-sorbitol solution (blank solution), adjust it to have the same content of antigen, aluminum hydroxide and sorbitol as in the test sample, measure 50 μl of the mixed solution and 4.95 ml of purified water, mix well, and dilute 100 times in total.

[0041] S4: The CpG content of the solution from step S3 was determined using steps S2-S3 of claim 1. The experimental results are shown in the table below:

[0042] Table 1: Results of Specificity Test

[0043]

[0044] In some embodiments, CpG content detection further includes a linearity test to determine the ability of the linear test result to be directly proportional to the concentration of the analyte in the sample within a designed range. The specific steps are as follows:

[0045] S1: Measure 20 μl, 30 μl, 40 μl, 50 μl, and 60 μl of the 2 mg / ml CpG reference working solution and place them in 5 ml EP tubes respectively. Add purified water to make up to 4 ml, which means that the CpG content is 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, and 30 μg / ml respectively.

[0046] S2: The CpG content of the solution from step S1 was determined using steps S2-S3 of claim 1. The experimental results are shown in the table below:

[0047] Table 2: Linearity Test Results

[0048]

[0049] In some embodiments, CpG content detection also includes repeatability and intermediate precision testing, which measures the similarity between results obtained from multiple sampling and determinations of the same homogeneous sample under specified determination conditions. The precision of results obtained by the same analyst is called repeatability, and the precision of results obtained under different conditions in the same laboratory is called intermediate precision. The specific steps are as follows:

[0050] S1: Take a batch of test samples. Place 1 ml of the test sample into a 10 ml EP tube, add 1 ml of 3% sodium hydroxide solution, ensure a tight seal, and heat in a boiling water bath for 15 minutes until clear. Shake well. Accurately measure 100 μl of the mixed solution into a 10 ml EP tube, add 4.9 ml of water, and shake well. This is the test solution, diluted 100 times. Prepare 6 parallel samples for each test sample.

[0051] S2: The CpG content of the solution from step S1 was determined using steps S2-S3 of claim 1, and the relative standard deviation (RSD) of the CpG content of the six test solutions was measured. The experimental results are shown in the table below:

[0052] Table 3: Repeatability Test Results

[0053]

[0054] S3: The CpG content of the solution from step S1 is determined using steps S2-S3 of claim 1. Experimenter A and Experimenter B respectively determine the RSD value of the CpG content for each test solution. The experimental results are shown in the table below:

[0055] Table 4: Intermediate Precision Test Results

[0056]

[0057] In some embodiments, CpG content detection further includes an accuracy test to determine the degree to which the result obtained by the established method approaches the true value or reference value, expressed as a recovery rate (%). The specific steps are as follows:

[0058] S1: Prepare test seedlings with CpG concentrations of 1 mg / ml, 2 mg / ml, and 3 mg / ml. Take 1 ml of the test sample and put it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution. After ensuring a good seal, heat in a boiling water bath for 15 minutes until clear. Shake well. Accurately measure 100 μl of the mixed solution and put it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This is the test sample solution. Dilute it 100 times. Prepare 3 parallel samples for each test sample.

[0059] S2: The CpG content of the solution from step S1 was determined using steps S2-S3 of claim 1. The experimental results are shown in the table below:

[0060] Table 5: Accuracy Test Results

[0061]

[0062] In some embodiments, CpG content detection also includes a robustness test, which tests the degree to which the measurement results are unaffected by minor changes in the measurement conditions, providing a basis for the established method to be used in routine testing. The boiling time and sodium hydroxide solution were varied, and the specific steps are as follows:

[0063] S1: The difference between this step and step S1 in claim 1 is that the boiling time is changed to 14 min and 16 min, and the concentration of sodium hydroxide solution is changed to 2.8% and 3.2%.

[0064] S2: The CpG content of the solution from step S1 was determined using steps S2-S3 of claim 1. The experimental results are shown in the table below:

[0065] Table 6: Durability Test Results

[0066]

[0067] In some embodiments, the vaccine contains the following components: 1.5 mg / dose of aluminum hydroxide, 1.0 mg / dose of CpG, 10% / dose of sorbitol, and 50 μg / dose of antigen.

Claims

1. A method for detecting CpG content in Pichia pastoris, a recombinant novel coronavirus vaccine with dual adjuvants, characterized in that: Includes the following steps: S1: Take 1 ml of the Pichia pastoris test sample for the novel coronavirus vaccine and place it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution, ensure a tight seal, and heat in a boiling water bath for 15 minutes until clear. Shake well, accurately measure 100 μl of the mixed solution and place it into a 10 ml EP tube. Add 4.9 ml of water and shake well. This is the test solution. Dilute it 100 times. S2: Set the wavelength of the UV-Vis spectrophotometer to 260 nm, using purified water as a blank control, and measure the absorbance values ​​of the standard curve solution, the test solution, and the diluted antigen. S3: Plot a standard curve using the absorbance values ​​corresponding to the CpG standard solution concentration (μg / ml), and derive the linear regression equation Conc = aX + b. Substitute the measured absorbance values ​​of the test sample into the linear regression equation. The linear regression equation is: 。 2. The method for detecting CpG content in Pichia pastoris of the dual-adjuvant recombinant novel coronavirus vaccine according to claim 1, characterized in that: The CpG content detection also includes specificity testing. The analytical method used can accurately determine the analyte's ability to react. The specific steps are as follows: S1: Accurately measure 1 ml of the test sample into a 10 ml EP tube, add 1 ml of 3% sodium hydroxide solution, ensure a tight seal, heat in a boiling water bath for 15 minutes until clear, shake well, accurately measure 100 μl of the mixed solution into a 10 ml EP tube, add 4.9 ml of water, shake well, and use this as the test solution. Dilute 100 times in total. S2: Measure 40 μl of the 2 mg / ml CpG reference working solution and place it in a 5 ml EP tube. Add purified water to a final volume of 4 ml, which indicates a CpG concentration of 20 μg / ml. S3: Prepare 1 ml of antigen-aluminum hydroxide-sorbitol solution, adjusting it to have the same content of antigen, aluminum hydroxide, and sorbitol as the test sample. Measure 50 μl of the mixed solution and 4.95 ml of purified water, mix well, and dilute 100 times. S4: The CpG content of the solution in step S3 is determined by steps S2-S3 in claim 1.

3. The method for detecting CpG content in Pichia pastoris of the dual-adjuvant recombinant novel coronavirus vaccine according to claim 1, characterized in that: The CpG content detection also includes a linearity test, which determines the ability of the linear test result to be directly proportional to the concentration of the analyte in the sample within a designed range. The specific steps are as follows: S1: Measure 20 μl, 30 μl, 40 μl, 50 μl, and 60 μl of the 2 mg / ml CpG reference working solution and place them into 5 ml EP tubes respectively. Add purified water to a final volume of 4 ml, which will give CpG concentrations of 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, and 30 μg / ml. S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1.

4. The method for detecting CpG content in Pichia pastoris of the dual-adjuvant recombinant novel coronavirus vaccine according to claim 1, characterized in that: The CpG content detection also includes repeatability and intermediate precision tests. These tests measure the similarity between results obtained from multiple sampling measurements of the same homogeneous sample under specified testing conditions. The precision of results obtained by the same analyst is called repeatability, while the precision of results obtained under different conditions within the same laboratory is called intermediate precision. The specific steps are as follows: S1: Take a batch of test samples. Place 1 ml of the test sample into a 10 ml EP tube, add 1 ml of 3% sodium hydroxide solution, ensure a tight seal, heat in a boiling water bath for 15 minutes until clear, shake well, accurately measure 100 μl of the mixed solution into a 10 ml EP tube, add 4.9 ml of water, shake well, and use this as the test solution. Dilute 100 times in total. Prepare 6 parallel samples for each test sample. S2: The CpG content of the solution from step S1 is determined using steps S2-S3 of claim 1, and the relative standard deviation (RSD) of the CpG content of the six test solutions is determined. S3: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1, and the RSD value of the CpG content of each test solution is determined by experimenter A and experimenter B respectively.

5. The method for detecting CpG content in Pichia pastoris of the dual-adjuvant recombinant novel coronavirus vaccine according to claim 1, characterized in that: The CpG content detection also includes an accuracy test, which measures the degree to which the result obtained by the established method is close to the true value or reference value, and is expressed as a recovery rate (%). The specific steps are as follows: S1: Prepare test samples with CpG concentrations of 1 mg / ml, 2 mg / ml, and 3 mg / ml. Take 1 ml of the test sample and place it into a 10 ml EP tube. Add 1 ml of 3% sodium hydroxide solution, ensuring a tight seal. Heat in a boiling water bath for 15 minutes until clear, shake well, and accurately measure 100 μl of the mixed solution into a 10 ml EP tube. Add 4.9 ml of water, shake well, and use this as the test sample solution. Dilute 100 times in total. Prepare 3 parallel samples for each test sample. S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1.

6. The method for detecting CpG content in Pichia pastoris of the dual-adjuvant recombinant novel coronavirus vaccine according to claim 1, characterized in that: The CpG content detection also includes a robustness test, which tests the degree to which the measurement results are unaffected by minor changes in the measurement conditions. This provides a basis for the application of the established method in routine testing. The boiling time and sodium hydroxide solution were varied, and the specific steps are as follows: S1: The difference between this step and step S1 in claim 1 is that the boiling time is changed to 14 min and 16 min, and the concentration of sodium hydroxide solution is changed to 2.8% and 3.2%. S2: The CpG content of the solution in step S1 is determined by steps S2-S3 in claim 1.

7. The method for detecting CpG content in Pichia pastoris of the dual-adjuvant recombinant novel coronavirus vaccine according to any one of claims 1-6, characterized in that: The vaccine contains the following components: 1.5 mg / dose of aluminum hydroxide, 1.0 mg / dose of CpG, 10% / dose of sorbitol, and 50 μg / dose of antigen.

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