Sample pretreatment method for detecting bacterial endotoxins in LNP-type mRNA vaccines

Abstract

This invention discloses a sample pretreatment method for detecting bacterial endotoxins in LNP-type mRNA vaccines. The method involves unpacking the sample with a 3-10% (v / v) Triton X-100 solution, followed by a 0.9% sodium chloride solution as the diluent in the sample gradient dilution step. This effectively eliminates interference from the positive control group in the gel limit method for detecting bacterial endotoxins in mRNA vaccines. The method provides accurate and reliable results and is suitable for the quality control of bacterial endotoxins in LNP-type mRNA vaccines.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a sample pretreatment method for detecting bacterial endotoxins in mRNA vaccines. Technical Background

[0002] mRNA vaccines, as a novel nucleic acid preparation, have been widely used in protein replacement therapy, infectious disease prevention, and tumor treatment due to their advantages such as good safety, high preparation efficiency, and rapid production. Because mRNA molecules are unstable, easily degraded by nucleases, and carry a negative charge that makes them difficult to cross membranes, lipid nanoparticles (LNPs) have become the most widely used mRNA delivery carriers in clinical practice. Their classic formulations mainly contain ionizable lipid compounds, cofactor phospholipid compounds, cholesterol, and polyethylene glycol (PEG) lipid compounds.

[0003] Bacterial endotoxin detection is a core mandatory test for vaccine safety evaluation. Excessive endotoxin levels can cause serious adverse reactions such as high fever and endotoxin shock. The Limulus Amebocyte Lysate (LAL) assay is currently the most widely used method for endotoxin detection. This method is mainly divided into gel electrophoresis and photometric methods. The gel electrophoresis method qualitatively or semi-quantitatively detects endotoxins through the gel state formed by the reaction; it is simple to operate, requires low equipment, and does not require the preparation of a standard curve. Because the lipid bilayer structure of lipid nanoparticles (LNPs) encapsulates mRNA molecules, the mRNA vaccine needs to be unencapsulated before detection to release the encapsulated mRNA molecules and endotoxins into the detection system for objective evaluation of the endotoxin content of the mRNA vaccine. Unencapsulation reagents are typically nonionic surfactants (such as Triton X-100, Tween-80) and organic solvents (such as dimethyl sulfoxide, ethanol). In practical applications, incorrect concentrations of unencapsulation reagents can lead to incomplete unencapsulation. After unpacking and sealing mRNA vaccines, bacterial endotoxin testing is usually performed by serially diluting the vaccine with water to the maximum effective dilution (MVD) and then using the gel limit method for bacterial endotoxin detection. In practical applications, it has been found that false negatives frequently occur in the positive control group of the test sample, which seriously affects the reliability of the test. Moreover, changing the manufacturer of the horseshoe crab reagent and adding conventional auxiliary reagents cannot solve this problem, which poses a challenge to the quality control of mRNA vaccines. Summary of the Invention

[0004] This invention provides a sample pretreatment method for detecting bacterial endotoxins in (lipid nanoparticle) mRNA vaccines, which solves the problem of false negatives in positive controls of test samples and effectively improves the accuracy of detecting bacterial endotoxins in LNP (lipid nanoparticle) mRNA products.

[0005] The sample pretreatment method of this invention for detecting bacterial endotoxins in LNP-type mRNA vaccines involves adding an equal volume of 3-10% Triton X-100 solution to the LNP-type mRNA vaccine being tested, mixing thoroughly to obtain a mixture, calculating the maximum effective dilution factor based on the sensitivity value of the Limulus amebocyte lysate (LAL) reagent used in the gel limit method and the standard for bacterial endotoxins in the sample, and diluting the mixture to the maximum effective dilution factor using 0.9% sodium chloride solution as a diluent to obtain the test sample for gel limit method detection.

[0006] The mixing method is vortex mixing; the mixing time is 30-40 seconds, and the mixture is left to stand at room temperature for 5 minutes after mixing.

[0007] The method of the present invention can also add an equal volume of Triton X-100 solution with a volume concentration of 3-10% to the LNP-type mRNA vaccine being tested, mix well to obtain a mixture, calculate the maximum effective dilution factor according to the sensitivity value of the Limulus amebocyte lysate (LAL) reagent used in the gel limit method and the standard of bacterial endotoxin in the sample, and dilute the mixture to the maximum effective dilution factor using water for bacterial endotoxin testing and 0.9% sodium chloride solution as diluents to obtain the test sample for gel limit method detection.

[0008] The mixture was first diluted with water for bacterial endotoxin testing, then diluted with 0.9% sodium chloride solution, and finally diluted again with water for bacterial endotoxin testing.

[0009] When using the gel limit method for detection, set up positive control, positive control and negative control of the test sample according to the conventional method.

[0010] Compared with the prior art, the technical solution of the present invention has the following advantages: Studies have found that adding 3-10% Triton X-100 solution can unpack LNP (lipid nanoparticle) mRNA vaccine products, and adding 0.9% sodium chloride solution as a diluent plays a key role in inhibiting false negatives. This eliminates interference from mRNA vaccines in bacterial endotoxin limit detection, avoids the problem of false negative interference in existing technologies, and provides accurate and reliable test results. The operation is simple and reproducible, which is of great significance for ensuring the safety of mRNA vaccines. Attached Figure Description

[0011] Figure 1 OD detected by UV-Vis spectrophotometer 550 value. Detailed Implementation

[0012] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the reagents and methods used in the embodiments are all conventional reagents and conventional methods.

[0013] The reagents or instruments used in the examples are as follows: Triton X-100 reduced: MERCK-Sigma-Aldrich, specification: 25g / bottle, batch number: SHBN0734; Limulus amebocyte lysate (LAL) reagent: Zhanjiang Bokang Marine Biological Co., Ltd., specification: 0.25EU / mL, batch number: 2305252; Bacterial endotoxin working standard: Zhanjiang Andus Biotechnology Co., Ltd., specification: 1mL / vial, potency: 10EU / vial, batch number: 2301042; Water for bacterial endotoxin testing: Zhanjiang Andus Biotechnology Co., Ltd., specification: <0.003EU / mL, 2mL / vial, batch number: 2209270; Sodium chloride injection: Kunming Nanjiang Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: C22061507.

[0014] Main instruments: .

[0015] Example 1: Triton X-100 Unpacking Concentration Screening Test Because the unpacking concentration of mRNA vaccines affects the detection, a screening experiment on the unpacking concentration was conducted.

[0016] 1. Reagent preparation Take 1 mL of Triton X-100 and add it to different volumes of water for bacterial endotoxin testing. Mix thoroughly to obtain Triton X-100 solutions with concentrations of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.8%, and 0.4%. At the same time, set up a control without adding Triton X-10. 2. Delaying milk production Take 3 mL of each mRNA vaccine product, add 3 mL of Triton X-100 solution of different concentrations, vortex for 30 s, let stand at room temperature for 5 min, and set up 3 parallel samples for each concentration. 3. The OD of the above samples was detected using a UV-Vis spectrophotometer. 550 The values ​​and results are shown in the table below. Figure 1 ;

[0017] The results showed that, under the uniform demulsification conditions of 3 mL vaccine + 3 mL reagent, vortexing for 30 s, and standing at room temperature for 5 min, when the Triton X-100 concentration was ≥3%, the OD of the samples after demulsification was [not specified]. 550 Small changes in OD values ​​and stable readings indicate thorough and consistent demulsification; when the concentration is below 3%, OD... 550The values ​​fluctuated significantly, indicating incomplete demulsification and failure to eliminate emulsion interference in the system; the control group sample without Triton X-100 showed no demulsification, and the OD value was high. 550 The values ​​were significantly high. Based on the combined experimental results, 3%–10% Triton X-100 was determined to be the appropriate concentration range for unpacking and demulsification of this mRNA vaccine.

[0018] Example 2: Anti-interference verification experiment of the method of the present invention 1. Preparation of 6% Triton X-100 solution Add 1 mL of Triton X-100 to 15.7 mL of water for bacterial endotoxin testing, mix thoroughly to obtain a 6% Triton X-100 solution, and store in a pyrogen-free container at room temperature.

[0019] 2. Determine the maximum effective dilution factor (MVD) using the following formula: MVD = cL / λ = 1 × (10 / 0.15) / 0.25 = 264 In the formula: L is the bacterial endotoxin limit of the test sample; 10 EU / dose (i.e., 10 / 0.15≈66 EU / mL); c is the concentration of the test sample solution, 1 mL / mL; λ is the labeled sensitivity of the Limulus amebocyte lysate (LAL) reagent in the gel electrophoresis method (EU / mL): 0.25 EU / mL.

[0020] 3. Preparation of working standards for bacterial endotoxins Take one ampoule of bacterial endotoxin working standard, break it off at the top, add an appropriate amount of bacterial endotoxin test water according to the instructions, mix well on a vortex mixer for at least 15 minutes, and then dilute to 4λ or 2λ as required. Limulus amebocyte lysate (LAL) reagent with a sensitivity of 0.25 EU / mL (1λ) can be diluted with the bacterial endotoxin working standard as follows: ; 4. Use water to dilute the test sample for bacterial endotoxin testing (control experiment). Prepare the solutions in the table below using a test sample solution with a dilution factor not exceeding MVD: ; A. Preparation of the test solution Take 0.3 mL of the mRNA vaccine product, add 0.3 mL of 6% Triton X-100 solution, vortex for 30 s to obtain mixture S, let stand at room temperature for 5 min to unpack, this is a 2-fold dilution; take 0.3 mL of mixture S and dilute with 1.2 mL of bacterial endotoxin test water, vortex for 30 s to obtain S1, this is a 10-fold dilution; take 0.3 mL of S1 and dilute with 1.68 mL of bacterial endotoxin test water, vortex for 30 s to obtain S2, this is a 66-fold dilution; take 1.0 mL of S2 and dilute with 1.0 mL of bacterial endotoxin test water, vortex for 30 s to obtain S3, this is a 132-fold dilution; take 0.5 mL of S3 and dilute with 0.5 mL of bacterial endotoxin test water, vortex for 30 s to obtain the test solution, this is a 264-fold dilution.

[0021] B. Positive control of the test sample Take 0.3 mL of the S3 test sample dilution solution (diluted 132 times with water for bacterial endotoxin testing), add 0.3 mL of 4λ endotoxin standard solution, mix thoroughly, and the test sample positive control is obtained. C. Positive control A standard solution of 2λ endotoxin was used as a positive control. D. Negative control Water used for bacterial endotoxin testing was used as a negative control.

[0022] 5. Prepare test samples using water and 0.9% sodium chloride solution as diluents for bacterial endotoxin testing (Method 1 of this invention). Prepare the solutions in the table below using a test sample solution with a dilution factor not exceeding MVD:

[0023] A. Preparation of the test solution Take 0.3 mL of the mRNA vaccine product, add 0.3 mL of 6% Triton X-100 solution, vortex for 30 s to obtain mixture S, let stand at room temperature for 5 min to unpack, this is a 2-fold dilution; take 0.3 mL of mixture S and dilute with 1.2 mL of water for bacterial endotoxin testing, vortex for 30 s to obtain S1, this is a 10-fold dilution; take 0.3 mL of S1 and dilute with 1.68 mL of water for bacterial endotoxin testing, vortex for 30 s to obtain S2, this is a 66-fold dilution; take 1.0 mL of S2 and dilute with 0.9% sodium chloride solution, vortex for 30 s to obtain S3, this is a 132-fold dilution; take 0.5 mL of S3 and dilute with 0.5 mL of water for bacterial endotoxin testing, vortex for 30 s to obtain the test solution, this is a 264-fold dilution.

[0024] B. Positive control of the test sample Take 0.3 mL of the S3 test sample diluent diluted 132 times with water and 0.9% sodium chloride solution for bacterial endotoxin testing, add 0.3 mL of 4λ endotoxin standard solution, and mix thoroughly to obtain the final product.

[0025] C. Positive control A standard solution of 2λ endotoxin was used as a positive control.

[0026] D. Negative control 1 Water used for bacterial endotoxin testing was used as a negative control.

[0027] E. Negative Control 2 Use 0.9% sodium chloride solution as a negative control.

[0028] 6. Prepare the test sample using a 0.9% sodium chloride solution as a diluent (Method 2 of this invention). The method is the same as step A in step 5, except that 0.3 mL of the mixed solution S is diluted 4 times with 0.9% sodium chloride solution to obtain the test solution, which is a 264-fold dilution; the negative control is 0.9% sodium chloride solution, and the rest is the same as above.

[0029] 7. Take a Limulus amebocyte lysate reagent with a sensitivity of 0.25 EU / mL and a specification of 0.1 mL / vial, and redissolve each vial in 0.1 mL of water for bacterial endotoxin testing; 8. Take 0.1 mL of each sample group from steps 4, 5, and 6 and add it to the sample. Make two parallel tests for each test solution. Add 0.1 mL of Limulus Amebocyte Lysate (LAL) reagent to each test solution. Incubate at 37℃±1℃ for 60 minutes±2 minutes and then observe the results.

[0030] 9. Result Judgment A. If all parallel tubes of the negative control are negative, all parallel tubes of the positive control of the test sample are positive, and all parallel tubes of the positive control are positive, the test is valid.

[0031] B. If both parallel tubes of the test solution are negative, the test solution is deemed to be free of endotoxins and compliant with regulations, provided the test is valid. If both parallel tubes of the test solution are positive, the test solution is deemed to contain endotoxins and compliant with regulations.

[0032] C. If one of the two parallel tubes of the test solution is positive and the other is negative, a retest is required. For the retest, four parallel tubes of the test solution must be prepared. If all parallel tubes are negative, the test solution is deemed compliant; otherwise, it is deemed non-compliant.

[0033] 10. Test Results

[0034] The results showed that the control experiment was not valid, indicating that using water as a diluent in the bacterial endotoxin test could not eliminate interference. The method of the present invention, by using 0.9% sodium chloride solution as a diluent, can eliminate interference in the experimental process.

[0035] Example 3: Interference test of dilution with water + 0.9% sodium chloride solution as diluent for bacterial endotoxin detection of test sample. The purpose of this experiment was to dilute the sample with 0.9% sodium chloride injection to confirm whether there was any interference.

[0036] 1. Sample preparation Prepare the solution according to the table below: ; A. Preparation of the test solution Preparation of test sample stock solution: Measure 0.3 mL of mRNA vaccine product, add 0.3 mL of 6% Triton X-100 solution, vortex for 30 s to obtain mixture S, let stand at room temperature for 5 min to unpack, this is a 2-fold dilution; take 0.3 mL of mixture S and dilute with 1.2 mL of bacterial endotoxin test water, vortex for 30 s to obtain S1, this is a 10-fold dilution; take 0.3 mL of S1 and dilute with 1.68 mL of bacterial endotoxin test water, vortex for 30 s to obtain S2, this is a 66-fold dilution; take 1.0 mL of S2 and dilute with 0.9% sodium chloride solution, vortex for 30 s to obtain S3, this is a 132-fold dilution; take 0.5 mL of S3 and dilute with 0.5 mL of bacterial endotoxin test water, vortex for 30 s to obtain the test sample solution, this is a 264-fold dilution.

[0037] B. Preparation of Interference Test Series Solutions Take 0.3 mL of S3 test sample diluent diluted 132 times with water and 0.9% sodium chloride solution for bacterial endotoxin testing, and then add 0.3 mL of endotoxin standard solutions containing 4λ, 2λ, 1λ, and 0.5λ respectively to prepare test sample solutions containing 2λ, λ, 0.5λ, and 0.25λ endotoxins.

[0038] C. Preparation of Limulus Amebocyte Lysate (LAL) Reagent Label Sensitivity Control Series Take one vial of bacterial endotoxin standard and dilute it with water for bacterial endotoxin testing to prepare four standard solutions of different concentrations: 2λ, λ, 0.5λ, and 0.25λ, a sensitivity control series for the Limulus Amebocyte Lysate (LAL) reagent.

[0039] D. Negative control for test water: Use the reconstituted horseshoe crab reagent and the test water for bacterial endotoxin testing as negative controls for test water.

[0040] E. 0.9% sodium chloride solution negative control: Take a 0.9% sodium chloride solution diluted with the test sample as a 0.9% sodium chloride solution negative control.

[0041] 2. Preparation of Limulus Amebocyte Lysate (LAL) Reagent Take 30 ampoules of Limulus Amebocyte Lysate (LAL) reagent (0.1 mL / ampoule, sensitivity 0.25 EU / mL). Gently tap the ampoule to allow the powder to fall to the bottom. Wipe the ampoules with 75% alcohol swabs and open them for later use, preventing glass shards from falling into the ampoules. Add 0.1 mL of test water to each ampoule to dissolve it. Gently rotate the ampoule to ensure the contents are fully dissolved, avoiding the formation of air bubbles.

[0042] 3. Adding samples Take 12 of the prepared horseshoe crab reagents and arrange them into 6 columns, with 2 reagents in each column. Add 0.1 mL of 2λ, λ, 0.5λ, and 0.25λ endotoxin standard solutions to each of the reagents in 4 columns, respectively. Add 0.1 mL of bacterial endotoxin test water to each of the 2 reagents in the other column as a negative control for the test water. Add 0.1 mL of 0.9% sodium chloride solution to each of the 2 reagents in the last column as a negative control for the 0.9% sodium chloride solution.

[0043] The remaining 18 Limulus amebocyte lysate (LAL) reagents were arranged into 5 columns, with 4 columns containing 4 reagents each and 1 column containing 2 reagents. In the 4 columns, 0.1 mL of a test solution containing 2λ, λ, 0.5λ, and 0.25λ endotoxins was added to each reagent. In the other column, 0.1 mL of the test solution was added to each of the 2 reagents to form the test solution group.

[0044] After adding the sample, seal the opening with sealing film, gently shake to mix, avoid generating air bubbles, and place it vertically in a constant temperature water bath at 37℃±1℃ or a suitable thermostat, keep it at this temperature for 60±2 minutes, and observe and record the results.

[0045] 4. Calculation of experimental results The test is valid when all parallel tubes of the negative control, sample negative control, and 0.9% sodium chloride solution negative control are negative, and all tubes with the highest concentration (2λ) of the endotoxin standard solution are positive, and all tubes with the lowest concentration (0.25λ) are negative. The geometric mean (Es) of the endpoint concentration of the endotoxin standard solution prepared with test water and the geometric mean (Et) of the endpoint concentration of the endotoxin solution prepared with the test sample dilution are calculated using the following formulas: Es = antilg(∑Xs / 2) Et=antilg(∑Xt / 4) In the formula: Xs and Xt are the logarithmic values ​​(lg) of the endpoint concentration of the endotoxin solution prepared with test water and test sample dilution.

[0046] Acceptable criteria: The test is valid when all parallel tubes of test solution A, negative control D (water for testing), and negative control E (0.9% sodium chloride solution) are negative, and the results of the Limulus Amebocyte Lysate (LAL) reagent labeled sensitivity control series C are within the LAL reagent sensitivity verification range.

[0047] When Es is between 0.5λ and 2λ (inclusive) and Et is between 0.5Es and 2Es (inclusive), the test sample is considered to have no interfering effect at that concentration, and bacterial endotoxin testing (gel limit method) can be performed on the test sample at this concentration. If the test sample solution interferes with the test at a dilution factor less than MVD, the test sample solution should be further diluted by no more than MVD, and the interference test should be repeated.

[0048] 5. The experimental results are as follows:

[0049] The test results for the test solution, the negative control using water for testing, and the negative control using 0.9% sodium chloride injection were all negative, with Es and Et results both showing negative values. This demonstrates that 0.9% sodium chloride injection, as a diluent, does not interfere with the detection of bacterial endotoxins in mRNA vaccines.

Claims

1. A sample pretreatment method for detecting bacterial endotoxins in LNP-type mRNA vaccines, characterized in that: Add an equal volume of 3-10% Triton X-100 solution to the tested LNP-type mRNA vaccine, mix well to obtain a mixture, calculate the maximum effective dilution factor based on the sensitivity value of the Limulus amebocyte lysate (LAL) reagent used in the gel limit method and the standard of bacterial endotoxin in the sample, and dilute the mixture to the maximum effective dilution factor using 0.9% sodium chloride solution as a diluent to obtain the test sample for gel limit method detection.

2. A sample pretreatment method for detecting bacterial endotoxins in LNP-type mRNA vaccines, characterized in that: Add an equal volume of 3-10% Triton X-100 solution to the tested LNP-type mRNA vaccine, mix well to obtain a mixture, calculate the maximum effective dilution factor based on the sensitivity value of the Limulus amebocyte lysate (LAL) reagent used in the gel limit method and the standard of bacterial endotoxin in the sample, and dilute the mixture to the maximum effective dilution factor using water for bacterial endotoxin testing and 0.9% sodium chloride solution as diluents to obtain the test sample for gel limit method detection.

3. The sample pretreatment method for detecting bacterial endotoxins in LNP-type mRNA vaccines according to claim 2, characterized in that: The mixture was first diluted with water for bacterial endotoxin testing, then diluted with 0.9% sodium chloride solution, and finally diluted again with water for bacterial endotoxin testing.

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