Method for eliminating interference in LNP type sample bacterial endotoxin detection

Abstract

The invention discloses a method for eliminating interference in LNP type sample bacterial endotoxin detection. The method comprises the following steps: S1, determining a detection standard curve of a tachypleus amebocyte lysate dynamic color development method; s2, preparing a test sample solution A with different dispersing agent volume ratios and a test sample solution B added with the endotoxin with the midpoint concentration in the standard curve, and carrying out an interference elimination test by adopting the standard bacterial endotoxin solution C, the solution A and the solution B; s3, redissolving the tachypleus amebocyte lysate with bacterial endotoxin inspection water according to the specification of the tachypleus amebocyte S4, detecting by adopting a tachypleus amebocyte lysate dynamic color development method; and S5, judging the effectiveness of the experiment. According to the method for eliminating interference in bacterial endotoxin detection of the LNP type sample, the endotoxin dispersing agent is introduced, so that the multi-dimensional interference problems of endotoxin wrapped by lipid in the LNP sample, pH value deviation, osmotic pressure abnormity and the like are solved in a targeted manner.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial endotoxin detection, and in particular to a method for eliminating interference in bacterial endotoxin detection of LNP type samples. Background Art

[0002] In the biopharmaceutical field, LNPs, as an important drug delivery vehicle, are widely used in cutting-edge technologies such as mRNA vaccines and gene therapy. Bacterial endotoxins are components of the cell walls of Gram-negative bacteria. Once they enter the human body, they can cause severe inflammatory reactions such as fever and shock. Therefore, accurate detection of bacterial endotoxins in LNP samples is crucial.

[0003] Currently, the dynamic colorimetric method with Limulus amebocyte lysate (LA-lysate) is one of the most commonly used methods for detecting bacterial endotoxins. The principle is that bacterial endotoxins can specifically activate Factor C in the LA-lysate, which in turn activates Factor B, which in turn activates procoagulant. Coagulase then hydrolyzes the chromogenic substrate in the reaction, producing free p-nitroaniline (pNA), which in turn causes a change in absorbance. The concentration of bacterial endotoxins is quantified by dynamically detecting the rate of change in absorbance of the solution. However, this method is often interfered with by various factors when testing LNP-type samples.

[0004] The lipid components in the LNP sample may encapsulate endotoxins, preventing them from fully contacting the limulus amebocyte lysate (LAS), thereby affecting the activation of the LAS. For example, the lipid components may form micelle structures, encapsulating the endotoxins and rendering them undetectable. Furthermore, factors such as the pH value of the sample, the presence of enzymes or enzyme inhibitors in the sample, the osmotic pressure of the sample, detergents in the sample, and metal ions in the sample may inhibit the reaction between the LAS and endotoxins, leading to deviations in the test results and an inability to accurately reflect the true content of bacterial endotoxins in the LNP sample.

[0005] Therefore, there is an urgent need for a detection method that can eliminate the interference caused by LNP samples in dynamic colorimetric detection. Summary of the Invention

[0006] The main purpose of the present invention is to propose a method for eliminating interference in bacterial endotoxin detection of LNP type samples, aiming to solve the problem in the prior art that LNP samples are interfered with by multiple factors during bacterial endotoxin detection, resulting in inaccurate test results.

[0007] To achieve the above object, the present invention provides a method for eliminating interference in bacterial endotoxin detection of LNP type samples, comprising the following steps:

[0008] S1. Prepare at least three standard bacterial endotoxin solutions C with concentration gradients, and use the standard bacterial endotoxin solutions to perform a standard curve reliability test to determine the detection standard curve of the limulus amebocyte lysate kinetic colorimetric method;

[0009] S2. Prepare a test solution A with different dispersant volume ratios and a test solution B with endotoxin added at the midpoint concentration of the standard curve, and perform an interference elimination test using the standard bacterial endotoxin solution C, solution A, and solution B. The interference elimination test uses an endotoxin dispersant, which eliminates interference by at least two mechanisms: destroying the micelle structure formed by the lipid components in the LNP sample, adjusting the pH value of the sample to the optimal reaction range of 6.0 to 8.0, changing the physical state of the endotoxin, competitively binding to specific groups on the surface of the endotoxin, promoting the Brownian motion of the endotoxin, affecting the charge distribution of the endotoxin, and destroying the hydrogen bonds between endotoxin molecules;

[0010] S3. Reconstitute the limulus amebocyte lysate with bacterial endotoxin test water according to the limulus amebocyte lysate specifications;

[0011] S4. Add negative control solution D, standard bacterial endotoxin solution C, solution A, and solution B to the reaction plate, respectively. Set up at least two parallel tubes for each sample, then add the reconstituted limulus amebocyte lysate (LA-lysate) and perform detection using the limulus amebocyte lysate kinetic colorimetric method.

[0012] S5. Determine the validity of the experiment based on the following criteria: the correlation coefficient of the standard curve |r| ≥ 0.980, the recovery rate of solution B between 50% and 200%, the negative control test value less than the test value at the lowest point of the standard curve or the reaction time greater than the reaction time at the lowest point of the standard curve, and the CV of each parallel tube result ≤ 10%.

[0013] Preferably, when preparing the test solution A and solution B, the volume proportion of the endotoxin dispersant is set to five different gradient groups of 0, 5%, 10%, 25%, and 50%.

[0014] Preferably, the test solution A is a test solution with a dilution factor not exceeding MVD.

[0015] Preferably, the solution B is a test solution to which endotoxin at a concentration at or near the midpoint of the standard curve is added and which has the same dilution factor as solution A.

[0016] Preferably, when preparing the standard bacterial endotoxin solution C, the concentrations of the three standard bacterial endotoxin solutions in the concentration gradients are 0.5 EU / mL, 0.05 EU / mL, and 0.005 EU / mL, and the concentration of the positive solution is 0.2 EU / mL.

[0017] Preferably, when the limulus amebocyte lysate dynamic colorimetric method is used for detection, the preset OD value is 0.1, the detection wavelength is 405 nm, and the reaction time is set according to the reliability experiment of the standard curve.

[0018] Preferably, the endotoxin dispersant comprises a combination of components capable of achieving at least two functions of destroying micelle structure, adjusting pH, changing the physical state of endotoxin, competitive binding, promoting Brownian motion, adjusting charge distribution, and destroying hydrogen bonds.

[0019] Preferably, the endotoxin dispersant is a phosphate surfactant.

[0020] Preferably, the volume proportion of the endotoxin dispersant is 10%.

[0021] When performing dynamic colorimetric analysis on LNP (lipid nanoparticle) samples, the test results may be suppressed. This suppression may be caused by a variety of factors, including the following:

[0022] 1. Interference of lipid components:

[0023] The lipid components in the LNP sample may encapsulate endotoxins, preventing them from fully contacting the LAL, thus affecting the activation of the LAL. For example, the lipid components may form micelle structures that encapsulate the endotoxins, making them undetectable.

[0024] 2. pH value of the sample:

[0025] When using the kinetic colorimetric method to detect endotoxins, the sample pH significantly affects the activity of the limulus amebocyte lysate (LAS). If the pH of the LNP sample is outside the optimal reaction range of the LAS (typically 6.0-8.0), the reaction between the LAS and endotoxin may be inhibited. For example, a pH that is too high or too low can result in decreased sensitivity of the LAS.

[0026] 3. Enzymes or enzyme inhibitors in the sample:

[0027] If the LNP sample contains serine proteases or their inhibitors, it may affect the enzymatic reaction in the limulus amebocyte lysate (LAL). Serine proteases may enhance the reaction, while their inhibitors may inhibit it. For example, if the test sample contains serine protease inhibitors, it may cause a false negative test result.

[0028] 4. Osmotic pressure of the sample:

[0029] High concentrations of sugars or salts may inhibit the reaction of the limulus amebocyte lysate. If the LNP sample contains a high concentration of osmotic substances, it may inhibit the reaction of the limulus amebocyte lysate.

[0030] 5. Detergents in the sample:

[0031] High concentrations of detergents may inactivate the proteins in the LAL and inhibit the LAL reaction. For example, if detergents are used during the preparation of LNP samples, this may result in false negative results.

[0032] 6. Metal ions in the sample:

[0033] Divalent cations (such as Mg 2+ or Ca 2+ ) have an important influence on the reaction of the limulus amebocyte lysate. If these ions are lacking in the LNP sample, the reaction of the limulus amebocyte lysate may be inhibited. For example, Mg 2+ Insufficient ion concentration may prevent endotoxin from activating the enzyme in the LAL reagent.

[0034] In order to eliminate the interference caused by LNP samples in the dynamic colorimetric detection, the present invention uses an endotoxin dispersant, the working principle of which mainly includes the following points:

[0035] 1. Increase the accessibility of endotoxins:

[0036] Endotoxin dispersants can disrupt the micelle structure formed by the lipid components in the LNP sample, exposing the encapsulated endotoxin molecules and increasing the contact between the endotoxin and the limulus amebocyte lysate. For example, the dispersant may interact with the lipid molecules, disrupting their encapsulation structure.

[0037] 2. Adjust the pH value of the sample:

[0038] The endotoxin dispersant may contain a buffer component, which can adjust the pH value of the LNP sample to the optimal reaction range of the limulus amebocyte lysate (6.0-8.0), thereby improving the sensitivity of the limulus amebocyte lysate.

[0039] 3. Change the physical state of endotoxins:

[0040] Endotoxin dispersants may enhance the reaction of endotoxin with the limulus amebocyte lysate (LAL) by altering the physical state of the endotoxin, such as increasing its solubility or changing its aggregation behavior. For example, dispersants may form complexes with endotoxin molecules, altering their physical properties.

[0041] 4. Competitive Binding:

[0042] Endotoxin dispersants may reduce interference by competitively binding to specific groups on the surface of endotoxins, reducing the interaction between endotoxins and other components in the sample.

[0043] 5. Promote Brownian motion of endotoxins:

[0044] Endotoxin dispersants may increase the freedom of movement of endotoxin molecules and promote their Brownian motion in the solution, thereby increasing the chance of contact with the limulus amebocyte lysate.

[0045] 6. Impact on the charge distribution of endotoxins:

[0046] Endotoxin dispersants may promote the dispersion of endotoxin by changing the charge distribution of endotoxin and reducing the electrostatic interaction between endotoxin molecules.

[0047] 7. Destroy hydrogen bonds between endotoxin molecules:

[0048] Endotoxin dispersants may promote the dispersion of endotoxin molecules by destroying the hydrogen bonds between endotoxin molecules and reducing the aggregation of endotoxin molecules.

[0049] Through the above mechanism, endotoxin dispersants can effectively eliminate the interference caused by LNP samples in dynamic colorimetric detection, improving the accuracy and reliability of detection. In actual operation, the appropriate endotoxin dispersant should be selected according to the specific characteristics of the LNP sample and the interference situation, and its usage conditions should be optimized to achieve the best detection effect.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The method for eliminating interference in bacterial endotoxin detection of LNP type samples provided by the present invention, by introducing an endotoxin dispersant, specifically solves the multi-dimensional interference problems such as lipid-encapsulated endotoxins, pH deviation, and abnormal osmotic pressure in LNP samples. The dispersant releases the encapsulated endotoxin by destroying the lipid micelle structure, and at the same time adjusts the sample pH to the optimal reaction range of the Limulus amebocyte lysate (6.0-8.0), and ensures that the endotoxin is in full contact with the Limulus amebocyte lysate through multiple mechanisms such as changing the physical state of the endotoxin and promoting Brownian motion. Compared with traditional single pretreatment methods (such as simple dilution or heating), it can eliminate interference more comprehensively, avoid false negative or false positive results, and make the detection value closer to the actual endotoxin content, which is particularly suitable for LNP samples with complex lipid components.

[0052] (2) The method provided by the present invention constructs a standardized detection system through strict experimental design (such as setting up 5 dispersant volume ratio gradients and at least 2 parallel tubes) and clear effectiveness judgment criteria (standard curve correlation coefficient |r| ≥ 0.980, recovery rate 50% to 200%, etc.). At the same time, the optimal dosage of the dispersant is verified through interference testing to ensure the consistency of the test results of different batches of LNP samples. This process not only meets the strict requirements of the pharmacopoeia for bacterial endotoxin detection, but also provides repeatable technical support for the quality control of LNP drugs (such as mRNA vaccines). DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0054] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0055] The preparations and instruments used in the following examples are:

[0056] 1. Reagents and consumables

[0057] Reagent / consumables name Manufacturer Item No. Bacterial endotoxin working standard Zhanjiang Andus Biological Co., Ltd. E-010 Bacterial endotoxin test reaction plate Zhanjiang Andus Biological Co., Ltd. S-0520 Kinetic colorimetric limulus amebocyte lysate Zhanjiang Andus Biological Co., Ltd. KC125005 Water for bacterial endotoxin testing Zhanjiang Andus Biological Co., Ltd. W-005 Non-pyrogenic reaction tubes Zhanjiang Andus Biological Co., Ltd. S-1007 Pyrogen-free ampoules Zhanjiang Andus Biological Co., Ltd. S-1003

[0058] 2. Instruments and equipment

[0059] Instrument name factory Model / Article Number Instrument name factory model Biological safety cabinets Haier HR30-IIA2 Endotoxin detector Thermo Fisher Multiskan ET Vortex mixer Qilin Bell V0RTEX-5 pipette Eppendorf 10μL-100μL pipette Eppendorf 100μL-1000μL

[0060] Example 1

[0061] 1. Prepare the experimental solution according to the table below.

[0062]

[0063] ①A is the test solution with a dilution factor not exceeding MVD.

[0064] ②B is a test solution with a known bacterial endotoxin concentration at the midpoint (or nearby point) of the standard curve and the same dilution factor as solution A, i.e., the sample recovery solution.

[0065] ③C is the standard bacterial endotoxin solution used to prepare the standard curve as described under "Standard Curve Reliability Experiment".

[0066] ④D is the negative control.

[0067] 2. The specific preparation methods of solutions A, B, and C are as follows:

[0068] Solution C: Dilute the endotoxin working standard into three concentration gradients, and record the three points of (0.5, 0.05, 0.005) EU / mL as E0.5 、E 0.05 、E 0.005 , the positive solution is 0.2EU / mL, recorded as E 0.2 .

[0069] Solution A:

[0070]

[0071] Solution B:

[0072]

[0073] S 10 Sample preparation method: (the sample stock solution is marked as S, and the sample is finally diluted 40 times)

[0074] Diluted 10 times, recorded as S10 Take 100 μL of the stock solution and add 900 μL of BET water

[0075] 3. Reconstitution of Limulus Amebocyte Lysate

[0076] Reconstitute the limulus amebocyte lysate (LAL) according to the specifications indicated on the kinetic colorimetric method (KC125005) with test water and allow to stand for clarification.

[0077] 4. On-machine testing

[0078] Before loading the sample onto the machine, open the Bioprobe software and enter the relevant information for the sample and reagent (the appropriate method should be selected based on the type of endotoxin). The default OD value is 0.1, the detection wavelength is 405nm, and the time is set according to the calibration curve reliability experiment. Using KC125005 Limulus amebocyte lysate reagent, first take 100μL of solution D / solution C (concentration from low to high) and solution A / solution B and add them to the reaction plate. All samples should be made into at least 2 parallel tubes. Then use a continuous injector or pipette to add 100μL of the reconstituted Limulus amebocyte lysate reagent to each well, click "Export Plate", place the reaction plate into the machine, click "Input Plate", and then click "Start". After the test reaction is completed, export the test data through the software.

[0079] 5. Criteria for judging experimental effectiveness

[0080] The results of series C of solution must meet the requirements of the "standard curve reliability experiment", that is, the standard curve correlation coefficient |r| ≥ 0.980, for the experiment to be valid.

[0081] The recovery rate R of solution B satisfies: 50%≤R≤200%.

[0082] The detection value of the negative control is lower than the detection value at the lowest point of the standard curve or the reaction time is higher than the reaction time at the lowest point of the standard curve.

[0083] The CV of each replicate tube is ≤10%. If the time limit of each replicate tube for the negative control or sample is greater than the time limit of each replicate tube for the lowest concentration of the calibration curve, the CV validity judgment is not performed and the result is displayed as "valid".

[0084] Example 2 Interference elimination experiment (gel limit method, using a gel-based limulus amebocyte lysate with a sensitivity of 0.125 EU / mL, i.e., λ = 0.125 EU / mL)

[0085]

[0086] Note: A is the test solution; B is the test solution positive control; C is the positive control; D is the negative control.

[0087] Solution A:

[0088]

[0089] Solution B:

[0090]

[0091] Solution C:

[0092] 2λ bacterial endotoxin standard solution, i.e. endotoxin solution with a concentration of 0.25EU / ml

[0093] Solution D:

[0094] Endotoxin test water

[0095] Note: In solution B, E 1.0 Indicates a concentration of 1.0 EU / ml endotoxin solution.

[0096] Take 2 limulus amebocyte lysate (specification 0.65mL / tube) with a sensitivity of 0.125EU / ml, add 0.65mL of endotoxin test water to each tube for reconstitution, combine the 2 limulus amebocyte lysate after reconstitution, mix them together, and dispense them into 24 reaction tubes (or ampoules), 0.05mL each, and then according to the gel limit method, take 2 portions of the test solution with the volume proportion of the dispersant being 0%, 5%, 10%, 25%, and 50%, 0.05mL each, and add them to 10 packaged limulus amebocyte lysate reaction tubes in sequence, and take 2 portions of the test solution with the volume proportion of the dispersant being 0%, 5%, 10%, 25%, and 50%, 0.05mL each, and add them to 10 packaged limulus amebocyte lysate reaction tubes in sequence, and take 2 portions of the test solution with the volume proportion of the dispersant being 0%, 5%, 10%, 25%, and 50%, respectively, in solution B. Two portions of 0.05 mL each of the positive control solution for each sample were added sequentially to 10 other aliquoted limulus amebocyte lysate reaction tubes. Two portions of 0.05 mL each of the 2λ bacterial endotoxin standard solution for solution C were added to two aliquoted limulus amebocyte lysate reaction tubes as the positive control tubes for the sample. Two portions of 0.05 mL each of the endotoxin test water for solution D were added to two aliquoted limulus amebocyte lysate reaction tubes as the negative control tubes. After the addition of the samples, the ampoule was sealed with sealing film and gently shaken to mix to avoid bubbles. The ampoule was placed in a thermostat and kept at 37°C ± 1°C for 60 ± 2 minutes. The results were observed and recorded. The results are shown in Table 1.

[0097]

[0098]

[0099] The test results showed that the negative control was negative and the positive control was positive, confirming the validity of the test results. When the volume percentage of the dispersant added was 0%, the standard curve correlation coefficient |r| was less than 0.980, and the recovery rate of Solution B exceeded the range of 50%-200%, making the experiment invalid. This indicates that it is difficult to accurately detect bacterial endotoxins in LNP samples without the use of an endotoxin dispersant. The optimal volume percentage of the dispersant was 10% in Group 3.

[0100] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A method for eliminating interference in bacterial endotoxin detection of LNP type samples, characterized in that: The following steps are involved: S1. Prepare at least three standard bacterial endotoxin solutions C with concentration gradients, and use the standard bacterial endotoxin solutions to perform a standard curve reliability test to determine the detection standard curve of the limulus amebocyte lysate kinetic colorimetric method; S2. Prepare a test solution A with different dispersant volume ratios and a test solution B with endotoxin added at the midpoint concentration of the standard curve, and perform an interference elimination test using the standard bacterial endotoxin solution C, solution A, and solution B. The interference elimination test uses an endotoxin dispersant, which eliminates interference by at least two mechanisms: destroying the micelle structure formed by the lipid components in the LNP sample, adjusting the pH value of the sample to the optimal reaction range of 6.0 to 8.0, changing the physical state of the endotoxin, competitively binding to specific groups on the surface of the endotoxin, promoting the Brownian motion of the endotoxin, affecting the charge distribution of the endotoxin, and destroying the hydrogen bonds between endotoxin molecules; S3. Reconstitute the limulus amebocyte lysate with bacterial endotoxin test water according to the limulus amebocyte lysate specifications; S4. Add negative control solution D, standard bacterial endotoxin solution C, solution A, and solution B to the reaction plate, respectively. Set up at least two parallel tubes for each sample, then add the reconstituted limulus amebocyte lysate (LA-lysate) and perform detection using the limulus amebocyte lysate kinetic colorimetric method. S5. Determine the validity of the experiment based on the following criteria: the correlation coefficient of the standard curve |r| ≥ 0.980, the recovery rate of solution B between 50% and 200%, the negative control test value less than the test value at the lowest point of the standard curve or the reaction time greater than the reaction time at the lowest point of the standard curve, and the CV of each parallel tube result ≤ 10%.

2. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, When preparing the test solution A and solution B, the volume proportion of the endotoxin dispersant was set to five different gradient groups of 0, 5%, 10%, 25%, and 50%.

3. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, The test solution A is a test solution with a dilution factor not exceeding MVD.

4. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, The solution B is a test solution to which endotoxin at a concentration at or near the midpoint of the standard curve is added and which has the same dilution factor as solution A.

5. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, When preparing the standard bacterial endotoxin solution C, the concentrations of the three standard bacterial endotoxin solutions in the concentration gradients are 0.5 EU / mL, 0.05 EU / mL, and 0.005 EU / mL, and the concentration of the positive solution is 0.2 EU / mL.

6. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, When using the limulus amebocyte lysate dynamic colorimetric method for detection, the preset OD value is 0.1, the detection wavelength is 405 nm, and the reaction time is set according to the reliability experiment of the standard curve.

7. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, The endotoxin dispersant comprises a combination of components capable of achieving at least two functions of destroying micelle structure, adjusting pH value, changing physical state of endotoxin, competitive binding, promoting Brownian motion, adjusting charge distribution and destroying hydrogen bonds.

8. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, The endotoxin dispersant is a phosphate surfactant.

9. The method for eliminating interference in bacterial endotoxin detection of LNP type samples according to claim 1, characterized in that, The volume proportion of the endotoxin dispersant is 10%.

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