Lipid nanoparticle bacterial endotoxin detection method and demulsifier

Abstract

The invention discloses a bacterial endotoxin detection method of lipid nanoparticles and a demulsifier, and the detection method comprises the following steps: firstly preparing the demulsifier comprising polyoxyethylene lauryl ether, dimethyl sulfoxide and ethanol, then demulsifying a sample containing the lipid nanoparticles by using the demulsifier, and finally diluting the demulsified sample by using a diluent to obtain the bacterial endotoxin detection product. And determining the content of bacterial endotoxin. According to the invention, the solution containing polyoxyethylene lauryl ether, dimethyl sulfoxide and ethanol is used as the demulsifier of the lipid nanoparticles, the demulsification effect is good, the intracellular toxin wrapped in the demulsifier can be fully released, and the demulsifier does not influence the detection of the intracellular toxin; after the diluent is diluted, when the toxin in the cells is determined, the determination result meets the methodological requirements, and the accuracy of the detection result is ensured; in addition, the method provided by the invention is suitable for detecting different types of lipid nanoparticles.

Description

Technical Field

[0001] This invention relates to the field of biomedical analysis technology, and in particular to a method for detecting bacterial endotoxins in lipid nanoparticles and a demulsifier. Background Technology

[0002] In recent years, with the development of gene therapy and mRNA vaccines, lipid nanoparticles (LNPs) have been widely used as a safe and effective delivery vector. Lipid nanoparticles can effectively encapsulate and protect drug molecules, such as DNA vaccines, RNA vaccines, and other gene editing tools, ensuring their stable delivery to target cells in vivo. In particular, the success of two mRNA vaccines, BNT162b2 and mRNA-1273, produced by Pfizer-BioNTech and Moderna respectively, in combating COVID-19 demonstrates the value and potential of LNPs as a delivery vector (Barbier A., ​​et al. The clinical progress of mRNA vaccines and immunotherapies. NatBiotechnology. 2022 May; 40(6): 840-854).

[0003] However, while lipid nanoparticle-based drug delivery systems have achieved success, they also present unique challenges to drug safety, particularly the detection of bacterial endotoxins. Endotoxins are potent pyrogens found in the outer membranes of Gram-negative bacteria, capable of triggering a strong immune response in the host even at extremely low concentrations. The chemical nature of endotoxin is lipopolysaccharide (LPS), which is mainly composed of three parts: cell-specific polysaccharide, non-specific core polysaccharide, and lipid A (LipidA). Among them, lipid A is the main toxic component of endotoxin (Hannecart-Pokorni E., Dekegel D., Depuydt F., Macromolecular structure of lipopolysaccharides from gram-negative bacteria. Eur. J. Biochem. 1973 Sep; 38(1): 6-13; Rietschel E., et al. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994 Feb; 8(2): 217-225; Rietschel E., et al. The chemical structure of bacterial endotoxin in relation to bioactivity. Immunobiology.1993 Apr; 187(3-5): 169-190). When it enters the human body, endotoxins act on macrophages, neutrophils, and other cells in the body, causing them to produce a variety of cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can lead to severe inflammatory reactions, resulting in fever, circulatory disorders, and even shock and death in the host (Mattern T., et al. Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes. J. Immunol., 1994 Oct;153(7): 2996-3004).Therefore, drug regulatory agencies such as the U.S. Food and Drug Administration (FDA) and China's National Medical Products Administration (NMPA) require endotoxin testing of raw materials, intermediates, and released products for injectable drugs and medical devices such as dialysis solutions and implantable devices used in humans and animals to ensure product safety.

[0004] However, the impact of lipid nanoparticles on endotoxin detection cannot be eliminated by simple dilution. On the one hand, lipid nanoparticles are typically composed of four components: ionizable cationic lipids, cholesterol, neutral phospholipids, and pegylated lipids (PEG-lipids). These lipids interact with LPS, potentially disrupting the aggregation structure of endotoxins and affecting the Limulus Amebocyte Lysate (LAL) reagent reaction. On the other hand, due to the inclusion effect of lipid nanoparticles, endotoxins may be encapsulated within them during LNP preparation and remain undetectable. In this regard, the Chinese Pharmacopoeia stipulates that for novel formulations employing inclusion technology, appropriate methods must be used to destroy the inclusion complex, ensuring complete release of the encapsulated endotoxins before detection (National Pharmacopoeia Commission. Guidelines for the Application of Bacterial Endotoxin Test Methods [M]. Chinese Pharmacopoeia [2020 Edition] (Part IV)).

[0005] The aforementioned issues not only affect the accurate assessment of lipid nanoparticle drug quality but also pose risks to its safety and reliability. Currently, there are no literature or patent reports on endotoxin detection methods for lipid nanoparticles. Therefore, there is an urgent need to develop a scientific, rational, stable, and universal method for detecting endotoxins in lipid nanoparticles, clarifying the processing and operation steps at each stage, eliminating sample interference, and improving the accuracy and reliability of the detection results. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting bacterial endotoxins in lipid nanoparticles and a demulsifier, which can achieve good demulsification of lipid nanoparticles without affecting the detection of bacterial endotoxins.

[0007] To address the aforementioned technical problems, the first aspect of this invention provides a method for detecting bacterial endotoxins in lipid nanoparticles, comprising: S1. A demulsifier for preparing lipid nanoparticles, comprising polyoxyethylene lauryl ether, dimethyl sulfoxide and ethanol; S2. Use the demulsifier obtained in S1 to demulsify the lipid nanoparticle sample; S3. After diluting the sample after demulsification in S2 with a diluent, determine the bacterial endotoxin content.

[0008] In this invention, polyoxyethylene lauryl ether has minimal interference with bacterial endotoxins, while dimethyl sulfoxide and ethanol, although effective at demulsifying lipid nanoparticles, can significantly interfere with endotoxin detection at higher concentrations. This invention uses a solution containing polyoxyethylene lauryl ether, dimethyl sulfoxide, and ethanol as a demulsifier for lipid nanoparticles, achieving excellent demulsification and facilitating the full release of encapsulated endotoxins. Furthermore, the demulsifier does not affect endotoxin detection. After dilution, the endotoxin test results meet methodological requirements, ensuring accuracy. Additionally, the method provided by this invention is applicable to the detection of different types of lipid nanoparticles.

[0009] In one specific embodiment, in S1, the demulsifier is prepared by mixing an aqueous solution of polyoxyethylene lauryl ether with dimethyl sulfoxide and ethanol. Preferably, the concentration of the aqueous solution of polyoxyethylene lauryl ether is 5%-25% (w / w) (e.g., 5%, 10% (w / w), 20% (w / w), 25% (w / w)); the volume ratio of the aqueous solution of polyoxyethylene lauryl ether to dimethyl sulfoxide and ethanol is 2:(0.5-2):(0-1.5) (e.g., 2:0.5:0.5, 2:1:1, 2:1.5:1.5, 2:0.5:1, 2:1:1.5, 2:2:1.5, etc.).

[0010] In one specific embodiment, in step S2, the method for demulsifying the lipid-containing nanoparticle sample involves mixing the lipid-containing nanoparticle sample with bacterial endotoxin water and the demulsifier obtained in step S1, followed by vortex demulsification. Preferably, the volume ratio of the lipid-containing nanoparticle sample, bacterial endotoxin water, and demulsifier is 1:(0.8-1.2):(6-10) (e.g., 1:1:8); the demulsification temperature is 10℃-50℃; the demulsification time is 35s-120s; and the vortexing condition is 1500-2500rpm.

[0011] In one specific embodiment, in step S3, the diluent is an aqueous solution of polyoxyethylene lauryl ether with a concentration of 0.5%-4% (w / w). Preferably, in step S3, the final dilution factor of the lipid nanoparticle sample is 200-800 times.

[0012] In one specific embodiment, the lipid nanoparticle sample contains one or more of the following: ALC-0315, DSPC, cholesterol, ALC-0159, SM102, and PEG-DMG.

[0013] To address the aforementioned technical problems, a second aspect of the present invention provides a demulsifier for lipid nanoparticles, comprising polyoxyethylene lauryl ether, dimethyl sulfoxide, and ethanol.

[0014] The demulsifier provided by this invention can demulsify lipid nanoparticles, fully release the intracellular toxins encapsulated within them, and will not affect the detection of intracellular toxins.

[0015] In one specific embodiment, the demulsifier is obtained by mixing an aqueous solution of polyoxyethylene lauryl ether with dimethyl sulfoxide and ethanol, wherein the concentration of the aqueous solution of polyoxyethylene lauryl ether is 5%-5% (w / w) (e.g., 5% (w / w), 10% (w / w), 20% (w / w), 25% (w / w)); and the volume ratio of the aqueous solution of polyoxyethylene lauryl ether to dimethyl sulfoxide and ethanol is 2:(0.5-2):(0-1.5) (e.g., 2:0.5:0.5, 2:1:1, 2:1.5:1.5, 2:0.5:1, 2:1:1.5, 2:2:1.5, etc.). Detailed Implementation

[0016] The technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0017] The experimental instruments used in this embodiment of the invention are as follows: MS 3 Digital Vortex Oscillator, Manufacturer: IKA; AS2-4S1 Biosafety Cabinet, Manufacturer: ESCO; SYNERGY HTX microplate reader, manufacturer: Biotek; MY-E3 electric dispenser, manufacturer: Eppendorf; Single-channel pipette, manufacturer: Eppendorf; Electronic balance, manufacturer: Mettler Toledo Instruments (Shanghai) Co., Ltd.

[0018] The experimental reagents used in this embodiment of the invention are as follows: Limulus amebocyte lysate (LAL) reagent, source: Charles River, detection range: 50-0.005 EU / mL; Bacterial endotoxin standards, source: Charles River; Water used for bacterial endotoxin testing, source: Charles River; Anhydrous ethanol, source: Shanghai Husheng; Dimethyl sulfoxide, source: Sigma-Aldrich; Polyoxyethylene lauryl ether, source: Yuanye Biotechnology; Qulaton X-100 (TX-100), source: Sigma-Aldrich; Lipid nanoparticle samples, sourced from Hangzhou Mingde Biomedical Technology Co., Ltd., batch numbers: M1010501, M1010406, M1010901, M1010601, M1011004, M1011104, M1010613, M1010702. Among them, lipid nanoparticles with batch numbers M1010501, M1010406, M1010901, M1010601, and M1011004 contain 4.2 mg / mL ALC-0315, 0.9 mg / mL DSPC, 1.8 mg / mL cholesterol, 0.51 mg / mL ALC-0159, and an mRNA concentration of 0.3 mg / mL. Lipid nanoparticles with batch number M1011104 contain 4.2 mg / mL ALC-0315, 0.9 mg / mL DSPC, 1.8 mg / mL cholesterol, and 0.51 mg / mL ALC-0159. Lipid nanoparticles with batch number M1010613 contain 1.46 mg / mL ALC-0315, 0.32 mg / mL DSPC, 0.63 mg / mL cholesterol, 0.18 mg / mL ALC-0159, and an mRNA concentration of 0.105 mg / mL. Lipid nanoparticles with batch number M1010702 contain 0.39 mg / mL SM102, 0.09 mg / mL DSPC, 0.16 mg / mL cholesterol, and 0.04 mg / mL PEG-DMG, with an mRNA concentration of 0.03 mg / mL.

[0019] Example 1: Screening of Demulsification Conditions 1) Preparation of bacterial endotoxin standard solution: According to the reconstitution volume provided on the standard CoA, add water for bacterial endotoxin test to the lyophilized bacterial endotoxin standard powder (CSE) to reconstitute the lyophilized product to a concentration of 50 EU / mL. For freshly reconstituted standards, vortex at approximately 2000 rpm for 5-10 minutes; for reconstituted standards, vortex for 1-5 minutes before use.

[0020] 2) Reagent preparation Preparation of 1% (v / v) TX-100 solution: Add 0.05 mL of TX-100 to 4.95 mL of water for bacterial endotoxin testing, and vortex thoroughly to mix to obtain a 1% (v / v) TX-100 solution.

[0021] Preparation of 9% (w / w) polyoxyethylene lauryl ether solution: Weigh 3.6 g of polyoxyethylene lauryl ether into 36.4 mL of bacterial endotoxin test water, and vortex thoroughly to dissolve it, thus obtaining a 9% (w / w) polyoxyethylene lauryl ether solution.

[0022] Preparation of 15% (w / w) polyoxyethylene lauryl ether solution: Weigh 3.0 g of polyoxyethylene lauryl ether into 17 mL of water for bacterial endotoxin testing, and vortex thoroughly to dissolve it, thus obtaining a 15% (w / w) polyoxyethylene lauryl ether solution.

[0023] Preparation of 20% (w / w) polyoxyethylene lauryl ether solution: Weigh 5.0 g of polyoxyethylene lauryl ether into 20 mL of water for bacterial endotoxin testing, and vortex thoroughly to dissolve it, thus obtaining a 20% (w / w) polyoxyethylene lauryl ether solution.

[0024] Preparation of 0.18% (w / w) polyoxyethylene lauryl ether solution: In an endotoxin-free glass test tube, dilute the 9% (w / w) polyoxyethylene lauryl ether solution 50 times with water for bacterial endotoxin testing to obtain a 0.18% (w / w) polyoxyethylene lauryl ether solution.

[0025] Preparation of 0.5% (w / w) polyoxyethylene lauryl ether solution: In an endotoxin-free glass test tube, dilute the 20% (w / w) polyoxyethylene lauryl ether solution 40 times with water for bacterial endotoxin testing to obtain a 0.5% (w / w) polyoxyethylene lauryl ether solution.

[0026] Preparation of 1% (w / w) polyoxyethylene lauryl ether solution: In an endotoxin-free glass test tube, dilute the 20% (w / w) polyoxyethylene lauryl ether solution 20 times with water for bacterial endotoxin testing to obtain a 1% (w / w) polyoxyethylene lauryl ether solution.

[0027] Demulsifier DC: In an endotoxin-free glass test tube, add 1 mL of 9% (w / w) polyoxyethylene lauryl ether solution and 1 mL of dimethyl sulfoxide, vortex and mix well to obtain demulsifier DC.

[0028] Demulsifier EC: Weigh 2.5 g of polyoxyethylene lauryl ether into 50 mL of anhydrous ethanol, and vortex thoroughly to dissolve it, thus obtaining demulsifier EC.

[0029] Demulsifier DEC-9: In an endotoxin-free glass test tube, add 400 μL of 9% (w / w) polyoxyethylene lauryl ether solution, 200 μL of dimethyl sulfoxide and 200 μL of anhydrous ethanol, and vortex to mix well to obtain demulsifier DEC-9.

[0030] Demulsifier DEC-15: In an endotoxin-free glass test tube, add 400 μL of 15% (w / w) polyoxyethylene lauryl ether solution, 200 μL of dimethyl sulfoxide and 200 μL of anhydrous ethanol, and vortex to mix well to obtain demulsifier DEC-15.

[0031] Demulsifier DEC-20: In an endotoxin-free glass test tube, add 400 μL of 20% (w / w) polyoxyethylene lauryl ether solution, 200 μL of dimethyl sulfoxide and 200 μL of anhydrous ethanol, and vortex to mix well to obtain demulsifier DEC-20.

[0032] 3) Demulsification of lipid nanoparticle samples Condition 1 for breaking the milk: Take 100 μL of lipid nanoparticles as test sample into an endotoxin-free glass tube, add 100 μL of water for bacterial endotoxin testing, and then add 800 μL of 1% (v / v) TX-100 solution. Vortex thoroughly to obtain the demulsified test sample. Then, in an endotoxin-free glass tube, dilute 100 times with water for bacterial endotoxin testing (the final dilution factor of the test sample is 1000 times).

[0033] Take 100 μL of lipid nanoparticle test sample into an endotoxin-free glass tube, add 100 μL of 50 EU / mL bacterial endotoxin standard solution, and then add 800 μL of 1% (v / v) TX-100 solution. Vortex thoroughly to obtain the positive control solution of the test sample. Then, in an endotoxin-free glass tube, dilute 100 times with water for bacterial endotoxin testing (the final dilution factor of the test sample is 1000 times).

[0034] Condition 2 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 1 in that the dilution factor is 80 times (the final dilution factor of the test sample is 800 times).

[0035] Condition 3 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 1 in that the demulsifier is 400 μL of demulsifier DC, and the dilution factor is 40 times (the final dilution factor of the test sample is 240 times).

[0036] Condition 4 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 1 in that the demulsifier is 800 μL of demulsifier DC, and the dilution factor is 40 times (the final dilution factor of the test sample is 400 times).

[0037] Condition 5 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 4 in that, during demulsification, the sample is placed in a water bath and heated at 55°C for 15 minutes.

[0038] Condition 6 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 4 in that the demulsifier is 800 μL of demulsifier EC, and the diluent is 0.18% (w / w) polyoxyethylene lauryl ether solution.

[0039] Condition 7-1 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 4 in that the demulsifier is 800 μL of demulsifier DEC-9.

[0040] Condition 7-2 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 4 in that the demulsifier is 800 μL of demulsifier DEC-15.

[0041] Condition 7-3 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 4 in that the demulsifier is 800 μL of DEC-20.

[0042] Condition 7-4 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in the demulsification condition 7-3 in that, during dilution, the diluent is 0.5% (w / w) polyoxyethylene lauryl ether solution, and the dilution factor is 20 times (the final dilution factor of the test sample is 200 times).

[0043] Condition 7-5 for breaking the milk: The preparation process of the demulsified sample and the positive control solution differs from that in demulsification condition 7-4 in that the diluent used during dilution is a 1% (w / w) polyoxyethylene lauryl ether solution.

[0044] Under different demulsification conditions, the batch numbers of lipid nanoparticles used in preparing the demulsified test sample and the positive control solution of the test sample also differ, as shown in Table 1.

[0045] 4) Preparation of a series of standard curve solutions for bacterial endotoxins Take the above 50 EU / mL bacterial endotoxin standard solution and gradually prepare a series of endotoxin standard curve solutions of 5 EU / mL, 0.5 EU / mL, 0.05 EU / mL, and 0.005 EU / mL using water for bacterial endotoxin testing.

[0046] 5) Preparation of Limulus Amebocyte Lysate (LAL) Reagent Reconstitute the horseshoe crab reagent with water for bacterial endotoxin testing according to the manufacturer's instructions. Let it stand at room temperature for 5-10 minutes to dissolve the contents. Gently shake before use to ensure thorough mixing. The horseshoe crab reagent should be reconstituted close to the time of use. If multiple bottles of horseshoe crab reagent are to be used at once, they must be thoroughly reconstituted separately before combining all the reagents for use.

[0047] 6) Bacterial endotoxin detection Microplate reader parameter settings: Preheat the microplate reader to 37°C before reading the plate and maintain this temperature during the test. Set the instrument according to the following parameters: detection wavelength 405 nm, well interval 30 s, total reading time 4500 seconds, absorbance increment 0.1 OD.

[0048] Detection method: In a pyrogen-free endotoxin bacterial endotoxin test plate, add 100 μL of endotoxin solution standard curve series solutions (5 EU / mL, 0.5 EU / mL, 0.05 EU / mL, 0.005 EU / mL), negative control (water for bacterial endotoxin test), demulsified test sample, and positive control solution, in duplicate. Then add 100 μL of reconstituted Limulus Amebocyte Lysate (LAL) reagent to each well, and transfer the plate to a microplate reader for detection.

[0049] The samples and test results are shown in Table 1: Table 1

[0050] Table 1 shows that: TX-100 and anhydrous ethanol have a good demulsification effect on lipid nanoparticles, but their presence greatly interferes with the detection of endotoxins, causing the positive control recovery rate of the test sample to be far below the standard range of 50%-200% (demulsification condition 1 and demulsification condition 2).

[0051] The demulsifier DC does not interfere with the detection of endotoxins, and the recovery rate of the positive control of the test sample meets the standard requirement of 50%-200%. However, its demulsification effect on lipid nanoparticles is insufficient. The solution remains turbid after demulsification treatment, and it is impossible to make the solution completely clear after demulsification treatment by increasing the proportion of demulsifier DC (demulsification condition 3 and demulsification condition 4).

[0052] Heat treatment (demulsification condition 5) allows the demulsifier DC to completely demulsify the lipid nanoparticles, and the treated test solution becomes completely clear, but it interferes with endotoxin detection, and the positive control recovery rate of the test sample is less than 50%.

[0053] In summary, demulsification conditions 7-4 and 7-5 meet the recovery rate criteria and do not interfere with the detection of bacterial endotoxins.

[0054] Example 2: Dispersant Concentration Range and Sample Dilution Factor 1) Reagent preparation Preparation of demulsifier DEC-20: 5 g of polyoxyethylene lauryl ether was dissolved in 20 mL of water for bacterial endotoxin testing. The solution was thoroughly shaken to obtain a 20% (w / w) polyoxyethylene lauryl ether solution. Subsequently, 400 μL of the 20% (w / w) polyoxyethylene lauryl ether solution was taken, and 200 μL of dimethyl sulfoxide and 200 μL of anhydrous ethanol were added. The mixture was then stirred to obtain a lipid nanoparticle demulsifier.

[0055] Preparation of bacterial endotoxin standard solution: Same as in Example 1 (50 EU / mL); Preparation of demulsified test sample: Take 100 μL of lipid nanoparticles (sample batch number in this example is M1010501) into an endotoxin-free glass test tube, add 100 μL of water for bacterial endotoxin testing, then add 800 μL of demulsifier DEC-20, and vortex thoroughly to obtain the demulsified test sample. At this time, the test sample solution is diluted 10 times.

[0056] Preparation of the positive control solution: Take 100 μL of lipid nanoparticles (sample batch number in this example is M1010501) into an endotoxin-free glass test tube, add 100 μL of 50 EU / mL bacterial endotoxin standard solution, and then add 800 μL of demulsifier DEC-20. Vortex thoroughly to obtain the positive control solution. At this point, the test solution is diluted 10 times, and the bacterial endotoxin standard is spiked at a concentration of 5 EU / mL.

[0057] Dispersant preparation: In endotoxin-free glass tubes, 20% (w / w) polyoxyethylene lauryl ether solution was diluted to 0.4%, 0.5%, 1%, 2% and 4% (w / w) with water for bacterial endotoxin testing to obtain dispersants of different concentrations.

[0058] Sample dilution preparation: In an endotoxin-free glass test tube, take 100 μL of the demulsified sample / positive control solution of the test sample and add 900 μL of dispersant. Vortex thoroughly to obtain a 100-fold dilution of the demulsified sample / positive control solution of the test sample as a stock solution.

[0059] Take a 100-fold diluted stock solution, add a dispersant, and vortex thoroughly to obtain test sample demulsification samples / test sample positive control solutions with different dilution ratios. The diluent type and corresponding dilution ratio are shown in Table 2.

[0060] Preparation of endotoxin solution standard curve series solutions: Same as in Example 1; Preparation of horseshoe crab reagent: Same as in Example 1; 2) Bacterial endotoxin detection The parameters for the microplate reader and the detection method are the same as in Example 1. The samples and test results are shown in Table 2. Table 2

[0061] As shown in Table 2, after demulsifying the LNP samples using the demulsifier DEC-20, the samples were diluted using a 0.5%-4% (w / w) polyoxyethylene lauryl ether aqueous solution as a dispersant. At final dilution ratios of 200-fold or 400-fold, the positive control recoveries of the test samples ranged from 76% to 122%, all meeting the standard requirements (50%-200%). At a final dilution ratio of 400-fold, the positive control recoveries were closer to 100%. However, when the concentration of the polyoxyethylene lauryl ether aqueous solution in the dispersant was below 0.5% (w / w), the positive control recoveries were below 50%, failing to meet the standard requirements (50%-200%).

[0062] Example 3: Detection of bacterial endotoxin content in lipid nanoparticle samples 1) Reagent preparation Preparation of demulsifier DEC-20: Same as in Example 2; Preparation of bacterial endotoxin standard solution: Same as in Example 1 (50 EU / mL); Preparation of demulsified test sample: Take 100 μL of lipid nanoparticles (samples in this example are from different batches) into an endotoxin-free glass test tube, add 100 μL of water for bacterial endotoxin testing, then add 800 μL of demulsifier DEC-20, and vortex thoroughly to obtain the demulsified test sample. At this point, the test sample solution is diluted 10 times.

[0063] Preparation of the positive control solution: Take 100 μL of lipid nanoparticles (samples in this example are from different batches) into an endotoxin-free glass test tube, add 100 μL of 50 EU / mL bacterial endotoxin standard solution, and then add 800 μL of demulsifier DEC-20. Vortex thoroughly to obtain the positive control solution. At this point, the test solution is diluted 10 times, and the bacterial endotoxin standard is spiked at a concentration of 5 EU / mL.

[0064] Dispersant preparation: In an endotoxin-free glass test tube, a 20% (w / w) polyoxyethylene lauryl ether solution was diluted to 2% (w / w) with water for bacterial endotoxin testing to obtain the dispersant.

[0065] Sample dilution preparation: In an endotoxin-free glass test tube, take 100 μL of the demulsified sample / positive control solution of the test sample and add 900 μL of dispersant. Vortex thoroughly to obtain a 100-fold dilution of the demulsified sample / positive control solution of the test sample as a stock solution.

[0066] Take a 100-fold diluted stock solution, add more dispersant, and vortex thoroughly to obtain demulsified samples / positive control solutions of each batch of test samples at a 400-fold dilution. The sample batch numbers are shown in Table 3.

[0067] Preparation of endotoxin solution standard curve series solutions: Same as in Example 1; Preparation of horseshoe crab reagent: Same as in Example 1; 2) Bacterial endotoxin detection The parameters for the microplate reader and the detection method are the same as in Example 1. The samples and test results are shown in Table 3. Table 3

[0068] As can be seen from the results in Table 3, using the processing method of this embodiment, after demulsifying the LNP samples with the demulsifier DEC-20, the samples were diluted with a 2% (w / w) polyoxyethylene lauryl ether aqueous solution as a dispersant. At a final dilution factor of 400 times, the bacterial endotoxin detection recovery rates of different batches of lipid nanoparticle samples after demulsification were all between 70% and 118%, meeting the standard requirements (50%-200%). Furthermore, Table 3 also shows that this embodiment is applicable to lipid nanoparticle samples with different lipid compositions and containing different concentrations of mRNA, demonstrating good versatility.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the bacterial endotoxin test of a lipid nanoparticle, characterized in that, The method comprises the following steps: S1, preparing a demulsifier for lipid nanoparticles, which comprises polyoxyethylene lauryl ether, dimethyl sulfoxide and ethanol; S2, demulsifying a sample containing lipid nanoparticles with the demulsifier obtained in S1; S3, diluting the sample demulsified in S2 with a diluent, and then determining the content of bacterial endotoxin.

2. The liposomal nanoparticle bacterial endotoxin test method according to claim 1, wherein, In S1, the demulsifier is prepared by mixing an aqueous polyoxyethylene lauryl ether solution with dimethyl sulfoxide and ethanol.

3. The method of liposomal nanoparticle bacterial endotoxin test according to claim 2, wherein, The concentration of the aqueous polyoxyethylene lauryl ether solution is 5%-25% (w / w); The volume ratio of the aqueous polyoxyethylene lauryl ether solution to dimethyl sulfoxide and ethanol is 2: (0.5-2): (0-1.5).

4. The method of liposomal nanoparticle bacterial endotoxin test according to claim 1, wherein, In S2, the sample containing lipid nanoparticles is demulsified by mixing the sample with bacterial endotoxin water and the demulsifier obtained in S1, and then vortexing.

5. The method of liposomal nanoparticle bacterial endotoxin test according to claim 4, wherein, When demulsifying, the volume ratio of the sample containing lipid nanoparticles to bacterial endotoxin water and the demulsifier is 1: (0.8-1.2): (6-10); The demulsification temperature is 10-50℃; The demulsification time is 35-120s; The vortexing condition is 1500-2500rpm.

6. The liposomal nanoparticle bacterial endotoxin test method according to claim 1, wherein, In S3, the diluent is an aqueous polyoxyethylene lauryl ether solution with a concentration of 0.5%-4% (w / w).

7. The method of liponanoparticle bacterial endotoxin test according to claim 1, wherein, In S3, the final dilution multiple of the sample containing lipid nanoparticles is 200-800.

8. The liposomal nanoparticle bacterial endotoxin test method according to claim 1, wherein, The lipid nanoparticles in the sample containing lipid nanoparticles include one or more of ALC-0315, DSPC, cholesterol, ALC-0159, SM102 and PEG-DMG.

9. A demulsifier for lipid nanoparticles, characterized in that, The method comprises polyoxyethylene lauryl ether, dimethyl sulfoxide and ethanol.

10. The lipid nanoparticle with a demulsifier of claim 9, wherein, The demulsifier is obtained by mixing an aqueous polyoxyethylene lauryl ether solution with dimethyl sulfoxide and ethanol, wherein the concentration of the aqueous polyoxyethylene lauryl ether solution is 5%-25% (w / w); the volume ratio of the aqueous polyoxyethylene lauryl ether solution to dimethyl sulfoxide and ethanol is 2: (0.5-2): (0-1.5).

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