A method for determining the content of lipid components in lipid nanoparticles

Abstract

The present invention discloses a detection method for determining the content of lipid components in lipid nanoparticles, wherein a high performance liquid chromatography and a detector are used in series to determine the lipid components and content in the lipid nanoparticles, wherein the lipid nanoparticles include cholesterol, auxiliary phospholipids, pegylated lipids, cationic lipids, anionic lipids and ionizable lipids. The detection method provided by the present invention can simultaneously separate and detect multiple lipids, has a wide detection range, and can ensure good detection result quality, has good separation, high accuracy, and good repeatability, and can meet complex and diverse lipid detection needs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical analysis, and in particular to a detection method for determining the content of lipid components in lipid nanoparticles. Background Art

[0002] In recent years, nucleic acid drugs have achieved remarkable achievements in disease prevention and treatment, and the key to success depends in part on the development of nucleic acid delivery systems. At present, common nucleic acid drug carriers include lipid nanoparticles (LNP), viral vectors, polymer nanocarriers and exosomes. Among them, nucleic acid drugs are encapsulated by LNP to protect them from degradation by nucleases, and at the same time improve the efficiency of cell uptake and expression. In addition, compared with other types of nucleic acid drug delivery systems, LNP has many advantages, such as high nucleic acid encapsulation rate and effective cell transfection, strong tissue penetration, low cytotoxicity and immunogenicity, and more conducive to drug delivery. These advantages make LNP an excellent nucleic acid delivery system. Therefore, nucleic acid drugs loaded with LNP have higher bioavailability and tissue selectivity.

[0003] At present, mainstream LNPs are composed of four types of lipids, namely ionizable cationic lipids, auxiliary phospholipids, cholesterol and PEGylated lipids. Cationic lipids / ionizable lipids account for almost half of the total lipid content. They are positively charged under acidic conditions and can encapsulate nucleic acid drugs through electrostatic binding; they are electrically neutral under physiological conditions and therefore do not produce cytotoxicity. Cholesterol, as a natural lipid, promotes the fusion of LNPs with cell membranes by regulating the filling, fluidity and permeability of lipid membranes. Auxiliary phospholipids are also natural lipids that can stabilize the morphology and structure of LNPs. PEGylated lipids can shield the surface of LNPs, thereby protecting them from phagocytosis by mononuclear macrophages and preventing LNP aggregation. In addition, the content of PEGylated lipids can also determine the size of LNPs. However, lipid hydrolysis and / or oxidation may occur during the preparation, transportation and storage of LNP drugs, resulting in changes in the lipid ratio. Related literature studies have shown that slight changes in the ratio of the four lipids will have a significant impact on the properties and delivery efficiency of LNP (Buschmann MD, Carrasco MJ, Alisetty S, Paige M, GabrielAlameh M, Weissman D. Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines 2021, 9, 65.). Therefore, it is necessary to develop corresponding analytical methods to monitor the content of LNP lipid components during research and development and production.

[0004] In 2018, the US FDA issued industry guidance for liposome drug development, which determined that the identification of lipid species and the quantification of components are key quality attributes of LNP delivery systems. Since then, analytical methods have been developed to separate lipids in LNPs based on reversed-phase ion pair chromatography and then quantify the separated lipids using ELSD or CAD detectors.

[0005] There are problems in the detection methods in the prior art to varying degrees, such as the invention patent with publication number CN116359396A, which discloses a method for detecting lipid composition and content in lipid nanoparticles, however, the method can only detect phospholipids, and the detection range is limited and the practicality is poor. A document has been published on an analytical method for detecting lipid content and stability in lipid nanoparticles (Caleb Kinsey, Tian Lu, Alyssa Deiss, Kim Vuolo. Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector. Electrophoresis 2022.), however, using this method, different mobile phases need to be used when detecting different cationic lipids, and the mobile phase composition is complex, the preparation process is cumbersome, and the detection efficiency is reduced. In addition, the method can only detect 4 kinds of lipids at the same time, and there are also problems of small detection range and poor practicality, and the baseline of the detection result spectrum also has more burrs, and the stability and accuracy are poor.

[0006] In summary, there is an urgent need in this field for a detection method for determining the content of lipid components in lipid nanoparticles, which can simultaneously separate and detect multiple lipids, have a wide detection range and can ensure good quality of test results, have high separation and good accuracy, and can meet complex and diverse lipid detection needs. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a detection method for determining the content of lipid components in lipid nanoparticles, which uses a high performance liquid chromatography and a detector in series to determine the lipid components and content in the lipid nanoparticles, wherein the lipid nanoparticles include cholesterol, auxiliary phospholipids, pegylated lipids, cationic lipids, anionic lipids and ionizable lipids;

[0008] In the high performance liquid chromatography, the chromatographic column is Acclaim 300C18, with specifications of 150×2.1 mm, 3.0 um; the mobile phase A is triethylamine acetic acid aqueous solution, and the mobile phase B is triethylamine acetic acid methanol solution.

[0009] Specifically, the high performance liquid chromatography adopts a gradient elution method, and the procedure is:

[0010]

[0011] Preferably, the high performance liquid chromatography adopts a gradient elution method, and the procedure is:

[0012]

[0013] Specifically, the detector is an evaporative light scattering detector or a charged aerosol detector.

[0014] Specifically, the detection parameters of the charged aerosol detector are: filtration constant of 3.6-20s, atomization temperature of 30-70°C, and collection frequency of 2-20Hz.

[0015] Preferably, the detection parameters of the charged aerosol detector are: filtration constant of 10s, atomization temperature of 50°C, and collection frequency of 10Hz.

[0016] Specifically, in the lipid nanoparticles:

[0017] The auxiliary phospholipids include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE);

[0018] The PEGylated lipids include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), Chol-PEG10000, and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159);

[0019] The cationic lipids include (2,3-dioleoyl-propyl)trimethylamine (DOTAP) and 1,2-dioleyl-3-dimethylamino-propane (DODMA);

[0020] The anionic lipids include distearoylphosphatidic acid (DSPA);

[0021] The ionizable lipids include DLin-MC3-DMA, SM-102 and ALC0315.

[0022] Preferably, when the lipid nanoparticles include DMPC, cholesterol, DMG-PEG2000, ALC0159, DOPC, SM102, ALC0315, DSPC, DLin-MC3-DMA and DODMA, they can be detected simultaneously.

[0023] Specifically, the concentration of the ion pair reagent TEAA in the mobile phase A and the mobile phase B is 5-10 mM.

[0024] Preferably, the concentration of the ion pair reagent TEAA in the mobile phase A and the mobile phase B is 10 mM.

[0025] Specifically, the column temperature of the chromatographic column is 30-60° C., and the flow rate is 0.4 mL / min.

[0026] Preferably, the column temperature of the chromatographic column is 50°C.

[0027] Specifically, the injection volume was 10-20 μL.

[0028] Preferably, the injection volume is 10 μL.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The detection method provided by the present invention can simultaneously determine at least 10 lipid components, has strong specificity and high separation, and the peak shape of the detection result graph is symmetrical, which is conducive to accurate quantification and has high detection quality. It can quantify multiple lipid components in lipid nanoparticles at the same time, has a wide detection range, and is highly practical. It can more efficiently control the quality of lipid nanoparticle samples and meet the complex and diverse lipid detection needs.

[0031] 2. The detection method provided by the present invention has simple steps and is easy to operate. It does not require complicated pretreatment or pretreatment of the sample, and the detection process is convenient and efficient.

[0032] 3. The detection method provided by the present invention can adjust the analysis time according to different LNP detection requirements, thereby saving more time costs for subsequent steps of drug development and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the HPLC graph of 10 lipid component reference solutions;

[0034] Figure 2 is the HPLC profile of the LNP-F1 test solution;

[0035] Figure 3 is the HPLC graph of LNP-F2 test solution;

[0036] Figure 4 is the HPLC graph of LNP-F3 test solution;

[0037] Figure 5 is the HPLC profile of the LNP-F4 test solution;

[0038] Figure 6 It is the linear relationship diagram of DMPC;

[0039] Figure 7 It is a linear relationship graph of cholesterol;

[0040] Figure 8 It is the linear relationship graph of DMG-PEG2000;

[0041] Fig. 9 It is the linear relationship graph of ALC0159;

[0042] Fig.10 It is the linear relationship diagram of DOPC;

[0043] Fig.11 It is the linear relationship diagram of SM102;

[0044] Fig.12 It is the linear relationship graph of ALC0315;

[0045] Fig.13 It is the linear relationship diagram of DSPC;

[0046] Fig.14 It is the linear relationship diagram of DLin-MC3-DMA;

[0047] Fig.15 It is the linear relationship diagram of DODMA. DETAILED DESCRIPTION

[0048] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] Embodiment 1

[0050] In this example, the method is used to detect the lipid components and contents in lipid nanoparticle samples prepared from DMPC, cholesterol, DMG-PEG2000, DOPC, SM-102, DSPC, DODMA, ALC0315 and ALC0159 under different prescriptions. The specific operation is as follows:

[0051] 1. Preparation of lipid nanoparticles

[0052] According to the prescription in Table 1, appropriate amounts of DMPC, cholesterol, DMG-PEG2000, DOPC, SM-102, DSPC, DODMA, ALC0315 and ALC0159 were weighed and dissolved in anhydrous ethanol as the ethanol phase, and the mRNA / siRNA solution was used as the aqueous phase. The aqueous phase and the ethanol phase were mixed using a microfluidic preparation instrument to prepare lipid nanoparticles. The received mixed solution was diluted 40 times with phosphate buffer, and the diluted mixed solution was ultrafiltered and centrifuged using a Vivaspin Turbo 15 centrifugal filter. The collected samples were LNP samples of different lipid components in each group.

[0053] Table 1 Lipid formulations of different LNP samples

[0054] name LNP-F1 / molar concentration ratio LNP-F2 / molar concentration ratio LNP-F3 / molar concentration ratio LNP-F4 / molar concentration ratio DMPC 10 / / / cholesterol 38.5 38.5 38.5 38.5 DMG-PEG2000 1.5 / 1.5 1.5 DOPC / 10 / / SM102 50 / / 50 DSPC / / 10 10 DODMA / / 50 / ALC0315 / 50 / / ALC0159 / 1.5 / /

[0055] 2. Investigation of the separation degree of the detection method

[0056] 1. Experimental methods

[0057] Preparation of reference substances and sample pretreatment:

[0058] (1) Reference substance solution: Accurately weigh appropriate amounts of DMPC, cholesterol, DMG-PEG2000, DOPC, SM-102, DSPC, DODMA, ALC0315, and ALC0159 into a volumetric flask, add appropriate amount of anhydrous ethanol for ultrasonic dissolution, and then dilute to the mark with anhydrous ethanol to prepare a reference substance stock solution containing approximately 500 μg of DMPC, 750 μg of cholesterol, 200 μg of DMG-PEG2000, 500 μg of DOPC, 1500 μg of SM-102, 500 μg of DSPC, 1500 μg of DODMA, 1500 μg of ALC0315, and 150 μg of ALC0159 per 1 mL. Subsequently, dilute the reference substance stock solution 20 times with anhydrous ethanol to prepare the reference substance solution.

[0059] (2) Test solution: Take the four groups of LNP samples prepared, add 9 times the volume of anhydrous ethanol to break the emulsion and dissolve them, and use them as the test solution.

[0060] The content of each lipid component in the lipid nanoparticles was determined by high performance liquid chromatography, and the instrument and chromatographic conditions were as follows:

[0061] (1) Chromatography: Agilent 1260Ⅱ high performance liquid chromatograph;

[0062] (2) Chromatographic column: Acclaim 300C18, 150×2.1 mm, 3.0 μm;

[0063] (3) Mobile phase: Mobile phase A: 10 mM triethylamine acetic acid aqueous solution; Mobile phase B: 10 mM triethylamine acetic acid methanol solution;

[0064] (4) The elution procedure is shown in Table 2 below:

[0065] Table 2 Elution program

[0066]

[0067] (5) Flow rate: 0.4 mL / min;

[0068] (6) Column temperature: 50°C;

[0069] (7) Detector: CAD, filtration constant 10s, atomization temperature 50°C, acquisition frequency 10Hz;

[0070] (8) Inject the test sample and reference sample solutions into a high performance liquid chromatograph, perform the test under the above conditions, and record the chromatograms.

[0071] 2. Results Analysis

[0072] like Figure 1-5 As shown, in the reference solution, the chromatographic peak separation of the 10 lipid components is good; the lipid components in the four groups of LNP test solutions are consistent with the corresponding lipid peak time in the reference. The results show that the detection conditions provided by the present invention can simultaneously meet the separation and detection of multiple lipid components such as DMPC, cholesterol, DMG-PEG2000, DOPC, SM-102, DSPC, DODMA, DLin-MC3-DMA, ALC0315, and ALC0159, and the detection results are accurate and conducive to further analysis.

[0073] Embodiment 2

[0074] In this embodiment, the performance analysis of the method provided by the present invention was carried out to examine its linear range, accuracy and repeatability. The detection conditions of the performance analysis are the same as those in the first embodiment and will not be described in detail here.

[0075] 1. Investigation of the linear range of the detection method

[0076] 1. Experimental methods

[0077] (1) Preparation of linear stock solution:

[0078] The reference substance stock solution in Example 1 was used as the linear stock solution.

[0079] (2) The preparation flow chart of the linear solution is shown in Table 3 below:

[0080] Table 3 Linear solution preparation flow chart

[0081]

[0082] (3) Detect each linear solution according to the high performance liquid chromatography method in Example 1, and record the chromatogram and the peak area of ​​each lipid component.

[0083] 2. Results Analysis

[0084] Linear regression was performed based on the concentration and peak area of ​​each lipid component. The results are shown in Table 4 and Figure 6-15 shown.

[0085] Table 4 Results of linear range investigation of each lipid component

[0086]

[0087]

[0088] The results show that all lipid components have a good linear relationship in the concentration range of 12% to 150%.

[0089] 2. Accuracy of the detection method

[0090] 1. Experimental methods

[0091] (1) Preparation of test sample stock solution:

[0092] Accurately weigh appropriate amounts of DMPC, cholesterol, DMG-PEG2000, DOPC, SM-102, DSPC, DODMA, ALC0315, and ALC0159 into a volumetric flask, add appropriate amount of anhydrous ethanol for ultrasonic dissolution, and make up to the scale with anhydrous ethanol to prepare a mother solution of the test sample containing approximately 500 μg DMPC, 750 μg cholesterol, 200 μg DMG-PEG2000, 500 μg DOPC, 1500 μg SM-102, 500 μg DSPC, 1500 μg DODMA, 1500 μg ALC0315, and 150 μg ALC0159 per 1 mL.

[0093] (2) Preparation of test solution:

[0094] According to the preparation process of the linear solution in the linear range investigation experiment, the stock solution of the test sample was diluted to three concentrations of L2, L3 and L4, with 3 test sample solutions for each concentration.

[0095] (3) According to the high performance liquid chromatography method in Example 1, the test solution and the linear solution in the linear range investigation experiment were respectively detected, and the measurement results were evaluated.

[0096] 2. Results Analysis

[0097] The accuracy test results of each lipid component are shown in Table 5 below:

[0098] Table 5 Accuracy test results of each lipid component

[0099]

[0100]

[0101]

[0102]

[0103] The results showed that the recovery rates of the lipid components in the test solution were all between 90% and 110%, and the RSDs of the recoveries were all less than 5%, indicating that the detection method had good accuracy.

[0104] 3. Repeatability of the detection method:

[0105] 1. Experimental methods

[0106] Take the four groups of LNP samples in Example 1, add 9 times anhydrous ethanol and shake to mix, as test samples, prepare 6 test samples for each group of LNP samples, and calculate the lipid concentration and RSD of each lipid component of each group of LNP samples.

[0107] 2. Results Analysis

[0108] The results of the repeatability test of lipid content in different LNP samples are shown in Table 6 below:

[0109] Table 6 Repeatability test results of lipid content in different LNP samples

[0110]

[0111]

[0112] The results showed that the RSD values ​​of each lipid component in each group of LNP samples were less than 5%, indicating that the detection method had good repeatability.

[0113] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the content of lipid components in lipid nanoparticles, characterized in that: Using high performance liquid chromatography in tandem with a detector to measure lipid composition and content in lipid nanoparticles, the lipid nanoparticles comprising cholesterol, auxiliary phospholipids, pegylated lipids, cationic lipids, anionic lipids and ionizable lipids; In the high performance liquid chromatography, the chromatographic column is Acclaim 300C18, with specifications of 150×2.1 mm, 3.0 um; the mobile phase A is triethylamine acetic acid aqueous solution, and the mobile phase B is triethylamine acetic acid methanol solution.

2. The method for detecting the content of lipid components in lipid nanoparticles according to claim 1, characterized in that: The high performance liquid chromatography adopts the gradient elution method, and the procedure is:

3. The method for detecting the content of lipid components in lipid nanoparticles according to claim 2, characterized in that: The high performance liquid chromatography adopts the gradient elution method, and the procedure is:

4. The method for detecting the content of lipid components in lipid nanoparticles according to claim 1, characterized in that: The detector is an evaporative light scattering detector or a charged aerosol detector.

5. The method for detecting the content of lipid components in lipid nanoparticles according to claim 4, characterized in that: The detection parameters of the charged aerosol detector are: filtration constant of 3.6-20s, atomization temperature of 30-70°C, and collection frequency of 2-20Hz.

6. The method for detecting the content of lipid components in lipid nanoparticles according to claim 5, characterized in that: The detection parameters of the charged aerosol detector are as follows: filtration constant of 10 s, atomization temperature of 50° C., and acquisition frequency of 10 Hz.

7. The method for detecting the content of lipid components in lipid nanoparticles according to claim 1, characterized in that: In the lipid nanoparticles: The auxiliary phospholipids include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE); The PEGylated lipids include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), Chol-PEG10000, and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159); The cationic lipids include (2,3-dioleoyl-propyl)trimethylamine (DOTAP) and 1,2-dioleyl-3-dimethylamino-propane (DODMA); The anionic lipids include distearoylphosphatidic acid (DSPA); The ionizable lipids include DLin-MC3-DMA, SM-102 and ALC0315.

8. The method for detecting the content of lipid components in lipid nanoparticles according to claim 1, characterized in that: The concentration of the ion pair reagent TEAA in the mobile phase A and the mobile phase B is 5-10 mM.

9. The method for detecting the content of lipid components in lipid nanoparticles according to claim 1, characterized in that: The column temperature of the chromatographic column is 30-60° C., and the flow rate is 0.4 mL / min.

10. The method for detecting the content of lipid components in lipid nanoparticles according to claim 1, characterized in that: The injection volume was 10-20 μL.

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