Methods for detecting RNA-LNP samples

Abstract

The present application provides a method for detecting RNA-LNP samples, the method comprising: detecting the content of RNA entity and RNA impurities in RNA-LNP samples by reversed-phase ion pair chromatography. The method can simultaneously identify and quantitatively determine various RNA impurities in RNA-LNP samples, thereby improving the comprehensiveness and efficiency of the analysis. In addition, the method only requires one ion pair reagent to achieve the separation of various RNA impurities, thereby simplifying the preparation process of the mobile phase, reducing the variable factors and potential risks in the detection process, and making the method more optimized and simple. The method is particularly suitable for the stability study of RNA in RNA-LNP samples, and provides reliable technical support and basis for the analysis and quality control of RNA-LNP preparations.

Description

Technical Field

[0001] The present application relates to the field of biological analysis. Specifically, the present application relates to a method for detecting RNA-LNP samples. Background Art

[0002] As one of the most widely studied and applied nucleic acid drugs in the field of biopharmaceuticals, RNA has been used in a variety of medical fields such as COVID-19 vaccines and tumor disease treatment. Currently, a number of RNA vaccines have been approved for marketing. However, due to the instability of RNA molecules themselves, they usually need to be encapsulated by lipid nanoparticle (LNP) nanocarriers before administration. However, factors such as the interaction between RNA and LNP components and the storage environment may still lead to RNA degradation and aggregation, thereby generating impurities such as RNA fragments and RNA adducts. Based on the above, the FDA defines RNA fragments and RNA adducts as impurities in RNA-LNP, and lists them as one of the key quality attributes of RNA-LNP drug products to ensure their in vivo efficacy and biological activity.

[0003] As the core carrier of RNA-LNP, LNP is composed of ionizable lipids, cholesterol, phospholipids and PEG. Studies have shown that the ionizable lipids in LNP are easily oxidized to form nitrogen oxides, and further hydrolyzed into secondary amines and aldehyde compounds under acid-base catalysis. These aldehyde compounds can covalently bind to RNA molecules to form RNA adduct impurities. In addition, RNA may also be degraded during storage and use due to factors such as exogenous nucleases, metal ions and solution pH, thereby generating RNA fragments. By analyzing the above impurities, the stability of RNA in the development and production of RNA-LNP preparations can be evaluated.

[0004] At present, the detection technologies used for the analysis of RNA and its impurities mainly include capillary electrophoresis (CE), gel electrophoresis and liquid chromatography. For RNA fragment impurities, due to their small molecular weight and size, full penetration may occur in size exclusion chromatography (SEC-HPLC) detection, resulting in failure to be detected. Based on reversed-phase ion pair chromatography, the counter ions of ion pair reagents can generate neutral ions with the analyte, enhancing the interaction between the solute and the non-polar stationary phase, and reversed-phase ion pair chromatography can be selected to analyze RNA fragments. RNA adduct impurities are formed by the covalent bonding of aldehyde small molecules to RNA. High-concentration salt solutions are used to neutralize the charge on the RNA chain. Based on the difference in hydrophobicity with the RNA itself, reversed-phase ion pair chromatography can be used to separate the RNA body from the RNA adduct impurities, thereby identifying the RNA adduct impurities.

[0005] At present, Arezou Azarani et al. have used reversed-phase ion pair chromatography and ion exchange chromatography analysis methods for RNA, but only RNA fragment impurities were identified, and quantitative analysis and methodological verification of impurities were not achieved. Therefore, the current analysis technology related to RNA impurities still has certain shortcomings, and comprehensive analysis of RNA impurities usually requires a combination of multiple methods. It can be seen that this field is in urgent need of a new type of analysis method that can comprehensively characterize the various impurity components of RNA and achieve effective quantitative detection of impurity components in RNA-LNP samples, so as to more efficiently achieve quality control of RNA-LNP samples. Summary of the invention

[0006] In view of the problems existing in the prior art, the present application provides a method for detecting RNA-LNP samples, the method comprising: detecting the content of RNA entity and RNA impurities in the RNA-LNP sample by reverse phase ion pair chromatography.

[0007] Beneficial effects:

[0008] The present application provides a method for detecting the content of RNA and RNA impurities in RNA-LNP samples by reversed-phase ion pair chromatography, which can simultaneously identify and quantitatively determine various RNA impurities in RNA-LNP samples, thereby improving the comprehensiveness and efficiency of the analysis. In addition, the method only requires one ion pair reagent to separate various RNA impurities, thereby simplifying the preparation process of the mobile phase, reducing the variable factors and potential risks in the detection process, and making the method more optimized and simple. The method is particularly suitable for the stability study of RNA in RNA-LNP samples, and provides reliable technical support and basis for the analysis and quality control of RNA-LNP preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The chromatogram of the mRNA reference product in Example 2 of the present application is shown;

[0010] Figure 2 shows the chromatogram overlay in Example 3 of the present application;

[0011] Figure 3 The figure shows the repeated chromatograms of three consecutive injections of the mRNA-LNP sample extract with an injection volume of 1 μg in Example 5 of the present application;

[0012] Figure 4 The chromatogram overlay of three consecutive injections of repetitive mRNA-LNP sample extract with an injection volume of 2 μg in Example 5 of the present application is shown.

[0013] Figure 5A chromatogram overlay is shown in Example 7 of the present application in which mobile phase A (first mobile phase) is a 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution and mobile phase B (second mobile phase) is a 100 mmol / L triethylamineacetic acid (TEAA) methanol (75% by volume) solution.

[0014] Figure 6 Shown is a chromatogram overlay in Example 8 of the present application, in which mobile phase A (first mobile phase) is an aqueous solution of 100 mmol / L triethylamineacetic acid (TEAA) and 50 mmol / L dibutylammonium acetate (DBAA), and mobile phase B (second mobile phase) is an acetonitrile (75% by volume) solution of 100 mmol / L triethylamineacetic acid (TEAA) and 50 mmol / L dibutylammonium acetate (DBAA). DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of the present application. The embodiments of the present application should not be interpreted as limiting the present application.

[0016] For simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0017] In the description of the present application, unless otherwise specified, “above” and “below” include the number.

[0018] Unless otherwise specified, the terms used in this application have the well-known meanings commonly understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the examples of this application).

[0019] A list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0020] The present application provides a method for detecting an RNA-LNP sample, the method comprising: detecting the content of RNA entity and RNA impurities in the RNA-LNP sample by reverse phase ion pair chromatography.

[0021] The present application provides a method for detecting the content of RNA and RNA impurities in RNA-LNP samples by reversed-phase ion pair chromatography, which can simultaneously identify and quantitatively determine various RNA impurities in RNA-LNP samples, thereby improving the comprehensiveness and efficiency of the analysis. In addition, the method only requires one ion pair reagent to separate various RNA impurities, thereby simplifying the preparation process of the mobile phase, reducing the variable factors and potential risks in the detection process, and making the method more optimized and simple. The method is particularly suitable for the stability study of RNA in RNA-LNP samples, and provides reliable technical support and basis for the analysis and quality control of RNA-LNP preparations.

[0022] In some embodiments of the present application, the above detection process is a gradient elution process, and the first mobile phase and the second mobile phase are used in the above gradient elution process, and the first mobile phase includes at least one of triethylamine acetic acid (TEAA), dibutylammonium acetate (DBAA) and hexafluoroisopropanol (HFP), and water; the second mobile phase includes at least one of triethylamine acetic acid (TEAA), dibutylammonium acetate (DBAA) and hexafluoroisopropanol (HFP), and an organic solvent, water, and the organic solvent includes at least one of methanol and acetonitrile. In the above detection process, the selected ion pair reagent can combine with the ions on the analyte to form a complex that can be effectively retained in the chromatographic column. Under the action of the above ion pair, the retention time of the target molecule on the chromatographic column is optimized, thereby improving the sensitivity and selectivity of the detection.

[0023] In addition, the mobile phase of the existing method needs to contain two ion pair reagents to identify RNA adduct impurities. Through this gradient elution process, only one ion pair reagent is needed to achieve the separation and quantification of RNA entity and various RNA impurities. While further improving the separation effect of target compounds, it also simplifies the preparation process of the mobile phase, reduces the variable factors and potential risks in the detection process, and makes the method more optimized and simple.

[0024] In some embodiments of the present application, the first mobile phase is an aqueous solution of triethylamineacetic acid, wherein the concentration of triethylamineacetic acid is 90 to 100 mmol / L; exemplarily, the concentration of triethylamineacetic acid may be selected from 90 mmol / L, 91 mmol / L, 92 mmol / L, 93 mmol / L, 94 mmol / L, 95 mmol / L, 96 mmol / L, 97 mmol / L, 98 mmol / L, 99 mmol / L, 100 mmol / L, or a range formed by any of the above values; exemplarily, the concentration of triethylamineacetic acid is preferably 100 mmol / L.

[0025] In some embodiments of the present application, the pH of the first mobile phase is 6.0 to 8.5. For example, the pH of the first mobile phase can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a range between any two of the above values.

[0026] In some embodiments of the present application, the second mobile phase is a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 90 to 100 mmol / L, and based on the volume of the mixture, the volume proportion of acetonitrile is 25% to 90%. Exemplarily, the concentration of triethylamineacetic acid can be selected as 90mmol / L, 91mmol / L, 92mmol / L, 93mmol / L, 94mmol / L, 95mmol / L, 96mmol / L, 97mmol / L, 98mmol / L, 99mmol / L, 100mmol / L, or a range formed by any of the above values; Exemplarily, the concentration of triethylamineacetic acid is preferably 100mmol / L; Exemplarily, the volume proportion of acetonitrile can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or a range formed by any of the above values; Exemplarily, the volume proportion of acetonitrile is preferably 75%.

[0027] In some embodiments of the present application, the second mobile phase is a mixture of triethylamine acetic acid, methanol and water, wherein the concentration of triethylamine acetic acid is 90-100mmol / L, and based on the volume of the above-mentioned mixture, the volume proportion of methanol is 25%-90%. Exemplarily, the concentration of triethylamine acetic acid can be selected as 90mmol / L, 91mmol / L, 92mmol / L, 93mmol / L, 94mmol / L, 95mmol / L, 96mmol / L, 97mmol / L, 98mmol / L, 99mmol / L, 100mmol / L, or the range formed by any of the above values; Exemplarily, the concentration of triethylamine acetic acid is preferably 100mmol / L; Exemplarily, the volume proportion of methanol can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or the range formed by any of the above values; Exemplarily, the volume proportion of methanol is preferably 75%.

[0028] In some embodiments of the present application, the first mobile phase is an aqueous solution of triethylamineacetic acid and dibutylammonium acetate, wherein the concentration of triethylamineacetic acid is 90-100mmol / L; exemplarily, the concentration of triethylamineacetic acid can be selected from 90mmol / L, 91mmol / L, 92mmol / L, 93mmol / L, 94mmol / L, 95mmol / L, 96mmol / L, 97mmol / L, 98mmol / L, 99mmol / L, 100mmol / L, or a range formed by any of the above values ​​... The concentration of ethylamineacetic acid is preferably 100mmol / L; the concentration of dibutylammonium acetate is 40-50mmol / L. Exemplarily, the concentration of dibutylammonium acetate can be selected as 40mmol / L, 41mmol / L, 42mmol / L, 43mmol / L, 44mmol / L, 45mmol / L, 46mmol / L, 47mmol / L, 48mmol / L, 49mmol / L, 50mmol / L, or a range formed by any of the above values; Exemplarily, the concentration of dibutylammonium acetate is preferably 50mmol / L.

[0029] In some embodiments of the present application, the second mobile phase is a mixture of triethylamine acetic acid, dibutylammonium acetate, acetonitrile and water, wherein the concentration of triethylamine acetic acid is 90-100 mmol / L, and the concentration of dibutylammonium acetate is 40-50 mmol / L. Exemplarily, the concentration of triethylamine acetic acid can be 90 mmol / L, 91 mmol / L, 92 mmol / L, 93 mmol / L, 94 mmol / L, 95 mmol / L, 96 mmol / L, 97 mmol / L, 98 mmol / L, 99 mmol / L, 100 mmol / L, or any of the above values. range formed; exemplary, the concentration of triethylamine acetic acid is preferably 100mmol / L; exemplary, the concentration of dibutylammonium acetate can be selected as 40mmol / L, 41mmol / L, 42mmol / L, 43mmol / L, 44mmol / L, 45mmol / L, 46mmol / L, 47mmol / L, 48mmol / L, 49mmol / L, 50mmol / L, or the range formed by any of the above values; exemplary, the concentration of dibutylammonium acetate is preferably 50mmol / L; based on the volume of the above mixed solution, the volume proportion of acetonitrile is 25% to 90%. Exemplarily, the volume proportion of acetonitrile can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or the range formed by any of the above values; exemplary, the volume proportion of acetonitrile is preferably 75%.

[0030] In some embodiments of the present application, the above-mentioned gradient elution process includes the following four stages:

[0031] In the 0th to T1th minute, the volume percentage of the second mobile phase is any value between 2% and 5%, wherein the volume percentage of the second mobile phase refers to the percentage of the second mobile phase to the sum of the volumes of the first mobile phase and the second mobile phase; T1 is any value between 0 and 3; illustratively, T1 can be 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 , 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, or a range between any two of the above values; illustratively, the volume percentage of the second mobile phase can be 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, or a range between any two of the above values;

[0032] In the period from T1 to T2 minutes, the volume percentage of the second mobile phase increases to any value between 25% and 30%, and T2 is any value between 3 and 15; illustratively, T2 can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a range between any two of the above values; illustratively, the volume percentage of the second mobile phase can be 25%, 25.2%, 25.4%, 25 .6%, 25.8%, 26.0%, 26.2%, 26.4%, 26.6%, 26.8%, 27.0%, 27.2%, 27.4%, 27.6%, 27.8%, 28.0%, 28.2%, 28.4%, 28.6%, 28.8%, 29.0%, 29.2%, 29.4%, 29.6%, 29.8%, 30.0%, or a range between any two of the above values;

[0033] In T2 to T3 minutes, the volume percentage of the second mobile phase increases to 100%, and T3 is any value between T2+0.5 and T2+2; illustratively, T3 can be T2+0.5, T2+0.6, T2+0.7, T2+0.8, T2+0.9, T2+1.0, T2+1.1, T2+1.2, T2+1.3, T2+1.4, T2+1.5, T2+1.6, T2+1.7, T2+1.8, T2+1.9, T2+2.0, or a range between any two of the above values;

[0034] From T3 to T4 minutes, the volume percentage of the second mobile phase is maintained at 100%, and T4 is any value between T3+9 and 15; illustratively, T4 can be T3+9, T3+9.5, T3+10, T3+10.5, T3+11, T3+11.5, T3+12, T3+12.5, T3+13, T3+13.5, T3+14, T3+14.5, T3+15, or a range between any two of the above values.

[0035] In the above gradient elution process, within the 0th to T1th minute, the shorter RNA fragment impurities can be retained, within the T1th to T2th minute, most of the RNA fragment impurities are separated from the RNA body, and within the T2th to T4th minute, the RNA adduct impurities are relatively aggregated and eluted together.

[0036] In some embodiments of the present application, the above-mentioned gradient elution process is preferably the following four stages:

[0037] In the 0th to 3rd minute, the volume percentage of the second mobile phase is constant at 2%, wherein the volume percentage of the second mobile phase refers to the percentage of the second mobile phase to the sum of the volumes of the first mobile phase and the second mobile phase;

[0038] From the 3rd to the 15th minute, the volume percentage of the second mobile phase is increased to any value between 25% and 30%;

[0039] During the 15th to 15.5th minute, the volume percentage of the second mobile phase is increased to 100%;

[0040] During the period from 15.5 to 25 minutes, the volume percentage of the second mobile phase was maintained at 100%.

[0041] In some embodiments of the present application, the above detection process is carried out in a high performance liquid chromatograph equipped with a reverse phase ion pair chromatography column, and the above chromatography column is selected from one of a C8 chromatography column, a C18 chromatography column and a polymer resin nucleic acid dedicated column.

[0042] In some embodiments of the present application, the chromatographic column is preferably a polymer resin nucleic acid column, which uses a polymer resin as a filler. Mobile phase A is a 100mmol / L triethylamine acetic acid (TEAA) aqueous solution; mobile phase B is a mixed solution of triethylamine acetic acid, acetonitrile and water, wherein the concentration of triethylamine acetic acid is 100mmol / L, and based on the volume of the above-mentioned mixed solution, the volume of acetonitrile accounts for 75%.

[0043] The present invention uses a polymer resin stationary phase chromatographic column specially used for nucleic acid analysis in combination with a specific mobile phase, thereby being able to achieve simultaneous separation of RNA bulk, RNA fragment impurities and RNA adduct impurities in an RNA-LNP sample, and quantitatively detecting the percentage of each component.

[0044] In some embodiments of the present application, the above-mentioned polymer resin nucleic acid dedicated column is preferably DNAPac TM RP, column length is 100 mm, inner diameter is 2.1 mm, and pore size is 4 μm.

[0045] In some embodiments of the present application, the above-mentioned C8 chromatographic column is preferably a PhenomenexLuna C8 column produced by Luna, whose stationary phase is C8 silyl bonded to the silica gel surface, with a column length of 250 mm, an inner diameter of 4.6 mm, and a pore size of 5 μm.

[0046] In some embodiments of the present application, the above detection process is carried out in a high performance liquid chromatograph equipped with a reverse phase ion pair chromatography column, and the detection conditions are as follows:

[0047] The column temperature is preferably 5°C to 110°C, the injection volume is preferably 1μg to 2μg, the flow rate is preferably 0.2mL / min to 0.5mL / min, the pH range can be 0 to 14, the detector is a diode array detector, and the detection wavelength is 260nm. Exemplarily, the column temperature can be 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, or a range between any two of the above values. Exemplarily, the column temperature is preferably 65°C. Exemplarily, the injection volume is preferably 2μg. Exemplarily, the flow rate is preferably 0.35mL / min.

[0048] In some embodiments of the present application, the method further comprises extracting the RNA entity and RNA impurities from the RNA-LNP sample by ammonium acetate isopropanol precipitation method.

[0049] In some embodiments of the present application, the method comprises treating the RNA-LNP sample with an ammonium acetate isopropanol solution to obtain a precipitate, and treating the precipitate with isopropanol to obtain the RNA entity and RNA impurities. Exemplarily, the operation is performed on ice.

[0050] Among them, ammonium acetate isopropanol solution can destroy the LNP structure and remove the hydration layer on the RNA surface through the combined effects of charge interference, solvent property changes, ionic strength effects, and the lipophilicity of isopropanol, and then precipitate the RNA with isopropanol. Operating on ice can maintain a lower temperature, making the precipitation more uniform and slow, and improving the precipitation efficiency and purity of RNA.

[0051] In some embodiments of the present application, the ammonium acetate isopropanol solution is centrifuged to treat the RNA-LNP sample, wherein the centrifugation conditions are: a rotation speed of 13000 to 16000 rpm and a centrifugation time of 10 to 30 minutes. Exemplarily, the rotation speed may be 13000 rpm, 13500 rpm, 14000 rpm, 14500 rpm, 15000 rpm, 15500 rpm, 16000 rpm, or a range between any two of the above values. Exemplarily, the centrifugation time may be 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or a range between any two of the above values. Exemplarily, the rotation speed is preferably 14000 rpm and the centrifugation time is preferably 15 minutes.

[0052] In some embodiments of the present application, the above-mentioned isopropanol is processed by centrifugation to treat the above-mentioned precipitation, wherein the conditions of the above-mentioned centrifugation are: a rotation speed of 13000-16000rpm and a centrifugation time of 10-30 minutes. Exemplarily, the rotation speed can be 13000rpm, 13500rpm, 14000rpm, 14500rpm, 15000rpm, 15500rpm, 16000rpm, or a range between any two of the above values. Exemplarily, the centrifugation time can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or a range between any two of the above values. Exemplarily, the rotation speed is preferably 14000rpm and the centrifugation time is preferably 15 minutes.

[0053] In some embodiments of the present application, in the ammonium acetate isopropanol solution, the concentration of ammonium acetate is 48-72 mmol / L. Exemplarily, the concentration of ammonium acetate can be 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, 56 mmol / L, 57 mmol / L, 58 mmol / L, 59 mmol / L, 60 mmol / L, 61 mmol / L, 62 mmol / L, 63 mmol / L, 64 mmol / L, 65 mmol / L, 66 mmol / L, 67 mmol / L, 68 mmol / L, 69 mmol / L, 70 mmol / L, 71 mmol / L, 72 mmol / L, or a range between any two of the above values. Exemplarily, the concentration of ammonium acetate is preferably 60 mmol / L, that is, 0.4625 g of ammonium acetate is accurately weighed and dissolved in 100 mL of isopropanol solution, and after mixing, an ammonium acetate isopropanol solution of the desired concentration can be prepared.

[0054] In some embodiments of the present application, the volume ratio of the ammonium acetate isopropanol solution to the RNA-LNP sample is 1:(10-20). Exemplarily, the volume ratio of the ammonium acetate isopropanol solution to the RNA-LNP sample can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, or a range between any two of the above values.

[0055] In some embodiments of the present application, the RNA-LNP sample is selected from one of an mRNA-LNP sample and a siRNA-LNP sample.

[0056] In some embodiments of the present application, the above-mentioned RNA impurities include at least one of RNA fragment impurities and RNA adduct impurities; RNA entity refers to the effective RNA in the RNA-LNP sample.

[0057] Examples and Comparative Examples

[0058] Hereinafter, the present application will be described in more detail with reference to Examples and Comparative Examples, but the present application is not limited to these Examples unless departing from the gist of the present application.

[0059] The present invention takes mRNA-LNP samples as an example, and detects the contents of mRNA entity and various mRNA impurities in the mRNA-LNP samples by the method of the present invention.

[0060] The information of the instruments and reagents used in the embodiments of the present invention are as follows:

[0061] 1. Reagent Information

[0062] The preparation method of the mRNA-LNP sample is as follows: mRNA standards and different lipids are synthesized into mRNA-LNP by microfluidic technology, and then the mRNA-LNP sample is obtained by changing the liquid and concentrating.

[0063] The LNP samples were synthesized by microfluidic technology using SM102, CHOL, DSPC, and PEG2000-DMG in a certain proportion.

[0064] For other reagent information, please see Table 1 below.

[0065] Table 1.

[0066]

[0067]

[0068] 2. Instrument information

[0069] Table 2.

[0070] name model High performance liquid chromatography Agilent1260 High Throughput Concentration Particle Size Analyzer Stunner Vacuum Concentrator SPD2030-230

[0071] Example 1 Verification of whether the mRNA extraction process causes damage to mRNA

[0072] 1. Methods

[0073] (1) Extraction of mRNA from mRNA standards

[0074] Take 40 μL of 1 mg / mL mRNA standard and add it to 360 μL of 60 mmol / L ammonium acetate isopropanol solution and vortex to mix. Then, centrifuge at 14000 rpm for 15 minutes at 4°C, discard the supernatant, and collect the precipitate;

[0075] 400 μL of isopropanol was added to the precipitate, vortexed again, and centrifuged at 14,000 rpm for 15 min at 4 °C;

[0076] After centrifugation, the precipitate was dried in a vacuum concentrator, resuspended in 80 μL of DEPC water, and mixed thoroughly.

[0077] (2) Determination of mRNA concentration after extraction

[0078] The concentration of extracted mRNA was determined using a Stunner high-throughput particle size analyzer, and the sample was diluted with DEPC water to the same concentration as the unextracted mRNA standard for liquid chromatography analysis.

[0079] Chromatographic conditions:

[0080] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 2 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0081] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0082] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0083] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0084] Table 3. RP-HPLC method parameter settings

[0085]

[0086] 2. Experimental results

[0087] The treated mRNA standards were compared with the untreated mRNA standards, and the specific results are shown in Table 4.

[0088] Table 4. Peak area percentage results of mRNA standards before and after treatment

[0089]

[0090]

[0091] As can be seen from Table 4, by comparing the mRNA standards before and after treatment, there is no significant change in the peak area percentage, indicating that the extraction method of the present invention does not cause significant damage to mRNA.

[0092] Example 2: Determination of Chromatographic Column Efficiency

[0093] 1. Methods

[0094] Preparation of mRNA reference: Take 10 μL of mRNA reference, incubate at 70°C for 2 minutes, dilute 5 times with DEPC water, and mix well.

[0095] Detection: The above samples were detected using high performance liquid chromatography.

[0096] Chromatographic conditions:

[0097] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 0.6 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0098] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0099] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0100] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0101] Table 5. RP-HPLC method parameter settings

[0102]

[0103] 2. Experimental results

[0104] Test results such as Figure 1 The chromatogram shown. Figure 1 In the test, the separation degree of 0.5k and 0.2k chromatographic peaks is not less than 2.0, the symmetry factor of 0.5k chromatographic peak is not greater than 1.0, and the number of theoretical plates is greater than or equal to 2000, which meets the column efficiency requirements, indicating that the chromatographic column performance is qualified.

[0105] Example 3 Specificity Verification

[0106] 1. Methods

[0107] (1) Blank solution: DEPC water was used as the blank solution.

[0108] (2) mRNA standard solution: Take 20 μL of mRNA standard, add 180 μL of DEPC water, and vortex to mix to obtain the mRNA standard solution.

[0109] (3) mRNA-LNP extract (sample placed at 4°C): Take 800 μL of mRNA-LNP sample, add it to 7.2 mL of 60 mmol / L ammonium acetate isopropanol solution, vortex mix, centrifuge at 14000 rpm at 4°C for 15 min, discard the supernatant, and collect the precipitate; add 8 mL of isopropanol to the precipitate, vortex mix again, centrifuge at 14000 rpm at 4°C for 15 min, place in a vacuum concentrator to dry, and finally resuspend in 160 μL of DEPC water and mix.

[0110] The concentration of the extracted mRNA was determined using a Stunner high-throughput particle size analyzer, and diluted to 100 μg / mL with DEPC water for liquid chromatography detection.

[0111] (4) Empty LNP solution: Take 500 μL of empty LNP sample, add it to 4.5 mL of 60 mmol / L ammonium acetate isopropanol solution, vortex to mix, centrifuge at 14000 rpm at 4°C for 15 minutes, and discard the supernatant. Add 5 mL of isopropanol to the precipitate, vortex to mix again, centrifuge at 14000 rpm at 4°C for 15 minutes, dry it in a vacuum concentrator, and finally resuspend it with 100 μL of DEPC water and dilute it to the same multiple as the mRNA-LNP extract for liquid chromatography detection.

[0112] (5) Detection: All the above samples were detected by high performance liquid chromatography.

[0113] Chromatographic conditions:

[0114] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 2 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0115] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0116] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0117] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0118] Table 6. RP-HPLC method parameter settings

[0119]

[0120] 2. Experimental results

[0121] Test results such as Figure 2 The chromatograms shown are overlaid. Figure 2 In the results, there was no interference between the blank solution and the unloaded LNP solution at the main peak position, indicating that this method has good specificity.

[0122] Example 4 Extraction recovery rate investigation

[0123] 1. Methods

[0124] (1) Blank solution: DEPC water was used as the blank solution.

[0125] (2) mRNA standard solution: Take 20 μL of mRNA standard, add 180 μL of DEPC water, and mix well.

[0126] (3) mRNA standard extract: Take 20 μL of mRNA standard and add it to 180 μL of 60 mmol / L ammonium acetate isopropanol solution, vortex to mix, centrifuge at 14000 rpm at 4°C for 15 min, discard the supernatant and collect the precipitate; add 200 μL of isopropanol to the precipitate, vortex to mix again, centrifuge at 14000 rpm at 4°C for 15 min, place in a vacuum concentrator to dry, and finally resuspend in 80 μL of DEPC water and mix.

[0127] (4) Detection: The concentration of the extracted mRNA was measured using a Stunner high-throughput concentration particle size analyzer and detected using a high-performance liquid chromatography.

[0128] Chromatographic conditions:

[0129] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 2 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0130] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0131] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0132] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0133] Table 7. RP-HPLC method parameter settings

[0134]

[0135] 2. Experimental results

[0136] The extraction recovery results are shown in Tables 8 and 9.

[0137] Table 8. Extraction recovery rates detected by Stunner high-throughput concentration particle size analyzer

[0138]

[0139]

[0140] Table 9. Liquid chromatography extraction recovery

[0141] name Total peak area Peak area recovery (%) mRNA Standard Stock Solution 9614.54 NA mRNA Standard Extract-1 9209.33 95.79 mRNA Standard Extract-2 9781.71 101.74 mRNA Standard Extract-3 9453.98 98.33

[0142] According to the test results in Tables 8 and 9, the mRNA extraction recovery rates measured by Stunner high-throughput concentration particle size analyzer and liquid chromatography were between 90% and 110%, indicating that the recovery rate of this extraction method was within an acceptable range, ensuring the reliability and consistency of mRNA sample recovery.

[0143] Example 5 Investigation of sample injection volume range

[0144] 1. Methods

[0145] In this example, mRNA in mRNA standards and mRNA-LNP samples is detected, and the specific operation steps are as follows:

[0146] (1) Preparation of mRNA standard solution: Take 10 μL of mRNA standard, add 190 μL of DEPC water, and vortex to mix.

[0147] (2) Preparation of mRNA extract from mRNA-LNP samples (40°C stability samples): mRNA-LNP samples are composed of cationic lipids, cholesterol, polyethylene glycol (PEG) and EGFP mRNA, and are prepared by microfluidic technology.

[0148] Take 1mL of mRNA-LNP sample and add it to 9mL of 60mmol / L ammonium acetate isopropanol solution and vortex to mix. Then, centrifuge at 14000rpm at 4℃ for 15 minutes, discard the supernatant and collect the precipitate. Add 10mL of isopropanol to the precipitate, vortex to mix again, and centrifuge at 14000rpm at 4℃ for 15 minutes. After centrifugation, place the precipitate in a vacuum concentrator to dry, resuspend it in 200μL of DEPC water, and mix it thoroughly.

[0149] The concentration of extracted mRNA was measured using a Stunner high-throughput particle size analyzer.

[0150] (3) Injection volume investigation: The mRNA standard and mRNA-LNP sample extracts were subjected to liquid chromatography detection, respectively. The injection volume of the mRNA standard was 1 μg, and the injection volume of the mRNA-LNP sample extract was 1 μg and 2 μg, respectively, to evaluate the applicable injection volume range.

[0151] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with detection at 260 nm on a diode array detector, using a gradient elution mode.

[0152] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0153] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0154] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0155] Table 10. RP-HPLC method parameter settings

[0156]

[0157] 2. Experimental results

[0158] (1) For the mRNA-LNP sample extract, when the injection volume was 1 μg and 2 μg, 3 injections were performed continuously for each injection volume. The results are shown in Tables 11 and 12.

[0159] Table 11. Peak area percentage of mRNA-LNP sample extract (injection volume 1 μg)

[0160]

[0161]

[0162] Table 12. Peak area percentage of mRNA-LNP sample extract (injection volume 2 μg)

[0163]

[0164] The liquid chromatogram of the extract (injection volume 1 μg) of the mRNA-LNP sample (40°C stability sample) injected three times continuously is shown in Figure 3 The liquid chromatogram of the extract (injection volume 2 μg) of the mRNA-LNP sample (40°C stability sample) injected for 3 consecutive times is shown in Figure 4The results showed that under the conditions of injection volume of 1 μg and 2 μg, the relative standard deviation (RSD%) of the peak area percentage of mRNA, mRNA fragment impurities and mRNA adduct impurities in three consecutive injections of mRNA-LNP sample extract was less than 5.0%. This shows that the injection volume range of this method has good repeatability and accuracy.

[0165] Example 6 Precision Investigation

[0166] 1. Methods

[0167] (1) Blank solution: DEPC water was used as the blank solution.

[0168] (2) Preparation of mRNA-LNP extract: Take 800 μL of mRNA-LNP sample, add it to 7.2 mL of 60 mmol / L ammonium acetate isopropanol solution, vortex to mix, centrifuge at 14000 rpm at 4°C for 15 minutes, discard the supernatant, and collect the precipitate. Add 8 mL of isopropanol to the precipitate, vortex to mix again, and centrifuge at 14000 rpm at 4°C for 15 minutes. After centrifugation, place the precipitate in a vacuum concentrator to dry, resuspend it in 160 μL of DEPC water, and mix it thoroughly to obtain the mRNA-LNP extract.

[0169] (3) Concentration determination and detection: The concentration of mRNA in the extract was determined using a Stunner high-throughput concentration particle size analyzer, diluted to 100 μg / mL, divided into 6 parallel samples, and subjected to liquid chromatography detection.

[0170] Chromatographic conditions:

[0171] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 2 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0172] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0173] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0174] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0175] Table 13. RP-HPLC method parameter settings

[0176]

[0177] 2. Experimental results

[0178] The results of precision investigation are shown in Table 15.

[0179] Table 15. Sample precision investigation

[0180]

[0181]

[0182] The above results show that the precisions of mRNA entity, mRNA fragment impurities and mRNA adduct impurities are 0.3%, 7.2% and 4.1%, respectively, all within 10.0%, indicating that this method has good precision in determining the content of RNA entity and various RNA impurities in mRNA-LNP samples.

[0183] Example 7 Investigation of mobile phase components 1

[0184] 1. Methods

[0185] (1) Blank solution: DEPC water was used as the blank solution.

[0186] (2) Standard solution: Take 20 μL of the standard, dilute it to 100 μg / mL with DEPC water, and mix well.

[0187] (3) Preparation of mRNA-LNP extract: Take 800 μL of mRNA-LNP sample, add it to 7.2 mL of 60 mmol / L ammonium acetate isopropanol solution, vortex to mix, centrifuge at 14000 rpm at 4°C for 15 minutes, discard the supernatant, and collect the precipitate. Add 8 mL of isopropanol to the precipitate, vortex to mix again, and centrifuge at 14000 rpm at 4°C for 15 minutes. After centrifugation, place the precipitate in a vacuum concentrator to dry, resuspend it in 160 μL of DEPC water, and mix it thoroughly to obtain the mRNA-LNP extract.

[0188] (4) Concentration determination and detection: The concentration of mRNA in the extract was determined using a Stunner high-throughput concentration particle size analyzer, diluted to 100 μg / mL, and then detected by liquid chromatography.

[0189] Chromatographic conditions:

[0190] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 2 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0191] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0192] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) aqueous solution.

[0193] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, methanol and water, the concentration of triethylamineacetic acid is 100 mmol / L, and the volume proportion of methanol is 75% based on the volume of the mixture.

[0194] Table 16. RP-HPLC method parameter settings

[0195]

[0196] 2. Experimental results

[0197] Test results such as Figure 5 The chromatograms shown are overlaid. Figure 5 In this method, mRNA fragment impurities and mRNA adduct impurities can be detected simultaneously.

[0198] Example 8 Investigation of mobile phase components 2

[0199] 1. Methods

[0200] (1) Blank solution: DEPC water was used as the blank solution.

[0201] (2) Standard solution: Take 20 μL of the standard, dilute it to 100 μg / mL with DEPC water, and mix well.

[0202] (3) Preparation of mRNA-LNP extract: Take 800 μL of mRNA-LNP sample, add it to 7.2 mL of 60 mmol / L ammonium acetate isopropanol solution, vortex to mix, centrifuge at 14000 rpm at 4°C for 15 minutes, discard the supernatant, and collect the precipitate. Add 8 mL of isopropanol to the precipitate, vortex to mix again, and centrifuge at 14000 rpm at 4°C for 15 minutes. After centrifugation, place the precipitate in a vacuum concentrator to dry, resuspend it in 160 μL of DEPC water, and mix it thoroughly to obtain the mRNA-LNP extract.

[0203] (4) Concentration determination and detection: The concentration of mRNA in the extract was determined using a Stunner high-throughput concentration particle size analyzer, diluted to 100 μg / mL, and then detected by liquid chromatography.

[0204] Chromatographic conditions:

[0205] The RP-HPLC (reversed phase ion-pair high performance liquid chromatography) method is shown below, with 2 μg loading, detection at 260 nm on a diode array detector, using a gradient elution mode.

[0206] Chromatographic column: DNA PacTM RP, column length 100 mm, inner diameter 2.1 mm, pore size 4 μm.

[0207] Mobile phase A (first mobile phase): 100 mmol / L triethylamineacetic acid (TEAA) and 50 mmol / L dibutylammonium acetate (DBAA) aqueous solution.

[0208] Mobile phase B (second mobile phase): a mixture of triethylamineacetic acid, dibutylammonium acetate, acetonitrile and water, wherein the concentration of triethylamineacetic acid is 100 mmol / L, the concentration of dibutylammonium acetate is 50 mmol / L, and the volume proportion of acetonitrile is 75% based on the volume of the mixture.

[0209] Table 17. RP-HPLC method parameter settings

[0210]

[0211] 2. Experimental results

[0212] Test results such as Figure 6 The chromatograms shown are overlaid. Figure 6 In this method, mRNA fragment impurities and mRNA adduct impurities can be detected simultaneously.

[0213] The above-mentioned embodiments of the present application take mRNA-LNP samples as an example for detection, and achieve the technical effect of effectively separating and accurately quantifying the mRNA entity, mRNA fragment impurities and mRNA adduct impurities. The method of the above-mentioned embodiments of the present application is also applicable to the detection of siRNA-LNP samples, except that the column temperature applicable to siRNA-LNP samples is 25°C, and other detection conditions are the same as those of mRNA-LNP samples. The present invention anticipates that the method of the above-mentioned embodiments can effectively separate and accurately quantify the siRNA entity, siRNA fragment impurities and siRNA adduct impurities.

[0214] Although some exemplary embodiments of the present application have been illustrated and described, the present application is not limited to the disclosed embodiments. On the contrary, those skilled in the art will recognize that some modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present application as described in the appended claims.

Claims

1. A method for detecting RNA-LNP samples, characterized in that, The method comprises: The contents of RNA entity and RNA impurities in the RNA-LNP sample are detected by reverse phase ion pair chromatography.

2. The method according to claim 1, characterized in that The detection process is a gradient elution process, in which a first mobile phase and a second mobile phase are used, wherein the first mobile phase includes at least one of triethylamine acetic acid, dibutylammonium acetate and hexafluoroisopropanol, and water; the second mobile phase includes at least one of triethylamine acetic acid, dibutylammonium acetate and hexafluoroisopropanol, and an organic solvent, water, wherein the organic solvent includes at least one of methanol and acetonitrile.

3. The method according to claim 2, characterized in that The first mobile phase is an aqueous solution of triethylamine acetic acid, wherein the concentration of the triethylamine acetic acid is 90 to 100 mmol / L, and / or the pH of the first mobile phase is 6.0 to 8.5; and / or, The second mobile phase is a mixture of triethylamine acetic acid, an organic solvent and water, wherein the concentration of the triethylamine acetic acid is 90 to 100 mmol / L, based on the volume of the mixture, the volume proportion of the acetonitrile is 25% to 90%, and the organic solvent includes at least one of methanol or acetonitrile.

4. The method according to claim 2, characterized in that: The first mobile phase is an aqueous solution of triethylamine acetic acid and dibutylammonium acetate, wherein the concentration of the triethylamine acetic acid is 90-100 mmol / L, the concentration of the dibutylammonium acetate is 40-50 mmol / L, and the pH of the first mobile phase is 6.0-8.5; The second mobile phase is a mixture of triethylamine acetic acid, dibutylammonium acetate, acetonitrile and water, wherein the concentration of the triethylamine acetic acid is 90-100 mmol / L, the concentration of the dibutylammonium acetate is 40-50 mmol / L, and based on the volume of the mixed solution, the volume proportion of the acetonitrile is 25-90%.

5. The method according to claim 2, characterized in that: The gradient elution process includes the following four stages: In the 0th to T1th minute, the volume percentage of the second mobile phase is any value between 2% and 5%, wherein the volume percentage of the second mobile phase refers to the percentage of the second mobile phase to the sum of the volumes of the first mobile phase and the second mobile phase; T1 is any value between 0 and 3; During the period from T1 to T2 minutes, the volume percentage of the second mobile phase increases to any value between 25% and 30%, and T2 is any value between 3 and 15; From T2 to T3 minutes, the volume percentage of the second mobile phase increases to 100%, and T3 is any value between T2+0.5 and T2+2; From T3 to T4 minutes, the volume percentage of the second mobile phase is maintained at 100%, and T4 is any value between T3+9 and T3+15.

6. The method according to any one of claims 1 to 5, characterized in that: The method comprises treating the RNA-LNP sample with an ammonium acetate isopropanol solution to obtain a precipitate, and adding isopropanol to treat the precipitate to obtain the RNA entity and the RNA impurities.

7. The method according to claim 6, characterized in that The ammonium acetate isopropanol solution is used to treat the RNA-LNP sample by centrifugation, wherein the centrifugation conditions are: a rotation speed of 13000-16000 rpm and a centrifugation time of 10-30 minutes; and / or, The isopropanol is used to treat the precipitate by centrifugation, wherein the conditions of the centrifugation are: a rotation speed of 13000-16000 rpm and a centrifugation time of 10-30 minutes.

8. The method according to claim 6, characterized in that In the ammonium acetate isopropanol solution, the concentration of ammonium acetate is 48-72 mmol / L; and / or, The volume ratio of the ammonium acetate isopropanol solution to the RNA-LNP sample is 1:(10-20).

9. The method according to claim 1, characterized in that: The RNA-LNP sample is selected from one of an mRNA-LNP sample and a siRNA-LNP sample.

10. The method according to claim 1 or 9, characterized in that: The RNA impurities include at least one of RNA fragment impurities and RNA adduct impurities; the RNA entity refers to the effective RNA in the RNA-LNP sample.

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